TW202509219A - Cytokine encoding lentiviral vectors and uses thereof for making tumor infiltrating lymphocytes - Google Patents

Abstract

Provided herein are cytokine-encoding nucleic acid molecules, such as recombinant expression vectors, and uses thereof to make modified tumor infiltrating lymphocytes (TILs), wherein the modified TILs include one or more immunomodulatory agents ( e.g., cytokines) associated with their cell surface. The immunomodulatory agents associated with the TILs provide a localized immunostimulatory effect that can advantageously enhance TIL survival, proliferation and/or anti-tumor activity in a patient recipient. As such, the compositions and methods disclosed herein provide effective cancer therapies.

Description

Lentiviral vector encoding interleukin and its use in preparing tumor-infiltrating lymphocytes

The present invention relates to a lentiviral vector encoding interleukin and its use in preparing tumor-infiltrating lymphocytes.

TIL cell therapy has shown clinical benefits for patients with solid tumors (Chesney et al., Journal for ImmunoTherapy of Cancer 2022 ; 10: e005755; 2. Schoenfeld et al., SITC Annual Meeting 2021 ). However, the immunosuppressive tumor microenvironment (TME) may eliminate the full potential of TIL cell therapy (Granhøj et al., Expert Opin Biol Th. 2022 ; 1-15). The proinflammatory interleukin IL-12 is known for its ability to increase IFN-γ production and promote type 1 immune responses, which can reshape the TME and has the potential to enhance anti-tumor activity.

Current TIL expansion processes generate tumor-specific TIL (TS-TIL) and bystander TIL in the expanded TIL product. The frequency of neoantigen-specific pure lines in the TIL infusion product and their ability to mobilize during in vitro expansion are associated with the efficacy of TIL in melanoma (Kristensen NP et al., J Clin. Invest. 2022 ; 132: e150535; Chiffelle, J. et al., bioRxiv 2023 ; the contents of which are incorporated herein by reference in their entirety). Bystander T cells can have Treg-like effects, stealing IL-2 and homeostatic interleukins from effector TS-TIL. During REP, bystander TILs also outcompete TS-TILs for nutrients and oxygen, thus reducing their total number, because: a) bystander TILs tend to be younger and healthier (less differentiated) T cells, and therefore have greater proliferative potential; b) TIL growth depends on the metabolic and phenotypic state of the TILs, i.e., the expression of co-stimulatory receptors. Bystander TILs take up valuable tumor space after donor-acceptor transfer and create a "traffic jam" to reach the tumor. Allen et al., Science , 2022 , 378, eaba1624.

Therefore, there is still a need for improved TIL therapy to treat cancer. This article discloses gene-edited TILs with inducible and membrane-bound IL-12, which show excellent cytotoxic function. The present invention further provides an improved expansion method for preferentially generating TS-TILs rather than bystander TILs.

Some embodiments of the present disclosure provide a nucleic acid molecule comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT)-responsive promoter.

Some embodiments of the present disclosure provide a nucleic acid molecule comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT)-responsive promoter, and wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62.

Some embodiments of the present disclosure provide a nucleic acid molecule comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT)-responsive promoter, and a nucleotide sequence encoding tethered IL-15 (TeIL-15), wherein the TeIL-15 is under the control of an EF1α promoter.

Some embodiments of the present disclosure provide a nucleic acid molecule comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT)-responsive promoter, and a nucleotide sequence encoding tethered IL-15 (TeIL-15), wherein the TeIL-15 is under the control of an EF1α promoter, and wherein the TeIL-15 has an amino acid sequence of SEQ ID NO:73.

In some embodiments, TeIL-12 comprises a membrane anchor and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit. In some embodiments, the human IL-12 p35 subunit has an amino acid sequence of SEQ ID NO: 60 and the human IL-12 p40 subunit has an amino acid sequence of SEQ ID NO: 61. In some embodiments, TeIL-12 comprises an amino acid sequence as shown in SEQ ID NO: 62. In some embodiments, the nucleotide sequence encoding TeIL-12 is shown in SEQ ID NO: 162. In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding an interleukin selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN γ, TNFa, IFN α, IFN β, GM-CSF, GCSF or variants thereof. In some embodiments, the interleukin is under the control of a constitutive promoter. In some embodiments, the constitutive promoter is selected from the group consisting of EF1α promoter, CMV promoter, CAG promoter, MND promoter and SSFV promoter. In some embodiments, the interleukin is under the control of a constitutive promoter. In some embodiments, the constitutive promoter is an EF1α promoter. In some embodiments, the interleukin is IL-15 or a variant thereof. In some embodiments, IL-15 is human IL-15. In some embodiments, human IL-15 has an amino acid sequence of SEQ ID NO:64. In some embodiments, IL-15 is tethered IL-15 (TeIL-15). In some embodiments, TeIL-15 comprises a membrane anchor and human IL-15. In some embodiments, TeIL-15 has an amino acid sequence of SEQ ID NO:73. In some embodiments, the interleukin is IL-2 or a variant thereof. In some embodiments, IL-2 is human IL-2. In some embodiments, human IL-2 has an amino acid sequence of SEQ ID NO:138. In some embodiments, IL-2 is tethered IL-2 (TeIL-2). In some embodiments, TeIL-2 has the amino acid sequence of SEQ ID NO: 139. In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a truncated CD19 (tCD19). In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 148-150. In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding shRNA. In some embodiments, shRNA inhibits the expression of immune checkpoint genes. In some embodiments, the immune checkpoint genes are selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD9 6. CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the shRNA is PD-1 shRNA. In some embodiments, the PD-1 shRNA comprises the nucleic acid sequence shown in SEQ ID NO: 141-147. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 151.

Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleic acid molecule disclosed herein.

Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT)-responsive promoter.

Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT)-responsive promoter, and wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62.

Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT)-responsive promoter, and a nucleotide sequence encoding tethered IL-15 (TeIL-15), wherein the TeIL-15 is under the control of an EF1α promoter.

Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT)-responsive promoter, and a nucleotide sequence encoding tethered IL-15 (TeIL-15), wherein the TeIL-15 is under the control of an EF1α promoter, and wherein the TeIL-15 has an amino acid sequence of SEQ ID NO:73.

Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:167.

In some embodiments, the vector is a transfer vector. In some embodiments, the transfer vector is derived from human immunodeficiency virus-1 (HIV-1) and is replication-deficient. In some embodiments, the transfer vector is a pLenti-IRES-EGFP vector.

Some embodiments of the present disclosure provide a lentiviral expression system comprising a recombinant expression vector disclosed herein and one or more accessory plasmids encoding Env protein, Gag protein, Pol protein, and Rev protein. In some embodiments, the lentiviral expression system comprises an accessory plasmid encoding Env protein, Gag protein, Pol protein, and Rev protein. In some embodiments, the lentiviral expression system comprises an accessory plasmid encoding Env protein and an accessory plasmid encoding Gag protein, Pol protein, and Rev protein. In some embodiments, the lentiviral expression system comprises an accessory plasmid encoding Env protein, Gag protein, and Pol protein, and an accessory plasmid encoding Rev protein. In some embodiments, the lentiviral expression system comprises an accessory plasmid encoding Env protein and Rev protein and an accessory plasmid encoding Gag protein and Pol protein. In some embodiments, the Env protein is selected from the group consisting of: Ba-EVTR, VSV-G and RD114. In some embodiments, the Env protein comprises Ba-EVTR protein.

Some embodiments of the present disclosure provide a method for producing recombinant lentiviral particles, comprising: a) culturing a packaging cell population in a cell culture medium; b) contacting the packaging cell population with a lentiviral expression system disclosed herein; and c) harvesting a supernatant of the cell culture medium, wherein the supernatant contains recombinant lentiviral particles.

Some embodiments of the present disclosure provide a recombinant lentiviral RNA molecule comprising a nucleic acid molecule disclosed herein.

Some embodiments of the present disclosure provide a recombinant lentiviral proviral DNA molecule comprising a nucleic acid molecule disclosed herein.

Some embodiments of the present disclosure provide a recombinant lentiviral particle comprising a recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the recombinant lentiviral particle is produced by a packaging cell line disclosed herein. In some embodiments, the recombinant lentiviral particle comprises an Env protein selected from the group consisting of: Ba-EVTR, VSV-G, and RD114. In some embodiments, the recombinant lentiviral particle comprises an Env protein that is Ba-EVTR. In some embodiments, the recombinant lentiviral particle further comprises a reverse transcriptase. In some embodiments, the recombinant lentiviral particle further comprises a shell that encapsulates the recombinant lentiviral RNA molecule.

Some embodiments of the present disclosure provide a gene-edited tumor-infiltrating lymphocyte (TIL) comprising the recombinant lentiviral proviral DNA disclosed herein. In some embodiments, the recombinant lentiviral proviral DNA is integrated into the genome of the gene-edited TIL.

Some embodiments of the present disclosure provide a gene-edited tumor-infiltrating lymphocyte (TIL) that expresses exogenous tethered IL-12 (TeIL-12).

Some embodiments of the present disclosure provide a gene-edited tumor-infiltrating lymphocyte (TIL) expressing exogenous tethered IL-12 (TeIL-12), wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62.

Some embodiments of the present disclosure provide a gene-edited tumor-infiltrating lymphocyte (TIL) that expresses exogenous tethered IL-12 (TeIL-12) and exogenous tethered IL-15 (TeIL-15).

Some embodiments of the present disclosure provide a gene-edited tumor-infiltrating lymphocyte (TIL) that expresses exogenous tethered IL-12 (TeIL-12) and exogenous tethered IL-15 (TeIL-15), wherein the TeIL-15 has an amino acid sequence of SEQ ID NO:73.

Some embodiments of the present disclosure provide a gene-edited tumor-infiltrating lymphocyte (TIL) expressing exogenous tethered IL-12 (TeIL-12) and exogenous tethered IL-15 (TeIL-15), wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62, and wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:73.

In some embodiments, TeIL-12 comprises a membrane anchor and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit. In some embodiments, the human IL-12 p35 subunit has an amino acid sequence of SEQ ID NO: 60 and the human IL-12 p40 subunit has an amino acid sequence of SEQ ID NO: 61. In some embodiments, TeIL-12 comprises an amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the gene-edited TIL further expresses an interleukin selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN γ, TNFa, IFN α, IFN β, GM-CSF, GCSF and variants thereof. In some embodiments, the interleukin is IL-15 or a variant thereof. In some embodiments, IL-15 is human IL-15. In some embodiments, human IL-15 has the amino acid sequence of SEQ ID NO:64. In some embodiments, IL-15 is tethered IL-15 (TeIL-15). In some embodiments, TeIL-15 comprises a membrane anchor and human IL-15. In some embodiments, TeIL-15 has the amino acid sequence of SEQ ID NO:73.

In some embodiments, the gene-edited TIL comprises a nucleic acid molecule disclosed herein. In some embodiments, the gene-edited TIL comprises a recombinant lentiviral particle disclosed herein. In some embodiments, the gene-edited TIL comprises a recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the gene-edited TIL comprises a recombinant lentiviral proviral DNA disclosed herein. In some embodiments, the recombinant lentiviral proviral DNA is integrated into the genome of the gene-edited TIL.

Some embodiments of the present disclosure provide a method of producing a tumor infiltrating lymphocyte (TIL) population, comprising culturing the TIL population in a cell culture medium, wherein the cell culture medium comprises IL-15, IL-21 and L-arginine.

Some embodiments of the present disclosure provide a method of producing a tumor infiltrating lymphocyte (TIL) population, comprising culturing the TIL population in a cell culture medium, wherein the cell culture medium comprises IL-15, IL-21, L-arginine and NAD+.

Some embodiments of the present disclosure provide a method for producing a tumor infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium for about 3-14 days; b) performing a second expansion by culturing the TIL population in a second cell culture medium for about 7-14 days, wherein the second cell culture medium comprises IL-15, IL-21 and L-arginine.

Some embodiments of the present disclosure provide a method for producing a tumor infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium for about 3-14 days; b) performing a second expansion by culturing the TIL population in a second cell culture medium for about 7-14 days, wherein the second cell culture medium comprises IL-15, IL-21, L-arginine and NAD+.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) performing a second expansion by culturing the TIL population in a second cell culture medium; and c) at any time, transducing the TIL population with a recombinant lentiviral particle disclosed herein to produce the gene-edited TIL population.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises IL-15 and IL-21; and c) at any time, transducing the TIL population with a recombinant lentiviral particle disclosed herein to produce the gene-edited TIL population.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises IL-15, IL-21 and L-arginine; and c) at any time, transducing the TIL population with a recombinant lentiviral particle disclosed herein to produce the gene-edited TIL population.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) performing a second expansion by culturing the TIL population in a second cell culture medium for about 7-14 days, wherein the second cell culture medium comprises IL-15, IL-21, L-arginine and NAD+; and c) at any time, transducing the TIL population with a recombinant lentiviral particle disclosed herein to produce the gene-edited TIL population.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) transducing the TIL population with a recombinant lentiviral particle disclosed herein; and c) performing a second expansion by culturing the TIL population in a second cell culture medium to produce the gene-edited TIL population.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) transducing the TIL population with recombinant lentiviral particles, the recombinant lentiviral particles comprising a recombinant lentiviral RNA molecule comprising nucleotides encoding TeIL-12; and c) performing a second expansion by culturing the TIL population in a second cell culture medium to produce the gene-edited TIL population.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) transducing the TIL population with recombinant lentiviral particles, the recombinant lentiviral particles comprising a recombinant lentiviral RNA molecule comprising nucleotides encoding TeIL-12 and nucleotides encoding TeIL-15; and c) performing a second expansion by culturing the TIL population in a second cell culture medium to produce the gene-edited TIL population.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) performing a second expansion by culturing the TIL population in a second cell culture medium; and c) at any time, transducing the TIL population with a recombinant lentiviral particle disclosed herein to produce the gene-edited TIL population, wherein the gene-edited TIL population expresses TeIL-12.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) performing a second expansion by culturing the TIL population in a second cell culture medium; and c) at any time, transducing the TIL population with a recombinant lentiviral particle disclosed herein to produce the gene-edited TIL population, wherein the gene-edited TIL population expresses TeIL-12, and wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) performing a second expansion by culturing the TIL population in a second cell culture medium; and c) at any time, transducing the TIL population with the recombinant lentiviral particles disclosed herein to produce the gene-edited TIL population, wherein the gene-edited TIL population expresses TeIL-12 and TeIL-15, wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62, and wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:73.

In some embodiments, the method further comprises activating the TIL population for 1 or 2 days before transducing the TIL population with the recombinant lentiviral particles. In some embodiments, the activation step comprises contacting the TIL population with an interleukin selected from the group consisting of: IL-2, IL-15, IL-21, IL-7, and combinations thereof. In some embodiments, the activation step comprises contacting the TIL population with 20 ng/mL IL-15. In some embodiments, the activation step comprises contacting the TIL population with 10 ng/mL IL-7. In some embodiments, the activation step comprises contacting the TIL population with TransAct. In some embodiments, the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. In some embodiments, the transduction step is performed in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transduction step comprises centrifugation. In some embodiments, the transduction step is performed in the presence of Lentibooster. In some embodiments, the transduction step is performed after the first expansion step and before the second expansion step. In some embodiments, the activation step is performed after the first expansion step and before the second expansion step. In some embodiments, the method further comprises allowing the TIL population to rest for 2 or 3 days after the transduction step.

In some embodiments, the TIL population is obtained from a tumor digest. In some embodiments, the method further comprises an initiation step. In some embodiments, the initiation step is performed before the first expansion step. In some embodiments, the initiation step is performed in the presence of IFNγ. In some embodiments, IFNγ is present at a concentration of 200 ng/mL during the initiation step. In some embodiments, the initiation step is performed in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration during the initiation step is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the initiation step. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the initiation step. In some embodiments, the initiation step lasts for 24-48 hours. In some embodiments, the initiation step lasts for about 30 hours. In some embodiments, the method further comprises an enrichment step. In some embodiments, the enrichment step is performed after the initiation step and before the first expansion step. In some embodiments, the TIL population is enriched for CD137+ T cells. In some embodiments, the TIL population is enriched for CD137+ and CD39+ T cells. In some embodiments, the TIL population is enriched for CD137+ and CD200+ T cells. In some embodiments, the TIL population is enriched for CD137+ and OX40+ T cells. In some embodiments, the TIL population is enriched for CD137+, CD200+ and CD39+ T cells. In some embodiments, the TIL population is enriched for CD137+, CD200+ and OX40+ T cells. In some embodiments, the TIL population is enriched for CD137+, CD39+ and OX40+ T cells. In some embodiments, the TIL population is enriched for CD137+, CD200+, CD39+ and OX40+ T cells. In some embodiments, the first expansion is carried out in the presence of feeder cells. In some embodiments, feeder cells are PBMCs depleted of T cells. In some embodiments, the first expansion is carried out in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration is 3000 IU/mL or less during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the first expansion period. In some embodiments, the first expansion is carried out for a period of about 3-11 days. In some embodiments, the first expansion is carried out for a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine. In some embodiments, L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ enhancer. In some embodiments, the NAD+ enhancer is selected from the group consisting of: L-Trp, NR, NMN, NAD+, NAM, and P7C3 activator. In some embodiments, the second cell culture medium comprises a GSK-3α/β inhibitor. In some embodiments, the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed for a period of about 7-11 days. In some embodiments, the second expansion is performed for a period of about 10 days.

In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-2. In some embodiments, the IL-2 concentration is 3000 IU/mL or less. In some embodiments, the first cell culture medium or the second cell culture medium does not contain additional IL-2. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and/or IL-21 at a concentration of about 1 ng/mL to about 100 ng/mL. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and/or IL-21 at a concentration of about 10 ng/mL. In some embodiments, the first cell culture medium comprises IL-2 and IL-21. In some embodiments, the first cell culture medium comprises 3000 IU/mL of IL-2 and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Herbacetin, Tehranolide, Isoliquiritigenin, Scutellarin, and Honokiol. In some embodiments, the AKT inhibitor is AZD5363. In some embodiments, the AKT inhibitor concentration is about 0.1 μM to about 10 μM. In some embodiments, the AKT inhibitor concentration is about 1 μM. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 11 days.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) priming a tumor digest comprising a TIL population in the presence of IFNγ; b) enriching the TIL population for CD137+ T cells; c) performing a first expansion by culturing the TIL population enriched for CD137+ T cells in a first cell culture medium; d) transducing the TIL population enriched for CD137+ T cells with recombinant lentiviral particles comprising a nucleic acid sequence encoding TeIL-12 to produce a gene-edited TIL population; and e) performing a second expansion by culturing the gene-edited TIL population in a second cell culture medium.

In some embodiments, TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT) responsive promoter. In some embodiments, TeIL-12 comprises a membrane anchor and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit. In some embodiments, the human IL-12 p35 subunit has an amino acid sequence of SEQ ID NO: 60 and the human IL-12 p40 subunit has an amino acid sequence of SEQ ID NO: 61. In some embodiments, TeIL-12 comprises an amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the nucleotide sequence encoding TeIL-12 is shown in SEQ ID NO: 162. In some embodiments, the method further comprises activating the TIL population for 1 day or 2 days before transducing the TIL population with recombinant lentiviral particles. In some embodiments, the activation step comprises contacting the TIL population with TransAct. In some embodiments, the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. In some embodiments, the transduction step is performed at a TIL concentration of 10 ⁵ cells/ml. In some embodiments, the transduction step is performed at an infection multiplicity (MOI) of about 10 to about 40. In some embodiments, the transduction step is performed in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transduction step comprises centrifugation. In some embodiments, the transduction step is performed in the presence of Lentibooster. In some embodiments, the method further comprises allowing the TIL population to rest for 2 or 3 days after the transduction step. In some embodiments, IFNγ is present at a concentration of 200 ng/mL during the initiation step. In some embodiments, the initiation step is performed in the presence of IL-2, IL-15, and/or IL-21. In some embodiments, the IL-2 concentration during the initiation step is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the initiation step. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the initiation step. In some embodiments, the initiation step lasts for 24-48 hours. In some embodiments, the initiation step lasts for about 30 hours.

In some embodiments, the TIL population is obtained from a tumor digest. In some embodiments, the method further comprises an initiation step. In some embodiments, the initiation step is performed before the first expansion step. In some embodiments, the initiation step is performed in the presence of IFNγ. In some embodiments, IFNγ is present at a concentration of 200 ng/mL during the initiation step. In some embodiments, the initiation step is performed in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration during the initiation step is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the initiation step. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 30 hours. In some embodiments, the first expansion is performed in the presence of feeder cells. In some embodiments, the feeder cells are T cell-depleted PBMCs. In some embodiments, the first expansion is performed in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration during the first expansion period is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the first expansion period. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine. In some embodiments, L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ enhancer. In some embodiments, the NAD+ enhancer is selected from the group consisting of: L-Trp, NR, NMN, NAD+, NAM, and a P7C3 activator. In some embodiments, the second cell culture medium comprises a GSK-3α/β inhibitor. In some embodiments, the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO and 3F8. In some embodiments, the second expansion is performed for a period of about 7-11 days. In some embodiments, the second expansion is performed for a period of about 10 days.

Some embodiments of the present disclosure provide a method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) priming a tumor digest comprising a TIL population in the presence of IFNγ; b) enriching the TIL population for CD137+ T cells; c) performing a first expansion by culturing the TIL population enriched for CD137+ T cells in a first cell culture medium; d) transducing the TIL population enriched for CD137+ T cells with recombinant lentiviral particles comprising nucleic acid sequences encoding TeIL-12 and TeIL-15 to produce the gene-edited TIL population; and e) performing a second expansion by culturing the gene-edited TIL population in a second cell culture medium.

In some embodiments, TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT) responsive promoter. In some embodiments, TeIL-15 is under the control of a constitutive promoter. In some embodiments, the constitutive promoter is an EF1α promoter. In some embodiments, the NFAT promoter and the constitutive promoter are introduced into the same nucleic acid of the coding sequence. In some embodiments, TeIL-12 comprises a membrane anchor and a human IL-12 p40 subunit fused with a human IL-12 p35 subunit. In some embodiments, the human IL-12 p35 subunit has an amino acid sequence of SEQ ID NO: 60 and the human IL-12 p40 subunit has an amino acid sequence of SEQ ID NO: 61. In some embodiments, TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62. In some embodiments, the nucleotide sequence encoding TeIL-12 is shown in SEQ ID NO:162. In some embodiments, TeIL-15 comprises the amino acid sequence shown in SEQ ID NO:73. In some embodiments, the method further comprises activating the TIL population for 1 day or 2 days before transducing the TIL population with recombinant lentiviral particles. In some embodiments, the activation step comprises contacting the TIL population with TransAct. In some embodiments, the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. In some embodiments, the method further comprises allowing the TIL population to rest for 2 or 3 days after the transduction step. In some embodiments, IFNγ is present at a concentration of 200 ng/mL during the elicitation step. In some embodiments, the elicitation step is performed in the presence of IL-2, IL-15, and/or IL-21. In some embodiments, the IL-2 concentration during the elicitation step is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the elicitation step. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the elicitation step. In some embodiments, the elicitation step lasts for 24-48 hours. In some embodiments, the elicitation step lasts for about 30 hours.

In some embodiments, the TIL population is obtained from a tumor digest. In some embodiments, the method further comprises an initiation step. In some embodiments, the initiation step is performed before the first expansion step. In some embodiments, the initiation step is performed in the presence of IFNγ. In some embodiments, IFNγ is present at a concentration of 200 ng/mL during the initiation step. In some embodiments, the initiation step is performed in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration during the initiation step is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the initiation step. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 30 hours. In some embodiments, the first expansion is performed in the presence of feeder cells. In some embodiments, the feeder cells are T cell-depleted PBMCs. In some embodiments, the first expansion is performed in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration during the first expansion period is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the first expansion period. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine. In some embodiments, L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ enhancer. In some embodiments, the NAD+ enhancer is selected from the group consisting of: L-Trp, NR, NMN, NAD+, NAM, and a P7C3 activator. In some embodiments, the second cell culture medium comprises a GSK-3α/β inhibitor. In some embodiments, the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO and 3F8. In some embodiments, the second expansion is performed for a period of about 7-11 days. In some embodiments, the second expansion is performed for a period of about 10 days.

Some embodiments of the present disclosure provide a therapeutic TIL population produced by a method of making a gene-edited TIL population disclosed herein. In some embodiments, about 15-60% of the TIL population expresses TeIL-12. In some embodiments, more than 30% of the TIL population expresses TeIL-12. In some embodiments, more than 50% of the TIL population expresses TeIL-12. In some embodiments, about 15-60% of the TIL population expresses TeIL-15. In some embodiments, more than 30% of the TIL population expresses TeIL-15. In some embodiments, more than 50% of the TIL population expresses TeIL-15. In some embodiments, about 15-60% of the TIL population expresses TeIL-2. In some embodiments, more than 30% of the TIL population expresses TeIL-2. In some embodiments, more than 50% of the TIL population express TeIL-2. In some embodiments, more than 30% of the TIL population has reduced PD-1 expression. In some embodiments, more than 40% of the TIL population has reduced PD-1 expression. In some embodiments, more than 60% of the TIL population has reduced PD-1 expression.

Some embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutic TIL population disclosed herein.

Some embodiments of the present disclosure provide a method of treating cancer in a patient in need thereof, comprising: a) removing a tumor sample from the patient, wherein the tumor sample comprises a TIL population; b) performing a first expansion by culturing the TIL population in a first cell culture medium; c) performing a second expansion by culturing the TIL population in a second cell culture medium; d) at any time, transducing the TIL population with a recombinant lentiviral particle disclosed herein to produce a therapeutic gene-edited TIL population; and e) administering the therapeutic gene-edited TIL population to the patient. In some embodiments, the method further comprises activating the TIL population for 1 or 2 days prior to transducing the TIL population with the recombinant lentiviral particle. In some embodiments, the activation step comprises contacting the TIL population with an interleukin selected from the group consisting of IL-2, IL-15, IL-21, IL-7, and combinations thereof. In some embodiments, the activation step comprises contacting the TIL population with 20 ng/mL IL-15. In some embodiments, the activation step comprises contacting the TIL population with 10 ng/mL IL-7. In some embodiments, the activation step comprises contacting the TIL population with TransAct. In some embodiments, the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. In some embodiments, the transduction step is performed at a TIL concentration of 10 ⁵ cells/mL. In some embodiments, the transduction step is performed at an infection multiplicity (MOI) of about 10 to about 40. In some embodiments, the transduction step is performed in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transduction step comprises centrifugation. In some embodiments, the transduction step is performed in the presence of Lentibooster. In some embodiments, the transduction step is performed after the first expansion step and before the second expansion step. In some embodiments, the activation step is performed after the first expansion step and before the second expansion step. In some embodiments, the method further comprises allowing the TIL population to rest for 2 or 3 days after the transduction step.

In some embodiments, the TIL population is obtained from a tumor digest. In some embodiments, the method further comprises an initiation step. In some embodiments, the initiation step is performed before the first expansion step. In some embodiments, the initiation step is performed in the presence of IFNγ. In some embodiments, IFNγ is present at a concentration of 200 ng/mL during the initiation step. In some embodiments, the initiation step is performed in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration during the initiation step is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the initiation step. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the initiation step. In some embodiments, the initiation step lasts for 24-48 hours. In some embodiments, the initiation step lasts for about 30 hours. In some embodiments, the method further comprises an enrichment step. In some embodiments, the enrichment step is performed after the initiation step and before the first expansion step. In some embodiments, the TIL population is enriched for CD137+ T cells. In some embodiments, the TIL population is enriched for CD137+ and CD39+ T cells. In some embodiments, the TIL population is enriched for CD137+ and CD200+ T cells. In some embodiments, the TIL population is enriched for CD137+ and OX40+ T cells. In some embodiments, the TIL population is enriched for CD137+, CD200+ and CD39+ T cells. In some embodiments, the TIL population is enriched for CD137+, CD200+ and OX40+ T cells. In some embodiments, the TIL population is enriched for CD137+, CD39+ and OX40+ T cells. In some embodiments, the TIL population is enriched for CD137+, CD200+, CD39+ and OX40+ T cells. In some embodiments, the first expansion is carried out in the presence of feeder cells. In some embodiments, feeder cells are PBMCs depleted of T cells. In some embodiments, the first expansion is carried out in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration is 3000 IU/mL or less during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the first expansion period. In some embodiments, the first expansion is carried out for a period of about 3-11 days. In some embodiments, the first expansion is carried out for a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine. In some embodiments, L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ enhancer. In some embodiments, the NAD+ enhancer is selected from the group consisting of: L-Trp, NR, NMN, NAD+, NAM, and P7C3 activator. In some embodiments, the second cell culture medium comprises a GSK-3α/β inhibitor. In some embodiments, the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed for a period of about 7-11 days. In some embodiments, the second expansion is performed for a period of about 10 days.

In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-2. In some embodiments, the IL-2 concentration is 3000 IU/mL or less. In some embodiments, the first cell culture medium or the second cell culture medium does not contain additional IL-2. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and/or IL-21 at a concentration of about 1 ng/mL to about 100 ng/mL. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and/or IL-21 at a concentration of about 10 ng/mL. In some embodiments, the first cell culture medium comprises IL-2 and IL-21. In some embodiments, the first cell culture medium comprises 3000 IU/mL of IL-2 and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of: patasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, orthoclase A, herbicide, terhei lactone, isoliquiritigenin, baicalein and honokiol. In some embodiments, the AKT inhibitor is AZD5363. In some embodiments, the AKT inhibitor concentration is about 0.1 μM to about 10 μM. In some embodiments, the AKT inhibitor concentration is about 1 μM. In some embodiments, the first expansion is performed for a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 11 days.

In some embodiments, the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by fludarabine at a dose of 25 mg/m2/day for one day. In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg aldesleukin or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerated. In some embodiments, the method does not include the step of treating the patient with the IL-2 regimen. In some embodiments, the therapeutic TIL population comprises about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs.

Some embodiments of the present disclosure provide a packaging cell line comprising a recombinant expression vector disclosed herein and one or more recombinant DNA molecules, each of which encodes any one protein, any two proteins, any three proteins, or all four proteins selected from the group consisting of: encoding Env protein, Gag protein, Pol protein, and Rev protein, with the proviso that any protein selected from the group consisting of Env protein, Gag protein, Pol protein, and Rev protein is encoded by at least one recombinant DNA molecule of the one or more recombinant DNA molecules, wherein each of the one or more recombinant DNA molecules is integrated into the genome of the packaging cell line or is contained by a helper plasmid. In some embodiments, the one or more recombinant DNA molecules consist of a first recombinant DNA molecule. In some embodiments, the first recombinant DNA molecule is integrated into the genome of the packaging cell line or is contained by the auxiliary plasmid. In some embodiments, one or more recombinant DNA molecules are composed of a first recombinant DNA molecule and a second recombinant DNA molecule. In some embodiments, the first recombinant DNA molecule encodes a single protein selected from the group consisting of Env protein, Gag protein, Pol protein and Rev protein. In some embodiments, the first recombinant DNA molecule encodes Env protein. In some embodiments, the first recombinant DNA molecule encodes Gag protein. In some embodiments, the first recombinant DNA molecule encodes Pol protein. In some embodiments, the first recombinant DNA molecule encodes Rev protein. In some embodiments, the first recombinant DNA molecule encodes two proteins selected from the group consisting of Env protein, Gag protein, Pol protein and Rev protein. In some embodiments, the first recombinant DNA molecule encodes Env protein and Gag protein. In some embodiments, the first recombinant DNA molecule encodes Env protein and Pol protein. In some embodiments, the first recombinant DNA molecule encodes Env protein and Rev protein. In some embodiments, the first recombinant DNA molecule encodes Gag protein and Pol protein. In some embodiments, the first recombinant DNA molecule encodes Gag protein and Rev protein. In some embodiments, the first recombinant DNA molecule encodes Pol protein and Rev protein. In some embodiments, each of the first recombinant DNA molecule and the second recombinant DNA molecule is integrated into the genome of the packaging cell line or is contained by a helper plasmid. In some embodiments, one or more recombinant DNA molecules consist of the first recombinant DNA molecule, the second recombinant DNA molecule, and the third recombinant DNA molecule. In some embodiments, the first recombinant DNA molecule encodes a first protein and the second recombinant DNA molecule encodes a second protein, wherein the first protein and the second protein are independently selected from the group consisting of Env protein, Gag protein, Pol protein and Rev protein, with the proviso that the first protein and the second protein are not the same. In some embodiments, the first protein is Env protein and the second protein is Gag protein. In some embodiments, the first protein is Env protein and the second protein is Pol protein. In some embodiments, the first protein is Env protein and the second protein is Rev protein. In some embodiments, the first protein is Gag protein and the second protein is Pol protein. In some embodiments, the first protein is Gag protein and the second protein is Pol protein. In some embodiments, the first protein is Gag protein and the second protein is Rev protein. In some embodiments, the first protein is Pol protein and the second protein is Rev protein. In some embodiments, the first recombinant DNA molecule encodes a first protein and the second recombinant DNA molecule encodes two proteins, wherein the first protein and each of the two proteins are independently selected from the group consisting of Env protein, Gag protein, Pol protein and Rev protein, with the proviso that any protein is not the same as any other protein in the group consisting of the first protein and the two proteins. In some embodiments, the first protein is Env protein and the two proteins are Gag protein and Pol protein. In some embodiments, the first protein is Env protein and the two proteins are Gag protein and Rev protein. In some embodiments, the first protein is Env protein and the two proteins are Pol protein and Rev protein. In some embodiments, the first protein is Gag protein and the two proteins are Env protein and Pol protein. In some embodiments, the first protein is Gag protein and the two proteins are Env protein and Rev protein. In some embodiments, the first protein is Gag protein and the two proteins are Pol protein and Rev protein. In some embodiments, the first protein is Pol protein and the two proteins are Env protein and Gag protein. In some embodiments, the first protein is Pol protein and the two proteins are Env protein and Rev protein. In some embodiments, the first protein is Pol protein and the two proteins are Gag protein and Rev protein. In some embodiments, the first protein is Rev protein and the two proteins are Env protein and Gag protein. In some embodiments, the first protein is Rev protein and the two proteins are Env protein and Pol protein. In some embodiments, the first protein is Rev protein and the two proteins are Gag protein and Pol protein. In some embodiments, each of the first recombinant DNA molecule, the second recombinant DNA molecule, and the third recombinant DNA molecule is integrated into the genome of the packaging cell line or is contained by an accessory plasmid. In some embodiments, one or more recombinant DNA molecules are composed of a first recombinant DNA molecule, a second recombinant DNA molecule, a third recombinant DNA molecule, and a fourth recombinant DNA molecule. In some embodiments, each of the first recombinant DNA molecule, the second recombinant DNA molecule, the third recombinant DNA molecule, and the fourth recombinant DNA molecule is integrated into the genome of the packaging cell line or is contained by a helper plasmid. In some embodiments, the Env protein comprises the Ba-EVTR protein. In some embodiments, the nucleic acid molecule encoding TeIL-12 is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA molecule encoding one or more of the Env protein, the Gag protein, the Pol protein, and the Rev protein is under the control of an inducible promoter. In some embodiments, the packaging cell line is a 293T cell line.

Some embodiments of the present disclosure provide a method for enriching tumor-reactive tumor-infiltrating lymphocytes (TILs), comprising: a) eliciting a TIL population obtained from a tumor digest in the presence of IFNγ; b) enriching the TIL population for CD137+ TILs; c) performing a first expansion by culturing the TIL population enriched for CD137+ T cells in a first cell culture medium; and d) expanding the TIL population enriched for CD137+ TILs in a second cell culture medium to produce a TIL population enriched for tumor-reactive TILs. In some embodiments, the TIL population enriched for tumor-reactive TILs comprises an increased percentage of tumor-reactive TILs compared to TILs expanded using a reference expansion procedure.

In some embodiments, IFNγ is present at a concentration of 200 ng/mL during the initiation step. In some embodiments, the initiation step is performed in the presence of IL-2, IL-15, and/or IL-21. In some embodiments, the IL-2 concentration during the initiation step is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the initiation step. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the initiation step. In some embodiments, the initiation step lasts for 24-48 hours. In some embodiments, the initiation step lasts for about 30 hours. In some embodiments, the first expansion is carried out in the presence of feeder cells. In some embodiments, the feeder cells are PBMCs depleted of T cells. In some embodiments, the first expansion is carried out in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration is 3000 IU/mL or less during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the first expansion period. In some embodiments, the first expansion is carried out over a period of about 3-11 days. In some embodiments, the first expansion is performed for a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs) and L-arginine. In some embodiments, L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ enhancer. In some embodiments, the NAD+ enhancer is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM, and P7C3 activator. In some embodiments, the second cell culture medium comprises a GSK-3α/β inhibitor. In some embodiments, the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed for a period of about 7-11 days. In some embodiments, the second expansion is performed for a period of about 10 days. In some embodiments, enriching the TIL population for CD137+ TILs comprises contacting the TIL population with an anti-CD137 antibody immobilized on beads. In some embodiments, the method further comprises contacting the TIL population with an anti-CD39 antibody. In some embodiments, the method further comprises contacting the TIL population with an anti-CD200 antibody. In some embodiments, the method further comprises contacting the TIL population with an anti-OX40 antibody.

Some embodiments of the present disclosure provide a method for enriching tumor-reactive tumor-infiltrating lymphocytes (TILs), comprising: a) eliciting a TIL population obtained from a tumor digest in the presence of IFNγ; b) enriching the TIL population for CD103+ TILs; c) performing a first expansion by culturing the TIL population enriched for CD103+ T cells in a first cell culture medium; and d) performing a second expansion by culturing the TIL population enriched for CD103+ T cells in a second cell culture medium to produce a TIL population enriched for tumor-reactive TILs. In some embodiments, a population of TILs enriched for tumor-reactive TILs comprises an increased percentage of tumor-reactive TILs compared to TILs expanded using a reference expansion procedure.

In some embodiments, IFNγ is present at a concentration of 200 ng/mL during the initiation step. In some embodiments, the initiation step is performed in the presence of IL-2, IL-15, and/or IL-21. In some embodiments, the IL-2 concentration during the initiation step is 3000 IU/mL or less. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the initiation step. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the initiation step. In some embodiments, the initiation step lasts for 24-48 hours. In some embodiments, the initiation step lasts for about 24 hours. In some embodiments, selecting CD103+ TIL includes sorting the second TIL population using flow cytometry. In some embodiments, step (b) includes selecting CD103+CD31- TIL. In some embodiments, the first expansion is carried out in the presence of feeder cells. In some embodiments, the feeder cells are PBMCs depleted of T cells. In some embodiments, the first expansion is carried out in the presence of IL-2, IL-15 and/or IL-21. In some embodiments, the IL-2 concentration is 3000 IU/mL or less during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 1 ng/mL to about 100 ng/mL during the first expansion period. In some embodiments, IL-15 and/or IL-21 are present at a concentration of about 10 ng/mL during the first expansion period. In some embodiments, the first expansion is performed for a period of about 3-11 days. In some embodiments, the first expansion is performed for a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs) and L-arginine. In some embodiments, L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ enhancer. In some embodiments, the NAD+ enhancer is selected from the group consisting of: L-Trp, NR, NMN, NAD+, NAM, and P7C3 activator. In some embodiments, the second cell culture medium comprises a GSK-3α/β inhibitor. In some embodiments, the GSK-3α/β inhibitor is selected from the group consisting of: SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed for a period of about 7-11 days. In some embodiments, the second expansion is performed for a period of about 10 days. Some embodiments of the present disclosure provide a method of treating cancer in a patient in need thereof, comprising: a. removing a tumor sample from the patient; b. digesting the tumor sample to obtain a tumor digest; c. generating a TIL population enriched for tumor-reactive TILs from the tumor digest using the methods disclosed herein; and d. administering the therapeutic gene-edited TIL population to the patient.

In some embodiments, the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by fludarabine at a dose of 25 mg/m2/day for one day. In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg aldesleukin or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerated. In some embodiments, the method does not include the step of treating the patient with the IL-2 regimen. In some embodiments, the therapeutic TIL population comprises about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs.

Some embodiments of the present disclosure provide a method for producing a tumor infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; and b) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, and L-arginine.

Some embodiments of the present disclosure provide a method for producing a tumor infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; and b) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, L-arginine, and NAD+.

In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ enhancer. In some embodiments, the NAD+ enhancer is selected from the group consisting of: L-Trp, NR, NMN, NAD+, NAM, and P7C3 activator. In some embodiments, the NAD+ enhancer is NAD+. In some embodiments, NAD+ is present at a concentration of about 50-75 μM. In some embodiments, the first expansion is performed over a period of about 3-14 days. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 7-14 days. In some embodiments, the second expansion is performed over a period of about 11 days.

Some embodiments of the present disclosure provide a therapeutic TIL population produced by the methods disclosed herein.

Some embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutic TIL population disclosed herein.

Some embodiments of the present disclosure provide a method for treating cancer in a patient in need thereof, comprising: a) removing a tumor sample from the patient, wherein the tumor sample comprises a TIL population; b) performing a first expansion by culturing the TIL population in a first cell culture medium; c) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, and L-arginine; and d) administering the therapeutic gene-edited TIL population to the patient.

Some embodiments of the present disclosure provide a method for treating cancer in a patient in need thereof, comprising: a) removing a tumor sample from the patient, wherein the tumor sample comprises a TIL population; b) performing a first expansion by culturing the TIL population in a first cell culture medium; c) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, L-arginine, and NAD+; and d) administering the therapeutic gene-edited TIL population to the patient.

In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ enhancer. In some embodiments, the NAD+ enhancer is selected from the group consisting of: L-Trp, NR, NMN, NAD+, NAM, and P7C3 activator. In some embodiments, the NAD+ enhancer is NAD+. In some embodiments, NAD+ is present at a concentration of about 50-75 μM. In some embodiments, the first expansion is performed over a period of about 3-14 days. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 7-14 days. In some embodiments, the second expansion is performed over a period of about 11 days.

In some embodiments, the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by fludarabine at a dose of 25 mg/m2/day for one day. In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg aldesleukin or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerated. In some embodiments, the method does not include the step of treating the patient with the IL-2 regimen.

Some embodiments of the present disclosure provide a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, comprising: (a) adding tumor fragments processed from a tumor resected from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) The second TIL population is cultured in a cell culture medium containing IL-21, IL-15, L-arginine, OKT-3 and antigen presenting cells (APC) to perform a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (b) to step (c) is performed without opening the system; (d) Harvesting the therapeutic TIL population obtained from step (c), wherein the transfer from step (c) to step (d) is performed without opening the system, and wherein the harvested therapeutic TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (e) transferring the TIL population harvested from step (d) to an infusion bag, wherein the transfer from step (d) to (e) is performed without opening the system, and (f) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process.

Some embodiments of the present disclosure provide a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, comprising: (a) adding tumor fragments processed from a tumor resected from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) The second TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, NAD+, OKT-3 and antigen presenting cells (APC) to perform a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (b) to step (c) is performed without opening the system; (d) Harvesting the therapeutic TIL population obtained from step (c), wherein the transfer from step (c) to step (d) is performed without opening the system, and wherein the harvested therapeutic TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (e) transferring the TIL population harvested from step (d) to an infusion bag, wherein the transfer from step (d) to (e) is performed without opening the system, and (f) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process.

Some embodiments of the present disclosure provide a method for producing a therapeutic gene-edited TIL population, comprising: (a) adding tumor fragments processed from a tumor removed from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) Transducing the second TIL population with the recombinant lentiviral particles disclosed herein to generate a gene-edited TIL population, wherein the transformation from step (b) to step (c) is performed without opening the system; (d) The gene-edited TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, OKT-3 and antigen presenting cells (APC) for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is the therapeutic gene-edited TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (c) to step (d) is performed without opening the system; (e) Harvesting the therapeutic gene-edited TIL population obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system, and wherein the harvested therapeutic gene-edited TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (f) transferring the therapeutic gene-edited TIL population harvested from step (e) to an infusion bag, wherein the transfer from step (e) to (f) is performed without opening the system, and (g) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process.

Some embodiments of the present disclosure provide a method for producing a therapeutic gene-edited TIL population, comprising: (a) adding tumor fragments processed from a tumor removed from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) Transducing the second TIL population with the recombinant lentiviral particles disclosed herein to generate a gene-edited TIL population, wherein the transformation from step (b) to step (c) is performed without opening the system; (d) The gene-edited TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, NAD+, OKT-3 and antigen presenting cells (APC) for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is the therapeutic gene-edited TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (c) to step (d) is performed without opening the system; (e) Harvesting the therapeutic gene-edited TIL population obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system, and wherein the harvested therapeutic gene-edited TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (f) transferring the therapeutic gene-edited TIL population harvested from step (e) to an infusion bag, wherein the transfer from step (e) to (f) is performed without opening the system, and (g) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process.

Some embodiments of the present disclosure provide a method for producing a therapeutic gene-edited TIL population, comprising: (a) processing a tumor resected from a patient to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and wherein the first expansion is performed for about 3-11 days to obtain the second TIL population; (c) transducing the second TIL population with the recombinant lentiviral particles disclosed herein to produce a gene-edited TIL population; (d) The gene-edited TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, OKT-3 and antigen presenting cells (APC) for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is the therapeutic gene-edited TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (e) Harvesting the therapeutic gene-edited TIL population obtained from step (d), wherein the harvested therapeutic gene-edited TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (f) transferring the therapeutic gene-edited TIL population harvested from step (e) to an infusion bag, and (g) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process.

Some embodiments of the present disclosure provide a method for producing a therapeutic gene-edited TIL population, comprising: (a) processing a tumor resected from a patient to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and wherein the first expansion is performed for about 3-11 days to obtain the second TIL population; (c) transducing the second TIL population with the recombinant lentiviral particles disclosed herein to produce a gene-edited TIL population; (d) The gene-edited TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, NAD+, OKT-3 and antigen presenting cells (APC) for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is the therapeutic gene-edited TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (e) Harvesting the therapeutic gene-edited TIL population obtained from step (d), wherein the harvested therapeutic gene-edited TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (f) transferring the therapeutic gene-edited TIL population harvested from step (e) to an infusion bag, and (g) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process.

In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ enhancer. In some embodiments, the NAD+ enhancer is selected from the group consisting of: L-Trp, NR, NMN, NAD+, NAM, and P7C3 activator. In some embodiments, the NAD+ enhancer is NAD+. In some embodiments, NAD+ is present at a concentration of about 50-75 μM. In some embodiments, the first expansion is performed over a period of about 3-14 days. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 7-14 days. In some embodiments, the second expansion is performed over a period of about 11 days.

In some embodiments, the method further comprises activating the TIL population for 1 or 2 days before transducing the TIL population with the recombinant lentiviral particles. In some embodiments, the activation step comprises contacting the TIL population with TransAct. In some embodiments, the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. In some embodiments, the transduction step is performed at a TIL concentration of 10 ⁵ cells/ml. In some embodiments, the transduction step is performed at an infection multiple (MOI) of about 10 to about 40. In some embodiments, the transduction step is performed in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transduction step comprises centrifugation. In some embodiments, the transduction step is performed in the presence of Lentibooster. In some embodiments, the method further comprises allowing the TIL population to rest for 2 or 3 days after the transduction step.

Some embodiments of the present disclosure provide therapeutic gene-edited TIL populations produced by the methods disclosed herein.

Some embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutic gene-edited TIL population disclosed herein.

Some embodiments of the present disclosure provide for use of a therapeutic TIL population disclosed herein or a therapeutic gene-edited TIL population disclosed herein for treating cancer in a patient in need thereof, comprising administering the therapeutic TIL population or the therapeutic gene-edited TIL population to the patient.

In some embodiments, the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by fludarabine at a dose of 25 mg/m2/day for one day. In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg aldesleukin or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerated. In some embodiments, the method does not include the step of treating the patient with the IL-2 regimen.

Cross-references to related applications

This application claims priority to U.S. Provisional Patent Application No. 63/513,550 filed on July 13, 2023, U.S. Provisional Patent Application No. 63/622,008 filed on January 17, 2024, U.S. Provisional Patent Application No. 63/562,210 filed on March 6, 2024, and U.S. Provisional Patent Application No. 63/649,302 filed on May 17, 2024, which are incorporated herein by reference in their entirety. Sequence Listing

This application contains a sequence listing that has been submitted electronically in XML ST.26 format and is incorporated herein by reference in its entirety. The XML copy was created on July 10, 2024, is named 116983-5126-WO_Sequence_Listing, and is 209,365 bytes in size. I. Introduction

Adoptive cell therapy using TILs is an effective method for inducing tumor regression in various cancers, including leukemia and melanoma. It has been found that the use of adjuvants including immunostimulatory agents can enhance the efficacy of adoptive cell therapy and extend this type of therapy to other solid tumors. However, co-administration of immunomodulators such as cytokines (e.g., interleukins) can cause undesirable toxicity due to the high doses required. Therefore, providing such adjuvants at the correct time and location is crucial to avoid such undesirable effects.

Provided herein are compositions and methods for treating cancer using modified TILs, wherein the modified TILs include one or more immunomodulators (e.g., interleukins) bound to their cell surface. The immunomodulators bound to the TILs provide local immune stimulation, which can advantageously enhance TIL survival and/or anti-tumor activity in a patient's recipient. Thus, the compositions and methods disclosed herein provide effective cancer treatments. II. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

As used herein, the terms "co-administration", "administered in combination with", "administered in combination with", "simultaneous" and "concurrent" encompass the administration of two or more active pharmaceutical ingredients (in preferred embodiments of the present invention, for example, a plurality of TILs) to an individual such that both the active pharmaceutical ingredients and/or their metabolites are present in the individual at the same time. Co-administration includes simultaneous administration as separate compositions, administration as separate compositions at different times, or administration in the form of a composition in which two or more active pharmaceutical ingredients are present. Administration in the form of a composition in which two agents are present and simultaneous administration as separate compositions are preferred.

The term "in vivo" refers to events that occur inside the body of an individual.

The term "in vitro" refers to events that occur outside the body of an individual. In vitro assays encompass cell-based assays that utilize living or dead cells, and may also encompass cell-free assays that do not utilize intact cells.

The term "ex vivo" refers to events involving treatment or procedures performed on cells, tissues and/or organs that have been removed from an individual's body. Where appropriate, the cells, tissues and/or organs may be returned to the individual's body by surgery or therapeutic means.

The term "rapid expansion" means that the number of antigen-specific TILs increases by at least about 3 times (or 4 times, 5 times, 6 times, 7 times, 8 times or 9 times) in one week, more preferably by at least about 10 times (or 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times or 90 times) in one week, or most preferably by at least about 100 times in one week. Various rapid expansion schemes are disclosed below.

"Tumor infiltrating lymphocytes" or "TILs" herein means a population of cells originally obtained as white blood cells that have left an individual's bloodstream and migrated into a tumor. TILs include, but are not limited to, CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and secondary TILs. "Primary TILs" are TILs obtained from a patient tissue sample as described herein (sometimes referred to as "fresh harvest"), and "secondary TILs" are any expanded or proliferated TIL cell populations as discussed herein, including, but not limited to, primary TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

"Cell population" (including TIL) herein means a number of cells with common characteristics. Typically, the number of populations is in the range of 1×10 ⁶ to 1×10 ¹⁰ , with different TIL populations containing different numbers. For example, initial growth of primary TIL in the presence of IL-2 produces a primary TIL population of approximately 1×10 ⁸ cells. REP expansion is generally performed to provide 1.5×10 ⁹ to 1.5×10 ¹⁰ cell populations for infusion.

As used herein, "cryopreserved TILs" means TILs, whether primary, primary or expanded (REP TILs), that have been processed and stored at a temperature in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that can be used as a source of primary TILs.

As used herein, "thawed cryopreserved TILs" means a population of TILs that have been previously cryopreserved and subsequently treated to return to room temperature or higher temperature (including but not limited to cell culture temperature or a temperature at which the TILs can be administered to a patient).

TILs can often be defined biochemically (using cell surface markers) or functionally (based on their ability to infiltrate tumors and effect therapy). TILs can often be classified by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors following reintroduction into a patient.

The term "cryopreservation media" refers to any medium that can be used to cryopreserve cells. Such medium may include a medium comprising 7% to 10% DMSO. Exemplary mediums include CryoStor CS10, HypoThermosol, and combinations thereof. The term "CS10" refers to a cryopreservation medium obtained from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CryoStor®CS10". CS10 medium is a serum-free, animal-free medium that includes DMSO.

The term "central memory T cells" refers to a subset of T cells that are CD45R0+ and constitutively express CCR7 (CCR7 ^hi ) and CD62L (CD62 ^hi ) in humans. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2 and BMI1. Central memory T cells mainly secrete IL-2 and CD40L as effector molecules after TCR priming. Central memory T cells are mainly present in the CD4 compartment of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

The term "effector memory T cells" refers to a subset of human or mammalian T cells, such as central memory T cells, which are CD45R0+ but have lost constitutive expression of CCR7 (CCR7 ^lo ) and are heterogeneous or low for CD62L expression (CD62L ^lo ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL4- and IL-5, after antigen stimulation. Effector memory T cells are mainly found in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestines in humans. CD8+ effector memory T cells carry large amounts of perforin.

The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, for example (but not limited to), closed G containers. Once the tumor segment is added to the closed system, the system is not open to the external environment until the TIL is ready to be administered to the patient.

As used herein, the terms "fragmenting," "fragment," and "fragmented" describe the process of disrupting a tumor, including mechanical fragmentation methods such as crushing, slicing, dividing, and pulverizing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMC" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen presenting cells (PBMC is a type of antigen presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

The term "anti-CD3 antibody" refers to an antibody or variant thereof directed against the CD3 receptor in the T cell antigen receptor of mature T cells, such as a monoclonal antibody, and includes human, humanized, chimeric, murine or mammalian antibodies. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include UHCT1 clones, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab and visilizumab.

The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or a biosimilar or variant thereof directed against the CD3 receptor in the T cell antigen receptor of mature T cells, including human, humanized, chimeric or murine antibodies, and includes commercially available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, San Diego, CA, USA) and muromonab or a variant, conservative amino acid substitution, glycosylated form or biosimilar thereof. The amino acid sequences of the heavy chain and light chain of muromonab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). The hybridoma capable of producing OKT-3 is deposited at the American Type Culture Collection and assigned the ATCC accession number CRL 8001. The OKT-3-producing fusion tumor was also deposited in the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biosimilars and variants thereof. IL-2 is described in, for example, Nelson's J. Immunol . 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of a recombinant human IL-2 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, available from multiple suppliers, 22 million IU per single-use vial) and recombinant IL-2 forms supplied by CellGenix, Inc., Secondmouth, New Hampshire, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene, Inc., East Brunswick, New Jersey, USA (Catalog No. CYT-209-b) and other commercial equivalents from other suppliers. Aldesleukin (des-propylamine-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is given in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses PEGylated forms of IL-2 as described herein, including the PEGylated IL2 prodrug bempegaldesleukin (NKTR-214, a PEGylated human recombinant IL-2 as in SEQ ID NO: 4, wherein an average of 6 lysine residues are substituted at N ⁶ by [(2,7-bis{[methylpoly(oxyethylene)]aminoformyl}-9H-fluoren-9-yl)methoxy]carbonyl), which is commercially available from Nektar Therapeutics (South San Francisco, CA, USA), or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO 2018/132496 A1 or U.S. Patent Application Publication No. US 2018/132496 A1. The method described in Example 1 of 2019/0275133 A1, the disclosures of which are incorporated herein by reference. Bepealdesleukin (NKTR-214) and other PEGylated IL2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. US 2014/0328791 A1 and International Patent Application Publication No. WO 2012/065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

In some embodiments, a form of IL-2 suitable for use in the present invention is THOR-707, available from Synthorx. The preparation and properties of THOR-707 and other alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US 2020/0181220 A1 and US 2020/0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, the form of IL-2 suitable for use in the present invention is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a binding portion that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from the group consisting of K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the number of the amino acid residues corresponds to SEQ ID NO: 5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72 and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72 and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62 and E68. In some embodiments, the amino acid position is at E62. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72 and Y107 further mutates into lysine, cysteine or histidine. In some embodiments, the amino acid residue mutates into cysteine. In some embodiments, the amino acid residue mutates into lysine. In some embodiments, the amino acid residue mutates into non-natural amino acids. In some embodiments, the unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyl tetrahydrolysine, allyloxycarbonyl lysine, 2-amino-8-oxono-nonanoic acid, 2-amino- 8-Oxyoctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine , isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl-tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine , phosphonyltyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonylserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)seleno)propionic acid, 2-amino-3-(phenylseleno)propionic acid or selenocysteine. In some embodiments, the IL-2 binder has reduced affinity for the IL-2 receptor α (IL-2Rα) subunit relative to the wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater than 99% reduced binding affinity to IL-2Rα relative to wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold or greater relative to wild-type IL-2 polypeptide. In some embodiments, the binding moiety weakens or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the binding moiety comprises a water-soluble polymer. In some embodiments, the additional binding moiety comprises a water-soluble polymer. In some embodiments, the water-soluble polymers each independently comprise polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(enols), poly(vinyl pyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymers each independently comprise PEG. In some embodiments, PEG is a linear PEG or a branched chain PEG. In some embodiments, the water-soluble polymers each independently comprise a polysaccharide. In some embodiments, the polysaccharide comprises polydextrose, polysialic acid (PSA), hyaluronic acid (HA), linear starch, heparin, heparan sulfate (HS), dextrin or hydroxyethyl starch (HES). In some embodiments, the water-soluble polymers each independently comprise a polysaccharide. In some embodiments, the water-soluble polymers each independently comprise a polyamine. In some embodiments, the binding moiety comprises a protein. In some embodiments, the additional binding moiety comprises a protein. In some embodiments, the protein each independently comprises albumin, transferrin or transthyretin. In some embodiments, the protein each independently comprises an Fc portion. In some embodiments, the protein each independently comprises an Fc portion of IgG. In some embodiments, the binding moiety comprises a polypeptide. In some embodiments, the additional binding moiety comprises a polypeptide. In some embodiments, the polypeptides each independently comprise an XTEN peptide, a glycine-rich high amino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamidation. In some embodiments, the binding moiety is directly bound to the isolated and purified IL-2 polypeptide. In some embodiments, the binding moiety is indirectly bound to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant's reagent (Lomant's reagent) dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl suberate) (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDST), glycosyl bis(succinimidyl Ethyl succinimidyl ester (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl diimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di(3'-( 2'-pyridyldisulfide)propionamido)butane (DPDPB), bis(cis-butylenediimidohexane) (BMH), compounds containing aryl halides (DFDNB) (such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene), 4,4'-difluoro-3,3'-dinitrophenylsulfonate (DFDNPS), bis-[β-(4-hydroxy- [0063] In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises 3-(2-pyridyldithio) propionate N-succinimidyl ester (sPDP), long-chain 3-(2-pyridyldithio) propionate N-succinimidyl ester (LC-sPDP), water-soluble long-chain 3-(2-pyridyldithio) propionate N-succinimidyl ester (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio) toluene (sMPT), sulfosuccinimidyl-6-\-[α-methyl-α-(2-pyridyldithio) toluamido] hexanoate ( Sulfonyl-LC-sMPT), succinimidyl-4-(N-cis-butylenediimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-cis-butylenediimidomethyl)cyclohexane-1-carboxylate (sulfonyl-sMCC), m-cis-butylenediimidobenzyl-N-hydroxysuccinimidyl ester (MBs), m-cis-butylenediimidobenzyl-N-hydroxysulfosuccinimidyl ester (sulfonyl-MBs), (4-iodoacetyl)aminobenzoic acid N-succinimidyl ester (sIAB), (4-iodoacetyl)aminobenzyl Sulfosuccinimidyl formate (sulfo-sIAB), succinimidyl-4-(p-cis-1,2-diaminophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-cis-1,2-diaminophenyl)butyrate (sulfo-sMPB), N-(γ-cis-1,2-diaminobutyryloxy)succinimidyl ester (GMBs), N-(γ-cis-1,2-diaminobutyryloxy)sulfosuccinimidyl ester (sulfo-GMBs), 6-((iodoacetyl)amino)hexanoic acid succinimidyl ester (sIAX), 6-[6-(((iodoacetyl)amino) [0063] succinimidyl hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinking agents such as 4-(4-N-cis-butylenediimidophenyl)butyric acid hydrazide (MPBH), 4-(N-cis-butylenediimidomethyl)cyclohexane-1-carboxy-hydrazide-8 (M ₂ C ₂ H), 3-(2-pyridyldithio)propionylhydrazine (PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamidohexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(paraazidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB) 、N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzyloxybutanediamide (ANB-Nos), sulfosuccinimidyl-2-(m-azido-o-nitrobenzylamino)-ethyl-1,3'-dithiopropionate (s AND), N-succinimidyl-4 (4-azidophenyl) 1,3'-dithiopropionate (sADP), (4-azidophenyl)-1,3'-dithiopropionate N-sulfosuccinimidyl ester (sulfo-sADP), 4-(p-azidophenyl)butyric acid sulfosuccinimidyl ester (sulfo-sAPB), 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate sulfosuccinimidyl ester (sAED), 7-azido-4-methylcoumarin-3-acetate sulfosuccinimidyl ester (sulfo-sAMC), A), p-nitrophenyl diazopyruvate (ρNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalanylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalanylamido)butyl]-3'-(2'-pyridyldisulfide)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzylhydrazine (ABH), 4-(p-azidosalanylamido)butylamine (AsBA) or p-azidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a cis-butenediimido group, optionally comprising cis-butenediimidohexanoyl (mc), succinimidyl-4-(N-cis-butenediimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-cis-butenediimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative or analog thereof. In some embodiments, the binding moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional binding moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the present invention is a fragment of any IL-2 form described herein. In some embodiments, the IL-2 form suitable for use in the present invention is PEGylated as disclosed in U.S. Patent Application Publication No. US 2020/0181220 A1 and U.S. Patent Application Publication No. US 2020/0330601 A1. In some embodiments, the form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) covalently linked to a binding moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5; and an AzK substitution for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72 with reference to the amino acid positions in SEQ ID NO: 5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO: 5. In some embodiments, the form of IL-2 suitable for use in the present invention lacks IL-2R alpha chain binding, but retains normal binding to the intermediate affinity IL-2R beta-gamma signaling complex. In some embodiments, the form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK) covalently linked to a binding moiety comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5; and an AzK substitution for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72 with reference to the amino acid positions in SEQ ID NO:5. In some embodiments, the form of IL-2 suitable for use in the present invention is an IL-2 conjugate, comprising: an IL-2 polypeptide comprising N6-azidoethoxy-lysine (AzK), which is covalently linked to a binding portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5; and an AzK substitution for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72 with reference to the amino acid positions in SEQ ID NO:5. In some embodiments, the form of IL-2 suitable for use in the present invention is an IL-2 conjugate, comprising: an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK), which is covalently linked to a binding portion comprising polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5; and with reference to the amino acid positions in SEQ ID NO:5, AzK substitutions of amino acids at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72.

In some embodiments, the form of IL-2 suitable for use in the present invention is Nemvaleukin alfa (also known as ALKS-4230 (SEQ ID NO: 6), which is commercially available from Alkermes). Naivasha alpha is also known as human interleukin 2 fragment (1-59) variant (Cys ¹²⁵ ＞Ser ⁵¹ ), which is fused to human interleukin 2 fragment (62-132) via a peptidyl linker ( ⁶⁰ GG ⁶¹ ), which is fused to human interleukin 2 receptor α chain fragment (139-303) via a peptidyl linker ( ¹³³ GSGGGS ¹³⁸ ), produced in Chinese hamster ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133)-peptide [Cys 125 (51) ＞Ser]-mutant (1-59), which is fused to human interleukin 2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS 138 ), produced in Chinese hamster ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133)-peptide [Cys ¹²⁵ (51) ＞Ser]-mutant (1-59), which is fused to human interleukin 2 receptor α chain fragment (139-303) via a peptidyl linker ( 133 GSGGGS 138 ), ₂ peptide linker (60-61) fused to human interleukin 2 (IL-2) (4-74)-peptide (62-132) and fused to human interleukin 2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) via GSG ₃ S peptide linker (133-138), produced in Chinese hamster ovary (CHO) cells, glycoform alpha. The amino acid sequence of nivanovin alpha is provided in SEQ ID NO:6. In some embodiments, nivanovin alpha exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using the numbering in SEQ ID NO: 6), and glycosylation sites at the following positions: N187, N206, T212 (using the numbering in SEQ ID NO: 6). The preparation and properties of nivanovin alpha and other alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. US 2021/0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, the form of IL-2 suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with SEQ ID NO: 6. In some embodiments, the form of IL-2 suitable for use in the present invention has an amino acid sequence provided in SEQ ID NO: 6 or a conservative amino acid substitution thereof. In some embodiments, the form of IL-2 suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO: 7, or a variant, fragment, or derivative thereof. In some embodiments, the form of IL-2 suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity with amino acids 24-452 of SEQ ID NO: 7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Patent No. 10,183,979, the disclosure of which is incorporated herein by reference. Optionally, in some embodiments, the IL-2 form suitable for use in the present invention is a fusion protein comprising a first fusion partner, the first fusion partner being linked to a second fusion partner via a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity of IL-Rα, and wherein the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, wherein the mucin domain polypeptide linker comprises SEQ ID NO: 8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8, and wherein the half-life of the fusion protein is improved compared to fusion of the first fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker.

In some embodiments, IL-2 forms suitable for use in the present invention include antibody-cytokine-grafted proteins, which include: a heavy chain variable region ( _VH ) comprising complementation determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region ( _VL ) comprising LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof grafted into the CDRs of _VH or _VL , wherein the antibody-cytokine-grafted protein preferentially expands T effector cells relative to regulatory T cells. In some embodiments, the antibody-cytokine-grafted protein comprises a heavy chain variable region ( _VH ) comprising complementary determining regions HCDR1, HCDR2, HCDR3; a light chain variable region ( _VL ) comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof, which is grafted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody-cytokine-grafted protein expands T effector cells prior to regulatory T cells. In some embodiments, the IL-2 regimen comprises administering an antibody described in U.S. Patent Application Publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine grafted protein comprises: a heavy chain variable region (VH) comprising complementary determining regions HCDR1, HCDR2, and HCDR3; a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3; and an IL-2 molecule or a fragment thereof grafted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, wherein the antibody cytokine grafted protein preferentially expands T effector cells relative to regulatory T cells, and wherein the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of: an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 38; an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 29; an IgG class light chain comprising SEQ ID NO: 39 and an IgG class heavy chain comprising SEQ ID NO: 38; an IgG class light chain comprising SEQ ID NO: 37 and an IgG class heavy chain comprising SEQ ID NO: 29; an IgG class light chain comprising SEQ ID NO: 38 NO:39 and an IgG class heavy chain comprising SEQ ID NO:29; and an IgG class light chain comprising SEQ ID NO:37 and an IgG class heavy chain comprising SEQ ID NO:38.

In some embodiments, the IL-2 molecule or its fragment is transplanted into the HCDR1 of V _H , wherein the IL-2 molecule is a mutant protein. In some embodiments, the IL-2 molecule or its fragment is transplanted into the HCDR2 of V _H , wherein the IL-2 molecule is a mutant protein. In some embodiments, the IL-2 molecule or its fragment is transplanted into the HCDR3 of V _H , wherein the IL-2 molecule is a mutant protein. In some embodiments, the IL-2 molecule or its fragment is transplanted into the LCDR1 of V _L , wherein the IL-2 molecule is a mutant protein. In some embodiments, the IL-2 molecule or its fragment is transplanted into the LCDR2 of V _L , wherein the IL-2 molecule is a mutant protein. In some embodiments, the IL-2 molecule or its fragment is transplanted into the LCDR3 of V _L , wherein the IL-2 molecule is a mutant protein.

Insertion of IL-2 molecules can be at or near the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody interleukin transplant protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody interleukin transplant protein comprises an IL-2 molecule incorporated into the CDR, wherein the IL-2 sequence replaces all or part of the CDR sequence. The IL-2 molecule replacement can be at the N-terminal region of the CDR, in the middle region of the CDR, or at or near the C-terminal region of the CDR. The IL-2 molecule replacement can be as little as one or two amino acids in the CDR sequence or the entire CDR sequence.

In some embodiments, the IL-2 molecule is directly grafted into a CDR without a peptide linker, wherein there are no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted into a CDR with a peptide linker, wherein there are one or more additional amino acids between the CDR sequence and the IL-2 sequence.

In some embodiments, the IL-2 molecule described herein is an IL-2 mutant protein. In some cases, the IL-2 mutant protein comprises an R67A substitution. In some embodiments, the IL-2 mutant protein comprises an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutant protein comprises an amino acid sequence in Table 1 of U.S. Patent Application Publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference.

In some embodiments, the antibody-cytokine grafting protein comprises a HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody-cytokine grafting protein comprises a HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody-cytokine grafting protein comprises a HCDR1 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26. In some embodiments, the antibody-cytokine grafting protein comprises a HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody cytokine graft protein comprises a _VH region comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the antibody cytokine graft protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody cytokine graft protein comprises a _VL region comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody cytokine graft protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine graft protein comprises a _VH region comprising the amino acid sequence of SEQ ID NO: 28; and a _VL region comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody cytokine grafting protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 29; and a light chain region comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine grafting protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 29; and a light chain region comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody cytokine grafting protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain region comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine grafting protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain region comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020/0270334 A1 or a variant, derivative or fragment thereof, or a conservative amino acid substitution thereof, or a protein having at least 80%, at least 90%, at least 95% or at least 98% sequence identity therewith. In some embodiments, the antibody component of the antibody cytokine transplant protein described herein comprises the immunoglobulin sequence, framework sequence or CDR sequence of palivizumab. In some embodiments, the serum half-life of the antibody cytokine transplant protein described herein is longer than that of the wild-type IL-2 molecule (such as (but not limited to) aldesleukin or similar molecules). In some embodiments, the antibody cytokine transplant protein described herein has a sequence as shown in Table 3.

The term "IL-4" (also referred to herein as "IL4") refers to an interleukin called interleukin 4, which is produced by Th2 T cells and eosinophils, eosinophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Following activation by IL-4, Th2 T cells then produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and MHC class II expression, and induces class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention can be purchased from a number of suppliers, including ProSpec-Tany TechnoGene, Inc. (East Brunswick, NJ, USA) (Catalog No. CYT-211) and ThermoFisher Scientific, Inc. (Waltham, MA, USA.) (human IL-15 recombinant protein, Catalog No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 9).

The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived interleukin called interleukin 7, which is obtained from stromal and epithelial cells and dendritic cells. Fry and Mackall , Blood 2002 , 99 , 3892-904 . IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor (a heterodimer composed of the IL-7 receptor alpha and the common gamma chain receptor), which belongs to a series of signals that are important for the development of T cells in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention can be purchased from a number of suppliers, including ProSpec-Tany TechnoGene, Inc. (East Brunswick, NJ, USA) (Catalog No. CYT-254) and ThermoFisher Scientific, Inc. (Waltham, MA, USA) (Human IL-15 recombinant protein, Catalog No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 10).

The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biosimilars and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001 , 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares the β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain containing 114 amino acids (and N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 can be purchased from a number of suppliers, including ProSpec-Tany TechnoGene, Inc. (East Brunswick, NJ, USA) (Catalog No. CYT-230-b) and ThermoFisher Scientific, Inc. (Waltham, MA, USA) (Human IL-15 Recombinant Protein, Catalog No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 11).

The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic interleukin protein called interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycosylated forms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is primarily produced by natural killer T cells and activated human CD4 ⁺ T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 can be purchased from a number of suppliers, including ProSpec-Tany TechnoGene, Inc. (East Brunswick, NJ, USA) (Catalog No. CYT-408-b) and ThermoFisher Scientific, Inc. (Waltham, MA, USA) (Human IL-21 Recombinant Protein, Catalog No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is provided in Table 2 (SEQ ID NO: 12).

When an "anti-tumor effective amount", "tumor suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (individual). It can be generally stated that the pharmaceutical compositions described herein comprising tumor infiltrating lymphocytes (e.g., inherited TILs or gene-modified cytotoxic lymphocytes) can be administered at a dose of 10 ⁴ to 10 ¹¹ cells/kg body weight (e.g., 10 ⁵ to 10 ⁶ , 10 ⁵ to 10 ¹⁰ , ^{10 5 to 10 11 ,} 10 ⁶ to 10 ¹⁰ , 10 ⁶ to 10 ¹¹ , 10 ⁷ to 10 ¹¹ , 10 ⁷ to 10 ¹⁰ , 10 ⁸ to 10 ¹¹ , 10 ⁸ to 10 ¹⁰ , 10 ^{9 to 10 11, or 10 9} to 10 ¹⁰ cells/kg body weight, including all integer values within those ranges. TIL (including genetically modified cytotoxic lymphocytes in some cases) compositions can also be administered multiple times at these doses. TIL (including genetically engineered TIL in some cases) can be administered using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 1988 , 319, 1676). The optimal dose and treatment regimen for a particular patient can be easily determined by a person skilled in the art of medicine by monitoring the patient's disease symptoms and adjusting treatment accordingly.

The term "hematological malignancy" or terms with related meanings refers to cancers and tumors of the hematopoietic and lymphoid tissues of mammals (including but not limited to the blood, bone marrow, lymph nodes, and tissues of the lymphatic system). Hematological malignancies are also called "fluid tumors". Hematological malignancies include but are not limited to acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), multiple myeloma, acute monocytic leukemia (aMoL), Hodgkin's lymphoma and non-Hodgkin's lymphoma. The term "B-cell malignancies" refers to blood malignancies that affect B cells.

The term "liquid tumor" refers to abnormal cell masses that are fluid in nature. Liquid tumor cancers include (but are not limited to) leukemias, myelomas and lymphomas, as well as other blood malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors (including liquid tumors circulating in peripheral blood) may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the type of tissue from which the cells are derived.

As used herein, the term "microenvironment" may refer to the physical or hematological tumor microenvironment as a whole or may refer to individual cell subsets within the microenvironment. As used herein, the tumor microenvironment refers to the complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical signals that promote mesenchymal transformation, support tumor growth and invasion, protect tumors from host immunity, encourage treatment resistance, and provide a niche for dominant metastasis to thrive," as described in Swartz et al., Cancer Res. , 2012 , 72 , 2473. Although tumors express antigens that should be recognized by T cells, clearance of the tumor by the immune system is rare due to the immunosuppressive microenvironment.

In some embodiments, the invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TIL according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (on days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m2/d for 5 days (on days 27 to 23 prior to TIL infusion). In some embodiments, following non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), patients receive an intravenous infusion of IL-2 at 720,000 IU/kg intravenously every 8 hours to achieve physiological tolerance.

Experimental findings indicate that lymphocyte depletion prior to the transfer of tumor-specific T lymphocytes plays a key role in enhancing therapeutic efficacy by eliminating regulatory T cells and elements of the competing immune system ("interleukin pool"). Therefore, some embodiments of the invention employ a lymphocyte depletion step (sometimes referred to as "immunosuppressive conditioning") in patients prior to the introduction of the TILs of the invention.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds as described herein that is sufficient to achieve the intended application, including but not limited to disease treatment. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the individual and disease condition being treated (e.g., the individual's weight, age and sex), the severity of the disease condition, or the mode of administration. The term also applies to an amount that will induce a specific response in the target cell (e.g., a decrease in platelet adhesion and/or cell migration). The specific amount will vary depending on the specific compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

The terms "treatment", "treating", "treat" and similar terms refer to obtaining a desired pharmacological and/or physiological effect. The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms, and/or therapeutic, in terms of partially or completely curing a disease and/or adverse effects attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, that is, arresting its development or progression; and (c) relieving the disease, that is, causing regression of the disease and/or relieving one or more symptoms of the disease. "Treatment" is also intended to encompass the delivery of an agent so as to provide a pharmacological effect, even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition (e.g., in the case of a vaccine).

The term "heterologous" when used with reference to a portion of an nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, a nucleic acid is typically produced recombinantly and has two or more sequences from unrelated genes that are arranged to make a new functional nucleic acid sequence, such as a promoter from one source and a coding region from another source or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

In the context of two or more nucleic acids or polypeptides, the terms "sequence identity", "percent identity", and "sequence percent identity" (or their synonyms, e.g., "99% identity") refer to two or more sequences or subsequences that are identical or have a specified percentage of nucleotides or amino acid residues that are identical when compared and aligned (introducing spacing if necessary) for maximum correspondence and not considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be performed using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign (available from DNASTAR) are other publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximum alignment using specific alignment software. In certain embodiments, the default parameters of the alignment software are used.

As used herein, the term "variant" encompasses, but is not limited to, antibodies or fusion proteins comprising an amino acid sequence that is different from the amino acid sequence of a reference antibody, the difference being that there are one or more substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. Variants may comprise one or more conservative substitutions in their amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may involve, for example, substitutions of similar charged or uncharged amino acids. Variants retain the ability to bind specifically to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

"Tumor infiltrating lymphocytes" or "TILs" herein means a population of cells originally obtained as white blood cells that have left an individual's bloodstream and migrated into a tumor. TILs include, but are not limited to, CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary TILs and secondary TILs. "Primary TILs" are cells obtained from a patient tissue sample as described generally herein (sometimes referred to as "fresh harvest"), and "secondary TILs" are any population of expanded or proliferated TIL cells as discussed herein, including, but not limited to, primary TILs and expanded TILs ("REP TILs") and "reREP TILs" as discussed herein. reREP TILs may include, for example, a second expanded TIL or a second additional expanded TIL (such as the TILs described in step D of FIG. 8 , including TILs referred to as reREP TILs).

TILs can generally be defined biochemically (using cell surface markers) or functionally (according to their ability to infiltrate tumors and effectuate therapy). TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45RA, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors following reintroduction into a patient. TILs can be further characterized by potency - for example, TILs can be considered potent if, for example, interferon (IFN) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL. TILs can be considered potent if, for example, interferon (IFNγ) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, greater than about 300 pg/mL, greater than about 400 pg/mL, greater than about 500 pg/mL, greater than about 600 pg/mL, greater than about 700 pg/mL, greater than about 800 pg/mL, greater than about 900 pg/mL, greater than about 1000 pg/mL.

The term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes in the sugar moiety, the base moiety and/or the linkages between deoxyribonucleotides in the oligonucleotide.

The term "RNA" defines a molecule comprising at least one ribonucleotide residue. "Ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of the b-D-ribofuranosyl moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA (such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. The nucleotides in the RNA molecules described herein may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Unless any known pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Additional active pharmaceutical ingredients such as other drugs may also be incorporated into the described compositions and methods.

The term "about" or "approximately" means within a statistically significant range of values. This range may be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The permissible differences encompassed by the term "about" or "approximately" depend on the specific system under study and are readily understood by those of ordinary skill in the art. In addition, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and features are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art. Generally, whether or not expressly stated as such, dimensions, sizes, formulations, parameters, shapes, or other quantities or characteristics are "about" or "approximately." It should be noted that embodiments of widely varying sizes, shapes, and dimensions may employ the described arrangements.

As used in the appended claims, in both original and amended form, the transition terms "comprising," "consisting essentially of," and "consisting of" define the claims relative to which unrecited additional claim elements or steps, if any, are excluded from the scope of the claims. The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of" does not include any elements, steps, or materials other than those specified in the claims and, in the latter case, excludes impurities normally associated with the specified materials. The term "consisting essentially of limits the scope of the claims to the specified elements, steps, or materials and those elements, steps, or materials that do not materially affect the basic and novel characteristics of the claimed invention. In alternative embodiments, all compositions, methods, and kits described herein that embody the invention may be more specifically defined by any of the transition terms "comprising," "consisting essentially of," and "consisting of."

The term "antibody" and its plural form "antibodies" refers to intact immunoglobulins and any antigen-binding fragments ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain comprises a heavy chain variable region (abbreviated herein as _VH ) and a heavy chain constant region. The heavy chain constant region comprises three domains (CH1, CH2 and CH3). Each light chain comprises a light chain variable region (abbreviated herein as _VL ) and a light chain constant region. The light chain constant region comprises one domain _CL . The _VH and _VL regions of an antibody can be further subdivided into hypervariable regions, which are called complement-determining regions (CDRs) or hypervariable regions (HVRs), and they may be interspersed with more highly conserved regions, called framework regions (FRs). Each _VH and _VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigenic determinants. The constant regions of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen" refers to a substance that induces an immune response. In some embodiments, if presented by a major histocompatibility complex (MHC) molecule, an antigen is a molecule that can be bound by an antibody or TCR. As used herein, the term "antigen" also encompasses T cell antigenic determinants. Antigens can also be recognized by the immune system. In some embodiments, antigens can induce humoral immune responses or cellular immune responses that cause activation of B lymphocytes and/or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell antigenic determinant. An antigen may also have one or more antigenic determinants (e.g., a B antigenic determinant and a T antigenic determinant). In some embodiments, an antigen will preferably react with its corresponding antibody or TCR, generally in a highly specific and selective manner, and will not react with the variety of other antibodies or TCRs that may be induced by other antigens.

The terms "monoclonal antibody", "mAb", "monoclonal antibody composition" or their plural forms refer to a preparation of antibody molecules in a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular antigenic determinant. Monoclonal antibodies specific for certain receptors can be made using the knowledge and techniques of the following techniques: injecting a test individual with an appropriate antigen, and then isolating the hybridoma expressing antibodies with the desired sequence or functional characteristics. The DNA encoding the monoclonal antibody is readily isolated and sequenced using known procedures (for example, by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA can be placed in an expression vector and then transfected into host cells that do not otherwise produce immunoglobulins (such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to achieve synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or, for short, "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the _VL , _VH , _CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the _VH and CH1 domains; (iv) a Fv fragment consisting of the _VL and _VH domains of a single arm of the antibody; (v) a domain antibody (dAb) fragment (Ward et al., Nature , 1989 , 341 , 544-546), which can consist of one _VH or one _VL domain; and (vi) isolated complementary determining regions (CDRs). In addition, although the two domains of the Fv fragment ( _VL and _VH ) are encoded by independent genes, they can be joined using recombinant methods by a synthetic linker that enables the two domains to be made into a single protein chain in which the _VL and _VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, e.g., Bird et al., Science 1988 , 242 , 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA 1988 , 85 , 5879-5883). Such scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises a _VH portion and a _VL portion. The scFv molecule is denoted _VL - _LVH when the VL domain is the N-terminal portion of _the scFv molecule, or _VH - _LVL when the _VH domain is the N-terminal portion of the scFv molecule. Methods for preparing scFv molecules and designing suitable peptide linkers are described in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; R. Raag and M. Whitlow, "Single Chain Fvs." FASEB, Vol. 9: 73-80 (1995); and RE Bird and BW Walker, "Single Chain Antibody Variable Regions", TIBTECH, Vol. 9: 132-137 (1991), the disclosures of which are incorporated herein by reference.

As used herein, the term "human antibody" is intended to include antibodies that meet the following conditions: having variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutations in vitro or introduced by in vivo somatic cell mutations). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as mice) have been transplanted onto human framework sequences.

The term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity with variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, the human monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal (e.g., a transgenic mouse) fused with an immortalized cell having a genome comprising a human heavy chain transgene and a light chain transgene.

As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from animals (such as mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, such as from transfectomas; (c) antibodies isolated from recombinant, combinatorial human antibody libraries; and (d) antibodies prepared, expressed, generated or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may have undergone in vitro mutation induction (or in vivo somatic cell mutation induction when transgenic animals expressing human Ig sequences are used), and thus the amino acid sequences of the _VH and _VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline _VH and _VL sequences, may not naturally exist within the human antibody germline in vivo.

As used herein, "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

The phrases "antibodies that recognize antigens" and "antibodies specific for antigens" are used interchangeably herein with the term "antibodies that specifically bind to antigens."

The term "human antibody derivative" refers to any modified form of a human antibody, including a conjugate of an antibody with another active pharmaceutical ingredient or antibody. The term "conjugate", "antibody-drug conjugate", "ADC" or "immunoconjugate" refers to an antibody or fragment thereof conjugated to another therapeutic moiety, which can be conjugated to the antibodies described herein using methods available in the art.

The terms "humanized antibody" (humanized antibodies) and "humanized" refer to antibodies in which CDR sequences derived from the germline of another mammalian species (such as mouse) have been grafted onto human framework sequences. Other framework region modifications may be made in the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (acceptor antibodies) in which residues from the hypervariable regions of the acceptor are replaced by residues from the 15 hypervariable regions of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues not found in the acceptor antibody or the donor antibody. Such modifications are performed to further optimize the antibody performance. In general, humanized antibodies will include substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies will also include at least a portion of the constant region (Fc) of an immunoglobulin, typically at least a portion of the constant region of a human immunoglobulin, as the case may be. For further details, see Jones et al., Nature 1986, 321 , 522-525; Riechmann et al., Nature 1988, 332 , 323-329; and Presta, Curr. Op. Struct. Biol. 1992, 2, 593-596. The antibodies described herein may also be modified to use any Fc variant known to provide improved (e.g., reduced) effector function and/or FcR binding. Fc variants may include, for example, any of the amino acid substitutions disclosed in International Patent Application Publication Nos. WO 1988/07089 A1, WO 1996/14339 A1, WO 1998/05787 A1, WO 1998/23289 A1, WO 1999/51642 A1, WO 99/58572 A1, WO 2000/09560 A2, WO 2000/32767 A1, WO 2000/42072 A2, WO 2002/44215 A2, WO 2002/060919 A2, WO 2003/074569 A2, WO 2004/016750 A2, WO WO 2004/029207 A2, WO 2004/035752 A2, WO 2004/063351 A2, WO 2004/074455 A2, WO 2004/099249 A2, WO 2005/040217 A2, WO 2005/070963 A1, WO 2005/077981 A2, WO 2005/092925 A2, WO 2005/123780 A2, WO 2006/019447 A1, WO 2006/047350 A2 and WO 2006/085967 A2; and U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the disclosures of which are incorporated herein by reference.

The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from mouse antibodies and the constant region sequences are derived from human antibodies.

"Bifunctional antibodies" are small antibody fragments with two antigen binding sites. The fragments comprise a heavy chain variable domain ( _VH ) linked to a light chain variable domain (VL) in the same polypeptide chain ( _VH - _VL or _VL - _{VH )} . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain and create two antigen binding sites. Bifunctional antibodies are described in more detail in, for example, European Patent No. EP 404,097, International Patent Publication No. WO 93/11161; and Bolliger et al., Proc. Natl. Acad. Sci. USA 1993, 90 , 6444-6448.

The term "glycosylation" refers to a modified derivative of an antibody. A deglycosylated antibody is not glycosylated. Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Deglycosylation can increase the affinity of an antibody for an antigen, as described in U.S. Patent Nos. 5,714,350 and 6,350,861. Alternatively or additionally, antibodies with altered glycosylation patterns can be generated, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the potency of the antibody. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expressing recombinant antibodies of the invention to generate antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 do not have the fucosyltransferase gene, FUT8 (α(1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines do not have fucose on their carbohydrates. Ms704, Ms705, and Ms709 FUT8-/- cell lines were generated by disruption of the FUT8 gene in targeted CHO/DG44 cells using two replacement vectors (see, e.g., U.S. Patent Publication No. 2004/0110704 or Yamane-Ohnuki et al., Biotechnol. Bioeng. , 2004, 87 , 614-622). As another example, European Patent No. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines are hypofucosylated by reducing or eliminating the α1,6 bond-related enzyme, and also describes a cell line having low or no enzyme activity for adding fucose to N-acetylglucosamine bound to the Fc region of an antibody, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). International Patent Publication WO 03/035835 describes a mutant CHO cell line, Lec 13 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also causing hypofucosylation of antibodies expressed in the host cells (see also Shields et al., J. Biol. Chem. 2002, 277 , 26733-26740. International Patent Publication WO 99/54342 describes a cell line engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit an increased bisected GlcNac structure, thereby increasing the ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 1999, 17 , 176-180). Alternatively, a fucosidase can be used to cleave the fucose residue of the antibody. For example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino et al., Biochem. 1975, 14, 5516-5523.

"PEGylation" refers to a modified antibody or fragment thereof, which is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. For example, PEGylation can increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is performed via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C ₁ -C ₁₀ ) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-cis-butylenediimide. The antibody to be PEGylated is a deglycosylated antibody. Pegylation methods are known in the art and can be applied to the antibodies of the present invention, as described, for example, in European Patent No. EP 0154316 and European Patent No. EP 0401384 and U.S. Patent No. 5,824,778, the disclosures of which are each incorporated herein by reference.

The term "biosimilar" means a biological product (including a monoclonal antibody or protein) that is closely similar to a U.S.-approved reference biological product, despite minor differences in clinically inactive components, and there are no clinically significant differences between the biological product and the reference product in terms of product safety, purity, and potency. In addition, a similar biological or "biosimilar" drug is a biological drug that is similar to another biological drug that has been authorized for use by the European Medicines Agency. The term "biosimilar" is also used synonymously by other national and regional regulatory agencies. A biological product or biopharmaceutical is a drug that is made or derived from a biological source, such as bacteria or yeast. It can be composed of relatively small molecules (such as human insulin or erythropoietin) or complex molecules (such as monoclonal antibodies). For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), the protein approved by the drug regulatory agency for the reference aldesleukin is "biosimilar to aldesleukin" or a "biosimilar of aldesleukin". In Europe, a similar biological or "biosimilar" drug is a biological drug that is similar to another biological drug that has been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for the use of similar biologicals in Europe is Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC, as amended and therefore in Europe, biosimilars may be authorised, approved for authorisation or be the subject of an application for authorisation pursuant to Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC. Authorised original biological medicinal products may be referred to as "reference medicinal products" in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guidance on similar biological medicinal products. In addition, product specific guidance, including guidance relevant to monoclonal antibody biosimilars, is provided by the EMA on a product-by-product basis and published on its website. Biosimilars as described herein may be similar to the reference drug in terms of quality characteristics, biological activity, mechanism of action, safety profile and/or efficacy. In addition, the biosimilars may be used or intended to be used to treat the same condition as the reference drug. Therefore, the biosimilars as described herein may be considered to have similar or very similar quality characteristics as the reference drug. Alternatively or additionally, the biosimilars as described herein may be considered to have similar or very similar biological activities as the reference drug. Alternatively or additionally, the biosimilars as described herein may be considered to have similar or very similar safety profiles as the reference drug. Alternatively or additionally, the biosimilars as described herein may be considered to have similar or very similar efficacy as the reference drug. As described herein, in Europe, biosimilars have been compared to reference drugs authorized by the EMA. However, in some cases, a biosimilar may be compared to a biopharmaceutical licensed outside the European Economic Area (a non-EEA licensed "comparator") in certain studies. Such studies include, for example, certain clinical and in vivo non-clinical studies. As used herein, the term "biosimilar" also relates to a biopharmaceutical that has been or can be compared to a non-EEA licensed comparator. Certain biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen binding portions), and fusion proteins. Protein biosimilars may have an amino acid sequence with minor modifications in the amino acid structure that do not significantly affect the function of the polypeptide (including, for example, deletions, additions and/or substitutions of amino acids). A biosimilar may comprise an amino acid sequence that has 97% or greater, such as 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of its reference drug. A biosimilar may comprise one or more post-translational modifications that are different from the post-translational modifications of the reference drug, such as, but not limited to, glycosylation, oxidation, deamidation, and/or truncation, provided that the differences do not cause a change in the safety and/or efficacy of the drug. A biosimilar may have the same or different glycosylation pattern as the reference drug. Specifically, but not exclusively, a biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety issues associated with the reference drug. In addition, a biosimilar may differ from a reference product in, for example, its strength, pharmaceutical form, formulation, excipient and/or mode of presentation, provided that the safety and efficacy of the drug are not compromised. A biosimilar may contain, for example, differences in the pharmacokinetic (PK) and/or pharmacodynamic (PD) profile compared to the reference product, but is still considered sufficiently similar to the reference product to be authorized or deemed suitable for authorization. In certain cases, a biosimilar presents different binding characteristics compared to the reference product, where regulatory agencies (such as the EMA) do not consider such different binding characteristics as a barrier to the authorization of a similar biological product. The term "biosimilar" is also used synonymously by other national and regional regulatory agencies.

The term "recombinant lentiviral RNA molecule" refers to a single-stranded RNA genome that can comprise at least a portion of a lentiviral genome, including 5' and 3' long terminal repeat (LTR) sequences. In some embodiments, the lentiviral genome can be modified to inhibit replication and limit pathogenicity while retaining function. For example, the env gene, gag gene, pol gene, and rev gene can be removed from the lentiviral genome comprised by the recombinant lentiviral RNA molecule. In some embodiments, the recombinant lentiviral RNA molecule is comprised by a lentiviral vector or recombinant lentiviral particle as described elsewhere in this disclosure.

The term "lentiviral particle" or "lentiviral virion" refers to lentiviruses, which are a subset of retroviruses. Lentiviruses can deliver large amounts of genetic information into host cells and integrate it into the cellular genome, making genetically engineered lentiviruses one of the most effective gene delivery tools. These lentiviruses contain a promoter to control the expression of the transgenic gene or shRNA, but do not contain virulence genes, making them safe to use in the laboratory.

The term "recombinant lentiviral particles" or "recombinant lentiviral virions" refers to lentiviral particles or lentiviral virions produced by genetic recombination techniques. Recombinant lentiviral particles or lentiviral virions can be produced using any suitable method, such as by transducing or transfecting a packaging cell line with nucleic acid encoding the viral genome, and then isolating the newly packaged viral particles. It should be understood that the recombinant technology can be performed at an upstream stage of the production of the viral vector itself. For example, a plasmid can be produced using recombinant technology, and then the plasmid can be produced on a larger scale, and finally the plasmid can be introduced into a cell line for packaging to produce a viral vector.

The term "lentiviral vector" refers to a recombinant lentiviral particle comprising a recombinant lentiviral RNA molecule, which contains at least a portion of the lentiviral genome, including the 5' and 3' LTRs, and nucleotide sequences encoding one or more genes of interest (GOI). The lentiviral genome can be modified to inhibit replication and limit pathogenicity while retaining function. For example, the env gene, gag gene, pol gene, and rev gene can be removed from the lentiviral genome and included in the helper plasmid.

The term "transfer vector" refers to a recombinant DNA plasmid containing a nucleotide sequence encoding a recombinant lentiviral RNA molecule, which contains at least a portion of the lentiviral genome, including the 5' and 3' LTRs, and a nucleotide sequence encoding one or more genes of interest (GOI), and one or more "helper plasmids" or "envelope plasmids" contain genes for proteins that appear on the surface of lentiviral particles or are essential for the function of lentiviral particles. The transfer vector can be transfected into a packaging cell line together with the helper plasmid to produce a lentiviral vector comprising a nucleotide sequence encoding one or more GOIs as part of the recombinant lentiviral RNA molecule.

The term "Env" refers to the envelope glycoprotein on the surface of the lentiviral particle encoded by the env gene in the lentiviral genome. The Env protein contains a surface subunit and a transmembrane subunit. In some embodiments, the Env protein is selected from the group consisting of: baboon retrovirus envelope (Ba-EVTR), vesicular stomatitis virus-G protein (VSV-G) and RD114.

The term "Gag" refers to the structural protein encoded by the gag gene in the lentiviral genome. The Gag protein is produced as a stand-alone protein or as a fusion protein with the Pol protein (Gag-Pol).

The term "Pol" refers to the reverse transcriptase and integrase encoded by the pol gene in the lentiviral genome. The Pol protein is produced as a stand-alone protein or as a fusion protein with the Gag protein (Gag-Pol).

The term "Rev" refers to the protein encoded by the rev gene in the lentiviral genome. Rev has a leucine-rich nuclear export signal (NES) and mediates the nuclear to cytoplasmic transport of partially spliced and unspliced RNA by binding to the Rev response element (RRE), thereby producing Gag, Gag-Pol, Env and accessory proteins (Pollard and Malim, Annu. Rev. Microbiol ., 1998 , 52, 491532). III. Nucleic acid molecules encoding interleukins

Provided herein are nucleic acid molecules comprising nucleotide sequences encoding one or more genes of interest (GOI), such as interleukins selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN γ, TNFa, IFN α, IFN β, GM-CSF, GCSF or variants thereof. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding tethered IL-12 (TeIL-12). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding TeIL-12 and tethered IL-15 (TeIL-15). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding TeIL-12 and tethered IL-2 (TeIL-2).

In some embodiments, a nucleotide sequence encoding one or more GOIs, such as an interleukin selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFNγ, TNFa, IFNα, IFNβ, GM-CSF, GCSF, or variants thereof, is provided by two or more nucleic acid molecules. For example, provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second nucleic acid molecule comprising a nucleotide sequence encoding IL-15 or a variant thereof. In some embodiments, provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second nucleic acid molecule comprising a nucleotide sequence encoding IL-2 or a variant thereof.

In some embodiments, the interleukin is a tethered interleukin. For example, the interleukin is linked to a cell membrane anchor portion that allows the interleukin to be tethered to the cell surface. Suitable cell membrane anchor portions include, for example, transmembrane domains of endogenous cell surface proteins and fragments thereof. Exemplary transmembrane domains that can be used include, for example, B7-1, B7-2, and CD8a transmembrane domains and fragments thereof. In some embodiments, the cell membrane anchor portion further includes the transmembrane and intracellular domains of endogenous cell surface proteins or fragments thereof. In some embodiments, the cell membrane anchor portion is a B7-1, B7-2 or CD8a transmembrane-intracellular domain or a fragment thereof. In some embodiments, the cell membrane anchor portion is a CD8a transmembrane domain having an amino acid sequence of IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 40). In some embodiments, the cell membrane anchor portion is a B7-1 transmembrane-intracellular domain having an amino acid sequence of LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 41).

In certain embodiments, the cell membrane anchoring moiety is a non-peptide cell membrane anchoring moiety. In exemplary embodiments, the non-peptide cell membrane anchoring moiety is a glycophosphatidylinositol (GPI) anchor. The GPI anchor has a structure including a phosphoethanolamine linker, a glycan core, and a phospholipid tail. In some embodiments, the glycan core is modified with one or more side chains. In some embodiments, the glycan core is modified with one or more of the following side chains: a phosphoethanolamine group, mannose, galactose, sialic acid, or other sugars.

The membrane-anchored interleukin may include a linker that allows for the attachment of components of the membrane-anchored interleukin (e.g., the interleukin and the cell membrane anchoring moiety). Suitable linkers include linkers that are at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues in length. In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 45-50, 50-60 amino acids in length. Suitable linkers include, but are not limited to, cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, or non-helical linkers. In some embodiments, the linker is a peptide linker, which optionally comprises Gly and Ser. In certain embodiments, the peptide linker utilizes a glycine-serine polymer, including, for example, (GS)n (SEQ ID NO: 42), (GSGGS)n (SEQ ID NO: 43), (GGGS)n (SEQ ID NO: 44), (GGGGS)n (SEQ ID NO: 45), (GGGGGS)n (SEQ ID NO: 46), and (GGGGGGS)n (SEQ ID NO: 47), wherein n is an integer of at least one (and typically 3 to 10). Other linkers that can be used with the compositions and methods of the invention are described in U.S. Patent Publication Nos. US 2006/0074008, US 20050238649, and US 2006/0024317, each of which is incorporated herein by reference in its entirety, particularly with respect to linkers. In some embodiments, the peptide linker is SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 48).

In some embodiments, the linker is a cleavable linker. In an exemplary embodiment, the cleavable linker allows the release of the interleukin into the tumor microenvironment. The cleavable linker is also applicable to embodiments in which two membrane-anchored interleukins are co-expressed in the same cell. In an exemplary embodiment, the linker is a self-cleaving 2A peptide. See, for example, Liu et al., Sci. Rep. 7(1):2193 (2017), the relevant parts of which are related to the 2A peptide are incorporated herein by reference. 2A peptide viral oligopeptide, which mediates the cleavage of the polypeptide during eukaryotic cell translation. In some embodiments, the 2A peptide includes a C-terminus having the amino acid sequence GDVEXiNPGP (SEQ ID NO: 49), wherein Xi is any naturally occurring amino acid residue. In some embodiments, the 2A peptide is porcine teschovirus-1 2A peptide (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 50). In some embodiments, the 2A peptide is equine rhinitis A virus 2A peptide (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO: 51). In some embodiments, the 2A peptide is foot-and-mouth disease virus 2A peptide: (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO: 52). In some embodiments, the cleavable linker includes a furin cleavable sequence. Exemplary furin cleavable sequences are described, for example, in Duckert et al., Protein Engineering, Design & Selection 17(1):107-112 (2004) and U.S. Patent No. 8,871,906, each of which is incorporated herein by reference, particularly with respect to the relevant portions relating to the furin cleavable sequence. In some embodiments, the linker comprises a 2A peptide and a furin cleavable sequence. In an exemplary embodiment, the furin cleavable 2A peptide comprises the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 53).

In some embodiments, the linker is a degradable linker (e.g., a disulfide linker), such that the linker degrades under physiological conditions, thereby releasing the interleukin. In some embodiments, the interleukin is reversibly linked to the functional group via a degradable linker, such that the linker degrades under physiological conditions and releases the interleukin. Suitable degradable linkers include (but are not limited to): protease-sensitive linkers that are sensitive to one or more enzymes present in the biological culture medium, such as proteases in the tumor microenvironment, such as matrix metalloproteinases present in the tumor microenvironment or inflamed tissue (e.g., matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9)).

In other embodiments, the interleukin is an enzyme-sensitive linker. Exemplary cleavable linkers include cleavable linkers recognized by one of the following enzymes: metalloproteinases MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, fibrolytic enzymes, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, apoptosis-1, apoptosis-2, apoptosis-3, apoptosis-4, apoptosis-5, apoptosis-6, apoptosis-7, apoptosis-8, apoptosis-9, apoptosis-10, apoptosis-11, apoptosis-12, apoptosis-13, apoptosis-14, and TACE. See, e.g., U.S. Patent Nos. 8,541,203 and 8,580,244, each of which is incorporated herein by reference in its entirety and relevant portions relating to cleavable linkers.

In certain embodiments, the membrane-anchored interleukin includes a signal peptide that promotes the translocation of the interleukin to the cell membrane. Any suitable signal peptide that promotes the localization of the interleukin to the cell membrane can be used. In some embodiments, the signal peptide does not interfere with the biological activity of the interleukin. Exemplary signal peptide sequences include (but are not limited to): human granulocyte-macrophage colony stimulating factor (GM-CSF), receptor signal sequence, human prolactin signal sequence and human IgE signal sequence. In certain embodiments, the fusion protein includes a human IgE signal sequence. In an exemplary embodiment, the human IgE signal sequence has the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 54). In some embodiments, the human IgE signal sequence comprises the amino acid sequence NIKGSPWKGSLLLLLVSNLLLCQSVAP (SEQ ID NO: 55). In some embodiments, the signal peptide sequence is an IL-2 signal sequence having the amino acid sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 56).

The nucleic acid molecule may further comprise a natural or standard promoter operably linked to a nucleotide sequence encoding one or more interleukins. In some embodiments, the nucleotide sequence encoding each interleukin is operably linked to the same promoter. In some embodiments, the nucleotide sequence encoding each interleukin is operably linked to different promoters. Preferably, the promoter is functional in T cells. The selection of promoters, such as strong promoters, weak promoters, induced promoters, tissue-specific promoters, and development-specific promoters, is within the ordinary technical scope of the technician. Similarly, the combination of nucleotide sequences and promoters is also within the technical scope of the technician. The promoter may be a non-viral promoter or a viral promoter, such as the nuclear factor of activated T cells (NFAT) promoter, the EF-1a promoter, the cytomegalovirus (CMV) promoter, the CAG promoter, the MND promoter, the SSFV promoter, the SV40 promoter, the RSV promoter, or a promoter found in the long terminal repeat sequence of the murine stem cell virus.

As used herein, "NFAT promoter" means one or more NFAT response elements connected to the minimal promoter of any gene expressed by T cells. Preferably, the minimal promoter of the gene expressed by T cells is the minimal human IL-2 promoter. The NFAT response element may include, for example, NFAT1, NFAT2, NFAT3 and/or NFAT4 response elements. The NFAT promoter (or its functional part or functional variant) may include any number of binding motifs, such as at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at most twelve binding motifs.

In a preferred embodiment, the NFAT promoter comprises six NFAT binding motifs. See, for example, U.S. Patent No. 8,556,882, which is incorporated herein by reference in its entirety and particularly with the relevant parts of the NFAT promoter. In some embodiments, the NFAT promoter system controls the expression of one or more interleukins. In certain embodiments, the interleukin is selected from the group consisting of: IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN γ, TNFa, IFN α, IFN β, GM-CSF, GCSF or its variants. In some embodiments, the interleukin is a tethered interleukin. In some embodiments, the NFAT activation subsystem controls the expression of IL-12 or its variants. In some embodiments, the NFAT activation subsystem controls the expression of IL-15 or its variants. In some embodiments, the NFAT activation subsystem controls the expression of IL-18 or its variants. In some embodiments, the NFAT activation subsystem controls the expression of TeIL-12. In some embodiments, the NFAT activation subsystem controls the expression of TeIL-15. In some embodiments, the NFAT activation subsystem controls the expression of TeIL-18.

As used herein, "interleukin 12", "IL-12" and "IL12" all refer to an interleukin that is a heterodimeric interleukin encoded by the IL-12A and IL-12B genes (Genbank accession numbers: NM_000882 (IL-12A) and NM_002187 (IL-12B)). IL-12 consists of a bundle of four α-helices and is involved in the differentiation of naive T cells into TH1 cells. It is encoded by two independent genes, IL-12A (p35) and IL-12B (p40). After protein synthesis, it forms an active heterodimer (called "p70") and a p40 homodimer. IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL-12R-β1 and IL-12R-β2. IL-12 is called a T cell stimulating factor, which can stimulate the growth and function of T cells. In detail, IL-12 can stimulate the production of interferon gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) by T cells and natural killer (NK) cells, and reduce IL-4-mediated IFN-γ inhibition. IL-12 can further mediate the enhancement of cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. In addition, IL-12 can also have anti-angiogenic activity by increasing the production of interferon gamma, which in turn increases the production of interleukin-inducing protein 10 (IP-10 or CXCL10). IP-10 then mediates this anti-angiogenic effect. Therefore, without being bound by any particular theory of operation, it is believed that IL-12 can increase the viability and/or anti-tumor effect of the TIL compositions provided herein.

In some embodiments, IL-12 is a full-length IL-12, a fragment or a variant of IL-12. In some embodiments, IL-12 is a human IL-12 or a variant human IL-12. In an exemplary embodiment, IL-12 is a biologically active human IL-12 variant. In some embodiments, IL-12 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to wild-type IL-12.

In some embodiments, IL-12 comprises an IL-12 p35 subunit or a variant thereof. In some embodiments, the IL-12 p35 subunit is a human IL-12 p35 subunit. In some embodiments, the IL-12 p35 subunit has the amino acid sequence of SEQ ID NO: 60. In certain embodiments, IL-12 comprises an IL-12 p40 subunit or a variant thereof. In some embodiments, the IL-12 p40 subunit has the amino acid sequence of SEQ ID NO: 61. In certain embodiments, IL-12 is a single-chain IL-12 polypeptide comprising an IL-12 p35 subunit attached to an IL-12 p40 subunit. Such IL-12 single-chain polypeptides advantageously retain one or more biological activities of wild-type IL-12. In some embodiments, the single chain IL-12 polypeptide described herein is according to the following formula: from N-terminus to C-terminus, (p40)-(L)-(p35), wherein "p40" is the IL-12 p40 subunit, "p35" is the IL-12 p35 subunit, and L is a linker. In other embodiments, the single chain IL-12 is according to the following formula: from N-terminus to C-terminus, (p35)-(L)-(p40). Any suitable linker can be used for the single chain IL-12 polypeptide, including those described herein. Suitable linkers can include, for example, a linker having the amino acid sequence (GGGGS) _x , wherein x is an integer from 1-10. Other suitable linkers include, for example, the amino acid sequence GGGGGGS. Exemplary single-chain IL-12 linkers that can be used with the subject single-chain IL-12 polypeptides are also described in Lieschke et al., Nature Biotechnology 15: 35-40 (1997), which is incorporated herein by reference in its entirety, and particularly for its teachings regarding IL-12 polypeptide linkers. In an exemplary embodiment, the single-chain IL-12 polypeptide is a single-chain human IL-12 polypeptide (i.e., it includes human p35 and p40 IL-12 subunits).

In an exemplary embodiment, the nucleic acid molecule disclosed herein encodes TeIL-12, which comprises the amino acid sequence of SEQ ID NO: 62 (amino acids 1-18: human IgE signal sequence peptide; amino acids 529-553: peptide linker; amino acids 554-601: membrane anchor), wherein the nucleic acid is operably linked to a NFAT promoter, an EF-1a promoter, a CMV promoter, a CAG promoter, a MND promoter, or a SSFV promoter, as described herein. See, e.g., U.S. Patent No. 8,556,882, which is incorporated herein by reference in its entirety and particularly with respect to the relevant portions of the NFAT promoter for IL-12 expression.

In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFNγ, TNFa, IFNα, IFNβ, GM-CSF, GCSF or variants thereof. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the cytokine is under the control of an EF1a promoter, a CMV promoter, a CAG promoter, a MND promoter or a SSFV promoter.

As used herein, "interleukin 15", "IL-15" and "IL15" all refer to interleukins that bind to and signal through a complex consisting of the IL-15-specific receptor α chain (IL-15Rα), the IL-2/IL-15 receptor β chain (CD122) and the common γ chain (γ-C, CD132) (e.g., Genbank accession numbers: NM_00000585, NP_000576 and NP_751915 (human); and NM_001254747 and NP_001241676 (mouse)). IL-15 has been shown to stimulate T cell proliferation in tumors. IL-15 can also enhance the survival of effector memory CD8+ T cells and is important for the development of NK cells. Thus, without being bound by any particular theory of operation, it is believed that the modified TILs combined with IL-15 described herein exhibit enhanced survival and/or anti-tumor effects.

IL-15 has a short half-life of less than 40 minutes in vivo. Modifications to the IL-15 monomer can improve its in vivo pharmacokinetic properties in the treatment of cancer. These modifications are generally focused on improving the trans-presentation of IL-15 by the α subunit of the IL-15 receptor, IL-15Rα. Such modifications include: 1) pre-binding of IL-15 to its soluble receptor α-subunit-Fc fusion to form an IL-15:IL-15Rα-Fc complex (see, e.g., Rubinstein et al., Proc Natl Acad Sci U.S.A. 103:9166-71 (2006)); 2) expression of the superagonist IL-15-sIL-15Rα-sushi protein (see, e.g., Bessard et al., Molecular cancer therapeutics 8: 2736-45 (2009)); and 3) pre-binding of the human IL-15 mutant IL-15N72D to the IL-15Rα-Fc sushi-Fc fusion complex (see, e.g., Zhu et al., Journal of Immunology 183: 3598-6007). (2009)).

In some embodiments, IL-15 is tethered IL-15 (TeIL-15). In some embodiments, TeIL-15 comprises the amino acid sequence of SEQ ID NO: 73 (amino acids 1-18: human IgE signal sequence peptide; amino acids 132-157: peptide linker; amino acids 158-205: membrane anchor).

In some embodiments, IL-15 is a full-length IL-15, a fragment or a variant of IL-15. In some embodiments, IL-15 is a human IL-15 or a variant human IL-15. In exemplary embodiments, IL-15 is a biologically active human IL-15 variant. In some embodiments, IL-15 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to wild-type IL-15. In certain embodiments, IL-15 comprises an N72D mutation compared to wild-type human IL-15. In some embodiments, the variant IL-15 exhibits IL-15Rα binding activity.

In some embodiments, IL-15 comprises the extracellular domain of IL-15 and IL-15Rα. In certain embodiments, IL-15 comprises IL-15 and IL-15Rα fused to an Fc domain (IL-15Rα-Fc).

In some embodiments, the interleukin is superagonist IL-15 (IL-15SA), which includes a complex of human IL-15 and soluble human IL-15Rα. The combination of human IL-15 and soluble human IL-15Rα forms an IL-15 SA complex, which has higher biological activity than human IL-15 alone. Soluble human IL-15Rα and truncated forms of the extracellular domain have been described in the art (Wei et al., 2001 J of Immunol. 167: 277-282). The amino acid sequence of human IL-15Rα is described in SEQ ID NO: 72. In some embodiments, IL-15SA includes a complex of human IL-15 and soluble human. IL-15Rα comprises all or part of the extracellular domain and does not have a transmembrane or cytoplasmic domain. In some embodiments, IL-15SA comprises a complex of human IL-15 and soluble human IL-15Rα, wherein the soluble human IL-15Rα comprises the complete extracellular domain or a truncated form of the extracellular domain that retains IL-15 binding activity.

In some embodiments, IL-15SA comprises a complex of human IL-15 and soluble human IL-15Rα, wherein the soluble human IL-15Rα comprises a truncated form of the extracellular domain that retains IL-15 binding activity. In some embodiments, the soluble human IL-15Rα comprises amino acids 1-60, 1-61, 1-62, 1-63, 1-64, or 1-65 of human IL-15Rα. In some embodiments, the soluble human IL-15Rα comprises amino acids 1-80, 1-81, 1-82, 1-83, 1-84, or 1-85 of human IL-15Rα. In some embodiments, the soluble human IL-15Rα comprises amino acids 1-180, 1-181, or 1-182 of human IL-15Rα.

In some embodiments, the interleukin is IL-15SA comprising a complex of human IL-15 and soluble human IL-15Rα, wherein the soluble human IL-15Rα comprises a truncated form of the extracellular domain that retains IL-15 binding activity and comprises a sushi domain. In this technology, the sushi domain of IL-15Rα is described as being approximately 60 amino acids in length and comprising 4 cysteines. (Wei et al., 2001). Truncated forms of soluble human IL-15Rα that retain IL-15 activity and comprise a sushi domain are suitable for use in the IL-15SA of the present invention.

In some embodiments, the interleukin comprises a complex comprising soluble human IL-15Rα and IL-15 expressed as a fusion protein, such as an Fc fusion (e.g., human IgG1 Fc) as described herein. In some embodiments, IL-15SA comprises a complex of a dimeric human IL-15RαFc fusion protein (e.g., human IgG1 Fc) and two human IL-15 molecules.

In some embodiments, the interleukin is an IL-15SA interleukin complex, which includes an IL-15 molecule comprising the amino acid sequence shown in SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the IL-15SA interleukin complex comprises a soluble IL-15Rα molecule comprising the sequence of SEQ ID NO: 66, SEQ ID NO: 70, or SEQ ID NO: 71.

In some embodiments, the interleukin is an IL-15SA interleukin complex, which includes a complex of a dimeric IL-15RαFc fusion protein and two IL-15 molecules. In some embodiments, IL-15-SA comprises a dimeric IL-15RαSu (sushi domain)/Fc (SEQ ID NO: 65) and two IL-15N72D (SEQ ID NO: 64) molecules (also known as ALT-803), as described in US20140134128, which is incorporated herein by reference. In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc molecule (SEQ ID NO: 65) and two IL-15 molecules (SEQ ID NO: 67). In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc molecule (SEQ ID NO: 65) and two IL-15 molecules (SEQ ID NO: 68). In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc molecule (SEQ ID NO: 65) and two IL-15 molecules (SEQ ID NO: 69).

In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc molecule (SEQ ID NO: 65) and two IL-15 molecules having an amino acid sequence selected from SEQ ID NOs: 64, 67, 68 and 69.

In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 66) and two IL-15 molecules (SEQ ID NO: 64). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 66) and two IL-15 molecules (SEQ ID NO: 67). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 66) and two IL-15 molecules (SEQ ID NO: 68). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 66) and two IL-15 molecules (SEQ ID NO: 69).

In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO: 64). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO: 67). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO: 68). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO: 69).

In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 71) and two IL-15 molecules (SEQ ID NO: 64). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 71) and two IL-15 molecules (SEQ ID NO: 67). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 165) and two IL-15 molecules (SEQ ID NO: 68). In some embodiments, IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 165) and two IL-15 molecules (SEQ ID NO: 69).

In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc (SEQ ID NO: 65) molecule and two IL-15 molecules (SEQ ID NO: 68). In some embodiments, IL-15SA comprises a dimeric IL-15RαSu/Fc (SEQ ID NO: 65) molecule and two IL-15 molecules (SEQ ID NO: 69).

In some embodiments, IL-15SA comprises SEQ ID NO: 65 and SEQ ID NO: 66. In some embodiments, IL-15SA comprises SEQ ID NO: 67 or SEQ ID NO: 68. In some embodiments, IL-15SA comprises SEQ ID NO: 67 and SEQ ID NO: 65. In some embodiments, IL-15SA comprises SEQ ID NO: 68 and SEQ ID NO: 65. In some embodiments, IL-15SA comprises SEQ ID NO: 69 and SEQ ID NO: 65. In some embodiments, IL-15SA comprises SEQ ID NO: 67 and SEQ ID NO: 66. In some embodiments, IL-15SA comprises SEQ ID NO: 68 and SEQ ID NO: 66.

As used herein, "interleukin 18", "IL-18", "IL18", "IGIF", "IL-1g", "interferon-γ-inducing factor" and "IL1F4" all refer to an interleukin, which is a heterodimeric cytokine encoded by the IL-18 gene (e.g., Genbank accession numbers: NM_001243211, NM_001562 and NM_001386420). IL-18, which is structurally similar to IL-1β, is a member of the IL-1 superfamily of cytokines. This cytokine, which is expressed by many human lymphoid and non-lymphoid cells, plays an important role in the inflammatory process. The combination of IL-18 and IL-12 can activate cytotoxic T cells (CTL) and natural killer (NK) cells to produce IFN-γ and thus, contribute to tumor immunity. Therefore, without being bound by any particular theory of operation, it is believed that IL-18 can enhance the anti-tumor effect of the TIL compositions provided herein.

In some embodiments, IL-18 is tethered IL-18 (TeIL-18). In some embodiments, TeIL-18 comprises the amino acid sequence of SEQ ID NO: 132 (amino acids 1-18: human IgE signal sequence peptide; amino acids 176-200: peptide linker; amino acids 201-248: membrane anchor). In some embodiments, TeIL-18 comprises the amino acid sequence of SEQ ID NO: 134 (amino acids 1-18: human IgE signal sequence peptide; amino acids 175-199: peptide linker; amino acids 200-247: membrane anchor).

In some embodiments, IL-18 is a full-length IL-18, a fragment or variant of IL-18. In some embodiments, IL-18 is human IL-18 or a variant human IL-18. In exemplary embodiments, IL-18 is a biologically active human IL-18 variant. In some embodiments, IL-18 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mutations compared to wild-type IL-18 (SEQ ID NO: 75). In some embodiments, the biologically active variant is an anti-attractant IL-18 variant ("DR-IL18" or "DR-IL-18"), which provides IL-18 signaling activity even in the presence of inhibitory molecules such as IL-18 binding protein (IL-18BP). Exemplary IL-18 variants that may be included in the subject modified TILs described herein are shown in Table 7 below. Other IL-18 variants that may be included in the subject modified TILs are described in WO 2022/094473, which is incorporated by reference in its entirety and specifically with respect to the disclosure related to variant DR-IL-18.

In some embodiments, the variant IL-18 comprises a stabilizing mutation pair selected from C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, and C38S/C68N [relative to human wild-type IL-18 - SEQ ID NO: 75]. In some embodiments, in addition to a stabilizing mutation pair selected from C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, and C38S/C68N [relative to human wild-type IL-18 - SEQ ID NO: 75], the variant IL-18 comprises amino acid positions M51 (e.g., M51E, M51R, M51K, M51T, M51D, or M51N), K53 (e.g., K53G, K53S, K53T, or K53R), Q56 (e.g., Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K), D110 (e.g., D110S, D110N, D110G, D110K, D110H, D110Q, or D110E) and N111 (e.g., N111G, N111R, N111S, N111D, N111H, or N111Y). In some such cases, the stabilized IL-18 variant polypeptide additionally comprises a mutation at amino acid position S105 (e.g., S105D, S105A, S105N, S105R, S105D, or S105K); and in some cases, further comprises mutations at amino acid positions P57 (e.g., P57A, P57L, P57G, or P57K) and M60 (e.g., M60L, M60R, M60K, or M60Q).

As used herein, "interleukin 21", "IL-21" and "IL21" (e.g., Genbank accession numbers: NM_001207006 and NP_001193935 (human); and NM_0001291041 and NP_001277970 (mouse)) all refer to members of the interleukin class that bind to the IL-21 receptor and have potent regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic cells, and bind to the IL-21 receptor that can destroy virally infected or cancerous cells. Therefore, without being bound by any particular theory of operation, it is believed that IL-21 can increase the viability and/or anti-tumor effects of the TIL compositions provided herein.

In some embodiments, IL-21 is tethered IL-21. In some embodiments, tethered IL-21 comprises the amino acid sequence of SEQ ID NO: 137 (amino acids 1-18: human IgE signal sequence peptide; amino acids 152-197: peptide linker; amino acids 198-245: membrane anchor).

In some embodiments, IL-21 is human IL-21 (SEQ ID NO: 136). In some embodiments, the IL-21 combined with the modified TIL is a fragment or variant of full-length IL-21, IL-21. In some embodiments, IL-21 is human IL-21 or variant human IL-21. In exemplary embodiments, IL-21 is a biologically active human IL-21 variant. In some embodiments, IL-21 includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to wild-type IL-21.

As used herein, "interleukin 2", "IL-2", "IL2" and "TCGF" (e.g., Genbank accession numbers: NM_000586 and NP_000577 (human)) all refer to members of the interleukin family that bind to the IL-2 receptor. IL-2 enhances activation-induced cell death (AICD). When naive T cells are also stimulated by antigens, IL-2 also promotes T cell differentiation into effector T cells and memory T cells, thereby helping the body fight infection. IL-2, together with other interleukins, stimulates naive CD4+ T cells to differentiate into Th1 and Th2 lymphocytes and prevents differentiation into Th17 and filtration Th lymphocytes. IL-2 also increases the cytotoxic activity of natural killer cells and cytotoxic T cells. Therefore, without being bound by any particular theory of operation, it is believed that IL-2 can increase the survival ability and/or anti-tumor effect of the TIL compositions provided herein.

In some embodiments, the IL-2 is tethered IL-2. In some embodiments, the tethered IL-2 comprises the amino acid sequence of SEQ ID NO: 139 (amino acids 1-20: human IL-2 signal peptide; amino acids 154-178: peptide linker; amino acids 179-226: membrane anchor).

In some embodiments, IL-2 is human IL-2 (SEQ ID NO: 138). In some embodiments, the IL-2 bound to the modified TIL is a full-length IL-2, a fragment or a variant of IL-2. In some embodiments, IL-2 is human IL-2 or a variant human IL-2. In exemplary embodiments, IL-2 is a biologically active human IL-2 variant. In some embodiments, IL-2 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to wild-type IL-2.

In some embodiments, the nucleic acid molecules provided herein further comprise a nucleotide sequence encoding a truncated CD-19 (tCD19).

In some embodiments, the nucleic acid molecules provided herein further comprise a nucleotide sequence encoding shRNA. In some embodiments, shRNA inhibits the expression of immune checkpoint genes.

Non-limiting examples of immune checkpoint genes that can be silenced or inhibited by shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, S KIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. For example, immune checkpoint genes that can be silenced or inhibited by shRNA can be selected from the group comprising: PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ and PKA. BAFF (BR3) is described in Bloom et al., J. Immunother. , 2018, in press. According to another example, immune checkpoint genes that can be silenced or inhibited by shRNA can be selected from the group comprising: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3) and combinations thereof. Exemplary PD-1 shRNA sequences are provided in the table below.

In some embodiments of the present invention, the nucleic acid molecules disclosed herein further comprise long terminal repeat (LTR) sequences, such as 5' LTR sequences and 3' LTR sequences (FIG. 28). LTR sequences are crucial for the integration of viral genomes into host cell genomes and the expression of viral genes in host cells.

In one embodiment of the invention, the nucleic acid molecules disclosed herein are in the form of recombinant lentiviral RNA molecules. For example, the recombinant lentiviral RNA molecule may comprise at least a portion of the lentiviral genome, including the 5' and 3' LTRs. In some embodiments, the lentiviral genome may be modified to inhibit replication and limit pathogenicity while retaining function. For example, the env gene, gag gene, pol gene, and rev gene may be removed from the lentiviral genome and included in the helper plasmid. In some embodiments, the recombinant lentiviral RNA molecule is comprised by a lentiviral vector or recombinant lentiviral particle as described elsewhere in this disclosure.

In another embodiment of the present invention, the nucleic acid molecules disclosed herein are in the form of recombinant lentiviral proviral DNA molecules. For example, a host cell, such as a gene-edited TIL as disclosed herein, comprises a recombinant lentiviral proviral DNA molecule. In some embodiments, the recombinant lentiviral proviral DNA molecule is integrated into the genome of a host cell, such as a gene-edited TIL as disclosed herein. In some embodiments, the recombinant lentiviral proviral DNA molecule is comprised by a packaging cell line as described elsewhere in this disclosure. In some embodiments, the recombinant lentiviral proviral DNA molecule is integrated into the genome of the packaging cell line. IV. Recombinant expression vectors, lentiviral expression systems, and packaging cell lines

In one embodiment of the present invention, the nucleic acid molecules of the present invention disclosed herein are carried in a recombinant expression vector. Therefore, one embodiment of the present invention provides a recombinant expression vector comprising any nucleic acid molecule of the present invention described herein with respect to other aspects of the present invention.

In some embodiments, a nucleotide sequence encoding one or more GOIs, such as an interleukin selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFNγ, TNFa, IFNα, IFNβ, GM-CSF, GCSF, or variants thereof, is provided by two or more recombinant expression vectors. For example, a recombinant expression vector is provided herein, comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second recombinant expression vector comprising a nucleotide sequence encoding IL-15 or a variant thereof. In some embodiments, provided herein is a recombinant expression vector comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second recombinant expression vector comprising a nucleotide sequence encoding IL-2 or a variant thereof.

For the purposes of this article, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that allows the cell to express the mRNA, protein, polypeptide or peptide when the construct contains a nucleotide sequence encoding the mRNA, protein, polypeptide or peptide and the vector is in contact with the cell under conditions sufficient for the mRNA, protein, polypeptide or peptide to be expressed in the host cell. The vectors of the present invention are not naturally occurring as a whole. However, portions of the vector may be naturally occurring. The recombinant expression vector may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from a natural source, and it may contain natural, non-natural or altered nucleotides. The recombinant expression vector may contain naturally occurring or non-natural internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not prevent transcription or replication of the vector. The vector may contain regulatory nucleic acid sequences that provide for expression of the nucleic acid of the invention.

The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include vectors designed for propagation and expansion or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Bumie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden) and the pEX series (Clontech, Palo Alto, CA).

Phage vectors such as λGT10, λGT11, λZap II (Stratagene), λEMBL4 and λNMI 149 may also be used. Examples of plant expression vectors that can be used in the context of the present invention include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the context of the present invention include pEUK-Cl, pMAM and pMAMneo (Clontech).

In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, but are not limited to, lentivirus, retrovirus, alphavirus, vaccine, adenovirus, adeno-associated virus, herpes virus, and fowlpox virus vectors, and preferably have native or engineered ability to transform T cells.

Recombinant expression vectors can be prepared using standard recombinant DNA techniques, which are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual, (4th edition) Cold Spring Harbor Laboratory Press, New York (2012). Circular or linear expression vector constructs can be prepared to contain replication systems that function in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

Recombinant expression vectors may contain regulatory sequences, such as transcription and transfer start and stop codons, which are specific for the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, and depending on whether the vector is DNA-based or RNA-based.

The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected hosts. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc., complementation in a nutrient-deficient host to provide trophotypes, and the like. Suitable marker genes for recombinant expression vectors include, for example, neomycin/G418 resistance genes, hygromycin resistance genes, histamine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector may comprise a natural or standard promoter operably linked to a nucleic acid molecule. Preferably, the promoter is functional in T cells. The selection of promoters, such as strong promoters, weak promoters, inducible promoters, tissue-specific promoters, and development-specific promoters, is within the ordinary skill of the skilled person. Similarly, the combination of nucleotide sequences and promoters is also within the skill of the skilled person. The promoter can be a non-viral promoter or a viral promoter, such as the NFAT promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, or a promoter found in the long terminal repeat sequence of the mouse stem cell virus.

The present invention further provides a lentiviral expression system, which includes: a transfer vector comprising a nucleic acid molecule disclosed herein, which can be transcribed into a recombinant lentiviral RNA molecule to be packaged into lentiviral particles; and one or more auxiliary vectors or auxiliary plasmids encoding Env protein, Gag protein, Pol protein and Rev protein. In some embodiments, the Env protein is selected from the group consisting of: Ba-EVTR, VSV-G and RD114.

In some embodiments, the recombinant lentiviral RNA molecule may comprise at least a portion of the lentiviral genome, including the 5' and 3' LTRs. In some embodiments, the lentiviral genome may be modified to inhibit replication and limit pathogenicity while retaining function. For example, the env gene, gag gene, pol gene, and rev gene may be removed from the lentiviral genome and included in the helper plasmid. In some embodiments, the recombinant lentiviral RNA molecule is comprised by a lentiviral vector or recombinant lentiviral particle as described elsewhere in this disclosure.

In some embodiments, two auxiliary vectors or auxiliary plastids encode Env protein, Gag protein, Pol protein and Rev protein. In some embodiments, three auxiliary vectors or auxiliary plastids encode Env protein, Gag protein, Pol protein and Rev protein. In some embodiments, four auxiliary vectors or auxiliary plastids encode Env protein, Gag protein, Pol protein and Rev protein. Any combination of auxiliary vectors or auxiliary plastids can be used to encode Env protein, Gag protein, Pol protein and Rev protein. For example, in some embodiments, one auxiliary vector or auxiliary plastid encodes Env protein, Gag protein and Pol protein. In some embodiments, one auxiliary vector or auxiliary plastid encodes Gag protein, Pol protein and Rev protein. In some embodiments, a helper vector or helper plasmid encodes Gag protein and Pol protein. In some embodiments, a helper vector or helper plasmid encodes Env protein and Rev protein.

The packaging cell line can be used to package nucleic acids (e.g., RNA encoding a transgene) into a lentiviral vector. Thus, the systems and methods described herein can include, for example, a lentiviral packaging cell line comprising at least one plasmid suitable for producing a lentiviral vector, such as a lentiviral vector optionally comprising a transgene. Various lentiviral components that can be used to produce lentiviral vectors are known in the art. See, for example, Zufferey et al., 1997, Nat. Biotechnol. 15:871-875 and Dull et al., 1998, J. Virol. 72(11): 8463-8471. Different functions of packaging cells suitable for producing lentiviral vectors can be provided in a lentiviral packaging system, the lentiviral packaging system comprising one or more nucleic acids (e.g., plasmids), such as at least one, two, three, or four plasmids, wherein one plasmid encodes a retroviral envelope protein (Env plasmid), one plasmid encodes one or more retroviral packaging proteins, such as Gag and Pol proteins (packaging plasmid or Gag-Pol plasmid), one plasmid encodes a lentiviral Rev protein (Rev plasmid) and one or more plasmids comprising at least one gene of interest (GOI) expression cassette (transfer vector). In some embodiments, the lentiviral packaging system further comprises at least one, two, three, or four plasmids, or the methods described herein comprise the use of at least one, two, three, or four plasmids. In some embodiments, the lentiviral packaging system further comprises a fifth plasmid, or the methods described herein include using a fifth plasmid. In certain embodiments, the methods described herein include transfecting five plasmids into packaging cells, wherein the fifth plasmid does not encode a protein of the lentiviral vector packaging system. In some embodiments, the lentiviral packaging system comprises one or more nucleic acids (e.g., plasmids), such as five plasmids, wherein one plasmid encodes an expression vector, one plasmid encodes Tat (e.g., pcDNATat), one plasmid encodes Rev protein (e.g., pHCMV-Rev), one plasmid encodes gagpol (e.g., pHCMV-gagpol), and one plasmid encodes VSV-G (e.g., pVSVG), such as Rout-Pitt et al., J Biol. Methods 5(2): 1-9, 2018. In some embodiments, the plasmid may comprise a dual gene expression cassette, such as a bicistronic cassette, such as a bicistronic construct encoding two transgenes of interest. In some embodiments, the first transgene of interest encodes a first interleukin, such as TeIL-12, and the second transgene of interest encodes a second interleukin, such as IL-15. In some embodiments, the retroviral packaging protein is derived from a lentivirus, such as a lentiviral packaging protein, such as lentiviral gag and pol proteins.

In some embodiments, the lentiviral gag protein is a wild-type lentiviral gag protein, and in other embodiments, it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral pol protein is a wild-type lentiviral pol protein, and in other embodiments, it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the rev protein is a wild-type rev protein, and in other embodiments, it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral vector packaging system can be a pseudotyped lentiviral vector packaging system, which includes a modified envelope protein, such as an envelope protein derived from a different virus or a chimeric envelope protein, such as an Env plasmid that can encode a Ba-EVTR Env protein.

In some embodiments, a lentiviral vector is generated using a packaging system comprising pMDLgpRRE, pRSV-Rev, and pMD.G plasmids (Dull et al., supra), but a kanamycin resistance marker, e.g., a marker that confers resistance to both kanamycin and neomycin, e.g., neomycin phosphotransferase II, is used instead of the ampicillin gene.

Therefore, the present invention further provides a packaging cell line comprising a recombinant expression vector disclosed herein, such as a transfer vector, comprising a nucleic acid molecule comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12) and, optionally, a cytokine selected from the group consisting of: IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFNγ, TNFa, IFNα, IFNβ, GM-CSF, GCSF or variants thereof. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding TeIL-12 and tethered IL-15 (TeIL-15). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding TeIL-12 and tethered IL-18 (TeIL-18).

In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding shRNA. In some embodiments, shRNA inhibits the expression of immune checkpoint genes. In some embodiments, shRNA inhibits the expression of PD-1.

Non-limiting examples of immune checkpoint genes that can be silenced or inhibited by shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, S KIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. For example, immune checkpoint genes that can be silenced or inhibited by shRNA can be selected from the group comprising: PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ and PKA. BAFF (BR3) is described in Bloom et al., J. Immunother. , 2018 , in press. According to another example, immune checkpoint genes that can be silenced or inhibited by shRNA can be selected from the group comprising: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3) and combinations thereof.

In some embodiments, the packaging cell line is a human cell line. In some embodiments, the packaging cell line is a 293T cell line.

In some embodiments, the packaging cell line is a stable packaging cell line produced by the EuLV® system (Shenzhen Eureka Biotechnology Co., Ltd., Shenzhen, China) described in Xue et al., Cell & Gene Therapy Insights 2022 ; 8(2), 199-209 (the content is incorporated by reference in its entirety).

Any suitable protocol known in the art can be used to develop a stable packaging cell line. For example, Broussau et al., Mol. Thera. , 2008 , 16:500-507 describes an induced packaging cell line 293SF-PacLV for producing lentiviral vectors in serum-free culture, the contents of which are incorporated herein by reference in their entirety. First, a cell line derived from 293SF cells was established that expresses a repressor of the cumate switch (CymR) and a reverse transactivator of the tetracycline (Tet) switch (rtTA2S-M2). Next, a pure line that stably expresses the Gag/Pol and Rev genes of human immunodeficiency virus-1 and the glycoprotein of bronchiolitis virus (VSV-G) was generated. The expression of Rev and VSV-G is tightly regulated by the cumate and Tet switches in the 293SF-PacLV cell line. Two approaches were used to generate the packaging cell line. In the first approach (two-step), stable clones expressing Gag/Pol and Rev genes were first generated and characterized, and then clones expressing VSV-G and additional Rev were generated. In the first approach (one-shot), all LV components (Gag/Pol, rev, and VSV-G) were added simultaneously through a single transfection event.

Two-step approach: Generate a cell line derived from a 293 cell pure line (293SF) that is adapted for growth in suspension and in serum-free medium expressing CymR. Transfect 293SF with pMPGBFP/CMV5-CymR/tk-neo and isolate the resistant cell pool in the presence of neomycin. Then isolate the pure line by limiting dilution of the pool. Maintain the pure line for 6 weeks without selective pressure to test its stability. Analyze CymR function using an adenoviral vector (AdV) expressing β-galactosidase (β-gal) regulated by the CMV5-CuO promoter. The optimal β-gal on/off ratio in the presence and absence of cumate was used to select the clone producing the optimal amount of CymR. Clone G showed an on/off ratio of 14 and was selected as the receptor for the rtTA2 ^S -M2 transactivator. 293SF-CymR-G cells were transfected with the plasmid pUDHrtTA2 ^S -M2.hygro. Hygromycin-resistant cell pools were generated and clones were obtained by limiting dilution pools without selection. The functionality of CymR and rtTA2 ^S -M2 produced by the clones was tested using AdV encoding green fluorescent protein (GFP) regulated by the TR5 and CuO promoters. The clone with the best on/off ratio was selected for development of the packaging cell line.

This was done by transfection with pMPG-RSV-Rev/CMV-Gag/ polRRE, a plasmid encoding the Rev, Gag and pol genes of human immunodeficiency virus-1 and resistance to phleomycin as a fusion protein with GFP. Phleomycin-resistant clones were isolated in 96-well plates. The clone with the best GFP expression level was analyzed for LV production by transient transfection with pCSII-CMV5-GFPq and VSV-G. The titers obtained with the best clones were 10- to 55-fold lower than those obtained when the same clones were transfected with a plasmid (pRSV-Rev) encoding Rev regulated by Rous sarcoma virus (RSV). Two clones (#19 and #64) were then subcloned by limiting dilution and analyzed for LV production by transient transfection. As observed for the parental clones, significantly higher LV titers were obtained in the presence of additional Rev.

Because previous results showed that 293SF-Rev-Gag-Pol cells produced suboptimal amounts of Rev, the two best subclones (#19-17 and #64-8) were co-transfected with plasmids encoding puromycin resistance (pPuro), Rev (pkCMV5-CuO-Rev), and VSV-G (pTR5-CuO-VSVg-IRES-GFPq) to generate cell lines expressing VSV-G and more Rev. VSV-G is regulated by both the cumate switch and the Tet switch, while Rev is regulated only by the cumate switch. Puromycin-resistant clones were first screened for VSV-G expression by measuring GFP (via IRES-GFP) after Dox and cumate induction. LV production was then analyzed by transient transfection with pCSII-CMV5-GFPq. Co-transfection of the best clones with pRSV-Rev only increased LV titer by two to four times, below early levels. The three best clones were sub-selected without selective pressure. LV production was analyzed by transient transfection as described above. After the addition of Rev, the production of the six best sub-clones increased only by two to three times, indicating that the amount of Rev was close to optimal. The efficacy of the double switch was studied by measuring the increase in GFP expression (from the stably integrated VSV-G-IRES-GFPq cassette) after induction of clones #16-22. Induction factors of more than 2,500 were observed in the presence of cumate and Dox. GFP expression in the presence of both inducers was much higher than when either inducer was used alone. In the presence of cumate alone, GFP expression levels increased 4.5-fold, indicating that the cumate switch increases the density of the Tet switch by this fold.

One-shot approach: Since Rev is required for efficient expression of the protease from the Gag gene, which has been reported to be cytotoxic, even relatively small amounts of Rev may be harmful to cells. Therefore, a packaging cell line was generated that expresses Rev under very tight regulation. pTR5-CuO-Rev was constructed, which contains the Rev coding sequence that is doubly regulated by the Tet switch and the cumate switch. The 293SF-CymR-rtTA2 ^S -M2 cell line was co-transfected with plasmids encoding Rev, Gag, Pol, VSV-G and puromycin resistance. Stable clones were selected in the presence of puromycin. The clones with the highest GFP-inducing factor after the addition of Dox and cumate were analyzed for LV production by transfection with the transfer vector pCSII-CMV5-GFPq. The two best clones were then subcloned without selection and analyzed for LV production. Transfection of the best subclone with pCSIICMV5-GFPq and pRSV-Rev did not increase the amount of LV produced, indicating that the clones were not limiting in the amount of Rev produced.

The levels of LV production obtained in the best clones using the two-step (#16-22) and one-step (#29-6) methods were then compared. The titers for #16-22 and #29-6 were 8.6×10 ⁶ and 2.6×10 ⁷ TU/ml, respectively. The amount of p24 produced after induction was also assessed by enzyme-linked immunosorbent assay (ELISA). The amounts of p24 produced by #16-22 and #29-6 were 39 ng/ml and 571 ng/ml, respectively. The fact that the specific activity (TU/ng of p24) was 219,000 for #16-22 and 45,000 for #29-6 suggests that the latter clone produces more defective or empty virions.

Briefly, Ba-EVTR, gag/pol and rev are stably inserted into 293T cells to obtain a packaging cell population, and the best packaging cell line is obtained by single cell and titer screening. Then, a transfer vector comprising a nucleic acid molecule disclosed herein, i.e., a transfer vector encoding a GOI, is integrated into the best packaging cell line, such as a transfer vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12) and, optionally, a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFNγ, TNFa, IFNα, IFNβ, GM-CSF, GCSF or variants thereof. In some embodiments, the interleukin is a tethered interleukin. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding TeIL-12 and tethered IL-15 (TeIL-15). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding TeIL-12 and tethered IL-18 (TeIL-18).

In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding shRNA. In some embodiments, shRNA inhibits the expression of immune checkpoint genes. In some embodiments, shRNA inhibits the expression of PD-1.

Non-limiting examples of immune checkpoint genes that can be silenced or inhibited by shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, S KIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. For example, immune checkpoint genes that can be silenced or inhibited by shRNA can be selected from the group comprising: PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ and PKA. BAFF (BR3) is described in Bloom et al., J. Immunother. , 2018, in press. According to another example, immune checkpoint genes that can be silenced or inhibited by shRNA can be selected from the group comprising: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3) and combinations thereof.

In some embodiments, the packaging cell line comprises recombinant DNA encoding Env protein, Gag protein, Pol protein and Rev protein. In some embodiments, the recombinant DNA encoding one or more of Env protein, Gag protein, Pol protein and Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding Env protein, Gag protein and Pol protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding Env protein, Gag protein and Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding Env protein, Pol protein and Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding Gag protein, Pol protein and Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the Env protein is selected from the group consisting of: Ba-EVTR, VSV-G and RD114. In some embodiments, the Env protein is a Ba-EVTR Env protein. In some embodiments, the recombinant DNA encoding one or more of the Env protein, Gag protein, Pol protein and Ba-EVTR Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Ba-EVTR Env protein, Gag protein and Pol protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Ba-EVTR Env protein, Gag protein and Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Ba-EVTR Env protein, Pol protein and Rev protein is integrated into the genome of the packaging cell line.

In some embodiments, the recombinant DNA encoding Env protein, Gag protein, Pol protein and Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the nucleic acid molecules disclosed herein, such as recombinant lentiviral proviral DNA molecules, are integrated into the genome of the packaging cell line.

In some embodiments, different functions for producing lentiviral vectors are provided to a plurality of host cells, such as mammalian cells, such as HEK293 cells, such as Expi293F cells (e.g., a plurality of Expi293F cells grown in suspension under serum-free conditions), by transfection, such as transient or stable transfection, of a lentiviral packaging system suitable for producing lentiviral vectors. In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the host cells, such as HEK293 cells, such as Expi293F cells, are transfected. Methods of transfection or infection are well known to those skilled in the art. In some embodiments, at least 0.3 pg, at least 0.4 pg, at least 0.5 pg, at least 0.6 pg, at least 0.7 pg, at least 0.8 pg cells, at least 0.9 pg, or at least 1.0 pg of the lentiviral packaging system is provided per million cells for transfection. In some embodiments, the transfection reagent is used to transfect a host cell, such as a mammalian cell, such as a HEK293 cell, such as an Expi293F cell. In some embodiments, a transfection reagent is used. Transfection reagents are well known in the art and can be obtained from commercial suppliers. Examples of transfection reagents include, but are not limited to, Lipofectamine™ (Invitrogen), Polifectamine, LentiTran (Origene), PEIpro® (Polyplus), FectoVIR® - AAV (Polyplus), and ProFection® (Promega). V. Recombinant Lentivirus Particles and Methods for Preparing Them

In some embodiments, the present disclosure provides methods for making recombinant lentiviral particles. The following general steps can be used. First, a packaging cell population is cultured in a cell culture medium. Once a sufficient number of packaging cells are obtained, the desired nucleic acid, such as a lentiviral vector comprising a nucleic acid molecule encoding a cytokine disclosed herein, can be introduced into the packaging cells. Additional nucleic acids that can be introduced into the packaging cells include plasmids that promote packaging, such as plasmids encoding viral gag, pol, env, and rev. The packaging cells then begin to produce recombinant lentiviral particles. After transfection, a nuclease such as Benzonase can be added to the culture medium.

In some embodiments, a stable packaging cell line with VSV-G, gag/pol and rev integrated into the genome can be used to produce recombinant lentiviral particles. In some embodiments, the stable packaging cell line further comprises a lentiviral vector comprising a nucleic acid molecule encoding a cytokine disclosed herein integrated into the genome.

Any suitable protocol known in the art can be used to produce recombinant lentiviral particles using stable packaging cell lines. For example, Broussau et al. (supra) describes the production of lentiviral particles using stable packaging cell lines, the contents of which are incorporated herein by reference in their entirety.

If cell lines (producers) are available that synthesize all essential viral functions including viral RNA, there is no need to prepare large amounts of plasmids and no need for transfection procedures. Using such cell lines, LV production can be initiated simply by adding inducers such as Dox and cumate. The ability of packaging cells to generate stable producers was assessed. Lines #29-6 and #16-22 were transduced with conditional-SIN-LV, which was generated by transfecting packaging cells with the transfer vector pLVR2-GFP23. Lines were isolated by limiting dilution of the transduced cell pool. The line with the highest GFP expression after induction with Dox was depleted. The best pure lines from parental cells #16-22 (16-22-22) and #29-6 (#29-6-14) were tested for production in serum-free suspension culture. The medium was changed daily and the number of infectious particles was determined by flow cytometry.

The amount of p24 produced was also analyzed by ELISA. The behavior of the two clones was very similar, differing only in the absolute amount of LV produced, which was two to three times higher for clone #29-6-14. The number of infectious particles produced increased every day until it reached a maximum on the 4th day. It then gradually decreased. When using #29-6-14, the highest titer (3.4×10 ⁷ TU/ml) was obtained on the 4th day. The amount of p24 produced by the cells at different time points followed the same pattern. On the 1st day, the specific activity (TU/ng of p24) of the virus preparations of #16-22-22 and #29-6-14 were 54,000 and 166,000, respectively, and gradually decreased. At the end of production (day 6 or 7), the specific activity decreased 3- to 4-fold, indicating that more defective particles were produced at later time points.

The relative stability of four different production clones derived from packaging cell #29-6 was evaluated. The LV yields were compared after 4 and 18 weeks of culture without selective pressure. After 18 weeks of culture, the titers obtained with these four clones did not drop significantly. These data indicate that using the packaging cells described in this study, production can be obtained with long-term stability sufficient for large-scale production processes. In addition, the presence of replication-competent lentivirus was tested by infecting 293 cells with concentrated LV stocks. No replication-competent lentivirus was detected by ELISA analysis of P24, and no VSV-G was detected by polymerase chain reaction (PCR).

Since subcloning and analysis of individual clones is a time-consuming process, it was investigated whether cell pools could be used to obtain high levels of LV. For this experiment, packaging cells #29-6 were transduced with conditional-SIN-LV, which was produced by transfection of packaging cells with pTet07-CSII-CMVGFPq. The production efficiency of the production pool was then tested in serum-free suspension culture in shake flasks. The medium was changed every day and the number of infectious particles was determined by flow cytometry. The production pattern of the pool was very similar to that of the two clones, except that the highest titer (1.9×10 ⁷ TU/ml) was obtained on day 3 instead of day 4, and the duration of useful production was one day shorter.

Without wishing to be bound by theory, in some embodiments, the cell culture medium is a source of contaminating nucleic acids in the final lentiviral preparation, for example, the culture medium may contain packaging cell DNA from lysed packaging cells. Therefore, adding benzonase to the cell culture medium can degrade contaminating nucleic acids, thereby improving the purification of recombinant lentiviral particles.

Next, the recombinant lentiviral particles can be harvested from the packaging cell culture to initiate purification of the recombinant lentiviral particles. In some embodiments, harvesting the recombinant lentiviral particles includes separating the supernatant or cell culture medium from the packaging cells. In some embodiments, the packaging cells are not lysed prior to clarification. In some embodiments, the packaging cells can be lysed and the lysate can be clarified.

Naturally occurring lentiviruses are a type of virus in the retroviridae family that is characterized by a long latent period. Lentiviruses can usually deliver large amounts of genetic information into the DNA of host cells. Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), the causative agent of acquired immunodeficiency syndrome (AIDS) in humans; visna-maedi virus (causing encephalitis (visna) or pneumonia (maedi) in sheep), caprine arthritis encephalitis virus (causing immunodeficiency, arthritis, and encephalopathy in goats); equine infectious anemia virus, which causes autoimmune hemolytic anemia and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immunodeficiency in cats; bovine immunodeficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possible central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in primates inferior to humans. Diseases caused by these viruses are characterized by a long incubation period and a prolonged course. Usually, the virus latently infects monocytes and macrophages, which spread to other cells. HIV, FIV and SIV also easily infect T lymphocytes (also known as T cells).

The present invention further provides a recombinant lentiviral particle comprising the recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the recombinant lentiviral particle is produced by the packaging cell line disclosed herein.

In some embodiments, the recombinant lentiviral particle comprises a recombinant lentiviral vector disclosed herein. In some embodiments, the recombinant lentiviral vector comprises a recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the recombinant lentiviral particle further comprises a capsid encapsulating the recombinant RNA viral genome. In some embodiments, the recombinant lentiviral particle further comprises an Env protein selected from the group consisting of: Ba-EVTR, VSV-G, and RD114. In some embodiments, the recombinant lentiviral particle further comprises a reverse transcriptase.

Added section on the production of non-gene-edited TILs VI. Methods for preparing gene-edited TILs

In some embodiments of the present invention for the method for expanding TIL groups, the methods include one or more gene editing at least a portion of TIL to enhance its therapeutic effect. As used herein, "gene editing (gene-editing/gene editing)" and "genome editing" refer to a kind of gene modification, wherein DNA is permanently modified in the genome of a cell, such as inserting, deleting, modifying or replacing DNA in the genome of a cell. In some embodiments, gene editing causes silencing (sometimes referred to as gene knockout) or inhibition/reduction (sometimes referred to as gene attenuation) of the expression of a DNA sequence. In other embodiments, gene editing causes enhancement of the expression of a DNA sequence (e.g., by causing overexpression). According to embodiments of the present invention, gene editing technology is used to enhance the effectiveness of therapeutic TIL groups.

In some embodiments of the present invention, a method for producing a gene-edited TIL population is provided herein, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) performing a second expansion by culturing the TIL population in a second cell culture medium; and c) at any time, transducing the TIL population with the recombinant lentiviral particles disclosed herein to produce the gene-edited TIL population, wherein the gene-edited TIL population expresses one or more interleukins, such as IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFNγ, TNFa, IFNα, IFN β, GM-CSF, GCSF or their variants.

Cell surface markers PD-1, CD39, and CD103 are the main biomarkers for identifying tumor-specific T cells, but these TILs are terminally differentiated. Upregulation of CD137 (4-1BB; TNFSR9) on recently activated CD8+ T cells has been used to identify tumor antigen-specific T cells (Draghi A. et al., Front Immunol 2021 ; 12:705422, the contents of which are incorporated herein by reference in their entirety), which are antigen-specific and tend to have a more fit/younger phenotype (c-fos and c-jun expression). CD137 provides enhanced survival, proliferation, and effector functions and metabolic advantages to antigen-induced T cells through costimulatory effects.

Thus, the present invention provides a method for enriching tumor-specific CD8 TILs (preserving the breadth of the TCR tumor-reactive repertoire) while providing CD4 TILs in the same process. In some embodiments, the method comprises initiating a tumor and using CD137 as a marker to enrich and expand tumor-specific T cells without the need to understand epitope specificity. See, e.g., FIG. 48. A. Obtaining a Patient Tumor Sample

Generally, TILs are initially obtained from patient tumor samples ("primary TILs") and then expanded into larger populations for further manipulation as described herein.

Patient tumor samples can be obtained using methods known in the art, usually obtained by surgical resection, biopsy, core needle biopsy, small biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells. In some embodiments, multi-lesion sampling is used. In some embodiments, surgical resection, biopsy, core needle biopsy, small biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells include multi-lesion sampling (that is, one or more tumor sites and/or positions in the patient and one or more tumors in the same position or closely adjacent to obtain samples). In general, tumor samples can come from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. Tumor samples may also be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors may be any type of cancer, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from endometrial cancer, cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), neuroglioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, suitable TILs are obtained from melanoma.

Once obtained, tumor samples are typically fragmented using a sharp tool into small pieces between 1 and about 8 mm ³ , with about 2-3 mm ³ being particularly useful. In some embodiments, TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzymatic medium (e.g., Roswell Park Cancer Institute (RPMI) 1640 buffer, 2 mM glutamine, 10 mcg/mL tamiprocin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in an enzymatic medium and mechanically dissociating the tumor for about 1 minute, followed by incubation at 37°C in 5% _CO2 for 30 minutes, followed by repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched chain hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art may be used, such as the method described in U.S. Patent Application Publication No. 2012/0244133 A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the methods of expanding TILs or treating cancer described herein. B. Tumor fragmentation and / or digestion

As noted above, in some embodiments, TILs are derived from solid tumors. In some embodiments, solid tumors are not fragmented. In some embodiments, solid tumors are not fragmented and are enzymatically digested with the whole tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme, and neutral protease. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme, and neutral protease for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme, and neutral protease at 37°C, ₅ % CO2 for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme, and neutral protease at 37°C, 5% _CO2 , with rotation for 1 to 2 hours. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C, 5% _CO2 , under constant rotation. In some embodiments, the whole tumor is combined with an enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of collagenase is 100 mg/ml 10X working stock solution.

In some embodiments, the enzyme mixture comprises DNase. In some embodiments, the working stock solution of DNase is 10,000 IU/ml 10X working stock solution.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10 mg/ml 10X working stock solution.

In some embodiments, the enzyme mixture comprises 10 mg/ml collagenase, 1000 IU/ml DNase, and 1 mg/ml hyaluronidase.

In some embodiments, the enzyme mixture comprises 10 mg/ml collagenase, 500 IU/ml DNase, and 1 mg/ml hyaluronidase.

In some embodiments, the enzyme mixture comprises a neutral protease. In some embodiments, the working stock solution of the neutral protease is reconstituted at a concentration of 175 DMC U/mL.

In some embodiments, the enzyme mixture comprises a neutral protease, a DNase, and a collagenase.

In some embodiments, the enzyme mixture comprises 10 mg/ml collagenase, 1000 IU/ml DNase, and 0.31 DMC U/ml neutral protease. In some embodiments, the enzyme mixture comprises 10 mg/ml collagenase, 500 IU/ml DNase, and 0.31 DMC U/ml neutral protease.

Generally speaking, the harvested cell suspension is called the "primary cell population" or the "freshly harvested" cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, segmentation and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is segmentation. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially obtained from enzymatic tumor digests and tumor fragment cultures of patients. In some embodiments, TILs can be initially obtained from enzymatic tumor digests and tumor fragment cultures of patients before gene editing.

In some embodiments, when the tumor is a solid tumor, after obtaining the tumor sample, the tumor is physically fragmented. In some embodiments, the fragmentation occurs before cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after the tumor is obtained and without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm ³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 mm ³ to about 1500 mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total volume of about 1350 mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments. In some embodiments, the plurality of fragments comprises about to about 100 fragments.

In some embodiments, TILs are obtained from tumor fragments. In some embodiments, tumor fragments are obtained by sharpening. In some embodiments, tumor fragments are between about 1 mm ³ and 10 mm ^3. In some embodiments, tumor fragments are between about 1 mm ³ and 8 mm ^3. In some embodiments, tumor fragments are about 1 mm ^3. In some embodiments, tumor fragments are about 2 mm ^3. In some embodiments, tumor fragments are about 3 mm ^3. In some embodiments, tumor fragments are about 4 mm ^3. In some embodiments, tumor fragments are about 5 mm ^3. In some embodiments, tumor fragments are about 6 mm ^3. In some embodiments, tumor fragments are about 7 mm ³ . In some embodiments, a tumor fragment is about 8 mm ³ . In some embodiments, a tumor fragment is about 9 mm ³ . In some embodiments, a tumor fragment is about 10 mm ³ . In some embodiments, a tumor is 1 to 4 mm x 1 to 4 mm x 1 to 4 mm. In some embodiments, a tumor is 1 mm x 1 mm x 1 mm. In some embodiments, a tumor is 2 mm x 2 mm x 2 mm. In some embodiments, a tumor is 3 mm x 3 mm x 3 mm. In some embodiments, a tumor is 4 mm x 4 mm x 4 mm.

In some embodiments, tumors are resected to minimize the amount of hemorrhagic, necrotic, and/or fatty tissue on each slice. In some embodiments, tumors are resected to minimize the amount of hemorrhagic tissue on each slice. In some embodiments, tumors are resected to minimize the amount of necrotic tissue on each slice. In some embodiments, tumors are resected to minimize the amount of fatty tissue on each slice.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without using a scalpel to perform a sawing action. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are produced by cultivating in an enzymatic medium (such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg/mL genitamicin, 30 U/mL DNA enzyme, and 1.0 mg/mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After the tumor is placed in an enzymatic medium, the tumor can be mechanically dissociated for about 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes and then mechanically disrupted again for about 1 minute. After another 30 minutes of incubation at 37°C in 5% _CO2 , the tumor can be mechanically disrupted a third time for about 1 minute. In some embodiments, if large pieces of tissue still exist after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to the sample, regardless of whether or not the sample is incubated for another 30 minutes at 37°C in 5% _CO2 . In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

In some embodiments, the cell suspension harvested before the first expansion step is referred to as the "primary cell population" or "freshly harvested" cell population.

In some embodiments, cells are optionally frozen following sample harvest and stored frozen prior to expansion as described in further detail below.

In some embodiments, the TILs are obtained by thawing a cryopreserved tumor digest comprising a first TIL population derived from a tumor resected from an individual. In some embodiments, the first TIL population is obtained from a tumor resected from an individual by processing a tumor sample obtained from an individual into a tumor digest. In some embodiments, a tumor digest or tumor fragment comprises a first TIL population and is obtained from a tumor resected from an individual.

In some embodiments, the methods disclosed herein include adding a tumor digest or tumor fragment to a closed system, wherein the tumor digest or tumor fragment comprises a first TIL population and is obtained from a tumor excised from an individual. In some embodiments, the methods disclosed herein include performing a first expansion by thawing a cryopreserved tumor digest comprising a first TIL population derived from a tumor excised from an individual. In some embodiments, the methods disclosed herein include obtaining a first TIL population from a tumor excised from a patient by processing a tumor sample obtained from a patient into a tumor digest or multiple tumor fragments.

In some embodiments, tumor lysate can be further obtained from tumor digest through several freeze-thaw cycles or mass spectrometry procedures.

In some embodiments, tumor fragments and/or tumor digests and/or tumor lysates may be frozen and stored frozen prior to entering the priming step, as appropriate.

In some embodiments, the tumor is reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of collagenase is 100 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises DNA enzyme. In some embodiments, the working stock solution of DNA enzyme is 10,000 IU/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 1000 IU/mL DNase, and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

In some embodiments, fragmentation includes physical fragmentation, including, for example, segmentation and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is segmentation. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially obtained from enzymatic tumor digests and tumor fragment cultures of patients. In some embodiments, TILs can be initially obtained from enzymatic tumor digests and tumor fragment cultures of patients.

In some embodiments, the TILs are not obtained from a tumor digest. In some embodiments, the solid tumor core is not fragmented.

In some embodiments, obtaining the first TIL population comprises a multi-lesion sampling approach.

The tumor lytic enzyme cocktail may include one or more lytic (digestive) enzymes such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other lytic or proteolytic enzyme, and any combination thereof.

In some embodiments, the lyophilized enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a certain amount of sterile buffer (such as Hank's balanced salt solution (HBSS)).

In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 mL sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 2892 PZ U per vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, such as about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 150 PZ U/mL, about 200 PZ U/mL, about 210 PZ U/mL, about 220 PZ U/mL, about 230 PZ U/mL, about 240 PZ U/mL, about 250 PZ U/mL, about 260 PZ U/mL, about 270 PZ U/mL, about 280 PZ U/mL, about 299.2 PZ U/mL, about 300 PZ U/mL, about 310 PZ U/mL, about 320 PZ U/mL, about 330 PZ U/mL, about 340 PZ U/mL, about 350 PZ U/mL, about 360 PZ U/mL, about 370 PZ U/mL, about 380 PZ U/mL, about 390 PZ U/mL, about 400 PZ U/mL, about 410 PZ U/mL, about 420 PZ U/mL, about 430 PZ U/mL, about 440 PZ U/mL U/mL, about 300 PZ U/mL, about 350 PZ U/mL, or about 400 PZ U/mL.

In some embodiments, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after reconstitution, the neutral protease stock solution ranges from about 100 DMC/mL to about 400 DMC/mL, such as about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL DMC/mL or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, such as about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution may vary, therefore checking the concentration of the lyophilized stock solution and modifying the final amount of enzyme added to the digestion mixture accordingly.

In some embodiments, the enzyme mixture includes about 10.2 μl of neutral protease (0.36 DMC U/mL), 21.3 μl of collagenase (1.2 PZ/mL), and 250 μl of DNase I (200 U/mL) in about 4.7 mL of sterile HBSS .

In some embodiments, the method includes an initiation step, wherein the tumor digest is initiated in the presence of IFNγ (see Figure 48). In some embodiments, IFNγ is present at a concentration of about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 100 ng/mL, about 200 ng/mL, about 300 ng/mL, about 400 ng/mL, about 500 ng/mL, about 600 ng/mL, about 700 ng/mL, about 800 ng/mL, about 900 ng/mL, about 1,000 ng/mL. In some embodiments, IFNγ is present at a concentration of about 100 ng/mL. In some embodiments, IFNγ is present at a concentration of about 200 ng/mL. In some embodiments, IFNγ is present at a concentration of about 400 ng/mL.

In some embodiments, the priming step comprises a combination of two or more of IL-2, IL-15, and IL-21. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15, and IL-21, the latter of which has particular use in many embodiments. The use of a combination of interleukins is particularly advantageous for generating lymphocytes, and in particular T cells as described therein.

In some embodiments, the initiation step comprises IL-2. In some embodiments, the IL-2 concentration is 3000 IU/mL or less. In some embodiments, the initiation step does not comprise additional IL-2. In some embodiments, the initiation step comprises IL-15 and/or IL-21 at a concentration of about 1 ng/mL to about 100 ng/mL. In some embodiments, the initiation step comprises IL-15 and/or IL-21 at a concentration of about 10 ng/mL. In some embodiments, the initiation step comprises IL-2 and IL-21. In some embodiments, the initiation step comprises IL-2 at 3000 IU/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the initiation step comprises IL-15 and IL-21. In some embodiments, the priming step comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL.

The priming step can last about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 48 hours. In some embodiments, the priming step lasts 12 hours. In some embodiments, the priming step lasts 24 hours. In some embodiments, the priming step lasts 30 hours. In some embodiments, the priming step lasts 36 hours. In some embodiments, the priming step lasts 48 hours. D. Enrichment of Tumor Responsive TILs

The present invention encompasses methods for enriching and expanding a population of tumor-reactive T cells having a CD137+ phenotype. In some embodiments, the TIL population after the priming step is enriched for CD137+ TILs (see FIG. 48 ). For example, "CD137+" or "expressing CD137" cells are contrasted herein with CD137- or cells that do not express detectable levels of CD137. As used herein, "CD137 dark" cells have a lower detectable level of CD137 expression than CD137+ cells or "CD137 bright" cells. In one embodiment, the tumor-reactive T cells are positive for both CD137 and PD-1. For example, cells that are "CD137+PD-1+" or "express CD137 and PD-1" are contrasted herein with cells that are CD137-PD-1+, CD137+PD-1-, or do not express detectable levels of CD137 or PD-1.

The present invention encompasses methods of isolating CD137+ cells using a simple, direct antibody-based purification system for isolating such cells. However, any cell separation method may be used to isolate the cells of the present invention from a biological sample. For example, antibodies that bind to CD137 can be bound to a physical support, such as magnetic beads, Dynal beads, microbeads, columns, adsorption columns, and adsorption membranes. Binding antibodies to physical supports is well known in the art. Alternatively, antibodies bound to physical supports (such as magnetic beads) can be purchased from a variety of sources, such as Milteny Biotec (Auburn, CA).

The literature suggests that some tumor-reactive TILs are present in the CD137+ population. CD39 is more selective and is expressed by most of the putative CD4 tumor-reactive (PD1+ICOS+) population. Most of the putative tumor-reactive CD4+CXCL13 populations express CD200. Therefore, in some embodiments, the enrichment step may further include selecting TILs that express CD200 and/or CD39. In addition, OX40 is a known CD4 T cell activation marker that may be upregulated 24 hours after TCR stimulation in digestion culture. Therefore, in some embodiments, the enrichment step may further include selecting TILs that express CD200, CD39 and/or OX40.

By including anti-CD200 antibodies, anti-C39 antibodies, and/or anti-OX40 antibodies in addition to anti-CD137 antibodies, the yield of tumor-reactive TILs will be increased by the methods disclosed herein, which still removes a large portion of bystander TILs. In some embodiments, tumor-reactive TILs may be CD200+ cell populations. In some embodiments, tumor-reactive TILs may be CD39+ cell populations. In some embodiments, tumor-reactive TILs may be OX40+ cell populations.

In some embodiments, enriching the TIL population for CD137+ TILs includes contacting the TIL population with an anti-CD137 antibody fixed on beads. In some embodiments, the method further includes contacting the TIL population with an anti-CD39 antibody fixed on beads. In some embodiments, the method further includes contacting the TIL population with an anti-CD200 antibody fixed on beads. In some embodiments, the method further includes contacting the TIL population with an anti-OX40 antibody fixed on beads.

A variety of antibodies can be used in the present invention. As will be appreciated by those skilled in the art, any antibody that can recognize and bind to an antigen of interest (such as CD137, CD200, CD39 or OX40) can be used in the present invention. Methods for making and using such antibodies are well known in the art. For example, multiple antibodies that can be used in the present invention are generated by immunizing rabbits according to standard immunological techniques well known in the art. E. First Expansion

In some embodiments, the tumor-reactive TILs generated by the enrichment step are cultured in a second cell culture medium comprising feeder cells. In some embodiments, the feeder cells are T cell-depleted feeder cells, such as T cell-depleted PBMCs (see FIG. 48 ).

In some embodiments, the tumor-reactive TILs produced by the enrichment step are cultured in serum containing IL-2 under conditions that are favorable for TIL growth but unfavorable for tumor and other cell growth. In some embodiments, the tumor-reactive TILs produced by the enrichment step are cultured in a 2 mL well in a medium containing inactivated human AB serum with 6000 IU/mL IL-2. In some embodiments, the tumor-reactive TILs are cultured for a period of several days, typically 3 to 14 days. In some embodiments, the tumor-reactive TILs are cultured for a period of 5 to 11 days. In some embodiments, the tumor-reactive TILs are cultured for a period of 7 to 10 days. In some embodiments, tumor-reactive TILs are cultured for a period of about 9 days.

In embodiments where TIL cultures are initiated in a 24-well plate, e.g., using a Costar 24-well cell culture cluster flat bottom (Corning, Corning, NY), each well can be seeded with 1×10 ⁶ tumor digest cells or a tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 IU/mL; Chiron, Emeryville, CA).

In some embodiments, the first expansion medium is referred to as "CM" (abbreviation for medium). In some embodiments, the first expansion CM consists of RPMI 1640 supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL GlutaMAX. In embodiments where the culture is initiated in a gas-permeable bottle with a 40 mL capacity and a 10 ^cm2 gas-permeable silicon bottom (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN), each bottle can be loaded with 10-40 mL of CM with IL-2 containing 10-40× ¹⁰⁶ live tumor digest cells or 5-30 tumor fragments. Both G-Rex10 and 24-well plates can be cultured in a humidified incubator at 37°C, 5% _CO2 , and 5 days after the start of culture, half of the medium can be removed and replaced with fresh CM and IL-2, and after the 5th day, half of the medium can be replaced every 2-3 days.

In some embodiments, the medium used in the expansion process disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and/or reduce experimental variations due to batch-to-batch variations of serum-containing media.

In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the basal cell culture medium includes (but is not limited to) CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (Minimal Essential Medium, BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (αMEM), Glasgow's Minimal Essential Medium (Glasgow's Minimal Essential Medium, Medium, G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include (but are not limited to) one or more of the following: CTS™ OpTmizer T Cell Expander Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin replacements, one or more amino acids, one or more vitamins, one or more transferrins or transferrin replacements, one or more antioxidants, one or more insulins or insulin replacements, one or more collagen prodrivers, one or more antibiotics, and one or more trace elements. In some embodiments, the defined culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and trace element portions Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ^{6+ In} some embodiments, the culture medium further comprises ^L -glutamine, sodium bicarbonate and/ ^{or 2} -hydroxyethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with a learned growth medium, which includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% based on the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-hydroxyethanol in the culture medium is 55 μM.

In some embodiments, the defined medium described in International PCT Publication No. WO/1998/030679, which is incorporated herein by reference, can be used in the present invention. In the publication, a serum-free eukaryotic cell culture medium is described. The serum-free eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen prodrivers, one or more trace elements and one or more antibiotics. In some embodiments, the medium further comprises L-glutamine, sodium bicarbonate and/or β-hydroxyethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen pro-drivers and one or more trace elements. In some embodiments, the defined culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and trace element portions Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), Eagle's basal medium (BME), ^{RPMI 1640} , F- ^{10 ,} F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium and Iskoff's modified Dulbecco's medium.

In some embodiments, the concentration of glycine in the culture medium is determined to be in the range of about 5-200 mg/L, the concentration of L-histidine is about 5-250 mg/L, the concentration of L-isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-200 mg/L, the concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-proline is about 1-1000 mg/L, the concentration of L-hydroxyproline is about 1-45 mg/L, the concentration of L-serine is about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, and the concentration of L-tryptophan is about 2-110 mg/L, L-tyrosine at a concentration of about 3-175 mg/L, L-valine at a concentration of about 5-500 mg/L, thiamine at a concentration of about 1-20 mg/L, reduced glutathione at a concentration of about 1-200 mg/L, L-ascorbic acid-2-phosphate at a concentration of about 1-200 mg/L, iron-saturated transferrin at a concentration of about 1-50 mg/L, insulin at a concentration of about 1-100 mg/L, sodium selenite at a concentration of about 0.000001-0.0001 mg/L, and albumin (e.g., AlbuMAX® I) at a concentration of about 5000 to 50,000 mg/L.

In some embodiments, the non-trace element portion of the medium is determined to be present in the concentration range listed in the column titled "Concentration Range in 1X Medium" in Table 11 below. In other embodiments, the non-trace element portion of the medium is determined to be present in the final concentration listed in the column titled "Preferred Embodiments of 1X Medium" in Table 11. In other embodiments, the medium is determined to be a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace element portions of the type and concentration listed in the column titled "Preferred Embodiments of Supplements" in Table 11 below.

In some embodiments, the osmotic pressure of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmotic pressure is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-hydroxyethanol (final concentration of about 100 μM).

In some embodiments, the defined medium described in Smith et al., Clin Transl Immunology, 4(1) 2015 (doi: 10.1038/cti.2014.31) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ is used as the basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. Using an unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and/or second gas permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

After preparing the tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor TIL growth relative to tumor and other cells. In some embodiments, the tumor digest is cultured in a 2 mL well in a medium comprising inactivated human AB serum (or in some cases, as outlined herein, in the presence of APC cell populations) and 6000 IU/mL IL-2. This primary cell population is cultured for a period of several days, typically 10 to 14 days, to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells. In some embodiments, the growth medium during the first expansion period comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20-30×10 ⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20×10 ⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 25×10 ⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 30×10 ⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6×10 ⁶ IU/mg IL-2. In some embodiments, the first expansion medium comprises about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, between 1000 and 5000 IU/mL, or about 8000 IU/mL of IL-2.

In some embodiments, the first expansion may be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first expansion may be performed for 1 day to 14 days. In some embodiments, the first expansion may be performed for 2 days to 14 days. In some embodiments, the first expansion may be performed for 3 days to 14 days. In some embodiments, the first expansion may be performed for 4 days to 14 days. In some embodiments, the first expansion may be performed for 5 days to 14 days. In some embodiments, the first expansion may be performed for 6 days to 14 days. In some embodiments, the first expansion may be performed for 7 days to 14 days. In some embodiments, the first expansion may be performed for 8 days to 14 days. In some embodiments, the first expansion may be carried out for 9 to 14 days. In some embodiments, the first expansion may be carried out for 10 to 14 days. In some embodiments, the first expansion may be carried out for 11 to 14 days. In some embodiments, the first expansion may be carried out for 12 to 14 days. In some embodiments, the first expansion may be carried out for 13 to 14 days. In some embodiments, the first expansion may be carried out for 14 days. In some embodiments, the first expansion may be carried out for 1 to 11 days. In some embodiments, the first expansion may be carried out for 2 to 11 days. In some embodiments, the first expansion may be carried out for 3 to 11 days. In some embodiments, the first expansion may be carried out for 4 to 11 days. In some embodiments, the first expansion may be carried out for 5 to 11 days. In some embodiments, the first expansion may be carried out for 6 to 11 days. In some embodiments, the first expansion may be carried out for 7 to 11 days. In some embodiments, the first expansion may be carried out for 8 to 11 days. In some embodiments, the first expansion may be carried out for 9 to 11 days. In some embodiments, the first expansion may be carried out for 10 to 11 days. In some embodiments, the first expansion may be carried out for 11 days. In some embodiments, the first expansion may be carried out for 5 to 7 days. In some embodiments, the first expansion may be carried out for 6 to 7 days. In some embodiments, the first expansion may be carried out for 7 to 12 days. In some embodiments, the first expansion may be carried out for 8 to 12 days. In some embodiments, the first expansion may be carried out for 9 to 12 days. In some embodiments, the first expansion can be carried out for 10 to 12 days. In some embodiments, the first expansion can be carried out for 7 days. In some embodiments, the first expansion can be carried out for 9 days.

In some embodiments, the first expansion is performed in a closed system bioreactor. In some embodiments, a closed system is used to perform TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, the first cell culture medium comprises 6000 IU/mL IL-2. In some embodiments, the first cell culture medium comprises 3000 IU/mL IL-2. In some embodiments, the first cell culture medium comprises 2000 IU/mL IL-2. In some embodiments, the first cell culture medium comprises 1000 IU/mL IL-2. F. Activation

In some embodiments, after the first expansion (pre-REP) step, TILs are activated by adding anti-CD3 agonists and anti-CD28 agonists (e.g., TransAct) to the culture medium and cultured for about 1 to 3 days, wherein the TILs will be transduced with the recombinant lentiviral particles disclosed herein to generate a gene-edited TIL population.

In some embodiments, the step of activating the second TIL population (obtained from the first expansion or pre-REP step) can be performed for a period of about, less than, greater than 1 day, 2 days, 3 days, or a range between any of the above values. For example, in some embodiments, the step of activating the second TIL population is performed for about 1 day. In some embodiments, the step of activating the second TIL population is performed for about 2 days. In some embodiments, the step of activating the second TIL population is performed for about 3 days.

In some embodiments, the step of activating the second TIL population (obtained from the first expansion or pre-REP step) is performed using an anti-CD3 agonist and an anti-CD28 agonist, such as TransAct. In some embodiments, the step of activating the second TIL population is performed using TransAct at a dilution of 1:10, 1:17.5, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100.

In some embodiments, the step of activating the second TIL population (obtained from the first expansion or pre-REP step) can be performed by adding an anti-CD3 agonist and an anti-CD28 agonist, such as TransAct, to the first cell culture medium. In some embodiments, the step of activating the second TIL population can be performed by replacing the first cell culture medium with a cell culture medium comprising an anti-CD3 agonist and an anti-CD28 agonist, such as TransAct.

In some embodiments, the method may include activating the TIL population for 1 day, 2 days, 3 days, or 4 days before transducing the TIL population with the recombinant lentiviral particles. In some embodiments, the activation step includes contacting the TIL population with an interleukin selected from the group consisting of: IL-2, IL-15, IL-21, IL-7, and combinations thereof.

In some embodiments, the activation step comprises contacting the TIL population with 20 ng/mL IL-15. In some embodiments, the activation step comprises contacting the TIL population with 10 ng/mL IL-7. In some embodiments, the activation step comprises contacting the TIL population with TransAct. In some embodiments, the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. In some embodiments, the activation step is performed after the first expansion step and before the second expansion step. G. Lentiviral Particle Production

In some embodiments, recombinant lentiviral particles are produced using a recombinant expression vector, a helper plasmid encoding one or more of the viral gag, pol, env and rev, and/or a packaging cell line disclosed herein.

The following general steps can be used. First, a population of packaging cells is cultured in a cell culture medium. Once a sufficient number of packaging cells is obtained, a desired nucleic acid, such as a transfer vector comprising a nucleic acid molecule encoding a cytokine disclosed herein, can be introduced into the packaging cells. Additional nucleic acids that can be introduced into the packaging cells include plasmids that facilitate packaging, such as plasmids encoding viral gag, pol, env, and rev. The packaging cells then begin to produce recombinant lentiviral particles.

In some embodiments, HEK 293T cells are resuspended in viral medium (10% FBS, DMEM (high glucose, glutamine), sodium pyruvate (1% w/v), sodium bicarbonate (0.075%), or HEPES, no Pen/strep).

The next day, Gag/Pol helper plasmid, Rev helper plasmid, BaEVTR or VSV-G helper plasmid and transfer vector were mixed at a 1:1 molar ratio at a concentration of 1 µg/µL. After mixing, 30 µL of Trans IT ^® -Lenti reagent (Mirus Bio) was added and incubated in 1 mL of Opti-MEM™ medium per HEK 293T dish at room temperature for 10 minutes. HEK 293T cells were then incubated at 37°C, 5% CO ₂ for 2 days.

The culture supernatant is removed from the culture dishes and fresh medium is added to each dish, centrifuged at 200 g for 5 minutes to remove cellular debris, and then filtered using a 0.45 µM filter. The viral supernatant is then concentrated by ultracentrifugation at 60,000 g for 2 hours. After ultracentrifugation, the supernatant is removed and the viral pellet is re-dissolved in Opti-MEM™ medium. The viral particle harvesting step can be repeated to optimize viral production. H. Transduction

In some embodiments, after the activation step, TILs are transduced with the recombinant lentiviral particles disclosed herein.

In some embodiments, the transduction step is performed at a TIL concentration of about 10 ⁴ -10 ⁶ cells/ml. In some embodiments, the transduction step is performed at a TIL concentration of about 10 ⁴ cells/ml. In some embodiments, the transduction step is performed at a TIL concentration of about 10 ⁵ cells/ml. In some embodiments, the transduction step is performed at a TIL concentration of about 10 ⁶ cells/ml. In some embodiments, the transduction step is performed at an infection multiple (MOI) of about 10 to about 40. In some embodiments, the transduction step is performed at an MOI of about 10. In some embodiments, the transduction step is performed at an MOI of about 20. In some embodiments, the transduction step is performed at an MOI of about 30. In some embodiments, the transduction step is performed at an MOI of about 40.

In some embodiments, the transduction step is performed in a non-tissue culture 48-well plate coated with Retronectin (1:100 dilution in PBS) by adding 250 uL per well. The 48-well plate is then wrapped in paraffin film and then incubated overnight at 4°C.

Remove coating solution from Retronectin coated plates and replace with 250 uL 2% BSA fraction V. Incubate plates at room temperature for 30 minutes to block. Add the following: a. 100 uL TIL suspension (1e5 cells) b. 300 uL CM2 + IL-15 (20 ng/mL) + IL-7 (40 ng/mL) + Lentibooster 1:50 c. Lentivirus at an MOI range of 10-40 d. Adjust with CM2 without retronectin, total volume 600 uL per well

The plate was then centrifuged at 2000 × g for 45 min at 32°C.

In some embodiments, the transduction step is performed in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transduction step comprises centrifugation. In some embodiments, the transduction step is performed in the presence of Lentibooster. In some embodiments, the transduction step is performed in the presence of Lentibooster at a ratio of 1:50.

In some embodiments, the transduction step is performed after the first expansion step and before the second expansion step.

In some embodiments, the method further comprises allowing the TIL population to rest for 1 day, 2 days, 3 days, or 4 days after the transduction step. I. Second Expansion

In some embodiments, the number of TIL cell populations is expanded after initial bulk processing, pre-REP expansion, and gene modification, wherein the expanded TILs have been transduced with recombinant lentiviral particles disclosed herein to produce gene-edited TIL populations. This further expansion is referred to herein as the second expansion, which may include an expansion process generally referred to as a rapid expansion process (REP) in this art. The second expansion is typically performed in a gas permeable container using a culture medium comprising a variety of components, including feeder cells, a cytokine source, and an anti-CD3 agonist antibody.

In some embodiments, the second expansion of TIL (which may include expansion sometimes referred to as REP) can be performed using any TIL bottle or container known to those skilled in the art, wherein the expanded TIL has been transduced with the recombinant lentiviral particles disclosed herein to produce a population of gene-edited TIL. In some embodiments, the second expansion can be performed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second expansion can be performed for about 7 days to about 14 days. In some embodiments, the second expansion can be performed for about 7 days to about 12 days. In some embodiments, the second expansion can be performed for about 7 days to about 10 days. In some embodiments, the second expansion can be performed for about 7 days to about 9 days. In some embodiments, the second expansion can be performed for about 8 days to about 9 days. In some embodiments, the second expansion can be performed for about 9 days. In some embodiments, the second expansion can be performed for about 10 days. In some embodiments, the second expansion can be performed for about 11 days.

In some embodiments, the second expansion can be performed in a gas permeable container using the methods of the present disclosure (including, for example, expansion referred to as REP). For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulators can include, for example, anti-CD3 agonist antibodies, such as about 30 ng/ml OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil (Raritan, NJ) or Miltenyi Biotech (Auburn, CA)), or UHCT-1 (available from BioLegend, San Diego, CA, USA). TILs can be expanded by including one or more antigens (including antigenic portions thereof, such as antigenic determinants) during the second expansion period to induce further TIL in vitro stimulation, such antigens can be expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, such as 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217 (210M), in the presence of T cell growth factors (such as 300 IU/mL IL-2 or IL-15) as appropriate. Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same cancer antigen pulsed onto antigen presenting cells expressing HLA-A2. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or 8000 IU/mL of IL-2.

In some embodiments, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, or about 1 µg/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:100 and 1:200.

In some embodiments, REP and/or secondary expansion are performed in flasks where the primary TILs are mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 ml of medium. The medium is replaced (usually by aspirating fresh medium to replace 2/3 of the medium) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX bottles and gas permeable containers, as described more fully below.

In some embodiments, as discussed in the examples and figures, the second expansion (which may include a process called REP process) is shortened to 7 to 14 days, wherein the TILs expanded by such second expansion have been transduced with the recombinant lentiviral particles disclosed herein to generate a population of gene-edited TILs. In some embodiments, the second expansion is shortened to 9 days.

In some embodiments, REP and/or secondary expansion can be performed using T-175 flasks and gas-permeable bags as previously described (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother. 2003, 26, 332-42) or gas-permeable culture vessels (G-Rex flasks), wherein the TILs expanded by such secondary expansion have been transduced with recombinant lentiviral particles disclosed herein to generate gene-edited TIL populations. In some embodiments, secondary expansion (including expansion referred to as rapid expansion) is performed in T-175 flasks, and approximately 1×10 ⁶ TILs suspended in 150 mL of medium can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/mL IL-2 and 30 ng/ml anti-CD3. T-175 flasks can be incubated at 37°C, 5% _CO2 , where the TILs expanded by such a second expansion have been transduced with the recombinant lentiviral particles disclosed herein to produce a population of gene-edited TILs. Half of the medium can be replaced on day 5 with a 50/50 medium with 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks can be combined in a 3 L bag, and 300 mL of AIM V with 5% human AB serum and 3000 IU/mL IL-2 are added to 300 ml of TIL suspension. The number of cells in each bag was counted every day or every two days, and fresh medium was added to maintain the cell count between 0.5 and 2.0×10 ⁶ cells/mL.

In some embodiments, the second expansion (which may include expansion referred to as REP) can be performed in a 500 mL capacity gas permeable bottle with a 100 cm gas permeable silicon bottom (G-Rex 100, available from Wilson Wolf Manufacturing), 5×10 ⁶ or 10×10 ⁶ TILs can be cultured with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/ml anti-CD3 (OKT3), wherein the TILs expanded by such a second expansion have been transduced with the recombinant lentiviral particles disclosed herein to generate a gene-edited TIL population. The G-Rex 100 bottle can be incubated at 37° C., 5% CO ₂ . On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellet can be resuspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU/mL IL-2, and added back to the original G-Rex 100 bottle. As TILs are continuously expanded in G-Rex 100 bottles, on day 7, the TILs in each G-Rex 100 can be suspended in the 300 mL of medium present in each bottle, and the cell suspension can be divided into three 100 mL aliquots that can be used to inoculate three G-Rex 100 bottles. 150 mL of AIM-V with 5% human AB serum and 3000 IU/mL IL-2 can then be added to each bottle. The G-Rex 100 bottles can be incubated at 37°C, 5% _CO2 and after 4 days, 150 mL of AIM-V with 3000 IU/mL IL-2 can be added to each G-REX 100 bottle. Cells can be harvested on day 14 of culture.

In some embodiments, the second expansion (which may include expansion referred to as REP) can be performed in a 500 mL capacity gas permeable bottle with a 100 cm gas permeable silicon bottom (G-REX-100, available from Wilson Wolf Manufacturing, New Brighton, MN, USA), and 5×10 ⁶ or 10×10 ⁶ TILs can be cultured with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/mL anti-CD3 (OKT3). G-REX-100 (or G-REX100M) can be incubated at 37° C., 5% CO _2. On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellet can be resuspended with 150 mL of fresh medium with 5% human AB serum, 6000 IU/mL IL-2 and added back to the original GREX-100 bottle. When the TIL continues to expand in the GREX-100 bottle, on the 10th or 11th day, the TIL can be moved to a larger bottle, such as GREX-500 (or G-REX500M). The cells can be harvested on the 14th day of culture. The cells can be harvested on the 15th day of culture. The cells can be harvested on the 16th day of culture. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber. In some embodiments, 2/3 of the medium is replaced by aspirating the spent medium and replacing it with an equal volume of fresh medium. In some embodiments, the alternative growth chamber comprises a GREX bottle and a gas permeable container, as discussed more fully below. In some embodiments, the method employs different centrifugation speeds (400 g, 300 g, 200 g for 5 minutes) and different numbers of repetitions.

In some embodiments, the second expansion (including expansion referred to as REP) is performed in a flask, where the main TIL is mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 ml of medium. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber, where the TIL expanded by such a second expansion has been transduced with the recombinant lentiviral particles disclosed herein to produce a gene-edited TIL population. In some embodiments, 2/3 of the medium is replaced by aspirating the spent medium, followed by infusion of fresh medium. In some embodiments, the alternative growth chamber comprises a G-REX bottle and a gas permeable container, as discussed more fully below.

In some embodiments, the second expansion medium (e.g., sometimes referred to as CM2 or second cell medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the medium used in the expansion process disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and/or reduce experimental variations due to batch-to-batch variations of serum-containing media.

In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include (but are not limited to) one or more of the following: CTS™ OpTmizer T Cell Expander Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin replacements, one or more amino acids, one or more vitamins, one or more transferrins or transferrin replacements, one or more antioxidants, one or more insulins or insulin replacements, one or more collagen prodrivers, one or more antibiotics, and one or more trace elements. In some embodiments, the defined culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and trace element portions Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , C _O ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ^{6+ In} some embodiments, the culture medium further comprises ^L -glutamine, sodium bicarbonate and/ ^{or 2} -hydroxyethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with a learned growth medium, which includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% based on the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-hydroxyethanol in the culture medium is 55 μM.

In some embodiments, the defined medium described in International PCT Publication No. WO/1998/030679, which is incorporated herein by reference, is suitable for use in the present invention. In the publication, a serum-free eukaryotic cell culture medium is described. The serum-free eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen prodrivers, one or more trace elements and one or more antibiotics. In some embodiments, the medium further comprises L-glutamine, sodium bicarbonate and/or β-hydroxyethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen pro-drivers and one or more trace elements. In some embodiments, the defined culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and trace element portions Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , C _O ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), Eagle's basal medium (BME), ^{RPMI 1640} , F- ^{10 ,} F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium and Iskoff's modified Dulbecco's medium.

In some embodiments, the concentration of glycine in the culture medium is determined to be in the range of about 5-200 mg/L, the concentration of L-histidine is about 5-250 mg/L, the concentration of L-isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-200 mg/L, the concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-proline is about 1-1000 mg/L, the concentration of L-hydroxyproline is about 1-45 mg/L, the concentration of L-serine is about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, and the concentration of L-tryptophan is about 2-110 mg/L, L-tyrosine at a concentration of about 3-175 mg/L, L-valine at a concentration of about 5-500 mg/L, thiamine at a concentration of about 1-20 mg/L, reduced glutathione at a concentration of about 1-20 mg/L, L-ascorbic acid-2-phosphate at a concentration of about 1-200 mg/L, iron-saturated transferrin at a concentration of about 1-50 mg/L, insulin at a concentration of about 1-100 mg/L, sodium selenite at a concentration of about 0.000001-0.0001 mg/L, and albumin (e.g., AlbuMAX® I) at a concentration of about 5000-50,000 mg/L.

In some embodiments, the non-trace element portion of the medium is determined to be present in the concentration range listed in the column titled "Concentration Range in 1X Medium" in Table 12 below. In other embodiments, the non-trace element portion of the medium is determined to be present in the final concentration listed in the column titled "Preferred Embodiments of 1X Medium" in Table 12. In other embodiments, the medium is determined to be a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace element portions of the type and concentration listed in the column titled "Preferred Embodiments of Supplements" in Table 12 below.

In some embodiments, the osmotic pressure of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmotic pressure is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-hydroxyethanol (final concentration of about 100 μM).

In some embodiments, the defined medium described in Smith et al., Clin Transl Immunology , 4(1) 2015 (doi: 10.1038/cti.2014.31) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ is used as the basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. Using an unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and/or second gas permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, the second expansion is performed in a closed system bioreactor. In some embodiments, a closed system is used to perform TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, the steps of the method are completed within a time period of about 22 days. In some embodiments, the steps of the method are completed within a time period of about 8 days. In some embodiments, the steps of the method are completed within a time period of about 9 days. In some embodiments, the steps of the method are completed within a time period of about 10 days. In some embodiments, the steps of the method are completed within a time period of about 11 days. In some embodiments, the steps of the method are completed within a time period of about 12 days. In some embodiments, the steps of the method are completed within a time period of about 13 days. In some embodiments, the steps of the method are completed within a time period of about 14 days. In some embodiments, the steps of the method are completed within a time period of about 15 days. In some embodiments, the steps of the method are completed within a time period of about 16 days. In some embodiments, the steps of the method are completed within a time period of about 17 days. In some embodiments, the steps of the method are completed within a time period of about 18 days. In some embodiments, the steps of the method are completed within a time period of about 19 days. In some embodiments, the steps of the method are completed within a time period of about 20 days. In some embodiments, the steps of the method are completed within a time period of about 21 days. In some embodiments, the steps of the method are completed within a time period of about 22 days. In some embodiments, the steps of the method are completed within a time period of about 23 days. In some embodiments, the steps of the method are completed within a time period of about 24 days. In some embodiments, the steps of the method are completed within a time period of about 25 days. In some embodiments, the steps of the method are completed within a time period of about 26 days. In some embodiments, the steps of the method are completed within a time period of about 27 days. In some embodiments, the steps of the method are completed within a time period of about 28 days. In some embodiments, the steps of the method are completed within a time period of about 29 days. In some embodiments, the steps of the method are completed within a time period of about 30 days. In some embodiments, the steps of the method are completed within a time period of about 31 days.

In some embodiments, the antigen presenting cell (APC) is a PBMC. According to some embodiments, the PBMC is irradiated. According to some embodiments, the PBMC is allogeneic. According to some embodiments, the PBMC is irradiated and allogeneic. According to some embodiments, the antigen presenting cell is an artificial antigen presenting cell.

In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1000 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1500 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 2000 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 2500 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 3000 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 3500 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 4000 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 4500 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 5000 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 5500 IU/mL and 6000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1000 IU/mL and 5000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1500 IU/mL and 5000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 2000 IU/mL and 5000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 2500 IU/mL and 5000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 3000 IU/mL and 5000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 3500 IU/mL and 5000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 4000 IU/mL and 5000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 4500 IU/mL and 5000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1000 IU/mL and 4000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1500 IU/mL and 4000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 2000 IU/mL and 4000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 2500 IU/mL and 4000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 3000 IU/mL and 4000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 3500 IU/mL and 4000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1000 IU/mL and 3000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1500 IU/mL and 3000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 2000 IU/mL and 3000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 2500 IU/mL and 3000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1000 IU/mL and 2000 IU/mL in the first expansion. In some embodiments, IL-2 is present in the cell culture medium at an initial concentration of between 1500 IU/mL and 2000 IU/mL in the first expansion.

In some embodiments, in the second expansion step, IL-2 is present at an initial concentration between 1000 IU/mL and 6000 IU/mL and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL.

In some embodiments, the first expansion is performed using a breathable container. In some embodiments, the second expansion is performed using a breathable container.

In some embodiments, the first cell culture medium further comprises an interleukin selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the second cell culture medium and/or the third cell culture medium further comprises an interleukin selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. 1. Feeder Cells and Antigen Presenting Cells

In some embodiments, the second expansion process described herein requires excess feeder cells during the REP TIL expansion period and/or during the second expansion period. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

Generally, allogeneic PBMCs are irradiated or heat treated without activation and used in the REP procedure as described in the Examples, which provide an exemplary protocol for assessing the replication incompetence of irradiated allogeneic PBMCs.

In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to be replication-incompetent and are acceptable for use in the TIL expansion procedures described herein.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase compared to the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to be replication-incompetent and acceptable for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase compared to the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to be replication-incompetent and acceptable for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen presenting feeder cells are artificial antigen presenting feeder cells. In some embodiments, the ratio of TILs to antigen presenting feeder cells in the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion process described herein requires a ratio of about 2.5×10 ⁹ feeder cells to about 100×10 ⁶ TILs. In other embodiments, the second expansion process described herein requires a ratio of about 2.5×10 ⁹ feeder cells to about 50×10 ⁶ TILs. In other embodiments, the second expansion process described herein requires a ratio of about 2.5×10 ⁹ feeder cells to about 25×10 ⁶ TILs.

In some embodiments, the second expansion process described herein requires an excess of feeder cells during the second expansion period. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen presenting cells (aAPCs) are used instead of PBMCs.

In some embodiments, artificial antigen presenting cells are used in the second expansion to replace PBMCs or in combination with PBMCs. 2. Interleukins and other additives

The expansion methods described herein typically utilize media with high doses of interleukins, particularly IL-2, as is known in the art.

Alternatively, it is possible to use a combination of interleukins for rapid expansion and/or secondary expansion of TILs, as described in U.S. Patent Application Publication No. US 2017/0107490 A1 (the disclosure of which is incorporated herein by reference), using a combination of two or more of IL-2, IL-15, and IL-21. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, wherein the latter has specific uses in many embodiments. The use of a combination of interleukins is particularly advantageous for the generation of lymphocytes, and in particular T cells as described therein.

In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-2. In some embodiments, the IL-2 concentration is 3000 IU/mL or less. In some embodiments, the first cell culture medium or the second cell culture medium does not contain additional IL-2. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and/or IL-21 at a concentration of about 1 ng/mL to about 100 ng/mL. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and/or IL-21 at a concentration of about 10 ng/mL. In some embodiments, the first cell culture medium comprises IL-2 and IL-21. In some embodiments, the first cell culture medium comprises 3000 IU/mL of IL-2 and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of patasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oregano, herbicide, terheolactone, isoliquiritigenin, baicalein and holmium solani.

In some embodiments, the first expansion is performed for a period of about 7-11 days. In some embodiments, the second expansion is performed for a period of about 7-11 days. 3. T cell metabolism modifiers

In vitro expansion alters TIL cellular states, which correlate with ACT efficacy (Chiffelle, J. et al., bioRxiv 2023 , which is incorporated herein by reference in its entirety). Rapidly expanding TILs have bioenergetic and biosynthetic requirements. Increasing the availability of essential cofactors such as L-arginine and NAD+ may support the synthesis of major biomass components. Functionally reinvigorating cells during in vitro expansion may generate TILs with new effector profiles.

Thus, provided herein are T cell metabolic modifiers that increase the availability of essential cofactors L-arginine and NAD ⁺ to support the synthesis of major biomass components in TILs, which can be added to the second expansion (REP) of TILs.

L-arginine is a building block for protein synthesis and improves T cell function through metabolic modification (Geiger R. et al., Cell 2016 : 167; 829-842; Fultang, L., Blood 2020 ; 136: 1155-60; the contents of which are incorporated herein by reference in their entirety).

NAD+ (nicotinamide adenine dinucleotide) is an electron acceptor that is consumed in large quantities during biomass synthesis, and its supplementation can improve T cell function (Canto C et al., Cell Metabolism 2015 ; 22:31-53; Wang Y. et al., Cell Reports 2021 ; 36:1-12; the contents of which are incorporated herein by reference in their entirety).

NAD+ (nicotinamide adenine dinucleotide) is a central coenzyme in cellular metabolism, particularly in redox reactions, where it cycles between oxidized (NAD+) and reduced (NADH) states. In the context of TILs, NAD+ plays a key role in regulating their metabolic fitness and function. TILs function in the tumor microenvironment, which is often characterized by nutrient deprivation and hypoxia. In this challenging microenvironment, TILs must adjust their metabolism to maintain their anti-tumor activity.

NAD+ enhances metabolic fitness of T cells in multiple ways: Glycolysis and the pentose phosphate pathway (PPP): Although glycolysis does not use NAD+ directly, regeneration of NAD+ from NADH is critical for maintaining glycolytic flux, which is particularly important under hypoxic conditions where mitochondrial respiration is limited. The PPP is a branch of glycolysis that relies on NADP+ (the phosphorylated form of NAD+) to generate 5-phosphoribose and reduce glutathione, thereby contributing to nucleotide synthesis and antioxidant defense mechanisms. Mitochondrial function: NAD+ is critical in mitochondria as it is a key electron acceptor in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Enhanced mitochondrial function supports the differentiation and survival of memory T cells, which is critical for sustained anti-tumor immunity. Sirtuin activation: Sirtuins are a family of NAD+-dependent deacetylases that regulate cell metabolism, stress response, and inflammation. By activating sirtuins, NAD+ promotes the formation of memory T cells and enhances their anti-tumor function. PARP activity: NAD+ is a substrate for poly(ADP-ribose) polymerase (PARP), which is involved in DNA repair and genome stability. By promoting PARP function, NAD+ helps maintain TIL integrity in the presence of DNA damage in tumors.

To replicate and enhance the benefits that NAD+ provides to TILs, pharmacological and genetic approaches may be considered: Pharmacological Approaches: - NAD+ Precursors: Enhancing NAD+ by administering NAD+ precursors such as niacinamide riboside (NR) or niacinamide mononucleotide (NMN) can increase intracellular NAD+ levels, thereby supporting TIL metabolism and function. - NAD+: Increases the availability of NAD+ metabolic substrates directly to TILs - Sempervivin Activators: Compounds such as resveratrol or SRT1720 can activate sempervivin, thereby mimicking the effects of high NAD+ levels and promoting TIL anti-tumor activity. - PARP inhibitors: Although PARP activity is dependent on NAD+ and can be beneficial, overactivation can deplete cellular NAD+ stores. PARP inhibitors can help maintain NAD+ levels under stressful conditions, potentially supporting T cell function. - CD38 inhibitors: CD38 uses NAD+ to generate ADP-ribose and cyclic ADP-ribose, which are important for calcium signaling, but also consume NAD+. CD38 inhibitors may help maintain NAD+ levels in TILs. Genetic approaches: - NAMPT overexpression: This is the rate-limiting enzyme in the NAD+ salvage pathway. Overexpression of NAMPT can increase the intracellular concentration of NAD+ in TILs. - Manipulation of longevity protein genes: Genetic modification to overexpress longevity protein genes may mimic the effects of high NAD+ levels and enhance TIL function. - Gene silencing of NAD+ consuming enzymes: Silencing or attenuation of genes encoding enzymes that consume large amounts of NAD+ (such as CD38 or PARP family members) may preserve NAD+ for TIL essential metabolic processes. - TCA cycle and OXPHOS support: Enhancing genes involved in mitochondrial biogenesis and function can support the efficient use of NAD+ and maintain TIL metabolic fitness.

Pharmacological and genetic tools to enhance NAD+ utilization in TILs are centered on specific molecular targets and pathways. In some embodiments, exemplary tools for enhancing NAD+ utilization focus on one or more of the following functions: mitochondrial biogenesis, promoting efficient energy utilization, and cultivating anti-tumor phenotypes. In embodiments, such tools are implemented in a coordinated manner, paying attention to the balance between enhancing TIL function and avoiding overstimulation that may lead to exhaustion or apoptosis. In some embodiments, the heterogeneity within the TIL population and the unique microenvironment of each tumor are considered when designing such interventions. Therefore, regulating mitochondrial function through pharmacological and genetic methods may be a powerful strategy to enhance the efficacy of donor-acceptor TIL therapy. The tools for enhancing NAD+ utilization are further described below. Pharmacological manipulation of the NAD+ pathway: 1. NAD+ prodromal and NAD+ : ○ Nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) are NAD+ prodromal. They are converted to NAD+ via the salvage pathway, with NR involving nicotinamide riboside kinase (NRK1/2) and NMN involving NMN adenylyltransferase (NMNAT1/2/3). ○ Nicotinic acid (NA) can also be used, which is converted to NAD+ via the Preiss-Handler pathway, but it causes flushing due to prostaglandin-mediated vasodilation. ○ Nicotinamide dinucleotide (NAD+) itself can also be used, as it has been shown to cross the lipid bilayer and enter the mitochondria. 2. Longevity protein modulators : ○ Versatrol is a longevity protein activating compound (STAC) that can enhance SIRT1 activity, which in turn can deacetylate and activate peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis. ○ SRT1720 is another STAC that has been shown to selectively activate SIRT1, thereby enhancing mitochondrial function and potentially improving TIL survival and function. 3. PARP inhibitors : ○ Compounds such as Olaparib and Niraparib inhibit the PARP enzyme, thereby preserving NAD+ for other metabolic processes that are critical for T cell function and potentially reducing the metabolic competition between PARP and SIRT1 for NAD + . 4. CD38 inhibitors : ○ Isatuximab and Daratumumab are monoclonal antibodies that target CD38 , which is responsible for NAD+ degradation. Small molecule inhibitors of CD38 are also in development and can be repurposed to increase NAD+ levels in TILs. 5. Mitochondrial metabolism regulators : ○ Metformin has been shown to activate AMP-activated protein kinase (AMPK), which in turn activates PGC-1α, improving mitochondrial biogenesis and potentially restoring TIL function. ○ Pioglitazone is a PPARγ agonist that may also enhance mitochondrial biogenesis and function in TILs through PGC-1α induction. ○ Bezafibrate : A pan-PPAR agonist that can induce PGC-1α, enhancing mitochondrial function and fatty acid oxidation Mitochondrial biogenesis and kinetics : 1. PGC-1α activation : ○ Specific activators of PGC-1α or direct overexpression via gene delivery systems can be used to stimulate mitochondrial biogenesis. 2. Mitochondrial dynamics : ○ Manipulate genes involved in mitochondrial fusion (e.g., MFN1/2 , OPA1 ) and fission (e.g., DRP1 , FIS1 ) to maintain the integrity and function of the mitochondrial network. Genetic tools: 1. Overexpression of NAD+ synthesis genes: ○ NAMPT : Overexpression can increase salvage pathway flux and increase NAD+ levels. ○ NMNAT1/2/ 3: Increase NMN adenylyltransferase expression, catalyzing the conversion of NMN to NAD+. 2. Longevity protein gene manipulation: ○ SIRT1-7 : Overexpression of longevity protein genes, focusing on the role of SIRT1 in mitochondrial biogenesis, SIRT3 for mitochondrial protein deacetylation, and SIRT6 for maintaining genome stability. 3. Upregulation of TCA cycle and OXPHOS genes: ○ PC ( pyruvate carboxylase ) : Enhances the replenishment reaction to replenish TCA cycle intermediates. ○ PDK ( pyruvate dehydrogenase kinase ) inhibitor: silencing the PDK gene, increasing PDH activity and promoting glucose oxidation. 4. Downregulation of NAD+ consuming enzymes: ○ Using CRISPR/Cas9 or siRNA methods to weaken CD38 or PARP family members to retain NAD+ and thus enhance TIL function. 5. Promote mitochondrial DNA stability and biogenesis: ○ TFAM ( mitochondrial transcription factor A) : Overexpression can promote mitochondrial DNA replication and transcription, thereby enhancing mitochondrial function. ○ OGG1 or MUTY H: Overexpression to enhance base excision repair and maintain mitochondrial DNA integrity. 6. Redox balance: ○ GCLC/GCLM ( Glutamine - cysteine ligase catalytic and modifying subunit ) : Overexpression to increase glutathione synthesis, glutathione is a key antioxidant in mitochondria. 7. Metabolic reprogramming: ○ CPT1A ( Carnitine palmityl transferase 1A) : Gene modification can promote fatty acid oxidation and maintain TIL function under conditions of nutrient deficiency. 8. Mitochondrial autophagy and autophagy regulators: ○ PRKN (Parkin) and PINK1 : Enhance mitochondrial autophagy to remove dysfunctional mitochondria. ○ ATG5 or ATG7 : Regulate autophagy-related genes to balance energy and organelle quality control in TILs.

In some embodiments, these strategies can be optimized for dosage, timing, and delivery mechanisms to ensure that TILs are metabolically enhanced without inducing deleterious effects, such as apoptosis or aging. In embodiments, such strategies are also evaluated to avoid promoting tumor cell survival or immune escape. In some embodiments, the combination of these strategies provides effective and personalized enhancement of TIL function for donor-acceptor cell therapy based on specific metabolic defects observed in TILs isolated from individual tumors. Methods / Compounds Mechanism of action Target Process / Path Potential applications in TIL Considerations / Challenges Nicotinamide riboside (NR) NAD+ precursor; converted to NMN and then to NAD+ NAD+ Salvage Pathway Enhance TIL metabolism and function Dose optimization to avoid side effects Nicotinamide Mononucleotide (NMN) Direct precursor of NAD+; converted to NAD+ by NMNAT enzyme NAD+ Salvage Pathway Enhance the anti-tumor activity of TIL Stability and Cell Uptake Considerations Niacin (Vitamin B3) Converted to NAD+ via the Price-Handler pathway NAD+ biosynthetic pathway Supports TIL energy production and DNA repair Flushing reaction occurs at high doses NAMPT inhibitors Increase NAD+ levels by inhibiting NAD+ degradation NAD+ Salvage Pathway May prolong TIL lifespan Risks of Whole-Body NAD+ Depletion PARP inhibitors Prevents NAD+ depletion by inhibiting the PARP enzyme DNA repair pathways Preserve the metabolic function of NAD+ in TIL The balance between PARP inhibition and cancer therapy Longevity Protein Activator Improve NAD+ utilization efficiency Longevity protein-mediated deacetylation Enhance TIL mitochondrial function The long-term effects on TIL are not yet fully understood CD38 inhibitors Reduce NAD+ degradation ADP-ribosyl cyclase/cyclo-ADP-ribose hydrolase pathway Increase the bioavailability of NAD+ in TIL May affect calcium signaling Ketogenic diet May increase NAD+ levels by increasing fatty acid oxidation Metabolism converted into ketone bodies Can indirectly support TIL in vivo Feasibility in patients and its effects on immune cells Calorie restriction Can upregulate NAD+ biosynthetic pathway General metabolic downregulation Potential to improve TIL efficacy Patient compliance and nutritional issues Exercise Upregulation of NAD+ biosynthesis and salvage pathways Enhance muscle contraction and energy requirements Indirect systemic effects on immune function Direct impact on TIL is unclear NAD+ Infusion Directly replenish NAD+ Bypassing the NAD+ biosynthetic pathway Immediately replenish NAD+ in TIL Imbalances and risks of destabilization Gene therapy Overexpression of NAD+ biosynthetic enzymes Targeted increase in NAD+ production TIL NAD+ levels continue to increase Carrier design, delivery and integration risks

The evolutionarily conserved Wnt/β-catenin pathway is crucially involved in the multipotency and differentiation of embryonic stem cells by maintaining DNA methylation patterns, thereby promoting epigenetic stability necessary for correct cell fate decisions in a highly mitotic state (Clevers, H. and R. Nusse, Cell , 2012. 149(6): pp. 1192-205). In humans, rapid recall of memory T cells after re-exposure to antigen suggests the need for a stable epigenetic program. Most TIL-based ACT regimens for solid tumors are generated from terminally differentiated effector memory T cells that are generated by repeated rounds of TCR ligation in vitro (Hinrichs, CS and SA Rosenberg, Immunol Rev , 2014. 257(1): p56-71). However, the critical involvement of DNA methylation in shaping the expansion, memory state, and therefore function of antigen-specific TILs that undergo TCR ligation-dependent ex vivo expansion is unclear (Abdelsamed, HA et al., J Exp Med , 2017. 214(6): p1593-1606).

There is evidence that pharmacological activation of Wnt/β-catenin signaling reprograms tumor-specific CD8+ TILs in tumor-bearing mice by increasing the expression of Tcf1 (encoded by Tcf7) (Gattinoni, L. et al., Nat Med , 2009. 15(7): p. 808-13). In human melanoma, CD8+TCF7+ reveals a TIL subset with a memory progenitor-like cell state, which has bystander cytotoxic function and is associated with positive results of immune checkpoint blockade (Li, H. et al., Cell , 2019. 176(4): pp. 775-789 e18; Sade-Feldman, M. et al., Cell , 2018. 175(4): pp. 998-1013 e20). Wnt pathway components have been reported to be downregulated in PD1+CD8+ memory TILs in vivo and further downregulated in all T cell populations after ex vivo expansion (Lipp, JJ et al., Oncoimmunology , 2022. 11(1): p. 2019466), and compensating for the loss of Wnt signaling by pharmacological inhibition of GSK3β improved the effector function of the ex vivo expanded populations. Since Wnt signaling is known to prevent the development of effector CD8+ T cells, it may also be involved in epigenetically reprogramming TILs. GSK-3 is a positive regulator of PD-1 expression in CD8+ T cells, and inhibition of GSK-3 can enhance T cell function and effectively control tumor growth (Taylor, A. et al., Immunity , 2016. 44(2): pp. 274-86; Steele, L. et al., iScience , 2021. 24(6): p. 102555). In addition to being a central regulator of PD-1, GSK-3 also negatively regulates lymphocyte activation gene 3 (LAG-3) expression on CD4+ and CD8+ T cells, and small molecule inhibitors of GSK-3 are more effective than LAG-3 blockade alone in inhibiting tumor growth in a melanoma mouse model (Rudd, CE et al., Cell Rep , 2020. 30(7): p. 2075-2082e4). Recently, it has been shown that Wnt activation can promote the multifunctionality of human memory T cells via the epigenetic regulator PRMT1 (Sung, BY et al., J Clin Invest , 2022. 132 (2)).

Thus, in some embodiments, the second cell culture medium comprises a GSK-3α/β inhibitor. In some embodiments, the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO, and 3F8. J. Harvesting TILs

After the second expansion step, the cells can be harvested. TILs can be harvested in any appropriate and sterile manner, including, for example, centrifugation. Methods for harvesting TILs are well known in the art and any such known methods can be used with the process of the present invention. In some embodiments, TILs are harvested using an automated system.

Cell harvesters and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. In some embodiments, the methods of the present invention may employ any cell-based harvester. In some embodiments, the cell harvester and/or cell processing system is a membrane-based cell harvester. In some embodiments, cell harvesting is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a solution containing cells through a membrane or filter (such as a rotating membrane or rotating filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell harvester and/or cell processing system can perform cell separation, washing, fluid exchange, concentration and/or other cell processing steps in a closed sterile system.

In some embodiments, harvesting is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, the closed system is entered through a syringe under sterile conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the examples is used. K. Final preparation and transfer to an infusion container

After the steps as described above and in detail herein are completed, the TIL is transferred to a container for administration to a patient, such as an infusion bag or a sterile vial. In some embodiments, once a sufficient number of TILs for treatment is obtained using the expansion method described above, it is transferred to a container, such as an infusion bag, for administration to a patient. In some embodiments, the TIL is cryopreserved in an infusion bag. In some embodiments, the TIL is cryopreserved before being placed in an infusion bag. In some embodiments, the TIL is cryopreserved and is not placed in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is generally accomplished by placing the TIL population in a cryogenic solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in the solution are placed in a cryogenic vial and stored at -80°C for 24 hours, with transfer to a gaseous nitrogen freezer for cryopreservation as appropriate. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, cells are removed from the freezer and thawed in a 37°C water bath until approximately 4/5 of the solution is thawed. Cells are generally resuspended in complete medium and washed one or more times as appropriate. In some embodiments, thawed TILs may be counted and survival assessed as known in the art.

In some embodiments, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLife Solutions). In some embodiments, the TIL population is cryopreserved using a cryopreservation medium containing dimethyl sulfoxide (DMSO). In some embodiments, the TIL population is cryopreserved using a 1:1 (vol:vol) ratio of CS10 to cell culture medium. In some embodiments, the TIL population is cryopreserved using an approximately 1:1 (vol:vol) ratio of CS10 to cell culture medium (further comprising additional IL-2).

In some embodiments, TILs are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded by the methods described in this disclosure may be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration. L. Closed Systems for TIL Manufacturing

The present invention provides for the use of a closed system during the TIL culture process. Such a closed system allows for the prevention and/or reduction of microbial contamination, allows for the use of fewer flasks, and allows for cost reduction. In some embodiments, the closed system uses two containers.

Such closed systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

The aseptic connecting device (STCD) creates an aseptic weld between two compatible tubes. This procedure allows aseptic connection of multiple containers and tube diameters. In some embodiments, the closed system includes a luer lock and heat seal system as described in the examples. In some embodiments, the closed system is entered through a syringe under aseptic conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the examples is used. In some embodiments, the TIL is dispensed into the final product dispensing container according to the methods described in the examples herein.

In some embodiments, the closed system uses one container from the time the tumor fragment is obtained until the TIL is ready to be administered to the patient or cryopreserved. In some embodiments, when two containers are used, the first container is a closed G container (such as a G-rex 100M series or G-rex 500M series flask), and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HypoThermosol. The characteristic of a closed system or closed TIL cell culture system is that once the tumor sample and/or tumor fragment has been added, the system is tightly sealed from the outside to form a closed environment that is not invaded by bacteria, fungi and/or any other microbial contamination.

In some embodiments, the microbial contamination is reduced by between about 5% and about 100%. In some embodiments, the microbial contamination is reduced by between about 5% and about 95%. In some embodiments, the microbial contamination is reduced by between about 5% and about 90%. In some embodiments, the microbial contamination is reduced by between about 10% and about 90%. In some embodiments, the microbial contamination is reduced by between about 15% and about 85%. In some embodiments, the microbial contamination is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or about 100%.

The closed system allows TILs to grow in the absence of microbial contamination and/or with significantly reduced microbial contamination.

In addition, the pH value, carbon dioxide partial pressure, and oxygen partial pressure of the TIL cell culture environment each change with cell culture. Therefore, even if the culture medium suitable for cell culture is circulated, the closed environment still needs to be constantly maintained as the best environment for TIL proliferation. To this end, it is necessary to use sensors to monitor the physical factors of the pH value, carbon dioxide partial pressure, and oxygen partial pressure in the culture solution of the closed environment. The signal is used to control the gas exchanger installed at the entrance of the culture environment, and to adjust the gas partial pressure of the closed environment in real time according to the changes in the culture solution in order to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system that incorporates a gas exchanger equipped with a monitoring device for measuring the pH value, carbon dioxide partial pressure, and oxygen partial pressure of the closed environment at the entrance to the closed environment, and optimizes the cell culture environment by automatically adjusting the gas concentration based on the signal from the monitoring device.

In some embodiments, the pressure in the closed environment is controlled continuously or intermittently. That is, the pressure in the closed environment can be changed by means of, for example, a pressure maintenance device, thereby ensuring that the space is suitable for TIL growth under a positive pressure state or promoting exudate and thus cell proliferation under a negative pressure state. In addition, by intermittently applying negative pressure, it is possible to uniformly and effectively replace the circulating liquid in the closed environment by temporarily reducing the volume of the closed environment.

In some embodiments, additional equipment, such as an electroporator (e.g., a Neon electroporator) is a component of a fully closed system. In some embodiments, optimal culture components for TIL proliferation can be replaced or added, and factors including, for example, IL-2 and/or OKT3 and combinations can be added.

In other embodiments, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified, wherein each container referenced in the method is GREX-10. In other embodiments, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified, wherein each container referenced in the method is GREX-100M. In other embodiments, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified, wherein each container referenced in the method is GREX-500M. VII. Methods of Making TIL Populations

Methods for making TIL populations (e.g., Gen 2 methods) have been described in International Patent Application Publication No. WO 2018/182817 A1 and International Patent Application Publication No. WO 2019/190579 A1, the disclosures of which are incorporated herein by reference in their entirety.

Some embodiments of the present disclosure provide a method of producing a tumor infiltrating lymphocyte (TIL) population, comprising culturing the TIL population in a cell culture medium, wherein the cell culture medium comprises IL-15, IL-21 and L-arginine.

Some embodiments of the present disclosure provide a method of producing a tumor infiltrating lymphocyte (TIL) population, comprising culturing the TIL population in a cell culture medium, wherein the cell culture medium comprises IL-15, IL-21, L-arginine and NAD+.

Some embodiments disclosed herein provide a method for producing a tumor infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; and b) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, and L-arginine.

Some embodiments disclosed herein provide a method for producing a tumor infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; and b) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, L-arginine, and NAD+.

Some embodiments of the present disclosure provide a method for producing a tumor infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium for about 3-14 days; b) performing a second expansion by culturing the TIL population in a second cell culture medium for about 7-14 days, wherein the second cell culture medium comprises IL-15, IL-21 and L-arginine.

Some embodiments of the present disclosure provide a method for producing a tumor infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium for about 3-14 days; b) performing a second expansion by culturing the TIL population in a second cell culture medium for about 7-14 days, wherein the second cell culture medium comprises IL-15, IL-21, L-arginine and NAD+.

Some embodiments of the present disclosure provide a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, comprising: (a) adding tumor fragments processed from a tumor resected from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) The second TIL population is cultured in a cell culture medium containing IL-21, IL-15, L-arginine, OKT-3 and antigen presenting cells (APC) to perform a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (b) to step (c) is performed without opening the system; (d) Harvesting the therapeutic TIL population obtained from step (c), wherein the transfer from step (c) to step (d) is performed without opening the system, and wherein the harvested therapeutic TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (e) transferring the TIL population harvested from step (d) to an infusion bag, wherein the transfer from step (d) to (e) is performed without opening the system, and (f) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process.

Some embodiments of the present disclosure provide a method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, comprising: (a) adding tumor fragments processed from a tumor resected from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) (d) culturing the second TIL population in a second cell culture medium comprising IL-21, IL-15, L-arginine, NAD+, OKT-3 and antigen presenting cells (APCs) for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (b) to step (c) is performed without opening the system; (e) transferring the TIL population harvested from step (d) to an infusion bag, wherein the transfer from step (d) to (e) is performed without opening the system, and (f) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process. A. Obtaining a Patient Tumor Sample

Generally, TILs are initially obtained from patient tumor samples ("primary TILs") and then expanded into larger populations for further manipulation as described herein.

Patient tumor samples can be obtained using methods known in the art, usually obtained by surgical resection, biopsy, core needle biopsy, small biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells. In some embodiments, multi-lesion sampling is used. In some embodiments, surgical resection, biopsy, core needle biopsy, small biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells include multi-lesion sampling (that is, one or more tumor sites and/or positions in the patient and one or more tumors in the same position or closely adjacent to obtain samples). In general, tumor samples can come from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. Tumor samples may also be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors may be any type of cancer, including, but not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from endometrial cancer, cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), neuroglioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. In some embodiments, suitable TILs are obtained from melanoma.

Once obtained, tumor samples are typically fragmented using a sharp tool into small pieces between 1 and about 8 mm ³ , with about 2-3 mm ³ being particularly useful. In some embodiments, TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzymatic medium (e.g., Roswell Park Cancer Institute (RPMI) 1640 buffer, 2 mM glutamine, 10 mcg/mL tamiprocin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in an enzymatic medium and mechanically dissociating the tumor for about 1 minute, followed by incubation at 37°C in 5% _CO2 for 30 minutes, followed by repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched chain hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art may be used, such as the method described in U.S. Patent Application Publication No. 2012/0244133 A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the methods of expanding TILs or treating cancer described herein. B. Tumor fragmentation and / or digestion

As noted above, in some embodiments, TILs are derived from solid tumors. In some embodiments, solid tumors are not fragmented. In some embodiments, solid tumors are not fragmented and are enzymatically digested with the whole tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme, and neutral protease. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme, and neutral protease for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme, and neutral protease at 37°C, ₅ % CO2 for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme, and neutral protease at 37°C, 5% _CO2 , with rotation for 1 to 2 hours. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C, 5% _CO2 , under constant rotation. In some embodiments, the whole tumor is combined with an enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of collagenase is 100 mg/ml 10X working stock solution.

In some embodiments, the enzyme mixture comprises DNase. In some embodiments, the working stock solution of DNase is 10,000 IU/ml 10X working stock solution.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10 mg/ml 10X working stock solution.

In some embodiments, the enzyme mixture comprises 10 mg/ml collagenase, 1000 IU/ml DNase, and 1 mg/ml hyaluronidase.

In some embodiments, the enzyme mixture comprises 10 mg/ml collagenase, 500 IU/ml DNase, and 1 mg/ml hyaluronidase.

In some embodiments, the enzyme mixture comprises a neutral protease. In some embodiments, the working stock solution of the neutral protease is reconstituted at a concentration of 175 DMC U/mL.

In some embodiments, the enzyme mixture comprises a neutral protease, a DNase, and a collagenase.

In some embodiments, the enzyme mixture comprises 10 mg/ml collagenase, 1000 IU/ml DNase, and 0.31 DMC U/ml neutral protease. In some embodiments, the enzyme mixture comprises 10 mg/ml collagenase, 500 IU/ml DNase, and 0.31 DMC U/ml neutral protease.

Generally speaking, the harvested cell suspension is called the "primary cell population" or the "freshly harvested" cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, segmentation and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is segmentation. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially obtained from enzymatic tumor digests and tumor fragment cultures of patients. In some embodiments, TILs can be initially obtained from enzymatic tumor digests and tumor fragment cultures of patients before gene editing.

In some embodiments, when the tumor is a solid tumor, after obtaining the tumor sample, the tumor is physically fragmented. In some embodiments, the fragmentation occurs before cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after the tumor is obtained and without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm ³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 mm ³ to about 1500 mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total volume of about 1350 mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments. In some embodiments, the plurality of fragments comprises about to about 100 fragments.

In some embodiments, TILs are obtained from tumor fragments. In some embodiments, tumor fragments are obtained by sharpening. In some embodiments, tumor fragments are between about 1 mm ³ and 10 mm ^3. In some embodiments, tumor fragments are between about 1 mm ³ and 8 mm ^3. In some embodiments, tumor fragments are about 1 mm ^3. In some embodiments, tumor fragments are about 2 mm ^3. In some embodiments, tumor fragments are about 3 mm ^3. In some embodiments, tumor fragments are about 4 mm ^3. In some embodiments, tumor fragments are about 5 mm ^3. In some embodiments, tumor fragments are about 6 mm ^3. In some embodiments, tumor fragments are about 7 mm ³ . In some embodiments, a tumor fragment is about 8 mm ³ . In some embodiments, a tumor fragment is about 9 mm ³ . In some embodiments, a tumor fragment is about 10 mm ³ . In some embodiments, a tumor is 1 to 4 mm x 1 to 4 mm x 1 to 4 mm. In some embodiments, a tumor is 1 mm x 1 mm x 1 mm. In some embodiments, a tumor is 2 mm x 2 mm x 2 mm. In some embodiments, a tumor is 3 mm x 3 mm x 3 mm. In some embodiments, a tumor is 4 mm x 4 mm x 4 mm.

In some embodiments, tumors are resected to minimize the amount of hemorrhagic, necrotic, and/or fatty tissue on each slice. In some embodiments, tumors are resected to minimize the amount of hemorrhagic tissue on each slice. In some embodiments, tumors are resected to minimize the amount of necrotic tissue on each slice. In some embodiments, tumors are resected to minimize the amount of fatty tissue on each slice.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without using a scalpel to perform a sawing action. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are produced by cultivating in an enzymatic medium (such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg/mL genitamicin, 30 U/mL DNA enzyme, and 1.0 mg/mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After the tumor is placed in an enzymatic medium, the tumor can be mechanically dissociated for about 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes and then mechanically disrupted again for about 1 minute. After another 30 minutes of incubation at 37°C in 5% _CO2 , the tumor can be mechanically disrupted a third time for about 1 minute. In some embodiments, if large pieces of tissue still exist after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to the sample, regardless of whether or not the sample is incubated for another 30 minutes at 37°C in 5% _CO2 . In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

In some embodiments, the cell suspension harvested before the first expansion step is referred to as the "primary cell population" or "freshly harvested" cell population.

In some embodiments, cells are optionally frozen following sample harvest and stored frozen prior to expansion as described in further detail below.

In some embodiments, the TILs are obtained by thawing a cryopreserved tumor digest comprising a first TIL population derived from a tumor resected from an individual. In some embodiments, the first TIL population is obtained from a tumor resected from an individual by processing a tumor sample obtained from an individual into a tumor digest. In some embodiments, a tumor digest or tumor fragment comprises a first TIL population and is obtained from a tumor resected from an individual.

In some embodiments, the methods disclosed herein include adding a tumor digest or tumor fragment to a closed system, wherein the tumor digest or tumor fragment comprises a first TIL population and is obtained from a tumor excised from an individual. In some embodiments, the methods disclosed herein include performing a first expansion by thawing a cryopreserved tumor digest comprising a first TIL population derived from a tumor excised from an individual. In some embodiments, the methods disclosed herein include obtaining a first TIL population from a tumor excised from a patient by processing a tumor sample obtained from a patient into a tumor digest or multiple tumor fragments.

In some embodiments, tumor lysate can be further obtained from tumor digest through several freeze-thaw cycles or mass spectrometry procedures.

In some embodiments, tumor fragments and/or tumor digests and/or tumor lysates may be frozen and stored frozen prior to entering the priming step, as appropriate.

In some embodiments, the tumor is reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of collagenase is 100 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises DNA enzyme. In some embodiments, the working stock solution of DNA enzyme is 10,000 IU/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 1000 IU/mL DNase, and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

In some embodiments, fragmentation includes physical fragmentation, including, for example, segmentation and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is segmentation. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially obtained from enzymatic tumor digests and tumor fragment cultures of patients. In some embodiments, TILs can be initially obtained from enzymatic tumor digests and tumor fragment cultures of patients.

In some embodiments, the TILs are not obtained from a tumor digest. In some embodiments, the solid tumor core is not fragmented.

In some embodiments, obtaining the first TIL population comprises a multi-lesion sampling approach.

The tumor lytic enzyme cocktail may include one or more lytic (digestive) enzymes such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other lytic or proteolytic enzyme, and any combination thereof.

In some embodiments, the lyophilized enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a certain amount of sterile buffer (such as Hank's balanced salt solution (HBSS)).

In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 mL sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 2892 PZ U per vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, such as about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 150 PZ U/mL, about 200 PZ U/mL, about 210 PZ U/mL, about 220 PZ U/mL, about 230 PZ U/mL, about 240 PZ U/mL, about 250 PZ U/mL, about 260 PZ U/mL, about 270 PZ U/mL, about 280 PZ U/mL, about 299.2 PZ U/mL, about 300 PZ U/mL, about 310 PZ U/mL, about 320 PZ U/mL, about 330 PZ U/mL, about 340 PZ U/mL, about 350 PZ U/mL, about 360 PZ U/mL, about 370 PZ U/mL, about 380 PZ U/mL, about 390 PZ U/mL, about 400 PZ U/mL, about 410 PZ U/mL, about 420 PZ U/mL, about 430 PZ U/mL, about 440 PZ U/mL U/mL, about 300 PZ U/mL, about 350 PZ U/mL, or about 400 PZ U/mL.

In some embodiments, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after reconstitution, the neutral protease stock solution ranges from about 100 DMC/mL to about 400 DMC/mL, such as about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL DMC/mL or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, such as about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the enzyme stock solution may vary, therefore checking the concentration of the lyophilized stock solution and modifying the final amount of enzyme added to the digestion mixture accordingly.

In some embodiments, the enzyme mixture includes about 10.2 μl of neutral protease (0.36 DMC U/mL), 21.3 μl of collagenase (1.2 PZ/mL), and 250 μl of DNase I (200 U/mL) in about 4.7 mL of sterile HBSS. C. First Expansion

After the tumor fragments are divided or digested, the resulting cells are cultured in serum containing IL-2 under conditions that are favorable for TIL growth but unfavorable for tumor and other cell growth. In some embodiments, the tumor digest is cultured in a 2 mL well in a medium containing inactivated human AB serum with 6000 IU/mL IL-2. This primary cell population is cultured for a period of several days, typically 3 to 14 days, to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells. In some embodiments, the primary cell population is cultured for a period of 10 to 14 days to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells. In some embodiments, the primary cell population is cultured for a period of about 11 days to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells.

In some embodiments, expansion of TILs may be performed using an initial bulk TIL expansion step as described below and herein (which may include a process referred to as pre-REP), followed by a second expansion as described below and herein (including a process referred to as a rapid expansion protocol (REP) step), followed by optional cryopreservation, and then a second REP step as described below and herein. TILs obtained from this process may be characterized for phenotypic characteristics and metabolic parameters as described herein, as appropriate.

In some embodiments where TIL cultures are initiated in a 24-well plate, for example, using a Costar 24-well cell culture cluster flat bottom (Corning, Corning, NY), each well can be seeded with 1×10 ⁶ tumor digest cells or one tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 IU/mL; Chiron, Emeryville, CA). In some embodiments, the tumor fragment is between about 1 mm ³ and 10 mm ³ .

In some embodiments, the first expansion medium is referred to as "CM" (abbreviation for medium). In some embodiments, the CM of step B consists of RPMI 1640 supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL GlutaMAX. In some embodiments in which the culture is initiated in a gas-permeable bottle with a 40 mL capacity and a 10 ^cm2 gas-permeable silicon bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each bottle is loaded with 10-40 mL of CM with IL-2 containing 10-40×10 ⁶ live tumor digest cells or 5-30 tumor fragments. Both G-REX10 and 24-well plates were cultured in a humidified incubator at 37°C, 5% _CO2 and 5 days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2, and after the 5th day, half of the medium was replaced every 2-3 days.

In some embodiments, the medium used in the expansion process disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and/or reduce experimental variations due to batch-to-batch variations of serum-containing media.

In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include (but are not limited to) one or more of the following: CTS™ OpTmizer T Cell Expander Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin replacements, one or more amino acids, one or more vitamins, one or more transferrins or transferrin replacements, one or more antioxidants, one or more insulins or insulin replacements, one or more collagen prodrivers, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and a compound containing trace elements Ag+, Al3+, Ba2+, Cd2+, _CO2 +, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+, and Zr4+. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate and/or 2-hydroxyethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with a learned growth medium, which includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% based on the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-hydroxyethanol in the culture medium is 55 μM.

In some embodiments, the defined medium described in International PCT Publication No. WO/1998/030679, which is incorporated herein by reference, can be used in the present invention. In the publication, a serum-free eukaryotic cell culture medium is described. The serum-free eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen prodrivers, one or more trace elements and one or more antibiotics. In some embodiments, the medium further comprises L-glutamine, sodium bicarbonate and/or β-hydroxyethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen pro-drivers and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and a compound containing trace elements Ag+, Al3+, Ba2+, Cd2+, _CO2 +, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+, and Zr4+. In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), Eagle's basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

In some embodiments, the concentration of glycine in the culture medium is determined to be in the range of about 5-200 mg/L, the concentration of L-histidine is about 5-250 mg/L, the concentration of L-isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-200 mg/L, the concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-proline is about 1-1000 mg/L, the concentration of L-hydroxyproline is about 1-45 mg/L, the concentration of L-serine is about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, and the concentration of L-tryptophan is about 2-110 mg/L, L-tyrosine at a concentration of about 3-175 mg/L, L-valine at a concentration of about 5-500 mg/L, thiamine at a concentration of about 1-20 mg/L, reduced glutathione at a concentration of about 1-200 mg/L, L-ascorbic acid-2-phosphate at a concentration of about 1-200 mg/L, iron-saturated transferrin at a concentration of about 1-50 mg/L, insulin at a concentration of about 1-100 mg/L, sodium selenite at a concentration of about 0.000001-0.0001 mg/L, and albumin (e.g., AlbuMAX® I) at a concentration of about 5000 to 50,000 mg/L.

In some embodiments, the non-trace element portion of the medium is determined to be present in the concentration range listed in the column titled "Concentration Range in 1X Medium" in Table 4 below. In other embodiments, the non-trace element portion of the medium is determined to be present in the final concentration listed in the column titled "Preferred Embodiments of 1X Medium" in Table 4. In other embodiments, the medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace element portions of the type and concentration listed in the column titled "Preferred Embodiments of Supplements" in Table 11 above.

In some embodiments, the osmotic pressure of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmotic pressure is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-hydroxyethanol (final concentration of about 100 μM).

In some embodiments, the defined medium described in Smith et al., Clin Transl Immunology, 4(1) 2015 (doi: 10.1038/cti.2014.31) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ is used as the basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. Using an unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and/or second gas permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

After preparing the tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor TIL growth relative to tumor and other cells. In some embodiments, the tumor digest is cultured in a 2 mL well in a medium comprising inactivated human AB serum (or in some cases, as outlined herein, in the presence of APC cell populations) and 6000 IU/mL IL-2. This primary cell population is cultured for a period of several days, typically 10 to 14 days, to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells. In some embodiments, the growth medium during the first expansion period comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20-30×10 ⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20×10 ⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 25×10 ⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 30×10 ⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first expansion medium comprises about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.

In some embodiments, the first expansion medium comprises about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 200 IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15.

In some embodiments, the first expansion medium comprises about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 2 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21.

In some embodiments, the cell culture medium comprises an anti-CD3 agonist antibody, such as OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 µg/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab. See, e.g., Table 1.

In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: urelumab, utomilumab, EU-101, fusion proteins and fragments thereof, derivatives, variants, biosimilars and combinations. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 µg/mL to 100 µg/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 µg/mL to 40 µg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises an initial concentration of about 3000 IU/mL of IL-2 and an initial concentration of about 30 ng/mL of OKT-3 antibody, and wherein the one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the first expansion medium is referred to as "CM" (abbreviation for medium). In some embodiments, it is referred to as CM1 (medium 1). In some embodiments, CM consists of RPMI 1640 supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL GlutaMAX. In some embodiments in which the culture is initiated in a 40 mL capacity, 10 cm ² gas-permeable silicon bottom gas-permeable bottle (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each bottle is loaded with 10-40 mL of CM with IL-2 containing 10-40×10 ⁶ live tumor digest cells or 5-30 tumor fragments. G-REX10 and 24-well plates are cultured in a humidified incubator at 37°C, 5% _CO2 and 5 days after the start of culture, half of the medium is removed and replaced with fresh CM and IL-2, and after the 5th day, half of the medium is replaced every 2-3 days. In some embodiments, CM is CM1 described in the examples, see Example 1. In some embodiments, the first expansion is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

In some embodiments, as discussed in the examples and figures, the first expansion (including processes such as those described in step B of FIG. 2, which may include processes sometimes referred to as pre-REP) is shortened to 3-14 days. In some embodiments, the first expansion (including processes such as those described in step B of FIG. 82, which may include processes sometimes referred to as pre-REP) is shortened to 7 to 14 days, such as including, for example, the expansion described in step B of FIG. 82. In some embodiments, the first expansion of step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days, such as discussed in the expansion described in step B of FIG. 82.

In some embodiments, the first TIL expansion may be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. In some embodiments, the first TIL expansion may be performed for 1 day to 14 days. In some embodiments, the first TIL expansion may be performed for 2 days to 14 days. In some embodiments, the first TIL expansion may be performed for 3 days to 14 days. In some embodiments, the first TIL expansion may be performed for 4 days to 14 days. In some embodiments, the first TIL expansion may be performed for 5 days to 14 days. In some embodiments, the first TIL expansion may be performed for 6 days to 14 days. In some embodiments, the first TIL expansion may be performed for 7 days to 14 days. In some embodiments, the first TIL expansion may be performed for 8 to 14 days. In some embodiments, the first TIL expansion may be performed for 9 to 14 days. In some embodiments, the first TIL expansion may be performed for 10 to 14 days. In some embodiments, the first TIL expansion may be performed for 11 to 14 days. In some embodiments, the first TIL expansion may be performed for 12 to 14 days. In some embodiments, the first TIL expansion may be performed for 13 to 14 days. In some embodiments, the first TIL expansion may be performed for 14 days. In some embodiments, the first TIL expansion may be performed for 1 to 11 days. In some embodiments, the first TIL expansion may be performed for 2 to 11 days. In some embodiments, the first TIL expansion may be performed for 3 to 11 days. In some embodiments, the first TIL expansion may be performed for 4 to 11 days. In some embodiments, the first TIL expansion may be performed for 5 to 11 days. In some embodiments, the first TIL expansion may be performed for 6 to 11 days. In some embodiments, the first TIL expansion may be performed for 7 to 11 days. In some embodiments, the first TIL expansion may be performed for 8 to 11 days. In some embodiments, the first TIL expansion may be performed for 9 to 11 days. In some embodiments, the first TIL expansion may be performed for 10 to 11 days. In some embodiments, the first TIL expansion may be performed for 11 days.

In some embodiments, the combination of IL-2, IL-7, IL-15 and/or IL-21 is used as the combination during the first expansion period. In some embodiments, during the first expansion period, including, for example, according to Figure 82 and step B process described herein, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included. In some embodiments, the combination of IL-2, IL-15 and IL-21 is used as the combination during the first expansion period. In some embodiments, according to Figure 82 and step B process as described herein, IL-2, IL-15 and IL-21 and any combination thereof may be included.

In some embodiments, as discussed in the examples and figures, the first expansion (including a process called pre-REP; e.g., step B according to FIG. 82) process is shortened to 3 to 14 days. In some embodiments, the first expansion of step B is shortened to 7 to 14 days. In some embodiments, the first expansion of step B is shortened to 10 to 14 days. In some embodiments, the first expansion is shortened to 11 days. In some embodiments, the first expansion is performed in a closed system bioreactor, e.g., step B according to FIG. 82. In some embodiments, a closed system is used to perform TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor. 1. Interleukins and other additives

The expansion methods described herein typically utilize media with high doses of interleukins, particularly IL-2, as is known in the art.

Alternatively, it is also possible to use a combination of interleukins for rapid expansion and/or secondary expansion of TILs, as described in U.S. Patent Application Publication No. US 2017/0107490 A1 (the disclosure of which is incorporated herein by reference), using a combination of two or more of IL-2, IL-15, and IL-21. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2 or IL-15 and IL-21, the latter of which has a specific use in many embodiments. The use of a combination of interleukins is particularly advantageous for the generation of lymphocytes, and in particular T cells as described therein.

In some embodiments, step B may also include adding OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step B may also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator factor I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference, may be used in the culture medium during step B. D. Second expansion

In some embodiments, the number of TIL cell populations is expanded after initial bulk processing and pre-REP expansion to produce TIL populations. This further expansion is referred to herein as the second expansion, which may include an expansion process generally referred to as a rapid expansion process (REP) in this technology. The second expansion is typically completed in a gas permeable container using a culture medium comprising a variety of components, including feeder cells, a cytokine source, and an anti-CD3 agonist antibody.

In some embodiments, the second expansion of TIL (which may include expansion sometimes referred to as REP) can be performed using any TIL bottle or container known to those skilled in the art, wherein the expanded TIL has been transduced with the recombinant lentiviral particles disclosed herein to produce a population of gene-edited TIL. In some embodiments, the second expansion can be performed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second expansion can be performed for about 7 days to about 14 days. In some embodiments, the second expansion can be performed for about 7 days to about 12 days. In some embodiments, the second expansion can be performed for about 7 days to about 10 days. In some embodiments, the second expansion can be performed for about 7 days to about 9 days. In some embodiments, the second expansion can be performed for about 8 days to about 9 days. In some embodiments, the second expansion can be performed for about 9 days. In some embodiments, the second expansion can be performed for about 10 days. In some embodiments, the second expansion can be performed for about 11 days.

In some embodiments, the second expansion can be performed in a gas permeable container using the methods of the present disclosure (including, for example, expansion referred to as REP). For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulators can include, for example, anti-CD3 agonist antibodies, such as about 30 ng/ml OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil (Raritan, NJ) or Miltenyi Biotech (Auburn, CA)), or UHCT-1 (available from BioLegend, San Diego, CA, USA). TILs can be expanded by including one or more antigens (including antigenic portions thereof, such as antigenic determinants) during the second expansion period to induce further TIL in vitro stimulation, such antigens can be expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, such as 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217 (210M), in the presence of T cell growth factors (such as 300 IU/mL IL-2 or IL-15) as appropriate. Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same cancer antigen pulsed onto antigen presenting cells expressing HLA-A2. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, or about 1 µg/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:100 and 1:200.

In some embodiments, REP and/or secondary expansion are performed in flasks where the primary TILs are mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 ml of medium. The medium is replaced (usually by aspirating fresh medium to replace 2/3 of the medium) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX bottles and gas permeable containers, as described more fully below.

In some embodiments, as discussed in the examples and figures, the second expansion (which may include a process called REP process) is shortened to 7 to 14 days, wherein the TILs expanded by such second expansion have been transduced with the recombinant lentiviral particles disclosed herein to generate a gene-edited TIL population. In some embodiments, the second expansion is shortened to 9 days.

In some embodiments, REP and/or secondary expansion can be performed using T-175 flasks and gas-permeable bags as previously described (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother. 2003, 26, 332-42) or gas-permeable culture vessels (G-Rex flasks), wherein the TILs expanded by such secondary expansion have been transduced with recombinant lentiviral particles disclosed herein to generate gene-edited TIL populations. In some embodiments, secondary expansion (including expansion referred to as rapid expansion) is performed in T-175 flasks, and approximately 1×10 ⁶ TILs suspended in 150 mL of medium can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/mL IL-2 and 30 ng/ml anti-CD3. T-175 flasks can be incubated at 37°C, 5% _CO2 , where the TILs expanded by such a second expansion have been transduced with the recombinant lentiviral particles disclosed herein to produce a population of gene-edited TILs. Half of the medium can be replaced on day 5 with a 50/50 medium with 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks can be combined in a 3 L bag, and 300 mL of AIM V with 5% human AB serum and 3000 IU/mL IL-2 are added to 300 ml of TIL suspension. The number of cells in each bag was counted every day or every two days, and fresh medium was added to maintain the cell count between 0.5 and 2.0×10 ⁶ cells/mL.

In some embodiments, the second expansion (which may include expansion referred to as REP) can be performed in a 500 mL capacity gas permeable bottle with a 100 cm gas permeable silicon bottom (G-Rex 100, available from Wilson Wolf Manufacturing), 5×10 ⁶ or 10×10 ⁶ TILs can be cultured with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/ml anti-CD3 (OKT3), wherein the TILs expanded by such a second expansion have been transduced with the recombinant lentiviral particles disclosed herein to generate a gene-edited TIL population. The G-Rex 100 bottle can be incubated at 37° C., 5% CO ₂ . On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellet can be resuspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU/mL IL-2, and added back to the original G-Rex 100 bottle. As TILs are continuously expanded in G-Rex 100 bottles, on day 7, the TILs in each G-Rex 100 can be suspended in the 300 mL of medium present in each bottle, and the cell suspension can be divided into three 100 mL aliquots that can be used to inoculate three G-Rex 100 bottles. 150 mL of AIM-V with 5% human AB serum and 3000 IU/mL IL-2 can then be added to each bottle. The G-Rex 100 bottles can be incubated at 37°C, 5% _CO2 and after 4 days, 150 mL of AIM-V with 3000 IU/mL IL-2 can be added to each G-REX 100 bottle. Cells can be harvested on day 14 of culture.

In some embodiments, the second expansion (which may include expansion referred to as REP) can be performed in a 500 mL capacity gas permeable bottle with a 100 cm gas permeable silicon bottom (G-REX-100, available from Wilson Wolf Manufacturing, New Brighton, MN, USA), and 5×10 ⁶ or 10×10 ⁶ TILs can be cultured with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/mL anti-CD3 (OKT3). G-REX-100 (or G-REX100M) can be incubated at 37° C., 5% CO _2. On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellet can be resuspended with 150 mL of fresh medium with 5% human AB serum, 6000 IU/mL IL-2 and added back to the original GREX-100 bottle. When the TIL continues to expand in the GREX-100 bottle, on the 10th or 11th day, the TIL can be moved to a larger bottle, such as GREX-500 (or G-REX500M). The cells can be harvested on the 14th day of culture. The cells can be harvested on the 15th day of culture. The cells can be harvested on the 16th day of culture. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber. In some embodiments, 2/3 of the medium is replaced by aspirating the spent medium and replacing it with an equal volume of fresh medium. In some embodiments, the alternative growth chamber comprises a GREX bottle and a gas permeable container, as discussed more fully below. In some embodiments, the method employs different centrifugation speeds (400 g, 300 g, 200 g for 5 minutes) and different numbers of repetitions.

In some embodiments, the second expansion (including expansion referred to as REP) is performed in a flask, where the main TIL is mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 ml of medium. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber, where the TIL expanded by such a second expansion has been transduced with the recombinant lentiviral particles disclosed herein to produce a gene-edited TIL population. In some embodiments, 2/3 of the medium is replaced by aspirating the spent medium, followed by infusion of fresh medium. In some embodiments, alternative growth chambers include G-REX bottles and gas permeable containers, as discussed more fully below.

In some embodiments, the second expansion medium (e.g., sometimes referred to as CM2 or second cell medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the medium used in the expansion process disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and/or reduce experimental variations due to batch-to-batch variations of serum-containing media.

In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include (but are not limited to) one or more of the following: CTS™ OpTmizer T Cell Expander Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin replacements, one or more amino acids, one or more vitamins, one or more transferrins or transferrin replacements, one or more antioxidants, one or more insulins or insulin replacements, one or more collagen prodrivers, one or more antibiotics, and one or more trace elements. In some embodiments, the defined culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and trace element portions Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ^{6+ In} some embodiments, the culture medium further comprises ^L -glutamine, sodium bicarbonate and/ ^{or 2} -hydroxyethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with a learned growth medium, which includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% based on the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-hydroxyethanol in the culture medium is 55 μM.

In some embodiments, the defined medium described in International PCT Publication No. WO/1998/030679, which is incorporated herein by reference, is suitable for use in the present invention. In the publication, a serum-free eukaryotic cell culture medium is described. The serum-free eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen prodrivers, one or more trace elements and one or more antibiotics. In some embodiments, the medium further comprises L-glutamine, sodium bicarbonate and/or β-hydroxyethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen pro-drivers and one or more trace elements. In some embodiments, the defined culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and trace element portions Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , Co ²⁺ , Cr ³⁺ , Ge ⁴⁺ , Se ⁴⁺ , Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), Eagle's basal medium (BME), ^{RPMI 1640} , F- ^{10 ,} F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium and Iskoff's modified Dulbecco's medium.

In some embodiments, the concentration of glycine in the culture medium is determined to be in the range of about 5-200 mg/L, the concentration of L-histidine is about 5-250 mg/L, the concentration of L-isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-200 mg/L, the concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-proline is about 1-1000 mg/L, the concentration of L-hydroxyproline is about 1-45 mg/L, the concentration of L-serine is about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, and the concentration of L-tryptophan is about 2-110 mg/L, L-tyrosine at a concentration of about 3-175 mg/L, L-valine at a concentration of about 5-500 mg/L, thiamine at a concentration of about 1-20 mg/L, reduced glutathione at a concentration of about 1-20 mg/L, L-ascorbic acid-2-phosphate at a concentration of about 1-200 mg/L, iron-saturated transferrin at a concentration of about 1-50 mg/L, insulin at a concentration of about 1-100 mg/L, sodium selenite at a concentration of about 0.000001-0.0001 mg/L, and albumin (e.g., AlbuMAX® I) at a concentration of about 5000-50,000 mg/L.

In some embodiments, the non-trace element portion of the medium is determined to be present in the concentration range listed in the column titled "Concentration Range in 1X Medium" in Table 12 below. In other embodiments, the non-trace element portion of the medium is determined to be present in the final concentration listed in the column titled "Preferred Embodiments of 1X Medium" in Table 12. In other embodiments, the medium is determined to be a basal cell culture medium containing a serum-free supplement. In some of these embodiments, the serum-free supplement contains non-trace element portions of the type and concentration listed in the column titled "Preferred Embodiments of Supplements" in Table 12 above.

In some embodiments, the osmotic pressure of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmotic pressure is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-hydroxyethanol (final concentration of about 100 μM).

In some embodiments, the defined medium described in Smith et al., Clin Transl Immunology , 4(1) 2015 (doi: 10.1038/cti.2014.31) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ is used as the basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. Using an unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and/or second gas permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, the second expansion is performed in a closed system bioreactor. In some embodiments, a closed system is used to perform TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, the steps of the method are completed within a time period of about 22 days. In some embodiments, the steps of the method are completed within a time period of about 8 days. In some embodiments, the steps of the method are completed within a time period of about 9 days. In some embodiments, the steps of the method are completed within a time period of about 10 days. In some embodiments, the steps of the method are completed within a time period of about 11 days. In some embodiments, the steps of the method are completed within a time period of about 12 days. In some embodiments, the steps of the method are completed within a time period of about 13 days. In some embodiments, the steps of the method are completed within a time period of about 14 days. In some embodiments, the steps of the method are completed within a time period of about 15 days. In some embodiments, the steps of the method are completed within a time period of about 16 days. In some embodiments, the steps of the method are completed within a time period of about 17 days. In some embodiments, the steps of the method are completed within a time period of about 18 days. In some embodiments, the steps of the method are completed within a time period of about 19 days. In some embodiments, the steps of the method are completed within a time period of about 20 days. In some embodiments, the steps of the method are completed within a time period of about 21 days. In some embodiments, the steps of the method are completed within a time period of about 22 days. In some embodiments, the steps of the method are completed within a time period of about 23 days. In some embodiments, the steps of the method are completed within a time period of about 24 days. In some embodiments, the steps of the method are completed within a time period of about 25 days. In some embodiments, the steps of the method are completed within a time period of about 26 days. In some embodiments, the steps of the method are completed within a time period of about 27 days. In some embodiments, the steps of the method are completed within a time period of about 28 days. In some embodiments, the steps of the method are completed within a time period of about 29 days. In some embodiments, the steps of the method are completed within a time period of about 30 days. In some embodiments, the steps of the method are completed within a time period of about 31 days.

In some embodiments, the antigen presenting cell (APC) is a PBMC. According to some embodiments, the PBMC is irradiated. According to some embodiments, the PBMC is allogeneic. According to some embodiments, the PBMC is irradiated and allogeneic. According to some embodiments, the antigen presenting cell is an artificial antigen presenting cell.

In some embodiments, the first expansion is performed using a breathable container. In some embodiments, the second expansion is performed using a breathable container.

In some embodiments, the second cell culture medium further comprises an interleukin selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the second cell culture medium and/or the third cell culture medium further comprises an interleukin selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. 1. Feeder cells and antigen presenting cells

In some embodiments, the second expansion process described herein requires excess feeder cells during the REP TIL expansion period and/or during the second expansion period. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

Generally, allogeneic PBMCs are irradiated or heat treated without activation and used in the REP procedure as described in the Examples, which provide an exemplary protocol for assessing the replication incompetence of irradiated allogeneic PBMCs.

In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to be replication-incompetent and are acceptable for use in the TIL expansion procedures described herein.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase compared to the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to be replication-incompetent and acceptable for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody, about 10 ng/mL concentration of IL-15, and about 10 ng/mL concentration of IL-21.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3, IL-15 and IL-21 on days 7 and 14 does not increase compared to the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to be replication-incompetent and acceptable for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody, about 10 ng/mL concentration of IL-15, and about 10 ng/mL concentration of IL-21. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody, about 10 ng/mL IL-15, and about 10 ng/mL IL-21. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody, about 10 ng/mL IL-15, and about 10 ng/mL IL-21. In some embodiments, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody, about 10 ng/mL IL-15, and about 10 ng/mL IL-21.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen presenting feeder cells are artificial antigen presenting feeder cells. In some embodiments, the ratio of TILs to antigen presenting feeder cells in the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion process described herein requires a ratio of about 2.5×10 ⁹ feeder cells to about 100×10 ⁶ TILs. In other embodiments, the second expansion process described herein requires a ratio of about 2.5×10 ⁹ feeder cells to about 50×10 ⁶ TILs. In other embodiments, the second expansion process described herein requires a ratio of about 2.5×10 ⁹ feeder cells to about 25×10 ⁶ TILs.

In some embodiments, the second expansion process described herein requires an excess of feeder cells during the second expansion period. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen presenting cells (aAPCs) are used instead of PBMCs.

In some embodiments, artificial antigen presenting cells are used in the second expansion to replace PBMCs or in combination with PBMCs. 2. Interleukins and other additives

The expansion methods described herein typically utilize media with high doses of interleukins, particularly IL-2, as is known in the art.

Alternatively, it is possible to use a combination of interleukins for rapid expansion and/or secondary expansion of TILs, as described in U.S. Patent Application Publication No. US 2017/0107490 A1 (the disclosure of which is incorporated herein by reference), using a combination of two or more of IL-2, IL-15, and IL-21. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, wherein the latter has specific uses in many embodiments. The use of a combination of interleukins is particularly advantageous for the generation of lymphocytes, and in particular T cells as described therein.

In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-2. In some embodiments, the IL-2 concentration is 3000 IU/mL or less. In some embodiments, the first cell culture medium or the second cell culture medium does not contain additional IL-2. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and/or IL-21 at a concentration of about 1 ng/mL to about 100 ng/mL. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and/or IL-21 at a concentration of about 10 ng/mL. In some embodiments, the first cell culture medium comprises IL-2 and IL-21. In some embodiments, the first cell culture medium comprises 3000 IU/mL of IL-2 and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of patasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oregano, herbicide, terheolactone, isoliquiritigenin, baicalein and holmium solani.

In some embodiments, the first expansion is performed for a period of about 7-11 days. In some embodiments, the second expansion is performed for a period of about 7-11 days. 3. T cell metabolism modifiers

In vitro expansion alters TIL cellular states, which correlate with ACT efficacy (Chiffelle, J. et al., bioRxiv 2023 , which is incorporated herein by reference in its entirety). Rapidly expanding TILs have bioenergetic and biosynthetic requirements. Increasing the availability of essential cofactors such as L-arginine and NAD+ may support the synthesis of major biomass components. Functionally reinvigorating cells during in vitro expansion may generate TILs with new effector profiles.

Thus, provided herein are T cell metabolic modifiers that increase the availability of essential cofactors L-arginine and NAD ⁺ to support the synthesis of major biomass components in TILs, which can be added to the second expansion (REP) of TILs.

L-arginine is a building block for protein synthesis and improves T cell function through metabolic modification (Geiger R. et al., Cell 2016 : 167; 829-842; Fultang, L., Blood 2020 ; 136: 1155-60; the contents of which are incorporated herein by reference in their entirety).

NAD+ (nicotinamide adenine dinucleotide) is an electron acceptor that is consumed in large quantities during biomass synthesis, and its supplementation can improve T cell function (Canto C et al., Cell Metabolism 2015 ; 22:31-53; Wang Y. et al., Cell Reports 2021 ; 36:1-12; the contents of which are incorporated herein by reference in their entirety).

NAD+ (nicotinamide adenine dinucleotide) is a central coenzyme in cellular metabolism, particularly in redox reactions, where it cycles between oxidized (NAD+) and reduced (NADH) states. In the context of TILs, NAD+ plays a key role in regulating their metabolic fitness and function. TILs function in the tumor microenvironment, which is often characterized by nutrient deprivation and hypoxia. In this challenging microenvironment, TILs must adjust their metabolism to maintain their anti-tumor activity.

NAD+ enhances T cell metabolic fitness in multiple ways:

Glycolysis and the Pentose Phosphate Pathway (PPP): Although glycolysis does not use NAD+ directly, regeneration of NAD+ from NADH is essential for maintaining glycolytic flux, especially under hypoxic conditions where mitochondrial respiration is limited. The PPP is a branch of glycolysis that relies on NADP+ (the phosphorylated form of NAD+) to generate 5-phosphate ribose and reduce glutathione, thereby contributing to nucleotide synthesis and antioxidant defense mechanisms.

Mitochondrial Function: NAD+ is critical in mitochondria as it is a key electron acceptor in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Enhanced mitochondrial function supports the differentiation and survival of memory T cells, which are critical for sustained anti-tumor immunity.

Sirtuin activation: Sirtuins are a family of NAD+-dependent deacetylases that regulate cell metabolism, stress response, and inflammation. By activating sirtuins, NAD+ can promote the formation of memory T cells and enhance their anti-tumor function.

PARP activity: NAD+ is a substrate for poly(ADP-ribose) polymerase (PARP), which is involved in DNA repair and genome stability. By promoting the function of PARP, NAD+ helps maintain TIL integrity in the presence of DNA damage in tumors.

To replicate and enhance the benefits that NAD+ provides to TILs, pharmacological and genetic approaches may be considered: Pharmacological Approaches: - NAD+ Precursors: Enhancing NAD+ by administering NAD+ precursors such as niacinamide riboside (NR) or niacinamide mononucleotide (NMN) can increase intracellular NAD+ levels, thereby supporting TIL metabolism and function. - NAD+: Increases the availability of NAD+ metabolic substrates directly to TILs - Sempervivin Activators: Compounds such as resveratrol or SRT1720 can activate sempervivin, thereby mimicking the effects of high NAD+ levels and promoting TIL anti-tumor activity. - PARP inhibitors: Although PARP activity is dependent on NAD+ and can be beneficial, overactivation can deplete cellular NAD+ stores. PARP inhibitors can help maintain NAD+ levels under stressful conditions, potentially supporting T cell function. - CD38 inhibitors: CD38 uses NAD+ to generate ADP-ribose and cyclic ADP-ribose, which are important for calcium signaling, but also consume NAD+. CD38 inhibitors may help maintain NAD+ levels in TILs. Genetic approaches: - NAMPT overexpression: This is the rate-limiting enzyme in the NAD+ salvage pathway. Overexpression of NAMPT can increase the intracellular concentration of NAD+ in TILs. - Manipulation of longevity protein genes: Genetic modification to overexpress longevity protein genes may mimic the effects of high NAD+ levels and enhance TIL function. - Gene silencing of NAD+ consuming enzymes: Silencing or attenuation of genes encoding enzymes that consume large amounts of NAD+ (such as CD38 or PARP family members) may preserve NAD+ for TIL essential metabolic processes. - TCA cycle and OXPHOS support: Enhancing genes involved in mitochondrial biogenesis and function can support the efficient use of NAD+ and maintain TIL metabolic fitness.

Pharmacological and genetic tools to enhance NAD+ utilization in TILs are centered on specific molecular targets and pathways. In some embodiments, exemplary tools for enhancing NAD+ utilization focus on one or more of the following functions: mitochondrial biogenesis, promoting efficient energy utilization, and cultivating anti-tumor phenotypes. In embodiments, such tools are implemented in a coordinated manner, paying attention to the balance between enhancing TIL function and avoiding overstimulation that may lead to exhaustion or apoptosis. In some embodiments, the heterogeneity within the TIL population and the unique microenvironment of each tumor are considered when designing such interventions. Therefore, regulating mitochondrial function through pharmacological and genetic methods may be a powerful strategy to enhance the efficacy of donor-acceptor TIL therapy. The tools for enhancing NAD+ utilization are further described below. Pharmacological manipulation of the NAD+ pathway: 6. NAD+ prodromal and NAD+ : ○ Nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) are NAD+ prodromal. They are converted to NAD+ via the salvage pathway, with NR involving nicotinamide riboside kinase (NRK1/2) and NMN involving NMN adenylyltransferase (NMNAT1/2/3). ○ Nicotinic acid (NA) can also be used, which is converted to NAD+ via the Preiss-Handler pathway, but it causes flushing due to prostaglandin-mediated vasodilation. ○ Nicotinamide dinucleotide (NAD+) itself can also be used, as it has been shown to cross the lipid bilayer and enter the mitochondria. 7. Longevity protein modulators : ○ Versatrol is a longevity protein activating compound (STAC) that can enhance SIRT1 activity, which in turn can deacetylate and activate peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis. ○ SRT1720 is another STAC that has been shown to selectively activate SIRT1, thereby enhancing mitochondrial function and potentially improving TIL survival and function. 8. PARP inhibitors : ○ Compounds such as Olaparib and Niraparib inhibit the PARP enzyme, thereby preserving NAD+ for other metabolic processes that are critical for T cell function and potentially reducing the metabolic competition between PARP and SIRT1 for NAD + . 9. CD38 inhibitors : ○ Isatuximab and Daratumumab are monoclonal antibodies that target CD38 , which is responsible for NAD+ degradation. Small molecule inhibitors of CD38 are also in development and can be repurposed to increase NAD+ levels in TILs. 10. Mitochondrial metabolism regulators : ○ Metformin has been shown to activate AMP-activated protein kinase (AMPK), which in turn activates PGC-1α, improving mitochondrial biogenesis and potentially restoring TIL function. ○ Pioglitazone is a PPARγ agonist that may also enhance mitochondrial biogenesis and function in TILs through PGC-1α induction. ○ Bezafibrate : A pan-PPAR agonist that induces PGC-1α, enhancing mitochondrial function and fatty acid oxidation Mitochondrial biogenesis and kinetics : 3. PGC-1α activation : ○ Specific activators of PGC-1α or direct overexpression via gene delivery systems can be used to stimulate mitochondrial biogenesis. 4. Mitochondrial dynamics : ○ Manipulate genes involved in mitochondrial fusion (e.g., MFN1/2 , OPA1 ) and fission (e.g., DRP1 , FIS1 ) to maintain the integrity and function of the mitochondrial network. Genetic tools: 9. Overexpression of NAD+ synthesis genes: ○ NAMPT : Overexpression can increase salvage pathway flux and increase NAD+ levels. ○ NMNAT1/2/ 3: Increase NMN adenylyltransferase expression, catalyzing the conversion of NMN to NAD+. 10. Longevity protein gene manipulation: ○ SIRT1-7 : Overexpression of longevity protein genes, focusing on the role of SIRT1 in mitochondrial biogenesis, SIRT3 for mitochondrial protein deacetylation, and SIRT6 for maintaining genome stability. 11. Upregulation of TCA cycle and OXPHOS genes: ○ PC ( pyruvate carboxylase ) : Enhances the replenishment reaction to replenish TCA cycle intermediates. ○ PDK ( pyruvate dehydrogenase kinase ) inhibitor: PDK gene silencing increases PDH activity and promotes glucose oxidation. 12. Downregulation of NAD+ consuming enzymes: ○ Use CRISPR/Cas9 or siRNA methods to weaken CD38 or PARP family members to preserve NAD+ and thus enhance TIL function. 13. Promote mitochondrial DNA stability and biogenesis: ○ TFAM ( mitochondrial transcription factor A) : Overexpression can promote mitochondrial DNA replication and transcription, thereby enhancing mitochondrial function. ○ OGG1 or MUTY H: Overexpression enhances base excision repair and maintains mitochondrial DNA integrity. 14. Redox balance: ○ GCLC/GCLM ( Glutamine - cysteine ligase catalytic and modifying subunit ) : Overexpression to increase glutathione synthesis, glutathione is a key antioxidant in mitochondria. 15. Metabolic reprogramming: ○ CPT1A ( Carnitine palmityl transferase 1A) : Genetic modification can promote fatty acid oxidation and maintain TIL function under conditions of nutrient deficiency. 16. Mitochondrial autophagy and autophagy regulators: ○ PRKN (Parkin) and PINK1 : Enhance mitochondrial autophagy to remove dysfunctional mitochondria. ○ ATG5 or ATG7 : Regulate autophagy-related genes to balance energy and organelle quality control in TILs.

In some embodiments, these strategies can be optimized for dosage, timing, and delivery mechanisms to ensure that TILs are metabolically enhanced without inducing deleterious effects, such as apoptosis or aging. In embodiments, such strategies are also evaluated to avoid promoting tumor cell survival or immune escape. In some embodiments, the combination of these strategies provides effective and personalized enhancement of TIL function for donor-acceptor cell therapy based on specific metabolic defects observed in TILs isolated from individual tumors. Methods / Compounds Mechanism of action Target Process / Path Potential applications in TIL Considerations / Challenges Nicotinamide riboside (NR) NAD+ precursor; converted to NMN and then to NAD+ NAD+ Salvage Pathway Enhance TIL metabolism and function Dose optimization to avoid side effects Nicotinamide Mononucleotide (NMN) Direct precursor of NAD+; converted to NAD+ by NMNAT enzyme NAD+ Salvage Pathway Enhance the anti-tumor activity of TIL Stability and Cell Uptake Considerations Niacin (Vitamin B3) Converted to NAD+ via the Price-Handler pathway NAD+ biosynthetic pathway Supports TIL energy production and DNA repair Flushing reaction occurs at high doses NAMPT inhibitors Increase NAD+ levels by inhibiting NAD+ degradation NAD+ Salvage Pathway May prolong TIL lifespan Risks of Whole-Body NAD+ Depletion PARP inhibitors Prevents NAD+ depletion by inhibiting the PARP enzyme DNA repair pathways Preserve the metabolic function of NAD+ in TIL The balance between PARP inhibition and cancer therapy Longevity Protein Activator Improve NAD+ utilization efficiency Longevity protein-mediated deacetylation Enhance TIL mitochondrial function The long-term effects on TIL are not yet fully understood CD38 inhibitors Reduce NAD+ degradation ADP-ribosyl cyclase/cyclo-ADP-ribose hydrolase pathway Increase the bioavailability of NAD+ in TIL May affect calcium signaling Ketogenic diet May increase NAD+ levels by increasing fatty acid oxidation Metabolism converted into ketone bodies Can indirectly support TIL in vivo Feasibility in patients and its effects on immune cells Calorie restriction Can upregulate NAD+ biosynthetic pathway General metabolic downregulation Potential to improve TIL efficacy Patient compliance and nutritional issues Exercise Upregulation of NAD+ biosynthesis and salvage pathways Enhance muscle contraction and energy requirements Indirect systemic effects on immune function Direct impact on TIL is unclear NAD+ Infusion Directly replenish NAD+ Bypassing the NAD+ biosynthetic pathway Immediately replenish NAD+ in TIL Imbalances and risks of destabilization Gene therapy Overexpression of NAD+ biosynthetic enzymes Targeted increase in NAD+ production TIL NAD+ levels continue to increase Carrier design, delivery and integration risks

The evolutionarily conserved Wnt/β-catenin pathway is crucially involved in the multipotency and differentiation of embryonic stem cells by maintaining DNA methylation patterns, thereby promoting epigenetic stability necessary for correct cell fate decisions in a highly mitotic state (Clevers, H. and R. Nusse, Cell , 2012. 149(6): pp. 1192-205). In humans, rapid recall of memory T cells after re-exposure to antigen suggests the need for a stable epigenetic program. Most TIL-based ACT regimens for solid tumors are generated from terminally differentiated effector memory T cells that are generated by repeated rounds of TCR ligation in vitro (Hinrichs, CS and SA Rosenberg, Immunol Rev , 2014. 257(1): p56-71). However, the critical involvement of DNA methylation in shaping the expansion, memory state, and therefore function of antigen-specific TILs that undergo TCR ligation-dependent ex vivo expansion is unclear (Abdelsamed, HA et al., J Exp Med , 2017. 214(6): p1593-1606).

There is evidence that pharmacological activation of Wnt/β-catenin signaling reprograms tumor-specific CD8+ TILs in tumor-bearing mice by increasing the expression of Tcf1 (encoded by Tcf7) (Gattinoni, L. et al., Nat Med , 2009. 15(7): p. 808-13). In human melanoma, CD8+TCF7+ reveals a TIL subset with a memory progenitor-like cell state, which has bystander cytotoxic function and is associated with positive results of immune checkpoint blockade (Li, H. et al., Cell , 2019. 176(4): pp. 775-789 e18; Sade-Feldman, M. et al., Cell , 2018. 175(4): pp. 998-1013 e20). Wnt pathway components have been reported to be downregulated in PD1+CD8+ memory TILs in vivo and further downregulated in all T cell populations after ex vivo expansion (Lipp, JJ et al., Oncoimmunology , 2022. 11(1): p. 2019466), and compensating for the loss of Wnt signaling by pharmacological inhibition of GSK3β improved the effector function of the ex vivo expanded populations. Since Wnt signaling is known to prevent the development of effector CD8+ T cells, it may also be involved in epigenetically reprogramming TILs. GSK-3 is a positive regulator of PD-1 expression in CD8+ T cells, and inhibition of GSK-3 can enhance T cell function and effectively control tumor growth (Taylor, A. et al., Immunity , 2016. 44(2): pp. 274-86; Steele, L. et al., iScience , 2021. 24(6): p. 102555). In addition to being a central regulator of PD-1, GSK-3 also negatively regulates lymphocyte activation gene 3 (LAG-3) expression on CD4+ and CD8+ T cells, and small molecule inhibitors of GSK-3 are more effective than LAG-3 blockade alone in inhibiting tumor growth in a melanoma mouse model (Rudd, CE et al., Cell Rep , 2020. 30(7): p. 2075-2082e4). Recently, it has been shown that Wnt activation can promote the multifunctionality of human memory T cells via the epigenetic regulator PRMT1 (Sung, BY et al., J Clin Invest , 2022. 132 (2)).

Therefore, in some embodiments, the second cell culture medium comprises a GSK-3α/β inhibitor. In some embodiments, the GSK-3α/β inhibitor is selected from the group consisting of: SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO and 3F8. E. Harvesting TILs

After the second expansion step, the cells can be harvested. TILs can be harvested in any appropriate and sterile manner, including, for example, centrifugation. Methods for harvesting TILs are well known in the art and any such known methods can be used with the process of the present invention. In some embodiments, TILs are harvested using an automated system.

Cell harvesters and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. In some embodiments, the methods of the present invention may employ any cell-based harvester. In some embodiments, the cell harvester and/or cell processing system is a membrane-based cell harvester. In some embodiments, cell harvesting is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a solution containing cells through a membrane or filter (such as a rotating membrane or rotating filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell harvester and/or cell processing system can perform cell separation, washing, fluid exchange, concentration and/or other cell processing steps in a closed sterile system.

In some embodiments, harvesting is performed in a closed system bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, the closed system is entered through a syringe under sterile conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the examples is used. F. Final preparation and transfer to an infusion container

After the steps as described above and in detail herein are completed, the TIL is transferred to a container for administration to a patient, such as an infusion bag or a sterile vial. In some embodiments, once a sufficient number of TILs for treatment is obtained using the expansion method described above, it is transferred to a container, such as an infusion bag, for administration to a patient. In some embodiments, the TIL is cryopreserved in an infusion bag. In some embodiments, the TIL is cryopreserved before being placed in an infusion bag. In some embodiments, the TIL is cryopreserved and is not placed in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is generally accomplished by placing the TIL population in a cryogenic solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in the solution are placed in a cryogenic vial and stored at -80°C for 24 hours, with transfer to a gaseous nitrogen freezer for cryopreservation as appropriate. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, cells are removed from the freezer and thawed in a 37°C water bath until approximately 4/5 of the solution is thawed. Cells are generally resuspended in complete medium and washed one or more times as appropriate. In some embodiments, thawed TILs may be counted and survival assessed as known in the art.

In some embodiments, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLife Solutions). In some embodiments, the TIL population is cryopreserved using a cryopreservation medium containing dimethyl sulfoxide (DMSO). In some embodiments, the TIL population is cryopreserved using a 1:1 (vol:vol) ratio of CS10 to cell culture medium. In some embodiments, the TIL population is cryopreserved using an approximately 1:1 (vol:vol) ratio of CS10 to cell culture medium (further comprising additional IL-2).

In some embodiments, TILs are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded by the methods described in this disclosure can be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration. G. Closed Systems for TIL Manufacturing

The present invention provides for the use of a closed system during the TIL culture process. Such a closed system allows for the prevention and/or reduction of microbial contamination, allows for the use of fewer flasks, and allows for cost reduction. In some embodiments, the closed system uses two containers.

Such closed systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

The aseptic connecting device (STCD) creates an aseptic weld between two compatible tubes. This procedure allows aseptic connection of multiple containers and tube diameters. In some embodiments, the closed system includes a luer lock and heat seal system as described in the examples. In some embodiments, the closed system is entered through a syringe under aseptic conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the examples is used. In some embodiments, the TIL is dispensed into the final product dispensing container according to the methods described in the examples herein.

In some embodiments, the closed system uses one container from the time the tumor fragment is obtained until the TIL is ready to be administered to the patient or cryopreserved. In some embodiments, when two containers are used, the first container is a closed G container (such as a G-rex 100M series or G-rex 500M series flask), and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HypoThermosol. The characteristic of a closed system or closed TIL cell culture system is that once the tumor sample and/or tumor fragment has been added, the system is tightly sealed from the outside to form a closed environment that is not invaded by bacteria, fungi and/or any other microbial contamination.

In some embodiments, the microbial contamination is reduced by between about 5% and about 100%. In some embodiments, the microbial contamination is reduced by between about 5% and about 95%. In some embodiments, the microbial contamination is reduced by between about 5% and about 90%. In some embodiments, the microbial contamination is reduced by between about 10% and about 90%. In some embodiments, the microbial contamination is reduced by between about 15% and about 85%. In some embodiments, the microbial contamination is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or about 100%.

The closed system allows TILs to grow in the absence of microbial contamination and/or with significantly reduced microbial contamination.

In addition, the pH value, carbon dioxide partial pressure, and oxygen partial pressure of the TIL cell culture environment each change with cell culture. Therefore, even if the culture medium suitable for cell culture is circulated, the closed environment still needs to be constantly maintained as the best environment for TIL proliferation. To this end, it is necessary to use sensors to monitor the physical factors of the pH value, carbon dioxide partial pressure, and oxygen partial pressure in the culture solution of the closed environment. The signal is used to control the gas exchanger installed at the entrance of the culture environment, and to adjust the gas partial pressure of the closed environment in real time according to the changes in the culture solution in order to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system that incorporates a gas exchanger equipped with a monitoring device for measuring the pH value, carbon dioxide partial pressure, and oxygen partial pressure of the closed environment at the entrance to the closed environment, and optimizes the cell culture environment by automatically adjusting the gas concentration based on the signal from the monitoring device.

In some embodiments, the pressure in the closed environment is controlled continuously or intermittently. That is, the pressure in the closed environment can be changed by means of, for example, a pressure maintenance device, thereby ensuring that the space is suitable for TIL growth under a positive pressure state or promoting exudate and thus cell proliferation under a negative pressure state. In addition, by intermittently applying negative pressure, it is possible to uniformly and effectively replace the circulating liquid in the closed environment by temporarily reducing the volume of the closed environment.

In some embodiments, additional equipment, such as an electroporator (e.g., a Neon electroporator) is a component of a fully closed system. In some embodiments, optimal culture components for TIL proliferation can be replaced or added, and factors including, for example, IL-2 and/or OKT3 and combinations can be added.

In other embodiments, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified, wherein each container referenced in the method is GREX-10. In other embodiments, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified, wherein each container referenced in the method is GREX-100M. In other embodiments, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified, wherein each container referenced in the method is GREX-500M. VIII. Gen 2 TIL Manufacturing Method

An exemplary family of TIL processes called Gen 2 (also referred to as Process 2A) containing some of these features is depicted in Figures 82 and 83. An embodiment of Gen 2 is shown in Figure 83.

As discussed herein, the invention may include steps associated with restimulating cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplantation into a patient, and methods of testing such metabolic health. As outlined herein, TILs are generally taken from a patient sample and manipulated to expand their number prior to transplantation into a patient. In some embodiments, TILs may be genetically manipulated as discussed below, as appropriate.

In some embodiments, TILs can be stored frozen. After thawing, they can also be re-stimulated to increase their metabolism before infusion into the patient.

In some embodiments, the first expansion (including the process called pre-REP and shown as step A in FIG. 82) is shortened to 3 to 14 days and the second expansion (including the process called REP and shown as step B in FIG. 82) is shortened to 7 to 14 days, as discussed in detail below and in the Examples and Figures. In some embodiments, the first expansion (e.g., the expansion described as step B in FIG. 82) is shortened to 11 days and the second expansion (e.g., the expansion described in step D in FIG. 82) is shortened to 11 days. In some embodiments, the combination of the first expansion and the second expansion (e.g., the expansion described in steps B and D in FIG. 82 ) is shortened to 22 days, as discussed in detail below and in the examples and figures.

The "steps" labeled A, B, C, etc. below refer to FIG. 82 and certain embodiments described herein. The order of the steps below and in FIG. 82 is exemplary, and the present application and the methods disclosed herein encompass any combination or order of steps, as well as additional steps, step repetitions, and/or step omissions. A. Step A : Obtain a patient tumor sample

Generally, TILs are initially obtained from patient tumor samples and subsequently expanded into larger populations for further manipulation as described herein, cryopreservation as appropriate, restimulation as outlined herein, and assessment of phenotypic and metabolic parameters as indicators of TIL health as appropriate.

Patient tumor samples can be obtained using methods known in the art, usually obtained by surgical resection, biopsy, core needle biopsy, small biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells. In some embodiments, multi-lesion sampling is used. In some embodiments, surgical resection, biopsy, core needle biopsy, small biopsy or other means for obtaining a sample containing a mixture of tumor and TIL cells include multi-lesion sampling (that is, one or more tumor sites and/or positions in the patient and one or more tumors in the same position or closely adjacent to obtain samples). In general, tumor samples can come from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a blood malignancy. A solid tumor may be lung tissue. In some embodiments, the applicable TIL is obtained from endometrial cancer. A solid tumor may be skin tissue. In some embodiments, the applicable TIL is obtained from melanoma.

Once obtained, tumor samples are typically fragmented using a sharp tool into small pieces between 1 and about 8 mm ³ , with about 2-3 mm ³ being particularly useful. In some embodiments, TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzymatic medium (e.g., Roswell Park Cancer Institute (RPMI) 1640 buffer, 2 mM glutamine, 10 mcg/mL tamiprocin, 30 units/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in an enzymatic medium and mechanically dissociating the tumor for about 1 minute, followed by incubation at 37°C in 5% _CO2 for 30 minutes, followed by repeating the mechanical dissociation and incubation cycle under the aforementioned conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched chain hydrophilic polysaccharides can be performed to remove these cells. Alternative methods known in the art may be used, such as the method described in U.S. Patent Application Publication No. 2012/0244133 A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the methods of expanding TILs or methods of treating cancer described herein.

The tumor lytic enzyme cocktail may include one or more lytic (digestive) enzymes such as, but not limited to, collagenase (including any blend or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other lytic or proteolytic enzyme, and any combination thereof.

In some embodiments, the lyophilized enzyme is reconstituted from a lyophilized enzyme. In some embodiments, the lyophilized enzyme is reconstituted in a certain amount of sterile buffer (such as HBSS).

In some cases, collagenase (such as animal-free type 1 collagenase) is reconstituted in 10 mL sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 289.2 PZ U per vial. In some embodiments, collagenase is reconstituted in 5 mL to 15 mL buffer. In some embodiments, after reconstitution, the collagenase stock solution ranges from about 100 PZ U/mL to about 400 PZ U/mL, such as about 100 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL to about 350 PZ U/mL, about 100 PZ U/mL to about 300 PZ U/mL, about 150 PZ U/mL to about 400 PZ U/mL, about 100 PZ U/mL, about 150 PZ U/mL, about 200 PZ U/mL, about 210 PZ U/mL, about 220 PZ U/mL, about 230 PZ U/mL, about 240 PZ U/mL, about 250 PZ U/mL, about 260 PZ U/mL, about 270 PZ U/mL, about 280 PZ U/mL, about 299.2 PZ U/mL, about 300 PZ U/mL, about 310 PZ U/mL, about 320 PZ U/mL, about 330 PZ U/mL, about 340 PZ U/mL, about 350 PZ U/mL, about 360 PZ U/mL, about 370 PZ U/mL, about 380 PZ U/mL, about 390 PZ U/mL, about 400 PZ U/mL, about 410 PZ U/mL, about 420 PZ U/mL, about 430 PZ U/mL, about 440 PZ U/mL U/mL, about 300 PZ U/mL, about 350 PZ U/mL, or about 400 PZ U/mL.

In some embodiments, the neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme can be 175 DMC U per vial. In some embodiments, after reconstitution, the neutral protease stock solution ranges from about 100 DMC/mL to about 400 DMC/mL, such as about 100 DMC/mL to about 400 DMC/mL, about 100 DMC/mL to about 350 DMC/mL, about 100 DMC/mL to about 300 DMC/mL, about 150 DMC/mL to about 400 DMC/mL, about 100 DMC/mL, about 110 DMC/mL, about 120 DMC/mL, about 130 DMC/mL, about 140 DMC/mL, about 150 DMC/mL, about 160 DMC/mL, about 170 DMC/mL, about 175 DMC/mL, about 180 DMC/mL, about 190 DMC/mL, about 200 DMC/mL, about 250 DMC/mL, about 300 DMC/mL, about 350 DMC/mL DMC/m or about 400 DMC/mL.

In some embodiments, DNase I is reconstituted in 1 mL of sterile HBSS or another buffer. The concentration of the lyophilized stock enzyme is 4 KU per vial. In some embodiments, after reconstitution, the DNase I stock solution ranges from about 1 KU/mL to 10 KU/mL, such as about 1 KU/mL, about 2 KU/mL, about 3 KU/mL, about 4 KU/mL, about 5 KU/mL, about 6 KU/mL, about 7 KU/mL, about 8 KU/mL, about 9 KU/mL, or about 10 KU/mL.

In some embodiments, the stock solution of the enzyme is variable and the concentration may need to be determined. In some embodiments, the concentration of the lyophilized stock solution can be tested. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined stock solution concentration.

In some embodiments, the enzyme mixture includes about 10.2 μL of neutral protease (0.36 DMC U/mL), 21.3 μL of collagenase (1.2 PZ/mL), and 250 μL of DNase I (200 U/mL) in about 4.7 mL of sterile HBSS.

As noted above, in some embodiments, TILs are derived from solid tumors. In some embodiments, solid tumors are not fragmented. In some embodiments, solid tumors are not fragmented and are enzymatically digested with the whole tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme and hyaluronidase for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme and hyaluronidase at 37°C, 5% _CO2 for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNA enzyme and hyaluronidase at 37°C, 5% _CO2 , with rotation for 1 to 2 hours. In some embodiments, the tumor is digested overnight under constant rotation. In some embodiments, the tumor is digested overnight at 37°C, 5% _CO2 , under constant rotation. In some embodiments, the whole tumor is combined with an enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and neutral protease. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and neutral protease for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and neutral protease at 37°C, 5% CO ₂ for 1 to 2 hours. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, DNase, and neutral protease at 37°C, 5% CO ₂ , with rotation for 1 to 2 hours. In some embodiments, the tumor is digested overnight with constant rotation. In some embodiments, the tumor is digested overnight at 37°C, 5% CO ₂ , with constant rotation. In some embodiments, a whole tumor is combined with an enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock solution of collagenase is 100 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises DNase. In some embodiments, the working stock solution of DNase is 10,000 IU/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock solution of hyaluronidase is 10 mg/mL 10X working stock solution.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 1000 IU/mL DNase, and 1 mg/mL hyaluronidase.

In some embodiments, the enzyme mixture comprises 10 mg/mL collagenase, 500 IU/mL DNase, and 1 mg/mL hyaluronidase.

Generally speaking, the harvested cell suspension is called the "primary cell population" or the "freshly harvested" cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, segmentation and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is segmentation. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained by digestion or fragmentation of tumor samples obtained from patients.

In some embodiments, when the tumor is a solid tumor, after obtaining a tumor sample, such as in step A (as provided in FIG. 82 ), the tumor is physically fragmented. In some embodiments, the fragmentation occurs before cryopreservation. In some embodiments, the fragmentation occurs after cryopreservation. In some embodiments, the fragmentation occurs after the tumor is obtained and without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for a first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for a first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm ³ . In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, with a total volume of about 1300 mm ³ to about 1500 mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total volume of about 1350 mm ³ . In some embodiments, the plurality of fragments comprises about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, TILs are obtained from tumor fragments. In some embodiments, tumor fragments are obtained by sharpening. In some embodiments, tumor fragments are between about 1 mm ³ and 10 mm ^3. In some embodiments, tumor fragments are between about 1 mm ³ and 8 mm ^3. In some embodiments, tumor fragments are about 1 mm ^3. In some embodiments, tumor fragments are about 2 mm ^3. In some embodiments, tumor fragments are about 3 mm ^3. In some embodiments, tumor fragments are about 4 mm ^3. In some embodiments, tumor fragments are about 5 mm ^3. In some embodiments, tumor fragments are about 6 mm ^3. In some embodiments, tumor fragments are about 7 mm ³ . In some embodiments, a tumor fragment is about 8 mm ³ . In some embodiments, a tumor fragment is about 9 mm ³ . In some embodiments, a tumor fragment is about 10 mm ³ . In some embodiments, a tumor is 1 to 4 mm x 1 to 4 mm x 1 to 4 mm. In some embodiments, a tumor is 1 mm x 1 mm x 1 mm. In some embodiments, a tumor is 2 mm x 2 mm x 2 mm. In some embodiments, a tumor is 3 mm x 3 mm x 3 mm. In some embodiments, a tumor is 4 mm x 4 mm x 4 mm.

In some embodiments, tumors are resected to minimize the amount of hemorrhagic, necrotic, and/or fatty tissue on each slice. In some embodiments, tumors are resected to minimize the amount of hemorrhagic tissue on each slice. In some embodiments, tumors are resected to minimize the amount of necrotic tissue on each slice. In some embodiments, tumors are resected to minimize the amount of fatty tissue on each slice.

In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without using a scalpel to perform a sawing action. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are produced by cultivating in an enzymatic medium (such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg/mL genitamicin, 30 U/mL DNA enzyme, and 1.0 mg/mL collagenase), followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After the tumor is placed in an enzymatic medium, the tumor can be mechanically dissociated for about 1 minute. The solution can then be incubated at 37°C in 5% _CO2 for 30 minutes and then mechanically disrupted again for about 1 minute. After another 30 minutes of incubation at 37°C in 5% _CO2 , the tumor can be mechanically disrupted a third time for about 1 minute. In some embodiments, if large pieces of tissue still exist after the third mechanical disruption, 1 or 2 additional mechanical dissociations are applied to the sample, regardless of whether or not the sample is incubated for another 30 minutes at 37°C in 5% _CO2 . In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

In some embodiments, the cell suspension harvested before the first expansion step is referred to as the "primary cell population" or "freshly harvested" cell population.

In some embodiments, cells may be frozen after sample harvest, as appropriate, and stored frozen prior to expansion as described in step B, which is described in further detail below and illustrated in FIG82. 1. Pleural effusion T cells, ascites fluid T cells, and TILs

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs expanded according to the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample derived from pleural effusion. In some embodiments, the source of T cells or TILs expanded according to the methods described herein is a sample derived from pleural effusion. See, for example, the methods described in U.S. Patent Publication US 2014/0295426, which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, the sample is an ascites fluid sample. In some embodiments, the source of the T cells or TILs expanded according to the methods described herein is an ascites fluid sample. In some embodiments, the sample is a sample derived from ascites. In any of the foregoing embodiments, the ascites fluid sample or a sample derived from ascites is obtained from an endometrial cancer patient. In some embodiments, ascites fluid from the abdomen of an endometrial cancer patient can be used to obtain TILs for expansion according to the methods described herein and use TILs and the synergistic therapy described herein, which is selected as appropriate, to treat an endometrial cancer patient.

In some embodiments, any pleural fluid or pleural exudate suspected of and/or containing TILs can be used. Such samples may be derived from primary or metastatic lung cancer, such as NSCLC or SCLC. In some embodiments, the sample may be derived from secondary metastatic cancer cells originating from another organ (e.g., breast, ovary, colon, or prostate). In some embodiments, the sample used in the expansion method described herein is a pleural exudate. In some embodiments, the sample used in the expansion method described herein is a pleural transudate. Other biological samples may include other slurries containing TILs, including, for example, ascites fluid from the abdomen or pancreatic cyst fluid. Ascites fluid and pleural fluid involve very similar chemistries; both the abdomen and lungs have mesothelial cell lines and fluid forms in the pleural and abdominal cavities in the same malignant tumor event, and in some embodiments, such fluids contain TILs. In some embodiments where the disclosed methods utilize pleural fluid, the same methods can be performed using ascites or other cystic fluids containing TILs to obtain similar results.

In some embodiments, pleural fluid or ascites fluid is directly removed from the patient in an unprocessed form. In some embodiments, before further processing steps, unprocessed pleural fluid or ascites fluid is placed in a standard blood collection tube (such as EDTA or heparin tube). In some embodiments, before further processing steps, unprocessed pleural fluid or ascites fluid is placed in a standard CellSave® tube (Veridex). In some embodiments, immediately after collecting from the patient, the sample is placed in a CellSave tube to avoid the number of live TILs from decreasing. If retained in unprocessed pleural fluid or ascites fluid, even at 4°C, the number of live TILs may be significantly reduced within 24 hours. In some embodiments, the sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours or at most 24 hours after being removed from the patient. In some embodiments, the sample is placed in an appropriate collection tube at 4°C 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient.

In some embodiments, the pleural or ascites fluid sample from the selected individual can be diluted. In some embodiments, the dilution is 1:10 pleural or ascites fluid to diluent. In other embodiments, the dilution is 1:9 pleural or ascites fluid to diluent. In other embodiments, the dilution is 1:8 pleural or ascites fluid to diluent. In other embodiments, the dilution is 1:5 pleural or ascites fluid to diluent. In other embodiments, the dilution is 1:2 pleural or ascites fluid to diluent. In other embodiments, the dilution is 1:1 pleural or ascites fluid to diluent. In some embodiments, the diluent comprises saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection and dilution from the patient to avoid reduction of viable TILs, which may be significantly reduced within 24 to 48 hours if retained in unprocessed pleural fluid, even at 4°C. In some embodiments, pleural or ascites fluid samples are placed in appropriate collection tubes 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal and dilution from the patient. In some embodiments, the pleural or ascites fluid sample is placed in an appropriate collection tube 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after being removed from the patient and diluted at 4°C.

In other embodiments, the pleural or ascites fluid sample is concentrated by known means prior to further processing steps. In some embodiments, pretreatment of the pleural or ascites fluid is preferred where the pleural or ascites fluid must be stored frozen for transport to a laboratory performing the method or for subsequent analysis (e.g., 24 to 48 hours after collection). In some embodiments, the pleural or ascites fluid sample is prepared by centrifuging the pleural or ascites fluid sample after it is removed from the individual and resuspending the centrate or pellet in a buffer. In some embodiments, pleural or ascites fluid samples are centrifuged and resuspended multiple times and then frozen for transport or subsequent analysis and/or processing.

In some embodiments, the pleural or ascites fluid sample is concentrated prior to further processing steps by using a filtration method. In some embodiments, the pleural or ascites fluid sample used in further processing is prepared by filtering the fluid through a filter containing a known and substantially uniform pore size that allows the pleural or ascites fluid to pass through the membrane but retains tumor cells. In some embodiments, the pore diameter in the membrane may be at least 4 μM. In other embodiments, the pore diameter may be 5 μM or greater, and in other embodiments, may be any of 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. After filtration, the cells (including TILs) retained by the membrane can be washed off the membrane into a suitable physiologically acceptable buffer. The cells (including TILs) concentrated in this way can then be used in further processing steps of the method.

In some embodiments, a pleural or ascites fluid sample (including, for example, untreated pleural or ascites fluid), diluted pleural or ascites fluid, or a resuspended cell pellet is contacted with a lysis reagent that differentially lyses the enucleated red blood cells present in the sample. In some embodiments, where the pleural or ascites fluid contains a large number of RBCs, this step is performed prior to further processing steps. Suitable lysis reagents include a single lysis reagent or a lysis reagent and a quenching reagent, or a lysis reagent, a quenching reagent, and a fixing reagent. Suitable lysis systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other dissolution systems include Versalyse™ system, FACSlyse™ system (Becton Dickenson), Immunoprep™ system or Erythrolyse II system (Beckman Coulter) or ammonium chloride system. In some embodiments, the dissolution reagent can vary with the main needs, which are the effective dissolution of red blood cells and the conservativeness of TIL and the phenotypic characteristics of TIL in pleural fluid or ascites fluid. In addition to using a single reagent for dissolution, the dissolution system suitable for the method described herein may include a second reagent, such as a second reagent that quenches or delays the effect of the dissolution reagent during the remaining steps of the method, such as Stabillyse™ reagent (Beckman Coulter). Depending on the selection of the dissolution reagent or the preferred implementation of the method, a known fixed reagent can also be used.

In some embodiments, the pleural fluid or ascites fluid sample, which has not been processed, diluted, or centrifuged or processed multiple times as described above, is stored frozen at a temperature of about -140°C and subsequently further processed and/or expanded as provided herein. B. Step B : First expansion

In some embodiments, the methods of the present invention provide for obtaining young TILs that are capable of increasing the replication cycle after administration to an individual/patient and thus may provide additional therapeutic benefits compared to older TILs (i.e., TILs that have undergone further rounds of replication prior to administration to an individual/patient). Characteristics of young TILs have been described in the literature, for example in Donia et al., Scand. J. Immunol. 2012, 75, 157-167; Dudley et al., Clin. Cancer Res. 2010, 16, 6122-6131; Huang et al., J. Immunother. 2005, 28, 258-267; Besser et al., Clin. Cancer Res. 2013, 19, OF1-OF9; Besser et al., J. Immunother. 2009, 32:415-423; Robbins et al., J. Immunol. 2004, 173, 7125-7130; Shen et al., J. Immunother., 2007, 30, 123-129; Zhou et al., J. Immunother. 2005, 28, 53-62; and Tran et al., J. Immunother., 2008, 31, 742-751, each of which is incorporated herein by reference.

The diverse antigen receptors of T lymphocytes and B lymphocytes are produced by somatic cell recombination of a limited but large number of gene segments. These gene segments: V (variable region), D (diversity region), J (joining region) and C (constant region) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCR). The present invention provides a method for producing TILs that exhibit and increase the diversity of T cell repertoires. In some embodiments, TILs obtained by the methods of the present invention exhibit increased T cell repertoire diversity. In some embodiments, compared to freshly harvested TIL and/or using other methods other than the methods provided herein, including, for example, TIL prepared by methods other than the methods implemented in Figure 82, TIL obtained by the method of the present invention exhibits increased T cell reservoir diversity. In some embodiments, compared to freshly harvested TIL and/or using TIL prepared by the method called process 1C as illustrated in Figure 5 and/or Figure 6, TIL obtained by the method of the present invention exhibits increased T cell reservoir diversity. In some embodiments, TIL obtained in the first expansion exhibits increased T cell reservoir diversity. In some embodiments, increasing diversity is to increase immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, in immunoglobulin heavy chains. In some embodiments, the diversity is present in immunoglobulins, in immunoglobulin light chains. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β increases. In some embodiments, the expression of T cell receptor (TCR) α increases. In some embodiments, the expression of T cell receptor (TCR) β increases. In some embodiments, the expression of TCRab (i.e., TCR α/β) increases.

After the segmentation or digestion of the tumor fragments described in, for example, step A of Figure 82, the resulting cells are cultured in serum containing IL-2 under conditions that are favorable for TIL growth but unfavorable for tumor and other cell growth. In some embodiments, the tumor digest is cultivated in a 2 mL well in a medium containing inactivated human AB serum with 6000 IU/mL IL-2. This primary cell population is cultured for a period of several days, typically 3 to 14 days, to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells. In some embodiments, the primary cell population is cultured for a period of 10 to 14 days to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells. In some embodiments, the primary cell population is cultured for a period of about 11 days to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells.

In some embodiments, expansion of TILs may be performed using an initial TIL expansion step as described below and herein (e.g., the step described in step B of FIG. 82 , which may include a process called pre-REP), followed by a second expansion as described below in step D and herein (step D, including a process called a rapid expansion protocol (REP) step), followed by optional cryopreservation, and then a second step D as described below and herein (including a process called a restimulation REP step). TILs obtained from this process may be characterized for phenotypic characteristics and metabolic parameters as described herein, as appropriate.

In some embodiments where TIL cultures are initiated in a 24-well plate, for example, using a Costar 24-well cell culture cluster flat bottom (Corning, Corning, NY), each well can be seeded with 1×10 ⁶ tumor digest cells or one tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 IU/mL; Chiron, Emeryville, CA). In some embodiments, the tumor fragment is between about 1 mm ³ and 10 mm ³ .

In some embodiments, the first expansion medium is referred to as "CM" (abbreviation for medium). In some embodiments, the CM of step B consists of RPMI 1640 supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL GlutaMAX. In some embodiments in which the culture is initiated in a gas-permeable bottle with a 40 mL capacity and a 10 ^cm2 gas-permeable silicon bottom (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each bottle is loaded with 10-40 mL of CM with IL-2 containing 10-40× ¹⁰⁶ live tumor digest cells or 5-30 tumor fragments. Both G-REX10 and 24-well plates were cultured in a humidified incubator at 37°C, 5% _CO2 , and 5 days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2, and after the 5th day, half of the medium was replaced every 2-3 days.

In some embodiments, the medium used in the expansion process disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and/or reduce experimental variations due to batch-to-batch variations of serum-containing media.

In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include (but are not limited to) one or more of the following: CTS™ OpTmizer T Cell Expander Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin replacements, one or more amino acids, one or more vitamins, one or more transferrins or transferrin replacements, one or more antioxidants, one or more insulins or insulin replacements, one or more collagen prodrivers, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and a compound containing trace elements Ag+, Al3+, Ba2+, Cd2+, _CO2 +, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+, and Zr4+. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate and/or 2-hydroxyethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with a learned growth medium, which includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% based on the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-hydroxyethanol in the culture medium is 55 μM.

In some embodiments, the defined medium described in International PCT Publication No. WO/1998/030679, which is incorporated herein by reference, can be used in the present invention. In the publication, a serum-free eukaryotic cell culture medium is described. The serum-free eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen prodrivers, one or more trace elements and one or more antibiotics. In some embodiments, the medium further comprises L-glutamine, sodium bicarbonate and/or β-hydroxyethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen pro-drivers and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and a compound containing trace elements Ag+, Al3+, Ba2+, Cd2+, _CO2 +, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+, and Zr4+. In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), Eagle's basal medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

In some embodiments, the concentration of glycine in the culture medium is determined to be in the range of about 5-200 mg/L, the concentration of L-histidine is about 5-250 mg/L, the concentration of L-isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-200 mg/L, the concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-proline is about 1-1000 mg/L, the concentration of L-hydroxyproline is about 1-45 mg/L, the concentration of L-serine is about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, and the concentration of L-tryptophan is about 2-110 mg/L, L-tyrosine at a concentration of about 3-175 mg/L, L-valine at a concentration of about 5-500 mg/L, thiamine at a concentration of about 1-20 mg/L, reduced glutathione at a concentration of about 1-200 mg/L, L-ascorbic acid-2-phosphate at a concentration of about 1-200 mg/L, iron-saturated transferrin at a concentration of about 1-50 mg/L, insulin at a concentration of about 1-100 mg/L, sodium selenite at a concentration of about 0.000001-0.0001 mg/L, and albumin (e.g., AlbuMAX® I) at a concentration of about 5000 to 50,000 mg/L.

In some embodiments, the non-trace element portion of the medium is determined to be present in the concentration range listed in the column titled "Concentration Range in 1X Medium" in Table 4 below. In other embodiments, the non-trace element portion of the medium is determined to be present in the final concentration listed in the column titled "Preferred Embodiments of 1X Medium" in Table 4. In other embodiments, the medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace element portions of the type and concentration listed in the column titled "Preferred Embodiments of Supplements" in Table 4 below.

In some embodiments, the osmotic pressure of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmotic pressure is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-hydroxyethanol (final concentration of about 100 μM).

In some embodiments, the defined medium described in Smith et al., Clin Transl Immunology, 4(1) 2015 (doi: 10.1038/cti.2014.31) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ is used as the basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. Using an unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and/or second gas permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

After preparing the tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor TIL growth relative to tumor and other cells. In some embodiments, the tumor digest is cultured in a 2 mL well in a medium comprising inactivated human AB serum (or in some cases, as outlined herein, in the presence of APC cell populations) and 6000 IU/mL IL-2. This primary cell population is cultured for a period of several days, typically 10 to 14 days, to produce a primary TIL population of typically about 1×10 ⁸ primary TIL cells. In some embodiments, the growth medium during the first expansion period comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20-30×10 ⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 20×10 ⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 25×10 ⁶ IU/mg. In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of 30×10 ⁶ IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6×10 ⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 5. In some embodiments, the first expansion medium comprises about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 9,000 IU/mL IL-2 to about 5,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 8,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 7,000 IU/mL IL-2 to about 6,000 IU/mL IL-2. In some embodiments, the first expansion medium comprises about 6,000 IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.

In some embodiments, the first expansion medium comprises about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the first expansion medium comprises about 200 IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15.

In some embodiments, the first expansion medium comprises about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the first expansion medium comprises about 2 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21.

In some embodiments, the cell culture medium comprises an anti-CD3 agonist antibody, such as OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 µg/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab. See, e.g., Table 1.

In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: urelulumab, utumumab, EU-101, fusion proteins and fragments thereof, derivatives, variants, biosimilars and combinations. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 µg/mL to 100 µg/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 µg/mL to 40 µg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises an initial concentration of about 3000 IU/mL of IL-2 and an initial concentration of about 30 ng/mL of OKT-3 antibody, and wherein the one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the first expansion medium is referred to as "CM" (abbreviation for medium). In some embodiments, it is referred to as CM1 (medium 1). In some embodiments, CM consists of RPMI 1640 supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL GlutaMAX. In some embodiments in which the culture is initiated in a 40 mL capacity, 10 cm ² gas-permeable silicon bottom gas-permeable bottle (e.g., G-REX10; Wilson Wolf Manufacturing, New Brighton, MN), each bottle is loaded with 10-40 mL of CM with IL-2 containing 10-40×10 ⁶ live tumor digest cells or 5-30 tumor fragments. G-REX10 and 24-well plates are cultured in a humidified incubator at 37°C, 5% _CO2 and 5 days after the start of culture, half of the medium is removed and replaced with fresh CM and IL-2, and after the 5th day, half of the medium is replaced every 2-3 days. In some embodiments, CM is CM1 described in the examples, see Example 1. In some embodiments, the first expansion is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

In some embodiments, as discussed in the examples and figures, the first expansion (including processes such as those described in step B of FIG. 2, which may include processes sometimes referred to as pre-REP) is shortened to 3-14 days. In some embodiments, the first expansion (including processes such as those described in step B of FIG. 82, which may include processes sometimes referred to as pre-REP) is shortened to 7 to 14 days, such as including, for example, the expansion described in step B of FIG. 82. In some embodiments, the first expansion of step B is shortened to 10-14 days. In some embodiments, the first expansion is shortened to 11 days, such as discussed in the expansion described in step B of FIG. 82.

In some embodiments, the first TIL expansion may be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. In some embodiments, the first TIL expansion may be performed for 1 day to 14 days. In some embodiments, the first TIL expansion may be performed for 2 days to 14 days. In some embodiments, the first TIL expansion may be performed for 3 days to 14 days. In some embodiments, the first TIL expansion may be performed for 4 days to 14 days. In some embodiments, the first TIL expansion may be performed for 5 days to 14 days. In some embodiments, the first TIL expansion may be performed for 6 days to 14 days. In some embodiments, the first TIL expansion may be performed for 7 days to 14 days. In some embodiments, the first TIL expansion may be performed for 8 to 14 days. In some embodiments, the first TIL expansion may be performed for 9 to 14 days. In some embodiments, the first TIL expansion may be performed for 10 to 14 days. In some embodiments, the first TIL expansion may be performed for 11 to 14 days. In some embodiments, the first TIL expansion may be performed for 12 to 14 days. In some embodiments, the first TIL expansion may be performed for 13 to 14 days. In some embodiments, the first TIL expansion may be performed for 14 days. In some embodiments, the first TIL expansion may be performed for 1 to 11 days. In some embodiments, the first TIL expansion may be performed for 2 to 11 days. In some embodiments, the first TIL expansion may be performed for 3 to 11 days. In some embodiments, the first TIL expansion may be performed for 4 to 11 days. In some embodiments, the first TIL expansion may be performed for 5 to 11 days. In some embodiments, the first TIL expansion may be performed for 6 to 11 days. In some embodiments, the first TIL expansion may be performed for 7 to 11 days. In some embodiments, the first TIL expansion may be performed for 8 to 11 days. In some embodiments, the first TIL expansion may be performed for 9 to 11 days. In some embodiments, the first TIL expansion may be performed for 10 to 11 days. In some embodiments, the first TIL expansion may be performed for 11 days.

In some embodiments, the combination of IL-2, IL-7, IL-15 and/or IL-21 is used as the combination during the first expansion period. In some embodiments, during the first expansion period, including, for example, according to Figure 82 and step B process described herein, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included. In some embodiments, the combination of IL-2, IL-15 and IL-21 is used as the combination during the first expansion period. In some embodiments, according to Figure 82 and step B process as described herein, IL-2, IL-15 and IL-21 and any combination thereof may be included.

In some embodiments, as discussed in the examples and figures, the first expansion (including a process called pre-REP; e.g., step B according to FIG. 82) process is shortened to 3 to 14 days. In some embodiments, the first expansion of step B is shortened to 7 to 14 days. In some embodiments, the first expansion of step B is shortened to 10 to 14 days. In some embodiments, the first expansion is shortened to 11 days. In some embodiments, the first expansion is performed in a closed system bioreactor, e.g., step B according to FIG. 82. In some embodiments, a closed system is used to perform TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor. 1. Interleukins and other additives

The expansion methods described herein typically utilize media with high doses of interleukins, particularly IL-2, as is known in the art.

Alternatively, it is also possible to use a combination of interleukins for rapid expansion and/or secondary expansion of TILs, as described in U.S. Patent Application Publication No. US 2017/0107490 A1 (the disclosure of which is incorporated herein by reference), using a combination of two or more of IL-2, IL-15, and IL-21. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2 or IL-15 and IL-21, the latter of which has a specific use in many embodiments. The use of a combination of interleukins is particularly advantageous for the generation of lymphocytes, and in particular T cells as described therein.

In some embodiments, step B may also include adding OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step B may also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step B may also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator factor I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference, may be used in the culture medium during step B. C. Step C : Transition from the first expansion to the second expansion

In some cases, the subject TIL population obtained from the first expansion, including, for example, the TIL population obtained from step B shown in, for example, FIG. 82 , can be immediately cryopreserved using the protocol discussed below. Alternatively, the TIL population obtained from the first expansion (referred to as the second TIL population) can undergo a second expansion (which may include an expansion sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, in the case where the genetically modified TILs are to be used in therapy, the first TIL population (sometimes referred to as the subject TIL population) or the second TIL population (which in some embodiments may include a population referred to as a REP TIL population) can be genetically modified for appropriate treatment prior to expansion or after the first expansion and prior to the second expansion.

In some embodiments, the TIL obtained from the first expansion (e.g., from step B as shown in Figure 82) is stored until phenotypic analysis is performed for selection. In some embodiments, the TIL obtained from the first expansion (e.g., from step B as shown in Figure 82) is not stored and directly continues to the second expansion. In some embodiments, the TIL obtained from the first expansion is not frozen after the first expansion and before the second expansion. In some embodiments, the transition from the first expansion to the second expansion occurs about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 3 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 4 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 4 days to about 10 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 7 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs about 14 days after fragmentation occurs.

In some embodiments, the transition from the first expansion to the second expansion occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 14 days after fragmentation occurs. In some embodiments, the first TIL expansion can be performed for 2 days to 14 days. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 to 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 2 days to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 to 11 days after fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days after fragmentation occurs.

In some embodiments, the TILs are not stored after the first expansion and before the second expansion, and the TILs proceed directly to the second expansion (e.g., in some embodiments, no storage is performed during the transition period from step B to step D as shown in FIG. 82 ). In some embodiments, the transition occurs in a closed system as described herein. In some embodiments, the TILs from the first expansion (the second TIL population) proceed directly to the second expansion without a transition period.

In some embodiments, the transition from the first expansion to the second expansion (e.g., according to step C of FIG. 82 ) is performed in a closed system bioreactor. In some embodiments, a closed system is used to perform TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100 bioreactor. In some embodiments, the closed system bioreactor is a single bioreactor. D. Step D : Second Expansion

In some embodiments, the number of TIL cell populations is expanded after harvest and initial bulk processing, for example, after step A and step B and a transition referred to as step C (as indicated in FIG82). This further expansion is referred to herein as the second expansion, which may include an expansion process generally referred to in the art as a rapid expansion process (REP); and as indicated in step D of FIG82. The second expansion is typically accomplished in a gas permeable container using a culture medium comprising a variety of components, including feeder cells, a source of cytokines, and an anti-CD3 antibody.

In some embodiments, the second expansion of TIL or the second TIL expansion (which may include expansion sometimes referred to as REP; and the process indicated in step D of Figure 82) can be performed using any TIL bottle or container known to those skilled in the art. In some embodiments, the second TIL expansion can be performed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second TIL expansion can be performed for about 7 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 8 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 9 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 10 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 11 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 13 days to about 14 days. In some embodiments, the second TIL expansion can be performed for about 14 days.

In some embodiments, the second expansion can be performed in a gas permeable container using the methods of the present disclosure (including, for example, expansion referred to as REP; and the process indicated in step D of FIG. 82 ). For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulators can include, for example, anti-CD3 antibodies, such as about 30 ng/mL OKT3, mouse monoclonal anti-CD3 antibodies (available from Ortho-McNeil (Raritan, NJ) or Miltenyi Biotech (Auburn, CA)), or UHCT-1 (available from BioLegend, San Diego, CA, USA). TILs can be expanded by including one or more antigens (including antigenic portions thereof, such as antigenic determinants) during the second expansion period to induce further TIL in vitro stimulation, such antigens can be expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, such as 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217 (210M), in the presence of T cell growth factors (such as 300 IU/mL IL-2 or IL-15) as appropriate. Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigens, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same cancer antigen pulsed onto antigen presenting cells expressing HLA-A2. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of a second expansion. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or 8000 IU/mL of IL-2.

In some embodiments, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 µg/mL OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: urelulumab, utumumab, EU-101, fusion proteins and fragments thereof, derivatives, variants, biosimilars and combinations. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 µg/mL to 100 µg/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 µg/mL to 40 µg/mL in the cell culture medium.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises an initial concentration of about 3000 IU/mL of IL-2 and an initial concentration of about 30 ng/mL of OKT-3 antibody, and wherein the one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15 and/or IL-21 is used as a combination during the second expansion period. In some embodiments, during the second expansion period, including, for example, according to Figure 82 and step D process described herein, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included. In some embodiments, a combination of IL-2, IL-15 and IL-21 is used as a combination during the second expansion period. In some embodiments, according to Figure 82 and step D process as described herein, IL-2, IL-15 and IL-21 and any combination thereof may be included.

In some embodiments, the second expansion can be carried out in a supplemented cell culture medium comprising IL-2, OKT-3, antigen presenting feeders, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen presenting feeders. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen presenting cells (APCs; also referred to as antigen presenting feeders). In some embodiments, the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen presenting feeder cells (ie, antigen presenting cells).

In some embodiments, the second expansion medium comprises about 500 IU/mL IL-15, about 400 IU/mL IL-15, about 300 IU/mL IL-15, about 200 IU/mL IL-15, about 180 IU/mL IL-15, about 160 IU/mL IL-15, about 140 IU/mL IL-15, about 120 IU/mL IL-15, or about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 500 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 400 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 300 IU/mL IL-15 to about 100 IU/mL IL-15. In some embodiments, the second expansion medium comprises about 200 IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15. In some embodiments, the cell culture medium further comprises IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL IL-15.

In some embodiments, the second expansion medium comprises about 20 IU/mL IL-21, about 15 IU/mL IL-21, about 12 IU/mL IL-21, about 10 IU/mL IL-21, about 5 IU/mL IL-21, about 4 IU/mL IL-21, about 3 IU/mL IL-21, about 2 IU/mL IL-21, about 1 IU/mL IL-21, or about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 20 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 15 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 12 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 10 IU/mL IL-21 to about 0.5 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 5 IU/mL IL-21 to about 1 IU/mL IL-21. In some embodiments, the second expansion medium comprises about 2 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU/mL IL-21. In some embodiments, the cell culture medium further comprises IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL IL-21.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1:100 and 1:200.

In some embodiments, REP and/or secondary expansion are performed in flasks where the primary TILs are mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. The medium is replaced (usually by aspirating fresh medium to replace 2/3 of the medium) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX bottles and gas permeable containers, as discussed more fully below.

In some embodiments, as discussed in the examples and figures, the second expansion (which may include a process called a REP process) is shortened to 7-14 days. In some embodiments, the second expansion is shortened to 11 days.

In some embodiments, REP and/or secondary expansion can be performed using T-175 bottles and gas-permeable bags as previously described (Tran et al., J. Immunother. 2008, 31, 742-51; Dudley et al., J. Immunother. 2003, 26, 332-42) or gas-permeable culture vessels (G-REX bottles). In some embodiments, secondary expansion (including expansion referred to as rapid expansion) is performed in T-175 bottles, and approximately 1×10 ⁶ TILs suspended in 150 mL of medium can be added to each T-175 bottle. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The T-175 flasks can be incubated at 37°C, 5% _CO2 . Half of the medium can be replaced on day 5 with a 50/50 medium with 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks can be combined in a 3 L bag, and 300 mL of AIM V with 5% human AB serum and 3000 IU/mL IL-2 can be added to 300 mL of TIL suspension. The number of cells in each bag is counted every day or every two days, and fresh medium is added to keep the cell count between 0.5 and 2.0× ¹⁰⁶ cells/mL.

In some embodiments, the second expansion (which may include the expansion referred to as REP and the expansion mentioned in step D of FIG. 82 ) can be carried out in a 500 mL capacity gas-permeable bottle with a 100 cm gas-permeable silicon bottom (G-REX-100, available from Wilson Wolf Manufacturing, New Brighton, MN, USA), and 5×10 ⁶ or 10×10 ⁶ TILs can be cultured with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/mL anti-CD3 (OKT3). G-REX-100 bottles can be incubated at 37° C., 5% CO ₂ . On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellet can be resuspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU/mL IL-2, and added back to the original G-REX-100 bottle. As the TILs are continuously expanded in the G-REX-100 bottles, on day 7, the TILs in each G-REX-100 can be suspended in the 300 mL of medium present in each bottle, and the cell suspension can be divided into three 100 mL aliquots that can be used to inoculate three G-REX-100 bottles. 150 mL of AIM-V with 5% human AB serum and 3000 IU/mL IL-2 can then be added to each bottle. The G-REX-100 bottles can be incubated at 37°C, 5% _CO2 and after 4 days, 150 mL of AIM-V with 3000 IU/mL IL-2 can be added to each G-REX-100 bottle. Cells can be harvested on day 14 of culture.

In some embodiments, the second expansion (including expansion referred to as REP) is performed in a flask, where the main TIL is mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of medium. In some embodiments, the medium is replaced until the cells are transferred to the alternative growth chamber. In some embodiments, 2/3 of the medium is replaced by aspirating fresh medium. In some embodiments, the alternative growth chamber includes a G-REX bottle and a gas permeable container, as discussed more fully below.

In some embodiments, a second expansion (including expansion referred to as REP) is performed, and further comprises a step in which TILs are selected for superior tumor responsiveness. Any selection method known in the art may be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058 A1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with superior tumor responsiveness.

Optionally, cell viability analysis can be performed after the second expansion (including expansions referred to as REP expansions) using standard assays known in the art. For example, a trypan blue exclusion assay can be performed on a subject TIL sample, which selectively marks dead cells and allows for viability assessment. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience (Lawrence, MA)). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

In some embodiments, the second expansion of TILs (including expansion referred to as REP) can be performed using T-175 bottles and breathable bags as previously described (Tran et al., 2008 , J Immunother. , 31 , 742-751, and Dudley et al. 2003 , J Immunother. , 26 , 332-342) or breathable G-REX bottles. In some embodiments, a flask is used for the second expansion. In some embodiments, a breathable G-REX bottle is used for the second expansion. In some embodiments, the second expansion is performed in a T-175 bottle, and about 1×10 ⁶ TILs are suspended in about 150 mL of medium and added to each T-175 bottle. TILs were cultured with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells at a ratio of 1:100 and cells were cultured in a 1:1 mixture of CM and AIM-V medium (50/50 medium) supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. T-175 flasks were incubated at 37°C, 5% _CO2 . In some embodiments, half of the medium was replaced on day 5 with 50/50 medium with 3000 IU/mL IL-2. In some embodiments, on day 7, cells from 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with 5% human AB serum and 3000 IU/mL IL-2 is added to 300 mL of TIL suspension. The number of cells in each bag can be counted every day or every two days, and fresh medium can be added to maintain the cell count between about 0.5 and about 2.0×10 ⁶ cells/mL.

In some embodiments, the second expansion (including the expansion referred to as REP) is performed in a 500 mL capacity gas permeable bottle with a 100 cm ² gas permeable silicon bottom (G-REX-100, Wilson Wolf), and approximately 5×10 ⁶ or 10×10 ⁶ TILs are cultured with irradiated allogeneic PBMCs at a ratio of 1:100 in 400 mL of 50/50 medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The G-REX-100 bottle is incubated at 37° C., 5% CO _2. In some embodiments, on day 5, 250 mL of supernatant is removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL IL-2 and added back to the original G-REX-100 bottle. In some embodiments where TILs are continuously expanded in G-REX-100 bottles, on day 7, the TILs in each G-REX-100 are suspended in 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots for inoculating 3 G-REX-100 bottles. 150 mL of AIM-V with 5% human AB serum and 3000 IU/mL IL-2 is then added to each bottle. The G-REX-100 flasks were incubated at 37°C, 5% _CO2 and after 4 days, 150 mL of AIM-V with 3000 IU/mL IL-2 was added to each G-REX-100 flask. The cells were harvested on day 14 of culture.

The diverse antigen receptors of T lymphocytes and B lymphocytes are produced by somatic cell recombination of limited but large number of gene segments. These gene segments: V (variable region), D (diversity region), J (joining region) and C (constant region) determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCR). The present invention provides a method for producing TILs that exhibit and increase T cell reservoir diversity. In some embodiments, TILs obtained by the method of the present invention exhibit increased T cell reservoir diversity. In some embodiments, TILs obtained in the second expansion exhibit increased T cell reservoir diversity. In some embodiments, increasing diversity is to increase immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity is present in immunoglobulins, in immunoglobulin heavy chains. In some embodiments, the diversity is present in immunoglobulins, in immunoglobulin light chains. In some embodiments, the diversity is present in T cell receptors. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of α, β, γ and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β increases. In some embodiments, the expression of T cell receptor (TCR) α increases. In some embodiments, the expression of T cell receptor (TCR) β increases. In some embodiments, the expression of TCRab (i.e., TCR α/β) increases.

In some embodiments, the second expansion medium (e.g., sometimes referred to as CM ² or a second cell medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the medium used in the expansion process disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and/or reduce experimental variations due to batch-to-batch variations of serum-containing media.

In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and/or a serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CTS™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow's Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iskoff's Modified Dulbecco's Medium.

In some embodiments, serum supplements or serum replacements include (but are not limited to) one or more of the following: CTS™ OpTmizer T Cell Expander Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumins or albumin replacements, one or more amino acids, one or more vitamins, one or more transferrins or transferrin replacements, one or more antioxidants, one or more insulins or insulin replacements, one or more collagen prodrivers, one or more antibiotics, and one or more trace elements. In some embodiments, the defined culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and trace element portions Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , CO ₂₊ , Cr ³⁺ , Ge ⁴⁺ , ^{Se 4+ ,} Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ^{6+ In} some embodiments, the culture medium further comprises ^L -glutamine, sodium bicarbonate and/ ^{or 2} -hydroxyethanol.

In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with a learned growth medium, which includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium, and Iskoff's modified Dulbecco's medium.

In some embodiments, the total serum replacement concentration (vol%) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% based on the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any CTS™ OpTmizer™ formulation can be used in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L CTS™ OpTmizer™ T Cell Expansion Base Medium and 26 mL CTS™ OpTmizer™ T Cell Expansion Supplement, which are mixed together before use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55 mM 2-hydroxyethanol, and 2 mM L-glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55 mM 2-hydroxyethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU/mL IL-2. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-hydroxyethanol in the medium is 55 µM.

In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-hydroxyethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-hydroxyethanol in the culture medium is 55 μM.

In some embodiments, the defined medium described in International PCT Publication No. WO/1998/030679, which is incorporated herein by reference, is suitable for use in the present invention. In the publication, a serum-free eukaryotic cell culture medium is described. The serum-free eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises one or more components selected from the group consisting of, or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen prodrivers, one or more trace elements and one or more antibiotics. In some embodiments, the medium further comprises L-glutamine, sodium bicarbonate and/or β-hydroxyethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen pro-drivers and one or more trace elements. In some embodiments, the defined culture medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron saturation and transferrin, insulin, and trace element portions Ag ⁺ , Al ³⁺ , Ba ²⁺ , Cd ²⁺ , CO ₂₊ , Cr ³⁺ , Ge ⁴⁺ , ^{Se 4+ ,} Br, T, Mn ²⁺ , P, Si ⁴⁺ , V ⁵⁺ , Mo ⁶⁺ In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), Eagle's basal medium (BME), ^{RPMI 1640} , F- ^{10 ,} F-12, minimum essential medium (αMEM), Glasgow's minimum essential medium (G-MEM), RPMI growth medium and Iskoff's modified Dulbecco's medium.

In some embodiments, the concentration of glycine in the culture medium is determined to be in the range of about 5-200 mg/L, the concentration of L-histidine is about 5-250 mg/L, the concentration of L-isoleucine is about 5-300 mg/L, the concentration of L-methionine is about 5-200 mg/L, the concentration of L-phenylalanine is about 5-400 mg/L, the concentration of L-proline is about 1-1000 mg/L, the concentration of L-hydroxyproline is about 1-45 mg/L, the concentration of L-serine is about 1-250 mg/L, the concentration of L-threonine is about 10-500 mg/L, and the concentration of L-tryptophan is about 2-110 mg/L, L-tyrosine at a concentration of about 3-175 mg/L, L-valine at a concentration of about 5-500 mg/L, thiamine at a concentration of about 1-20 mg/L, reduced glutathione at a concentration of about 1-20 mg/L, L-ascorbic acid-2-phosphate at a concentration of about 1-200 mg/L, iron-saturated transferrin at a concentration of about 1-50 mg/L, insulin at a concentration of about 1-100 mg/L, sodium selenite at a concentration of about 0.000001-0.0001 mg/L, and albumin (e.g., AlbuMAX® I) at a concentration of about 5000-50,000 mg/L.

In some embodiments, the non-trace element portion of the medium is determined to be present in the concentration range listed in the column titled "Concentration Range in 1X Medium" in Table 4. In other embodiments, the non-trace element portion of the medium is determined to be present in the final concentration listed in the column titled "Preferred Embodiments of 1X Medium" in Table 4. In other embodiments, the medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace element portions of the type and concentration listed in the column titled "Preferred Embodiments of Supplements" in Table 4.

In some embodiments, the osmotic pressure of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmotic pressure is between about 280 and 310 mOsmol. In some embodiments, the defined medium is supplemented with up to about 3.7 g/L or about 2.2 g/L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 μM), 2-hydroxyethanol (final concentration of about 100 μM).

In some embodiments, the defined medium described in Smith et al., Clin Transl Immunology , 4(1) 2015 (doi: 10.1038/cti.2014.31) can be used in the present invention. Briefly, RPMI or CTS™ OpTmizer™ is used as the basal cell culture medium and supplemented with 0, 2%, 5% or 10% CTS™ Immune Cell Serum Replacement.

In some embodiments, the cell culture medium in the first and/or second gas permeable container is unfiltered. Using an unfiltered cell culture medium can simplify the procedures required to expand the number of cells. In some embodiments, the cell culture medium in the first and/or second gas permeable container lacks β-mercaptoethanol (BME or βME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, the second expansion (e.g., according to step D of FIG. 82 ) is performed in a closed system bioreactor. In some embodiments, a closed system is used to perform TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, the rapid or secondary expansion step is divided into multiple steps to achieve vertical expansion of the culture scale by: (a) performing rapid or secondary expansion by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100 MCS container) for a period of about 3 to 7 days; and then (b) achieving transfer of the TILs in the small-scale culture to a second container (e.g., a G-REX-500-MCS container) that is larger than the first container and culturing the TILs from the small-scale culture in a larger-scale culture in the second container for a period of about 4 to 7 days.

In some embodiments, the rapid or second expansion step is divided into multiple steps to achieve horizontal expansion of the culture scale by: (a) by adding a second expansion vessel (e.g., G-REX-100 (a) culturing TILs in a first small-scale culture in an MCS container for a period of about 3 to 7 days for rapid or secondary expansion; and then (b) effecting transfer of TILs from the first small-scale culture and distributing them to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers of equal size to the first container, wherein in each second container, the portion of TILs from the first small-scale culture transferred to such second container is cultured in the second small-scale culture for a period of about 4 to 7 days.

In some embodiments, the first small-scale TIL culture is divided into a plurality of about 2 to 5 TIL subsets.

In some embodiments, the rapid or second expansion step is divided into multiple steps to achieve lateral and vertical expansion of the culture scale by: (a) by adding a plurality of cells to the first vessel (e.g., G-REX-100 (a) culturing TILs in a small-scale culture in a G-REX-500 MCS container for a period of about 3 to 7 days for rapid or secondary expansion; and then (b) effecting transfer of TILs from the small-scale culture and distributing them to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers (e.g., G-REX-500 MCS containers) that are larger in size than the first container, wherein in each second container, the portion of TILs from the small-scale culture transferred to such second container is cultured in the larger-scale culture for a period of about 4 to 7 days.

In some embodiments, the rapid or secondary expansion step is divided into multiple steps to achieve lateral and longitudinal expansion of the culture scale by: (a) performing rapid or secondary expansion by culturing TILs in a small-scale culture in a first container (e.g., a G-REX-100 MCS container) for a period of about 5 days; and then (b) achieving transfer of TILs from the small-scale culture and distributing them to 2, 3 or 4 second containers (e.g., G-REX-500 MCS containers) that are larger in size than the first container, wherein in each second container, the portion of TILs from the small-scale culture transferred to such second container is cultured in the larger-scale culture for a period of about 6 days.

In some embodiments, after the rapid or second expansion division, each second container comprises at least 10 ⁸ TILs. In some embodiments, after the rapid or second expansion division, each second container comprises at least 10 ⁸ TILs, at least 10 ⁹ TILs, or at least 10 ¹⁰ TILs. In an exemplary embodiment, each second container comprises at least 10 ¹⁰ TILs.

In some embodiments, the first small-scale TIL culture is distributed into a plurality of subpopulations. In some embodiments, the first small-scale TIL culture is distributed into a plurality of about 2 to 5 subpopulations. In some embodiments, the first small-scale TIL culture is distributed into a plurality of about 2, 3, 4, or 5 subpopulations.

In some embodiments, after the rapid or second expansion is completed, a plurality of subpopulations comprise a therapeutically effective amount of TILs. In some embodiments, after the rapid or second expansion is completed, one or more TIL subpopulations are pooled together to produce a therapeutically effective amount of TILs. In some embodiments, after the rapid expansion is completed, each TIL subpopulation comprises a therapeutically effective amount of TILs.

In some embodiments, the rapid or second expansion is carried out for a period of about 3 to 7 days before being divided into multiple steps. In some embodiments, the splitting of the rapid or second expansion occurs at about the 3rd day, 4th day, 5th day, 6th day or 7th day after the rapid or second expansion begins.

In some embodiments, the rapid or second expansion split occurs about day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, or day 18 after the start of the first expansion (i.e., pre-REP expansion). In an exemplary embodiment, the rapid or second expansion split occurs about day 16 after the start of the first expansion.

In some embodiments, after splitting, rapid or second expansion is further carried out for a period of about 7 to 11 days. In some embodiments, after splitting, rapid or second expansion is further carried out for a period of about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.

In some embodiments, the medium used for rapid or secondary expansion of cells before division contains the same components as the medium used for rapid or secondary expansion of cells after division. In some embodiments, the medium used for rapid or secondary expansion of cells before division contains different components than the medium used for rapid or secondary expansion of cells after division.

In some embodiments, the cell culture medium used for rapid or secondary expansion before division comprises IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion before division comprises IL-2, OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or secondary expansion before division comprises IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or second expansion before splitting is generated by replenishing the cell culture medium in the first expansion with a fresh medium comprising IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion before splitting is generated by replenishing the cell culture medium in the first expansion with a fresh medium comprising IL-2, OKT-3, and APC. In some embodiments, the cell culture medium used for rapid or second expansion before splitting is generated by replacing the cell culture medium in the first expansion with a fresh cell culture medium comprising IL-2, optionally OKT-3, and further optionally APC. In some embodiments, the cell culture medium used for rapid or second expansion before splitting is generated by replacing the cell culture medium in the first expansion with a fresh cell culture medium comprising IL-2, OKT-3, and APC.

In some embodiments, the cell culture medium used for rapid or secondary expansion after division comprises IL-2 and, optionally, OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after division comprises IL-2 and OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after division is generated by replacing the cell culture medium used for rapid or secondary expansion before division with a fresh medium comprising IL-2 and, optionally, OKT-3. In some embodiments, the cell culture medium used for rapid or secondary expansion after splitting is generated by replacing the cell culture medium used for rapid or secondary expansion before splitting with fresh culture medium comprising IL-2 and OKT-3.

In some embodiments, the rapid expansion splitting is performed in a closed system.

In some embodiments, the vertical expansion of the scale of TIL culture during the rapid or second expansion period includes adding fresh cell culture medium to the TIL culture (also referred to as feeding TIL). In some embodiments, feeding includes frequently adding fresh cell culture medium to the TIL culture. In some embodiments, feeding includes adding fresh cell culture medium to the TIL culture at regular intervals. In some embodiments, fresh cell culture medium is supplied to the TIL via constant flow. In some embodiments, rapid expansion and feeding are performed using an automated cell expansion system such as Xuri W25. 1. Feeder Cells and Antigen Presenting Cells

In some embodiments, the second expansion process described herein (e.g., including the following expansions, such as those described in step D of FIG. 82 and those expansions referred to as REP) requires excess feeder cells during the REP TIL expansion period and/or during the second expansion period. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

Generally, allogeneic PBMCs are irradiated or heat treated without activation and used in the REP procedure as described in the Examples, which provide an exemplary protocol for assessing the replication incompetence of irradiated allogeneic PBMCs.

In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to be replication-incompetent and are acceptable for use in the TIL expansion procedures described herein.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase compared to the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to be replication-incompetent and acceptable for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 does not increase compared to the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMCs are considered to be replication-incompetent and acceptable for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen presenting feeder cells are artificial antigen presenting feeder cells. In some embodiments, the ratio of TILs to antigen presenting feeder cells in the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:50 and 1:300. In some embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:100 and 1:200.

In some embodiments, the second expansion process described herein requires a ratio of about 2.5×10 ⁹ feeder cells to about 100×10 ⁶ TILs. In other embodiments, the second expansion process described herein requires a ratio of about 2.5×10 ⁹ feeder cells to about 50×10 ⁶ TILs. In other embodiments, the second expansion process described herein requires a ratio of about 2.5×10 ⁹ feeder cells to about 25×10 ⁶ TILs.

In some embodiments, the second expansion process described herein requires an excess of feeder cells during the second expansion period. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen presenting cells (aAPCs) are used instead of PBMCs.

Generally, allogeneic PBMCs are irradiated or heat treated without activation and used in the TIL expansion procedures described herein, including the exemplary procedures described in the Figures and Examples.

In some embodiments, artificial antigen presenting cells are used in the second expansion to replace PBMCs or in combination with PBMCs. 2. Interleukins and other additives

The expansion methods described herein typically use medium with high doses of interleukins, particularly IL-2, as is known in the art.

Alternatively, it is also possible to use a combination of cytokines for rapid expansion and/or secondary expansion of TILs, as described in U.S. Patent Application Publication No. US 2017/0107490 A1 (the disclosure of which is incorporated herein by reference), using a combination of two or more of IL-2, IL-15, and IL-21. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, the latter of which has a specific use in many embodiments. The use of a combination of cytokines is particularly advantageous for the generation of lymphocytes, and in particular T cells as described therein.

In some embodiments, step D may also include adding OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In some embodiments, step D may also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In some embodiments, step D may also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In addition, additives such as peroxisome proliferator-activated receptor gamma coactivator factor I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists, such as thiazolidinedione compounds, as described in U.S. Patent Application Publication No. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference, may be used in the culture medium during step D. E. Step E : Harvesting TIL

After the second expansion step, the cells can be harvested. In some embodiments, the TILs are harvested after one, two, three, four or more expansion steps, such as provided in FIG. 82. In some embodiments, the TILs are harvested after two expansion steps, such as provided in FIG. 82.

TILs can be harvested in any suitable and sterile manner, including, for example, centrifugation. Methods for harvesting TILs are well known in the art and any such known methods can be used with the process of the present invention. In some embodiments, TILs are harvested using an automated system.

Cell harvesters and/or cell processing systems are available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International. In some embodiments, the methods of the present invention may employ any cell-based harvester. In some embodiments, the cell harvester and/or cell processing system is a membrane-based cell harvester. In some embodiments, cell harvesting is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a solution containing cells through a membrane or filter (such as a rotating membrane or rotating filter) in a sterile and/or closed system environment, thereby allowing continuous flow and cell processing to remove supernatant or cell culture medium without clumping. In some embodiments, the cell harvester and/or cell processing system can perform cell separation, washing, fluid exchange, concentration and/or other cell processing steps in a closed sterile system.

In some embodiments, harvesting (e.g., according to step E of FIG. 82 ) is performed in a closed system bioreactor. In some embodiments, a closed system is used to perform TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, step E according to Figure 82 is performed according to the process described herein. In some embodiments, the closed system is entered through a syringe under sterile conditions to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the examples is used.

In some embodiments, TILs are harvested according to the methods described in the examples. In some embodiments, TILs are harvested between day 1 and day 11 using the methods described in the steps mentioned herein, such as the day 11 TIL harvest in the examples. In some embodiments, TILs are harvested between day 12 and day 24 using the methods described in the steps mentioned herein, such as the day 22 TIL harvest in the examples. In some embodiments, TILs are harvested between day 12 and day 22 using the methods described in the steps mentioned herein, such as the day 22 TIL harvest in the examples. F. Step F : Final Preparation and Transfer to Infusion Container

After steps A to E are completed as provided in an exemplary order in Figure 82 and as described in detail above and herein, the cells are transferred to a container for administration to a patient, such as an infusion bag or a sterile vial. In some embodiments, once a sufficient number of TILs for treatment is obtained using the expansion methods described above, they are transferred to a container for administration to a patient.

In some embodiments, TILs expanded using the APCs of the present disclosure are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using the PBMCs of the present disclosure may be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration. IX. Pharmaceutical Compositions, Dosages, and Dosage Regimens

In some embodiments, gene-edited TILs or non-gene-edited TILs prepared using the methods of the present disclosure are administered to a patient in the form of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure can be administered by any suitable route known in the art. In some embodiments, T cells are administered in the form of a single intra-arterial or intravenous infusion, which preferably lasts about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

In an exemplary embodiment, the gene-edited TIL provided herein includes one or more interleukins selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFNγ, TNFa, IFNα, IFNβ, GM-CSF, GCSF or variants thereof. In some embodiments, one or more interleukins are tethered interleukins.

In some embodiments, the gene-edited TIL provided herein further comprises a nucleotide sequence encoding shRNA. In some embodiments, shRNA inhibits the expression of immune checkpoint genes. Non-limiting examples of immune checkpoint genes that can be silenced or inhibited by shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two interleukins, wherein the first interleukin is IL- 12. In some embodiments, the second interleukin is IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN γ, TNFa, IFN α, IFN β, GM-CSF, GCSF or variants thereof.

In an exemplary embodiment, the gene-edited TILs provided herein include two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-15.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-2.

In an exemplary embodiment, the gene-edited TILs provided herein include two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-21.

In an exemplary embodiment, the gene-edited TILs provided herein include two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-18.

In an exemplary embodiment, the gene-edited TILs provided herein include two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-6.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-7.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-9.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-23.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-27.

In an exemplary embodiment, the gene-edited TILs provided herein include two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-33.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IFNγ.

In an exemplary embodiment, the gene-edited TILs provided herein include two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is TNFa.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IFNα.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IFNβ.

In an exemplary embodiment, the gene-edited TILs provided herein include two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is GM-CSF.

In an exemplary embodiment, the gene-edited TILs provided herein include two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is GCSF.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-15, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-2, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-21, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-18, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-6, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-7, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-9, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-23, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-27, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IL-33, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IFNγ, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is TNFa, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IFN α, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is IFNβ, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is GM-CSF, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

In an exemplary embodiment, the gene-edited TIL provided herein comprises two membrane-anchored interleukins, wherein the first interleukin is IL-12 and the second interleukin is GCSF, and the shRNA that inhibits the expression of immune checkpoint genes is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. In some embodiments, the gene-edited TIL provided herein further comprises PD-1 shRNA.

Further provided are gene-edited TILs or untreated gene-edited TIL populations disclosed herein, for example, gene-edited TILs or untreated gene-edited TIL populations produced using the methods of producing gene-edited TILs or untreated gene-edited TILs in the present disclosure. In some embodiments, gene-edited TILs or untreated gene-edited TIL populations can be administered to a patient in the form of a pharmaceutical composition.

Any suitable dose of TILs may be administered. In some embodiments, about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs are administered, with an average of about 7.8×10 ¹⁰ TILs, particularly when the cancer is NSCLC or melanoma. In some embodiments, about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TILs are administered. In some embodiments, about 3×10 ¹⁰ to about 12×10 ¹⁰ TILs are administered. In some embodiments, about 4×10 ¹⁰ to about 10×10 ¹⁰ TILs are administered. In some embodiments, about 5×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, about 6×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, about 7×10 ¹⁰ to about 8×10 ¹⁰ TILs are administered. In some embodiments, the therapeutically effective dose is about 2.3×10 ¹⁰ to about 13.7×10 ^10. In some embodiments, the therapeutically effective dose is about 7.8×10 ¹⁰ TILs, especially when the cancer is melanoma. In some embodiments, the therapeutically effective dose is about 7.8×10 ¹⁰ TILs, especially when the cancer is NSCLC. In some embodiments, the therapeutically effective dose is about 1.2×10 ¹⁰ to about 4.3×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3×10 ¹⁰ to about 12×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4×10 ¹⁰ to about 10×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5×10 ¹⁰ to about 8×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6×10 ¹⁰ to about 8×10 ¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7×10 ¹⁰ to about 8×10 ¹⁰ TILs.

In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is about 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some embodiments, the number of TILs provided in the pharmaceutical composition of the present invention is in the range of 1×10 ⁶ to 5×10 ⁶ , 5×10 ⁶ to 1×10 ⁷ , 1×10 ⁷ to 5×10 ⁷ , 5×10 ⁷ to 1×10 ⁸ , 1×10 ⁸ to 5×10 ⁸ , 5×10 ⁸ to 1×10 ⁹ , 1×10 ⁹ to 5×10 ⁹ , 5×10 ⁹ to 1×10 ¹⁰ , 1×10 ¹⁰ to 5×10 ¹⁰ , 5×10 ¹⁰ to 1×10 ¹¹ , 5×10 ¹¹ to 1×10 ¹² , 1×10 ¹² to 5×10 ¹² , and 5×10 ¹² to 1×10 ¹³ .

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 10 ... %, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.5 0%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 1 1.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8% ,7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75% , 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0. 0.002%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w, w/v, or v/v.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.0 % to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v/v.

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.

The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide range of dosages. The exact dosage will depend on the route of administration, the form of compound administration, the sex and age of the individual to be treated, the weight of the individual to be treated, and the preference and experience of the attending physician. Clinically determined dosages of TILs may also be used when appropriate. The amount of the pharmaceutical composition administered using the methods herein, such as the dosage of TILs, will depend on the human or mammal being treated, the severity of the disease or condition, the rate of administration, the disposition of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some embodiments, TILs may be administered in a single dose. Such administration may be performed by injection, such as intravenous injection. In some embodiments, TILs may be administered in multiple doses. Administration may be once, twice, three times, four times, five times, six times, or more than six times per year. Administration may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs may continue as needed.

In some embodiments, the effective amount of TIL is about 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ and 9×10 ¹³ . In some embodiments, the effective amount of TIL is in the range of 1×10 ⁶ to 5×10 ⁶ , 5×10 ⁶ to 1×10 ⁷ , 1×10 ⁷ to 5×10 ⁷ , 5×10 ⁷ to 1×10 ⁸ , 1×10 ⁸ to 5×10 ⁸ , 5×10 ⁸ to 1×10 ⁹ , 1×10 ⁹ to 5×10 ⁹ , 5×10 ⁹ to 1×10 ¹⁰ , 1×10 ¹⁰ to 5×10 ¹⁰ , 5×10 ¹⁰ to 1×10 ¹¹ , 5×10 ¹¹ to 1×10 ¹² , 1×10 ¹² to 5×10 ¹² , and 5×10 ¹² to 1×10 ¹³ .

In some embodiments, the effective amount of TIL is about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 In some embodiments, the present invention relates to an oral dosage form of at least about 1 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective amount of TIL is about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of TILs may be administered by any recognized mode of administration of agents with similar utilities, including intranasal and transdermal routes, by intraarterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topically, by implantation or by inhalation, in single or multiple doses.

In other embodiments, the present invention provides an infusion bag comprising a therapeutic TIL population as described above in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described above in any of the preceding paragraphs and a pharmaceutically acceptable carrier.

In other embodiments, the present invention provides an infusion bag comprising a TIL composition as described above in any preceding paragraph.

In other embodiments, the present invention provides a cryopreserved preparation of a therapeutic TIL population as described above in any of the preceding paragraphs.

In other embodiments, the present invention provides a tumor infiltrating lymphocyte (TIL) composition comprising a therapeutic TIL population as described above in any of the preceding paragraphs and a cryopreservation medium.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs above, wherein the cryopreservation medium contains DMSO.

In other embodiments, the present invention provides a modified TIL composition as described in any of the preceding paragraphs above, wherein the cryopreservation medium contains 7% to 10% DMSO.

In other embodiments, the present invention provides a cryopreserved formulation of a TIL composition as described above in any of the preceding paragraphs. X. Methods of treating cancer patients

In some embodiments, an untreated gene-edited TIL population produced using the methods of the present disclosure is administered to a patient to treat cancer. In some embodiments, provided herein is a method for treating cancer in a patient in need thereof, comprising: a) removing a tumor sample from the patient, wherein the tumor sample comprises a TIL population; b) performing a first expansion by culturing the TIL population in a first cell culture medium; c) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, and L-arginine; and d) administering the therapeutic gene-edited TIL population to the patient.

In some embodiments, provided herein is a method for treating cancer in a patient in need thereof, comprising: a) removing a tumor sample from the patient, wherein the tumor sample comprises a TIL population; b) performing a first expansion by culturing the TIL population in a first cell culture medium; c) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, L-arginine, and NAD+; and d) administering the therapeutic gene-edited TIL population to the patient.

In some embodiments, gene-edited TILs using the methods of the present disclosure are administered to a patient to treat cancer. In some embodiments, provided herein is a method of treating cancer in a patient in need thereof, comprising: a) removing a tumor sample from the patient, wherein the tumor sample comprises a TIL population; b) performing a first expansion by culturing the TIL population in a first cell culture medium; c) performing a second expansion by culturing the TIL population in a second cell culture medium; d) at any time, transducing the TIL population with a recombinant lentiviral particle disclosed herein to produce a therapeutic gene-edited TIL population; and e) administering the therapeutic gene-edited TIL population to the patient.

In some embodiments, the method further comprises activating the TIL population for 1 or 2 days before transducing the TIL population with recombinant lentiviral particles. In some embodiments, the activation step comprises contacting the TIL population with an interleukin selected from the group consisting of: IL-2, IL-15, IL-21, IL-7, and a combination thereof. In some embodiments, the activation step comprises contacting the TIL population with TransAct. In some embodiments, the transduction step is performed in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transduction step comprises centrifugation. In some embodiments, the transduction step is performed in the presence of Lentibooster. In some embodiments, the transduction step is performed after the first expansion step and before the second expansion step. In some embodiments, the activation step is performed after the first expansion step and before the second expansion step. In some embodiments, the method further comprises allowing the TIL population to rest for 1 or 2 days after the transduction step.

In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by fludarabine at a dose of 25 mg/m2/day for one day. In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the method further comprises the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg aldesleukin or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerated. In some embodiments, the method does not include the step of treating the patient with the IL-2 regimen. In some embodiments, the therapeutic TIL population comprises about 2.3×10 ¹⁰ to about 13.7×10 ¹⁰ TILs.

In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple-negative breast cancer), bone cancer, cancer caused by human papillomavirus (HPV), central nervous system-related cancer (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma and primitive neuroectodermal tumor), cervical cancer (including squamous cervical cancer, adenosquamous cervical cancer and cervical adenocarcinoma), colorectal cancer, colorectal cancer, endometrial cancer, esophageal cancer, esophageal gastric junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, neurofibromatosis glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), laryngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer and pharyngeal cancer), kidney cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer), melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, kidney cancer, renal cell carcinoma, sarcoma (including Ewing sarcoma sarcoma), osteosarcoma, rhabdomyosarcoma and other bone and soft tissue sarcomas), thyroid cancer (including anaplastic thyroid cancer), uterine cancer, and vaginal cancer.

In some embodiments, the cancer is one of the aforementioned cancers, including solid tumor cancers and hematological malignancies, which is relapsed or refractory to treatment with at least one prior therapy, including chemotherapy, radiation therapy, or immunotherapy. In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least two prior therapies, including chemotherapy, radiation therapy, and/or immunotherapy. In some embodiments, the cancer is one of the aforementioned cancers that is relapsed or refractory to treatment with at least three prior therapies, including chemotherapy, radiation therapy, and/or immunotherapy.

In some embodiments, the cancer is microsomal instability-high (MSI-H) or mismatch repair-deficient cancer. MSI-H and dMMR cancers and their detection have been described in Kawakami et al., Curr. Treat. Options Oncol. 2015, 16, 30, the disclosure of which is incorporated herein by reference.

In some embodiments, the cancer is metastatic melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma). In some embodiments, the cancer is lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer). In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is ovarian cancer. 1. Lymphocyte depletion preconditioning of patients

In some embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TIL according to the present disclosure. In some embodiments, the present invention includes a TIL population for treating cancer in a patient who has been pretreated with non-myeloablative chemotherapy. In some embodiments, the TIL population is administered by infusion. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (on days 27 and 26 before TIL infusion) and fludarabine 25 mg/m2/d for 5 days (on days 27 to 23 before TIL infusion). In some embodiments, following non-myeloablative chemotherapy and TIL infusion (day 0) according to the present disclosure, patients receive an intravenous infusion of IL-2 (aldesleukin, commercially available as PROLEUKIN) at 720,000 IU/kg every 8 hours to achieve physiological tolerance. In certain embodiments, TIL populations are used to treat cancer in combination with IL-2, wherein IL-2 is administered after the TIL population.

In some embodiments, the patient receives a reduced intensity non-myeloablative lymphocyte depletion regimen. In some embodiments, the reduced intensity non-myeloablative lymphocyte depletion regimen includes administering cyclophosphamide at a dose of about 250-750 mg/m2/day. In some embodiments, cyclophosphamide is administered at a dose of about 250 mg/m2/day. In some embodiments, cyclophosphamide is administered at a dose of about 500 mg/m2/day. In some embodiments, cyclophosphamide is administered at a dose of about 750 mg/m2/day. In some embodiments, cyclophosphamide is administered for three or four days. In some embodiments, cyclophosphamide is administered followed by fludarabine at a dose of 30 mg/m2/day. In some embodiments, fludarabine is administered for three, four, or five days. In some embodiments, cyclophosphamide is administered with mesnat. In some embodiments, the patient does not receive a non-myeloablative lymphocyte depletion regimen.

Experimental findings indicate that lymphocyte depletion prior to the transfer of tumor-specific T lymphocytes plays a key role in enhancing therapeutic efficacy by eliminating regulatory T cells and elements of the competing immune system (the "interleukin pool"). Therefore, some embodiments of the invention employ a lymphocyte depletion step (sometimes referred to as "immunosuppressive conditioning") in patients prior to the introduction of the TILs of the invention.

Generally, lymphocyte depletion is achieved using administration of fludarabine or cyclophosphamide (the active form is called mafosfamide), and combinations thereof. Such methods are described in Gassner et al., Cancer Immunol. Immunother 2011 , 60 , 75-85; Muranski et al., Nat. Clin. Pract. Oncol . , 2006, 3 , 668-681; Dudley et al., J. Clin. Oncol . 2008 , 26, 5233-5239; and Dudley et al., J. Clin. Oncol . 2005 , 23, 2346-2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/mL to 10 μg/mL fludarabine. In some embodiments, fludarabine is administered at a concentration of 1 μg/mL fludarabine. In some embodiments, fludarabine is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, fludarabine is administered at a dose of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 4 to 5 days. In some embodiments, fludarabine treatment is administered at 25 mg/kg/day for 4 to 5 days.

In some embodiments, cyclophosphamide is administered to obtain a concentration of 0.5 μg/mL to 10 μg/mL of mafosfamide, the active form of cyclophosphamide. In some embodiments, cyclophosphamide is administered to obtain a concentration of 1 μg/mL of mafosfamide, the active form of cyclophosphamide. In some embodiments, cyclophosphamide is administered for 1, 2, 3, 4, 5, 6, or 7 days or more. In some embodiments, cyclophosphamide is administered at a dose of 100 mg/m2/day, 150 mg/m2/day, 175 mg/m2/day, 200 mg/m2/day, 225 mg/m2/day, 250 mg/m2/day, 275 mg/m2/day, or 300 mg/m2/day. In some embodiments, cyclophosphamide is administered intravenously (i.e., i.v.). In some embodiments, cyclophosphamide treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, cyclophosphamide treatment is administered at 250 mg/m2/day intravenously for 4 to 5 days. In some embodiments, cyclophosphamide treatment is administered intravenously at 250 mg/m2/day for 4 days.

In some embodiments, lymphocyte depletion is performed by administering fludarabine and cyclophosphamide together to a patient. In some embodiments, fludarabine is administered intravenously at 25 mg/m2/day and cyclophosphamide is administered intravenously at 250 mg/m2/day for 4 days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by administration of fludarabine at a dose of 25 mg/m2/day for five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of 60 mg/m2/day for two days and fludarabine at a dose of 25 mg/m2/day for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days, and wherein lymphocyte depletion is performed for a total of five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of about 50 mg/m2/day for two days and fludarabine at a dose of about 25 mg/m2/day for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days, and wherein lymphocyte depletion is performed for a total of five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of about 50 mg/m2/day for two days and fludarabine at a dose of about 20 mg/m2/day for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days, and wherein lymphocyte depletion is performed for a total of five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of about 40 mg/m2/day for two days and fludarabine at a dose of about 20 mg/m2/day for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days, and wherein lymphocyte depletion is performed for a total of five days.

In some embodiments, lymphocyte depletion is performed by administering cyclophosphamide at a dose of about 40 mg/m2/day for two days and fludarabine at a dose of about 15 mg/m2/day for five days, wherein both cyclophosphamide and fludarabine are administered on the first two days, and wherein lymphocyte depletion is performed for a total of five days.

In some embodiments, lymphodepletion is performed by administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by fludarabine at a dose of 25 mg/m2/day for three days.

In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, mesna is administered at 15 mg/kg. In some embodiments, mesna is infused, and if infused continuously, over 24 hours, starting with the respective cyclophosphamide dose, mesna may be infused with cyclophosphamide over about 2 hours (Day -5 and/or Day -4), followed by an infusion at a rate of 3 mg/kg/hour for the remaining 22 hours.

In some embodiments, lymphocyte depletion comprises the step of treating the patient with an IL-2 regimen beginning the day after the third TIL population is administered to the patient.

In some embodiments, lymphocyte depletion comprises the step of starting treatment of the patient with an IL-2 regimen on the same day that the third TIL population is administered to the patient.

In some embodiments, lymphocyte depletion includes 5 days of preconditioning therapy. In some embodiments, the number of days is indicated as day -5 to day -1, or day 0 to day 4. In some embodiments, the regimen includes cyclophosphamide on day -5 and day -4 (i.e. day 0 and day 1). In some embodiments, the regimen includes intravenous cyclophosphamide on day -5 and day -4 (i.e. day 0 and day 1). In some embodiments, the regimen includes 60 mg/kg intravenous cyclophosphamide on day -5 and day -4 (i.e. day 0 and day 1). In some embodiments, cyclophosphamide is administered together with mesna. In some embodiments, the regimen further includes fludarabine. In some embodiments, the regimen further comprises intravenous fludarabine. In some embodiments, the regimen further comprises 25 mg/m ² intravenous fludarabine. In some embodiments, the regimen further comprises 25 mg/m ² intravenous fludarabine on days -5 and -1 (i.e., days 0 to 4). In some embodiments, the regimen further comprises 25 mg/m ² intravenous fludarabine on days -5 and -1 (i.e., days 0 to 4).

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for five days.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administering fludarabine at a dose of 25 mg/m2/day for five days.

In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administering fludarabine at a dose of 25 mg/m2/day for three days.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for one day.

In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administering fludarabine at a dose of 25 mg/m2/day for three days.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days.

In some embodiments, a non-myeloablative lymphodepleting regimen is administered according to Table 11.

In some embodiments, a non-myeloablative lymphodepleting regimen is administered according to Table 12.

In some embodiments, a non-myeloablative lymphodepleting regimen is administered according to Table 13.

In some embodiments, a non-myeloablative lymphodepleting regimen is administered according to Table 14.

In some embodiments, a non-myeloablative lymphodepleting regimen is administered according to Table 15.

In some embodiments, a non-myeloablative lymphodepleting regimen is administered according to Table 16.

In some embodiments, a non-myeloablative lymphodepleting regimen is administered according to Table 17.

In some embodiments, a non-myeloablative lymphodepleting regimen is administered according to Table 18.

In some embodiments, the TIL infusion used with the aforementioned myeloablative lymphocyte depletion regimen embodiments can be any of the TIL compositions described herein, as well as the addition of an IL-2 regimen and administration of co-therapy as described herein (e.g., PD-1 and PD-L1 inhibitors).

In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering melphalan to a total dose of 100 mg/m ² over the course of 1, 2, or 3 days prior to the TIL infusion day. In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering melphalan to a total dose of 200 mg/m ² over the course of 1, 2, or 3 days prior to the TIL infusion day. In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering melphalan to a total dose of 100 mg/m ² over the course of 1, 2, or 3 days prior to the TIL infusion day and administering fludarabine at a dose of 30 mg/m 2 /day. In some embodiments, the non-myeloablative lymphodepletion regimen comprises administering melphalan to a total dose of 200 mg/m ² and fludarabine at a dose of 30 mg/m 2 /day over the course of 1, 2, or 3 days prior to the TIL infusion day.

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administration of an anti-CD45 antibody. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administration of an anti-CD45 antibody-drug conjugate. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administration of an anti-CD45 antibody-radioactive isotope conjugate. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administration of apamistamab- ^131I . In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises administration of apamistamab- ^131I at a dose of 25 mCi, 50 mCi, 75 mCi, 100 mCi, 150 mCi, or 200 mCi between 2 days and 9 days prior to TIL infusion. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises apamizumab- ^131I administered at a dose of 25 mCi to 200 mCi between 2 days and 9 days prior to TIL infusion. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises apamizumab- ^131I administered at a dose of 50 mCi to 150 mCi between 4 days and 8 days prior to TIL infusion. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises apamizumab-131I administered at a dose of about 75 mCi about 6 days prior to TIL infusion. In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises apamizumab- ^131I administered at a dose of about 100 mCi about 7 days prior to TIL infusion ^.

In some embodiments, the TIL infusion used with the myeloablative lymphocyte depletion regimen of the foregoing embodiments may be any TIL composition described herein, including TIL products that have been genetically modified to include one or more immunomodulators attached to their surface, and may also include infusions of MIL and PBL to replace TIL infusions, as well as the addition of alternative lymphocyte depletion regimens, including the anti-CD52 antibody alemtuzumab or its variants, fragments, antibody-drug conjugates or biosimilars. 2. IL-2 regimen

In some embodiments, the IL-2 regimen includes a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen includes aldesleukin or a biosimilar or variant thereof, which is administered intravenously the same day after the therapeutically effective portion of the therapeutic TIL population is administered, wherein aldesleukin or a biosimilar or variant thereof is administered intravenously every eight hours using a 15-minute bolus intravenous infusion at a dose of 0.037 mg/kg or 0.044 mg/kg IU/kg (patient weight) until tolerated, up to 14 doses. After resting for 9 days, this schedule can be repeated for another 14 doses, for a total of up to 28 doses. In some embodiments, IL-2 is administered in 1, 2, 3, 4, 5, or 6 doses. In some embodiments, IL-2 is administered at a maximum dose of up to 6 doses.

In some embodiments, the IL-2 regimen comprises a reduced IL-2 regimen. Reduced IL-2 regimens have been described in O'Day et al., J. Clin. Oncol . 1999 , 17 , 2752-61 and Eton et al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporated herein by reference. In some embodiments, the debulking IL-2 therapy comprises 18×10 ⁶ IU/m ² intravenously administered over 6 hours, followed by 18×10 ⁶ IU/m ² intravenously administered over 12 hours, followed by 18×10 ⁶ IU/m ² intravenously administered over 24 hours, followed by 4.5×10 ⁶ IU/m ² of aldesleukin or a biosimilar or variant thereof intravenously administered over 72 hours. This treatment cycle can be repeated every 28 days for up to four cycles. In some embodiments, the tapering IL-2 regimen includes 18,000,000 IU/m ² on day 1, 9,000,000 IU/m ² on day 2, and 4,500,000 IU/m ² on days 3 and 4.

In some embodiments, the reduced-dose IL-2 regimen comprises reduced amounts, such as 1, 2, 3, 4, or 5 doses of 600,000 or 720,000 IU/kg of aldesleukin or a biosimilar or variant thereof, administered as a bolus intravenous infusion every 8 hours for 15 minutes until tolerated. In some embodiments, the patient does not receive the IL-2 regimen.

In some embodiments, the IL-2 regimen includes a dose-reducing IL-2 regimen. Any low-dose IL-2 regimen known in the art may be used, including Dominguez-Villar and Hafler, Nat. Immunology 2000, 19, 665-673; Hartemann et al., Lancet Diabetes Endocrinol . 2013 , 1 , 295-305; and Rosenzwaig et al., Ann. Rheum . Dis. 2019, 78, 209-217, the disclosures of which are incorporated herein by reference. In some embodiments, the low-dose IL-2 regimen includes 18×10 ⁶ IU/m ² of aldesleukin or a biosimilar or variant thereof every 24 hours as a continuous infusion for 5 days; followed by no IL-2 therapy for 2 to 6 days; optionally, aldesleukin or a biosimilar or variant thereof is then administered intravenously as a continuous infusion of 18×10 ⁶ IU/m ² every 24 hours for 5 days; optionally, no IL-2 therapy is then administered for 3 weeks, followed by other cycles of administration.

In some embodiments, IL-2 is administered at a maximum dose of up to 6 doses. In some embodiments, the high-dose IL-2 regimen is suitable for pediatric use. In some embodiments, 600,000 international units (IU)/kg of aldesleukin are used every 8 to 12 hours, up to 6 doses. In some embodiments, 500,000 international units (IU)/kg of aldesleukin are used every 8 to 12 hours, up to 6 doses. In some embodiments, 400,000 international units (IU)/kg of aldesleukin are used every 8 to 12 hours, up to 6 doses. In some embodiments, 500,000 international units (IU)/kg of aldesleukin is used every 8 to 12 hours, up to 6 doses. In some embodiments, 300,000 international units (IU)/kg of aldesleukin is used every 8 to 12 hours, up to 6 doses. In some embodiments, 200,000 international units (IU)/kg of aldesleukin is used every 8 to 12 hours, up to 6 doses. In some embodiments, 100,000 international units (IU)/kg of aldesleukin is used every 8 to 12 hours, up to 6 doses.

In some embodiments, the IL-2 regimen comprises administering pegylated IL-2 at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days. In some embodiments, the IL-2 regimen comprises administering bepegated interleukin or a fragment, variant, or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 regimen comprises administering THOR-707, or a fragment, variant, or biosimilar thereof, at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 regimen comprises administration of nivanovir alfa or a fragment, variant, or biosimilar thereof after administration of TILs. In certain embodiments, nivanovir is administered to the patient at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

In some embodiments, the IL-2 regimen comprises administering an IL-2 fragment grafted onto an antibody backbone. In some embodiments, the IL-2 regimen comprises administering an antibody cytokine grafted protein that binds to a low affinity receptor for IL-2. In some embodiments, the antibody cytokine grafted protein comprises a heavy chain variable region ( _VH ) comprising complementary determining regions HCDR1, HCDR2, HCDR3; a light chain variable region ( _VL ) comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into the CDRs of _VH or _VL , wherein the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody-cytokine-grafted protein comprises a heavy chain variable region ( _VH ) comprising complementation determining regions HCDR1, HCDR2, HCDR3; a light chain variable region ( _VL ) comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof grafted into the CDR of _VH or _VL , wherein the IL-2 molecule is a mutant protein, and wherein the antibody-cytokine-grafted protein expands T effector cells prior to regulatory T cells. In some embodiments, the IL-2 regimen comprises administering an antibody or a fragment, variant or biosimilar thereof at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14 or 21 days, wherein the antibody comprises a heavy chain selected from the group consisting of SEQ ID NO:29 and SEQ ID NO:38 and a light chain selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:39.

In some embodiments, the serum half-life of the anti-interleukin graft proteins described herein is longer than that of wild-type IL-2 molecules (such as but not limited to aldesleukin (Proleukin®) or comparable molecules).

In some embodiments, the TIL infusion used with the myeloablative lymphocyte depletion regimen of the foregoing embodiments can be any TIL composition described herein and can also include MIL and PBL infusions instead of TIL infusions, as well as the addition of an IL-2 regimen and the administration of co-therapy as described herein (such as PD-1 and/or PD-L1 inhibitors and/or CTLA-4 inhibitors).

Embodiments covered herein are now described with reference to the following examples. These examples are provided for illustrative purposes only and the present disclosure should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teachings provided herein. Example 1 : Preparation of culture medium for PRE-REP and REP processes

This example describes a procedure for preparing a tissue culture medium suitable for use in a protocol involving the culture of tumor infiltrating lymphocytes (TILs) derived from various solid tumors. This culture medium can be used to prepare any of the TILs described in this application and the examples.

Prepare CM1. Remove the following reagents from the freezer and warm them in a 37°C water bath: (RPMI1640, human AB serum, 200 mM L-glutamine). Prepare CM1 medium by adding the components to the top of a 0.2 µm filter unit appropriate for the volume to be filtered according to Table 19 below. Store at 4°C.

On the day of use, pre-warm the required amount of CM1 in a 37°C water bath and add 6000 IU/mL IL-2.

Additional supplements may be made as required according to Table 20. Preparation CM2

Remove prepared CM1 from refrigerator or prepare fresh CM1. Remove AIM-V® from refrigerator and prepare required amount of CM2 by mixing prepared CM1 with an equal volume of AIM-V® in a sterile medium bottle. Add 3000 IU/mL IL-2 to CM2 medium on day of use. Make up sufficient CM2 with 3000 IU/mL IL-2 on day of use. Label bottle of CM2 medium with name, preparer's initials, filter/preparation date, two-week expiration date, and store at 4°C until needed for tissue culture. Prepare CM3

Prepare CM3 on the day of use. CM3 is the same as AIM-V® medium, but supplemented with 3000 IU/mL IL-2 on the day of use. Prepare the amount of CM3 required for the experiment by adding IL-2 stock solution directly to the AIM-V bottle or bag. Mix thoroughly by gently vortexing. Immediately after adding AIM-V, label the bottle "3000 IU/mL IL-2." If excess CM3 is present, store it at 4°C in a bottle labeled with the medium name, the preparer's initials, the date the medium was prepared, and its expiration date (7 days after preparation). After 7 days of storage at 4°C, discard the IL-2-supplemented medium. Prepare CM4

CM4 is the same as CM3, but is additionally supplemented with 2 mM GlutaMAX ^TM (final concentration). Add 10 mL of 200 mM GlutaMAX™ per 1 L of CM3. Prepare the amount of CM4 required for your experiment by adding the IL-2 Stock Solution and the GlutaMAX™ Stock Solution directly to the AIM-V bottle or bag. Mix thoroughly by gently vortexing. Immediately after adding to the AIM-V, label the bottle "3000 IL/mL IL-2 and GlutaMAX". If there is excess CM4, store it at 4°C in a bottle labeled with the name of the medium, "GlutaMAX", and its expiration date (7 days after preparation). Discard the medium supplemented with IL-2 after storage at 4°C for more than 7 days. Example 2 : TIL activation using lentiviral particles to transduce TIL

Pre-REP treated TILs were suspended in 1e6/mL CM2 (without tamiprocin + 300 IU/mL IL-2) in a 24-well plate, 2 mL per well, and incubated overnight.

Prepare 20x IL-15, IL-7 mix and add 100 uL to each well. ○ Final concentration: IL-15 (10 ng/mL), IL-7 (20 ng/mL) in CM2 without tamiflu ○ Example: 10 wells required ■ 10 wells x 100 uL mix = 1 mL • Add IL-15 to a concentration of 200 ng/mL • Add IL-7 to a concentration of 400 ng/mL ○ Note: Remove 120 uL from each well before adding TransACT, IL-15, and IL-7 to a final volume of 2 mL

Add 100 uL of 20x interleukin mix to each TIL culture in a 24-well plate. Next, add 20 uL of TransACT to each well. Gently mix wells with a pipette to evenly disperse the TILs and mix with the stimulation mix. Incubate for 2 days. Gene transduction and static

Non-tissue culture 48-well plates were coated with Retronectin (1:100 dilution in PBS) by adding 250 uL per well. The plates were wrapped in parafilm and then incubated at 4°C overnight.

Remove coating solution from retronectin plate and replace with 250 uL 2% BSA Part V. NOTE: Add 2% BSA Part V immediately to prevent wells from drying out.

Incubate at room temperature for 30 minutes for blocking.

Remove blocking solution. Add calculated volume of viral supernatant and cover with parafilm. Centrifuge at 2000 × g for 90 min at 32°C. Note: Preheat the centrifuge before adding the culture plate.

Incubate at room temperature for 30 minutes to block. Remove blocking solution. Add the following: 1. 100 uL TIL suspension (1e5 cells) 2. 300 uL CM2 + IL-15 (20 ng/mL) + IL-7 (40 ng/mL) + Lentibooster 1:50 3. Lentivirus (produced using the lentiviral particle production protocol of Example 3), MOI in the range of 10-40 4. CM2 without lentimycin, total volume per well 600 uL

Centrifuge at 2000×g for 45 minutes at 32°C. Place in an incubator and incubate for 3 days. Example 3 : Lentiviral Particle Production Protocol

Resuspend 293T cells in virus production medium and place 6E6/10 ml HEK293T cells in a 10CM culture dish. Virus production medium: 10% FBS, DMEM (high glucose, glutamine), sodium pyruvate (1% W/v), sodium bicarbonate (0.075%), or HEPES (without Pen/strep).

The next day, Gag/Pol helper plasmid, Rev helper plasmid, BaEVTR or VSV-G helper plasmid and transfer vector were mixed at a concentration of 1 µg/µL in a 1:1 molar ratio. After mixing, 30 µL of Trans IT ^® -Lenti reagent (Mirus Bio) was added and incubated in 1 mL Opti-MEM™ medium per HEK 293T dish at room temperature for 10 minutes.

HEK293T cells were then incubated at 37°C, 5% CO ₂ for 2 days.

Virus Particle Harvest: Culture supernatants were removed from the culture dishes and fresh medium was added to each dish, centrifuged at 200 g for 5 minutes to remove cellular debris, and then filtered using a 0.45 µM filter. Virus supernatants were then concentrated by ultracentrifugation at 60,000 g for 2 hours. After ultracentrifugation, the supernatant was removed and the virus pellet was re-dissolved in Opti-MEM™ medium.

The virion harvesting step can be repeated the next day to optimize virus production. Example 4 : Preparation and Characterization of Modified TILs with Membrane-Anchored IL-15 and IL-21 Immunomodulatory Fusion Proteins Materials and Methods Virus Preparation and T Cell Transduction

To prepare lentivirus, pLenti-vectors containing tethered interleukin gene sequences (Table 21) and encapsulation helper vectors (VSV-G, Gag/Pol) were co-transfected into 293T cells. Lentiviral supernatants were collected from day 2-3 culture supernatants, followed by ultracentrifugation (120,000 g) to concentrate lentivirus for TIL transduction. Pre-REP cells were stimulated with TransACT (1:100) for 2 days prior to T cell transduction. 1E5 activated pre-REP cells were added to a 48-well plate pre-coated with Retronectin along with the concentrated lentivirus. Two days after gene transduction, pre-REP cells were harvested for REP procedures or other phenotypic characterization and functional assays. Flow cytometry

To examine the expression of mIL-15 in transduced cells, transduced cells were stained with biotin-conjugated IL-15 (biolgend, San Diego, CA) linked to avidin-BV421 (Biolegend, San Diego, CA). To examine the expression of mIL-21, transduced cells were stained with PE-conjugated IL-21 antibody. To examine T cell division, T cells were pre-labeled with CellTrace™ purple cell proliferation dye (ThermoFisher scientific, Waltham, MA). After 5 days, T cell division was analyzed in ex vivo culture in the presence or absence of IL-2 (20 IU/mL). To examine T cell activation and differentiation, phenotypic characterization was performed using antibodies from BD Bioscience, Biolegend, and Thermofisher. Data were acquired using a BioRad ZE5 flow cytometer and analyzed using FlowJo software (FlowJo, LLC, Ashland, OR). T cell count and viability

REP cells were harvested after TIL expansion. Viable cell counts were assessed by AOPI analysis using a Cellometer K2 cell counter (Nexcelom, Lawrence, MA). Pre-REP and REP Process

Human tumor samples were cut into approximately 3 mm pieces and cultured in Grex10 with the recommended medium containing IL-2 (6000 IU/mL). After 11 days of culture, pre-REP cells were harvested for 2 days of activation and 2 days of lentiviral transduction. The transduced pre-REP cells were then propagated for another 11 days in the REP expansion process with irradiated PBMC, anti-CD3 antibody, and 3,000 IU/mL IL-2. Results Expression of mIL-15/mIL-21 after gene transduction

After gene transduction, the transduced pre-REP cells were expanded ex vivo for another 5 days with 300 IU/mL IL-2. The transduced pre-REP cells showed expression of mIL-15 and mIL-21 by flow cytometry staining. As shown in Figure 1A, there was expression of 54.8% mIL-15 in TILs transduced with mIL-15 lentivirus, 53.5% mIL-21 in TILs transduced with mIL-21, and 36.5% mIL-15/mIL-21 double positive cells in TILs transduced with mIL-15/IL21. Untransduced cells were used as negative controls. mIL-15 pre-REP cells exhibit sustained activation of pSTAT5 signaling and display increased proliferation

After verification of surface interleukin expression, T cell functionality was examined. Phosphorylation of STAT5 is a marker of T cell activation after γ-chain interleukin stimulation. Under serum starvation conditions, cells transduced with mIL-15, mIL-21, and mIL-15/IL-21 lentiviruses showed sustained pSTAT5 signaling, indicating that mIL-15 is functional. As shown in Figure 1B, mIL-15 is superior to mIL-21 in inducing pSTAT5 activation. In addition, cell proliferation of TIL transduced with mIL-15 lentivirus was examined by CellTrace proliferation assay. TILs previously labeled with CellTrace Purple were cultured in vitro for 5 days in the presence or absence of IL-2. As shown in Figure 1C, mIL-15 TILs showed superior proliferation ability compared to mIL-21 or mIL-15/IL-21 TILs in the presence or absence of IL-2. mIL-15 TILs can proliferate in the absence of IL-2, indicating that mIL-15 can replace IL-2 to provide proliferation signals. Expression of mIL-15/mIL-21 after REP process

After lentiviral transduction, TILs were expanded for 11 days during REP. Surface expression of membrane-bound interleukin receptors was then assessed by flow cytometry. As shown in Figure 2A, there was expression of 31.1% mIL-15 in mIL-15 TILs, 63% mIL-21 in mIL-21 TILs, and 10.7% mIL-15/mIL-21 double positive cells in mIL-15/IL21 TILs. Non-transduced cells were used as negative controls. Expression of mIL-15/mIL-21 promotes CD8 T cell expansion during REP

In the harvested REP cells, an increase in the percentage of CD8+ T cells was detected in TILs transduced with mIL-15, mIL-21, and mIL-15/IL-21 lentiviruses (Figure 2B and Figure 2C). This is consistent with previous reports that demonstrated that IL-15 and IL-21 act as potent stimulators that preferentially stimulate the proliferation and survival of CD8 T cells. Phenotype of REP TILs expressing mIL-15/mIL-21

To characterize the immunomodulatory effects of mIL-15 and mIL-21 on REP cells, phenotypic analysis was performed using freshly thawed REP cells in selected CD8+CD3+ T cells (Figure 3) and CD4+CD3+ T cell subsets (Figure 4). We found that mbIL-15 significantly downregulated CD25 (IL-2Rα) expression. Similar to IL-2, IL-15 competes for the use of IL-2Rβ and IL-2Rγ, suggesting a negative feedback mechanism for the regulation of CD25. In addition, mIL-15 appears to activate T cells, characterized by higher expression of TIM3 and TOX, while IL-21 exhibits an antagonist effect. In addition, both mIL-15 and mIL-21 downregulated the expression of Eomes. No significant differences in the expression of other surface markers (such as PD-1, CD27, CXCR3, etc.) were observed. Example 5 : Preparation and characterization of modified TILs with membrane-anchored IL-15 and IL-21 immunomodulatory fusion proteins

For this study, modified TILs with membrane-anchored IL-15 and/or IL-21 immunoregulatory fusion proteins will be prepared, wherein the expression of membrane-anchored immunoregulatory IL-15 and IL-21 is controlled using the NFAT promoter, which includes a NFAT responsive element linked to a minimal human IL-2 promoter, see, e.g., Table 23. The modified TILs will be characterized as described below. Materials and Methods Virus Preparation and T Cell Transduction

To prepare lentivirus, pLenti-vectors containing tethered interleukin gene sequences (Table 22) and encapsulation helper vectors (BaEV-TR, Gag/Pol) were co-transfected into 293T cells. Lentiviral supernatants were collected from day 2-3 culture supernatants and then ultracentrifuged (120,000 g) to concentrate lentivirus for TIL transduction. 1E5 pre-REP cells were added to a 48-well plate pre-coated with Retronectin along with the concentrated lentivirus. Two days after gene transduction, pre-REP cells were harvested for REP procedures or other phenotypic characterization and functional assays. Flow cytometry

To examine the expression of mIL-15 in transduced cells, transduced cells were stained with biotin-conjugated IL-15 (biolgend, San Diego, CA) linked to avidin-BV421 (Biolegend, San Diego, CA). To examine the expression of mIL-21, transduced cells were stained with PE-conjugated IL-21 antibody. To examine T cell division, T cells were pre-labeled with CellTrace™ purple cell proliferation dye (ThermoFisher scientific, Waltham, MA). After 5 days, T cell division was analyzed in ex vivo culture in the presence or absence of IL-2 (20 IU/mL). To examine T cell activation and differentiation, phenotypic characterization was performed using antibodies from BD Bioscience, Biolegend, and Thermofisher. Data were acquired using a BioRad ZE5 flow cytometer and analyzed using FlowJo software (FlowJo, LLC, Ashland, OR). T cell count and viability

REP cells were harvested after TIL expansion. Viable cell counts were assessed by AOPI analysis using a Cellometer K2 cell counter (Nexcelom, Lawrence, MA). Pre-REP and REP Process

Human tumor samples were cut into approximately 3 mm pieces and cultured in Grex10 with the recommended medium containing IL-2 (6000 IU/mL). After 11 days of culture, pre-REP cells were harvested for 2 days of activation and 2 days of lentiviral transduction. The transduced pre-REP cells were then propagated for another 11 days in the REP expansion process with irradiated PBMC, anti-CD3 antibody, and 3,000 IU/mL IL-2. Results Expression of mIL-15/mIL-21 after gene transduction

After gene transduction, transduced pre-REP cells were expanded ex vivo for another 5 days with 300 IU/mL IL-2. Transduced pre-REP cells were evaluated for expression of mIL-15 and mIL-21 by flow cytometry staining. Non-transduced cells were used as negative controls. mIL-15 pre-REP cells exhibited sustained activation of pSTAT5 signaling and displayed increased proliferation

After verification of surface interleukin expression, T cell functionality was examined. Specifically, STAT5 phosphorylation of modified TILs was assessed under serum starvation conditions to determine whether the modified TILs expressed functional mIL-15 and/or IL-21. In addition, cell proliferation of mIL-15 lentivirally transduced TILs was examined by the CellTrace proliferation assay. Expression of mIL-15/mIL-21 after the REP process

Following lentiviral transduction, TILs were expanded for 11 days during the REP process and then assessed for surface expression of membrane-bound interleukin receptors by flow cytometry. Harvested REP cells were assessed for CD8+ T cells. Previous reports have demonstrated that IL-15 and IL-21 act as potent stimulators that preferentially stimulate the proliferation and survival of CD8 T cells. Phenotype of REP TILs expressing mIL-15/mIL-21

To characterize the immunomodulatory effects of mIL-15 and mIL-21 on REP cells, phenotypic analysis was performed using freshly thawed REP cells in the selected CD8+CD3+ T cell and CD4+CD3+ T cell pools. The following expressions will be evaluated: CD27, PD1, TIM3, CD62L, TOX, T-bet, CD38, CXCR3, Eomes, and CD25. Example 6 : Preparation and characterization of modified TIL expressing tethered IL-12 and reduced PD-1 expression under the control of the NFAT promoter

For this study, modified TILs with a membrane-anchored IL-12 immunoregulatory fusion protein and one or more shRNAs for reducing endogenous PD-1 expression will be prepared. FIG. 5 depicts an exemplary nucleic acid construct that allows expression of membrane-anchored IL-12 (TeIL-12) and PD-1 shRNA. As shown in FIG. 5 , expression of membrane-anchored immunoregulatory IL-12 is controlled using the NFAT promoter, which includes a NFAT response element linked to a minimal human IL-2 promoter, see, e.g., Table 23. The shRNA is under the control of the PolIII promoter (U6). Virus preparation and T cell transduction

To prepare lentivirus, pLenti-vectors containing tethered interleukin gene sequences (Table 23) and PD-1 shRNA (Table 9) and encapsulation helper vectors (BaEV-TR, Gag/Pol) were co-transfected into 293T cells. Lentiviral supernatants were collected from day 2-3 culture supernatants and then ultracentrifuged (120,000 g) to concentrate lentivirus for TIL transduction. 1E5 pre-REP cells were added to a 48-well plate pre-coated with Retronectin along with the concentrated lentivirus. Two days after gene transduction, pre-REP cells were harvested for use in the REP process described herein or other phenotypic characterization and functional analysis methods. Flow cytometry

Immunofluorescence staining and flow cytometry were used to evaluate the expression of TIL TeIL-12 and PD-1. T cell count and survival rate

REP cells were harvested after TIL expansion. Viable cell counts were assessed by AOPI analysis using a Cellometer K2 cell counter (Nexcelom, Lawrence, MA). Pre-REP and REP Process

Human tumor samples were cut into approximately 3 mm pieces and cultured in Grex10 with the recommended medium containing IL-2 (6000 IU/mL). After 11 days of culture, pre-REP cells were harvested for 2 days of activation and 2 days of lentiviral transduction. The transduced pre-REP cells were then propagated for another 11 days during REP expansion with irradiated PBMCs, anti-CD3 antibodies, and 3,000 IU/mL IL-2. Example 7 : Preparation and characterization of modified TIL expressing tethered IL-12 under the control of the NFAT promoter

FIG6 shows an exemplary TIL expansion process comprising transduction of a lentiviral vector comprising a nucleotide sequence encoding TeIL-12 controlled by an EF-1α promoter or a NFAT promoter comprising a NFAT response element linked to a minimal human IL-2 promoter.

After 11 days of pre-REP in cell culture medium including 6000 IU/mL IL-2, TILs were stimulated with TransACT at a ratio of 1:100 in the presence of 10 ng/mL IL-7 and 20 ng/mL IL-15 for 2 days.

Rotational transductions were performed in RetroNectin® coated plates using lentivirus in CM2 + IL-7 and IL-15 + Lentiboost-P at 1:100. Following transduction, TILs were then left to rest for 2 days before the REP process was performed in GREX-24 using 25K TILs, 5E6 feeder cells in CM2 + 3000 IU/mL IL-2 + 30 ng/mL OKT3 (4 mL total volume). After 5 days of incubation, an additional 4 mL of CM2 + 3000 IU/mL IL-2 was added to each well. Incubations were continued for a total of 11 days before TILs were harvested.

After REP harvest, the surface expression of IL-12 on TILs was examined by flow cytometry using IL-12P70 flow antibody (Figure 7A). TILs were also stimulated with TransACT or PMA. 48 hours after stimulation, the surface expression of IL-12 was examined by flow cytometry (Figure 7B).

The IL-12 activity of TIL expressing TeIL-12 was also analyzed (Figure 53). 5E4 HEK-IL18 reporter cells were seeded in DMEM + 10% FBS medium in a 96-well plate. After attachment, transduced TIL were seeded at 2.5E5, 5E4 and 2.5E4 cells/well at a ratio of 5:1, 1:1 and 0.5:1, respectively. Transwell was set up. After overnight incubation, 20 µl of culture supernatant was collected and transferred to another 96-well plate. 180 µL Quantiblue reagent was added to each well and incubated at 37°C until the highest IL-12 standard turned dark purple.

Five tumor samples (2 lung, 1 head and neck, 1 breast and 1 ovary) were used to generate TILs in this study. Figure 9 shows the expansion fold (A) and survival rate (B) of TILs after REP. Figure 10 shows the expression frequency of TeIL-12 detected by digital PCR (A) and the number of viral copies per cell (B).

Cytotoxicity of TIL expressing TeIL-12 was assessed by KILR® THP-1 assay. Post-REP TIL (1E5) and KILR-THP-1 cells (1E4) were co-cultured at a ratio of 10:1 in 96-well plates. After 24 hours of incubation, supernatants were harvested for cytotoxicity analysis. THP-1 target cell death was detected using KILR probe (Figure 11A and Figure 11B). IFN-γ production (Figure 11C and Figure 11D) and IL-12 shedding (Figure 56E) in culture supernatants were tested by ELISA. Both TeIL-12 and NFAT-driven induced TeIL-12 TILs showed improved killing activity in THP-1-based allogeneic cytotoxicity assays. A >30-fold increase in IFN-γ was detected in the co-culture assay. TeIL-12 shedding occurred during co-culture of TeIL-12 TILs and KILR® THP-1 cells.

The cytotoxicity of TIL expressing TeIL-12 was also assessed by the xCelligence RTCA assay. 10,000 target cells #1 were seeded in RTCA E plates. After overnight incubation, REP-post-TIL were added at a ratio of 10:1 or 3:1. Cell growth was dynamically monitored by the xCELLigence RTCA instrument (Figure 12A). The same assay was performed with target cells #2 (Figure 12B). TeIL-12 and NFAT TeIL-12 TILs exhibited excellent allogeneic cytotoxicity.

To test the ability of continuous killing, REP TILs were repeatedly stimulated with TransACT (1:100) and harvested 2 days after the last TransACT stimulation (Figure 13A). KILR® THP-1 cytotoxicity assay, IFN-γ quantification (Figure 13B), and xCELLigence RTCA killing assay (Figure 13C) were performed to evaluate TIL killing efficacy.

The CD8+, CD4+, and CD4+FoxP3+ T cell subsets of TILs after REP were analyzed by flow cytometry (Figure 14). No differences were observed in the distribution of CD8+, CD4+, and CD4+FoxP3+ T cell subsets.

The expression of Tcm (CCR7+ CCR45RA-), Tem (CCR7-CD45RA-), Tema (CCR7-CD45RA+) subsets and CD62L on TILs after REP was detected by flow cytometry (Figure 15). No statistically significant differences were observed in the distribution of Tcm, Tem and Tema. TeIL-12/NFAT-TeIL-12 TILs after REP showed less differentiation and increased CD62L expression.

The expression of T cell depletion markers PD-1, LAG-3, Tim-3, and TIGIT on TILs after REP was detected by flow cytometry (Figure 16). No significant changes in the expression of PD-1 and LAG-3 were observed. A decrease in the expression of Tim-3 and TIGIT was observed, which was more obvious in TIGIT.

The expression of T cell activation markers CD25, CD38, CD39, and CD69 on TIL after REP was detected by flow cytometry (Figure 17). Increased expression of activation marker CD25 was observed in TIL after TeIL-12 REP (CD8+ T cells), while decreased expression of CD25 was observed in TIL after NFAT-TeIL-12 REP. No change in CD38 expression was observed, while CD69 expression was decreased.

The expression of T cell function-related markers IFN-γ, TNF-α, CD107a and granzyme B in TIL after REP was detected by flow cytometry (Figure 18). Increased IFN-γ and granzyme B production was observed in TIL after TeIL-12 REP, and was more prominent in the CD8+ T cell population. Example 8 : Optimization of anti -CD3 antibody timing and dosage during REP and its effect on the expansion and surface expression of tethered IL-15/IL-21

This study tested the effect on TIL expansion and TeIL-15/TeIL21 surface expression by adding anti-CD3 antibody at different days after REP initiation. The above example was modified as described herein in terms of the choice of the time of adding anti-CD3 antibody; the time of adding anti-CD3 antibody is shown in the following paragraph.

Pre-REP TILs were prepared using the Gen 2 process described in Example 1, and then transduced with a lentiviral vector containing nucleotide sequences encoding TeIL-15 or TeIL-15 and TeIL-21 under the control of the EF-1α promoter using the following protocol. Viral transduction of TILs

TILs were thawed by adding 1 mL of thawed cell suspension to 9 mL of pre-warmed CM2 without tamiprocin and centrifuged at 500 × g for 4 min at room temperature (RT).

The supernatant was discarded and the TILs were suspended in 1 mL of CM2 without tamiprocin + 300 IU/mL IL-2 and counted.

TILs were suspended at 1e6/mL in CM2 without tamiprocin + 300 IU/mL IL-2 in a 24-well plate, 2 mL per well. Incubate overnight.

Prepare 20x IL-15, IL-7 mix and add 100 uL to each well. , add 100 uL to each well. ○ Final concentration: IL-15 (10 ng/mL), IL-7 (20 ng/mL) in CM2 without transactin ○ Example: 10 wells are needed ■ 10 wells x 100 uL mix = 1 mL ● Add IL-15 to a concentration of 200 ng/mL ● Add IL-7 to a concentration of 400 ng/mL ○ Note: Before adding TransACT, IL-15, and IL-7, remove 120 uL from each well to a final volume of 2 mL

Add 100 uL of 20x interleukin mix to each TIL culture in a 24-well plate. Next, add 20 uL of TransACT to each well. Gently mix each well with a pipette to evenly disperse the TILs and mix with the stimulation mix. Incubate for 2 days.

Non-tissue culture 48-well plates were coated with Retronectin (1:100 dilution in PBS) by adding 250 uL per well. Plates were wrapped with parafilm and then incubated overnight at 4°C.

Remove coating solution from retronectin plate and replace with 250 uL 2% BSA Part V. NOTE: Add 2% BSA Part V immediately to prevent wells from drying out.

Incubate at room temperature for 30 minutes for blocking.

Remove blocking solution. Add calculated volume of viral supernatant and cover with parafilm. Centrifuge at 2000 × g for 90 min at 32°C. Note: Preheat the centrifuge before adding the culture plate.

While the plate was spinning with the virus, TILs were collected and counted. TILs were suspended at 1e6/mL in CM2 without tamiprocin.

Once the plate has been spun, add the following: 5. 100 uL TIL suspension (1e5 cells) 6. 200 uL CM2 without tetanusin 7. 300 uL CM2 + IL-15 (20 ng/mL) + IL-7 (40 ng/mL) + Lentibooster 1:50 8. = 600 uL total per well

Centrifuge at 2000 × g for 20 minutes at 32°C. Place in an incubator and incubate for 3 days.

After transduction of pre-REP TILs with a lentiviral vector encapsulating a recombinant lentiviral RNA molecule capable of expressing TeIL-15, pre-REP TILs were treated with feeder cells, 3000 IU/ml IL-2, and anti-CD3 antibodies OKT3 or HIT3a for REP expansion. OKT3 (30 ng/ml) or HIT3a (30 ng/ml) was added to the REP medium at different days after the start of the REP process (day 0, day 2, and day 4). Post-REP TILs were harvested after 11 days of REP expansion. TIL expansion and surface expression of TeIL-15/TeIL-21 were analyzed using the following protocol. Addition of OKT3 or HIT3a on day 2 or day 4 resulted in increased surface expression of TeIL-15 (Figures 19A and 19B). Surface expression of tethered interleukins ( diluted 2x with 1E5 ) Maker 20 samples 25ul/sample X10 487.5ul DPBS Live/Dead 1:200 2.5ul FC Block 1:50 10ul 8 minutes at room temperature 50ul staining buffer 50ul staining buffer 20 samples 860ul CD3 BUV737 UCHT1 1.5ul 30ul CD45 PerCP HI30 1ul 20ul IL-21 PE 4BG1 2.5ul 50ul IL15 Biotin BH1543 2ul 40ul AB incubate at 4C for 45 minutes, wash twice 50ul staining buffer 20 samples 992ul Avidin-BV421 1:200 0.25ul 5ul AB Incubate at 4C for 20 minutes, wash twice, add 200ul staining buffer, and use for flow cytometry analysis

After transduction of pre-REP TILs with lentiviral vectors encapsulating recombinant lentiviral RNA molecules capable of expressing TeIL-15 and TeIL-21, pre-REP TILs were treated with feeder cells, 3000 IU/ml IL-2, and anti-CD3 antibodies OKT3 or HIT3a for REP expansion. OKT3 (30 ng/ml) or HIT3a (30 ng/ml) was added to the REP medium at different days after the start of the REP process (day 0, day 2, and day 4). Post-REP TILs were harvested after 11 days of REP expansion. TIL expansion and surface expression of TeIL-15/TeIL-21 were analyzed using the above protocol. Addition of OKT3 or HIT3a on day 2 or day 4 resulted in increased surface expression of TeIL-15 and TeIL-21 ( FIGS. 20A and 20B ).

After transduction of pre-REP TILs with lentiviral vectors encapsulating recombinant lentiviral RNA molecules capable of expressing TeIL-15, pre-REP TILs were treated with feeder cells, 3000 IU/ml IL-2, and anti-CD3 antibody OKT3 for REP expansion. OKT3 was added to REP medium at different concentrations (30 ng/mL, 10 ng/mL, 5 ng/mL, 3 ng/mL, and 1 ng/mL) on day 0. Post-REP TILs were harvested after 11 days of REP expansion. TIL expansion and surface expression of TeIL-15/TeIL-21 were analyzed using the above protocol. Addition of 30 ng/mL of OKT3 resulted in the highest surface expression of TeIL-15 (Figures 21A and 21B).

After transduction of pre-REP TILs with lentiviral vectors encapsulating recombinant lentiviral RNA molecules capable of expressing TeIL-15 and TeIL-21, pre-REP TILs were treated with feeder cells, 3000 IU/ml IL-2, and anti-CD3 antibody OKT3 for REP expansion. OKT3 was added to REP culture medium at different concentrations (30 ng/mL, 10 ng/mL, 5 ng/mL, 3 ng/mL, and 1 ng/mL) on day 0. Post-REP TILs were harvested after 11 days of REP expansion. TIL expansion and surface expression of TeIL-15/TeIL-21 were analyzed using the above protocol. Addition of OKT3 at all concentrations resulted in low surface expression of TeIL-15 and TeIL-21 (Figures 22A and 22B). Example 9 : Optimization of transduction conditions for TeIL -12 expression

Pre-REP TILs were activated with or without TransACT, then transduced with a lentiviral vector encapsulating a recombinant lentiviral RNA molecule capable of constitutively expressing TeIL-12, and then subjected to the REP process. Surface expression of TeIL-12 was examined by flow cytometry ( FIG. 23 ).

Activated pre-REP TIL were transduced with lentiviral vectors encapsulating recombinant lentiviral RNA molecules capable of constitutively expressing TeIL-12 in retronection or vectofusin coated plates, with or without centrifugation. After REP expansion, surface expression of TeIL-12 was examined by flow cytometry. Transduction efficiency was better with retronectin coated plates than with vectofusin coated plates (Figure 24). Transduction without centrifugation reduced transduction efficiency (Figure 24).

Lentiboost was added during the rotational transduction of activated pre-REP TILs with lentiviral vectors encapsulating recombinant lentiviral RNA molecules encoding NFAT-TeIL-12. Transduction efficiency was assessed by examination of surface TeIL-12 expression after TransACT or PMA-mycin stimulation. Transduction with or without Lentiboost showed similar TeIL-12 surface expression (Figure 25).

Lentiboost was added during the rotational transduction of activated pre-REP TILs with lentiviral vectors encapsulating recombinant lentiviral RNA molecules encoding NFAT-TeIL-12. Transduction efficiency was assessed by examination of surface TeIL-12 expression after TransACT or PMA-mycin stimulation. Transduction using BaEVTR Env plasmids resulted in more TeIL-12 surface expression compared to transduction using RD114 or VSV-G Env plasmids (Figure 26). Example 10 : NFAT-TeIL-12 engineered TILs show excellent cytotoxicity

Tumor tissues from several solid tumor types were fragmented and cultured, then transduced with lentivirus containing the gene encoding membrane-bound IL-12 (TeIL-12) via the NFAT promoter, followed by expansion using the rapid expansion protocol (REP). The expression, biological function, and shedding of the IL-12 molecule were evaluated in vitro. The immunophenotype and in vitro cytotoxic activity of TeIL-12 genetically engineered TILs were examined in various assays.

Lentiviral gene transfer caused TILs to express IL-12 on their surface via membrane anchoring. In assays using HEK-IL-12-Blue reporter cells, this modification enhanced activation of signaling downstream of the IL-12 receptor in a contact-dependent manner. TeIL-12-TILs exhibited increased IFN-γ production and superior cytotoxicity in the KILR® assay. xCELLigence-based cytotoxicity assays further confirmed the increased killing of unstimulated or stimulated TILs. In addition, phenotypic analysis revealed that TeIL-12-TILs were less differentiated, had reduced expression of immunosuppressive receptors, and increased production of cytotoxic molecules associated with anti-tumor activity. Shedding of IL-12 in co-culture supernatants was minimal.

KILR® THP-1 cells (1E4) were co-cultured with 5 individual REP-TILs (1E5) engineered with NFAT-TeIL-12, mock, pLV-ctrl, and mock control cells. After 24 hours, the supernatant was harvested. Cytotoxicity was then assessed using the KILR® THP-1 cytotoxicity assay ( FIG. 27A ), and IFN-g was quantified by ELISA ( FIG. 27B ).

Freshly thawed REP-TIL or REP-TIL stimulated 3 times with TransACT were added to E-plates pre-seeded with target cells (CaOV3). Target cell growth was dynamically monitored by xCELLigence RTCA analysis. TIL expressing NFAT-TeIL-12 showed excellent cytotoxicity (Figure 27C) and continuous killing efficacy (Figure 27D).

Enhanced in vitro killing activity, coupled with a less differentiated phenotype of TILs with inducible and membrane-bound IL-12, has been shown to improve clinical efficacy. In addition, minimal IL-12 shedding supports the possibility of reducing IL-12-related toxicity (Zhang et al., Clin Cancer Res . 2015 ; 21:2278-88). Example 11 : TILs engineered with NFAT-TEIL-12 and IL-15/IL-2 , PD-1 shRNA

To improve the in vivo expansion capacity of NFAT-TeIL-12 TILs and potentially avoid the use of aldesleukin in patients after TIL therapy, NFAT-TeIL-12 TILs were further engineered to co-express IL-15 or IL-2 (with or without membrane anchor) under the control of the EF-1α promoter (Figure 28). Co-expression of PD-1 shRNA was also tested to improve TIL function in the tumor microenvironment (TME). tCD19 was co-expressed under the same EF-1α promoter and can be used as an indicator of the expression of IL-15, IL-2 or PD-1 shRNA.

After lentiviral vector (LV) transduction and 11 days of REP expansion, gene-edited TILs were harvested. Viable cell counts were performed and fold expansion was calculated based on the initial transduction pre-REP TILs ( FIG. 29A ); survival was examined in Celica ( FIG. 29B ).

Transduction efficiency was assessed by flow cytometry analysis of the percentage of TeIL-12+/tCD19+ and TeIL-12-/tCD19+ TILs under unstimulated conditions ( FIG. 30A ). Virus copy number per cell (VCN) was detected by ddPCR ( FIG. 30B ).

TCR signaling activator TransACT (1:1000, 1:100, 1:10) and PMA/myosin were used to activate the downstream NFAT signaling pathway, thereby sequentially upregulating NFAT-driven TeIL-12 expression in gene-edited TILs. At the same time, tCD19 was used as an indicator of TeIL-2, TeIL-15, IL-2, IL-15, and PD-1 shRNA expression under the EF-1a promoter. Co-staining of the surface expression of TeIL-12 and tCD19 (known as induced TeIL-12-positive cells in gene-edited TILs) was analyzed by flow cytometry. The percentage of TeIL-12 and tCD19 double-positive T (TeIL12+CD19+) depends on the degree of TCR stimulation (Figure 31).

xCELLgeneRTCA killing assay was performed to evaluate the killing efficacy of gene-edited TIL targeting CaOV3 cells at an effector to target (E/T) ratio of 0.3:1. TIL engineered with NFAT-TeIL-12 and EF-1α-TeIL-15 exhibited excellent cytotoxicity (Figure 32).

Gene-edited TILs were co-cultured with CellTrace-labeled mock TILs (1:1) in serum-free AIM-V in 96-well U-bottomed plates overnight. After washing, fixation, and permeabilization, intracellular staining of phosphorylated STAT5 in gated cells was analyzed by flow cytometry. Simultaneously, mock cells treated with IL-2 or IL-15 were used as positive controls for phosphorylated STAT5 staining. TeIL-2 and TeIL-15 acted primarily in a cis-like manner (Figure 33).

Gene-edited TIL cells were labeled with CellTrace Blue and cultured in medium without IL-2. After 96 hours, cell divisions of tCD19+ (interleukin-transduced) and tCD19- (interleukin-untransduced) subsets were analyzed based on cell tracer. TeIL-2 and TeIL-15 showed skewed cis-proliferation, consistent with p-STAT5 activation (Figure 34).

Gen2 TILs were labeled and then stimulated with TILs expressing soluble IL-15 or TeIL-15. p-stat5 activation was examined by flow gating on Gen2 TILs and TeIL-15 TILs. The data showed that TeIL-15 activated T cells only in a cis-like manner (Figure 54).

Gene-edited TILs were washed and resuspended in CM2 without IL-2. 5E5 cells/well were added to GREX24-well plates. Treatments were divided into the following three groups: 1) TransACT plus IL-2; 2) TransACT alone; 3) no stimulation. Cell counts and cell viability were examined in Celica. EF-1a-driven IL-2/IL-15 improved T cell proliferation in the absence of IL-2 (Figures 35A and 35B).

The frequency of PD-1 expression on NFAT-TeIL-12 TILs expressing PD-1 shRNA under titrated TransACT stimulation conditions was compared with that of NFAT-TeIL-12 TILs expressing GFP shRNA as a control group. The gMFI of PD-1 expression on NFAT-TeIL-12 TILs expressing PD-1 shRNA under titrated TransACT stimulation conditions was compared with that of NFAT-TeIL-12 TILs expressing GFP shRNA as a control group. PD-1 shRNA effectively reduced PD-1 positivity in NFAT-TeIL-12 TILs expressing PD-1 shRNA (Figures 36A and 36B).

T cell survival and TVC were dynamically examined under different environments: TransACT stimulation (1:100) plus 3000 IU/ml IL-2 (Figure 37A); 3000 IU/ml IL-2 only (Figure 37B); TransACT stimulation only (1:100) (Figure 37C); and no TransACT stimulation, no IL-2 (Figure 37D). The data showed that TeIL-15 can promote TIL survival in vitro in the absence of IL-2. Example 12 : In the M1152 PDX model, NFAT-TeIL-12 genetically engineered TIL showed improved in vivo efficacy compared to Gen2 processed TIL

The M1152 PDX ACT model was used to test the in vivo efficacy of NFAT-teIL-12 TIL (see Figure 38A). 1E6 M1152 tumor cells were subcutaneously inoculated into NOG-hIL2 mice. 26 days later, 7.5E6 R-REP TILs with different processes were infused into M1152 mice via the intravenous route. The various processes are as follows: i. Gen2 ii. pLV-ctrl transduction followed by Gen2 iii. NFAT-TeIL-12-tCD19 transduction followed by Gen2 iv. NFAT-TeIL-12 transduction followed by Invigo-T+L-Arg process

Tumor size and mouse weight were checked twice a week until the mice reached the end point. On day 7 after TIL infusion, PB was collected for plasma interleukin examination (see Figure 38A).

Compared with the untreated group, TILs produced by the Gen2 process and the Gen2-pLV-ctrl process showed tumor inhibition effects; however, the tumor still grew progressively (see Figure 38B). However, NFAT-TeIL-12 genetically engineered TILs produced by the Gen2 process or the Invigo-T+L-arginine process controlled tumor growth (see Figure 38B). 3/7 mice in the NFAT-TeIL-12-tCD19 group showed complete regression, and 1/7 mice in the Invigo-T-modified-NFAT-TeIL-12 group showed complete regression (see Figure 38B).

The dynamic changes of body weight (Figure 39A) and the % body weight relative to baseline (Figure 39B) were examined. After TIL infusion, no significant changes in body weight were observed in the untreated group, Gen2 TIL, and TeIL-12 TIL-treated groups.

Peripheral blood was collected on day 7 after TIL infusion. Plasma IFNγ, TNFα, CCL4, and IL-12 p70 were examined by Bioplex analysis. Circulating IL-12 p70 and TNFα were not detected in NFAT-TeIL-12 TIL-infused mice. An increase in plasma IFNγ was observed in the NFAT-TeIL-12 TIL-treated group (47.9 pg/l) compared to the Gen2 control TIL (20.3 pg/ml). No significant difference in CCL4 was observed between the NFAT-TeIL-12 TIL-treated group (13.92 pg/ml) and the Gen2 TIL-treated group (13.27 pg/ml). See Figure 40. Example 13 : Tumor digests primed with IFNγ after the pre- REP phase significantly increase responsiveness

Tumors were digested into single cell suspensions and plated at 600k cells in 96-well flat bottom plates. Cells were then cultured for 11 days under the following conditions: IL2 (6000 IU/mL) control, ITIL interleukins (IL-2 (6000 IU/mL), IL-15 (10 ng/mL) and IL-21 (10 ng/mL)) or ITIL interleukins + IFNγ (200 ng/ml) or ITIL + anti-PD1 (pembrolizumab) 10 ug/mL or ITIL interleukins + anti-PD1 + IFNγ in complete medium. Medium was changed every 2-3 days. After 11 days, cells were counted and co-cultured with autologous tumor digest (100k TIL: 100k tumor digest cells) for 24 hours. To determine the percentage of tumor digest-reactive T cells, cells were stained for the activation marker 4-1BB (Biolegend, clones: 4B4-1, BV421), and flow cytometry was run on a Bio-Rad ZE5 flow cytometer to quantify the percentage of 4-1BB+ reactive T cells. As shown in Figure 41, tumor responsiveness was enhanced when TIL was cultured with ITIL interleukins in the presence of IFNγ. Example 14 : Sorting of tumor-reactive CD8 TIL using the T cell activation marker 4-1bb

4-1BB is known to be upregulated upon TCR triggering, particularly on CD8 T cells, but also on a subset of CD4 T cells. However, 4-1BB is only transiently expressed, so experiments were performed to determine the optimal time point for sorting TILs based on 4-1BB expression. In this experiment, tumor digests from five NSCLC tumors and five renal cell carcinoma tumors were plated at 6e5 cells in cell culture medium (AIM V) supplemented with IL2 (3000 IU/mL) and IFNγ (200 ng/mL) in 96-well flat bottom plates. As shown in Figure 42A, it was found that 4-1BB expression reached a maximum 24 hours after application of tumor digests and returned to the baseline (t = 0) level 48 hours after application. Therefore, 24 hours was identified as the optimal time point for sorting tumor-responsive TILs.

To demonstrate the importance of adding IFNγ to maximize 4-1BB expression on tumor digest-reactive TILs, tumor digest cells (600k in 96-well plates) from eight NSCLC tumors were plated in control medium (3000 IU/mL of IL-2) or TS-TIL medium (IL-2 3000 ng/mL, IL-21 10 ng/mL + IFNγ 200 ng/mL). 24 hours later, 4-1BB expression on CD8+ TILs was measured again using flow cytometry. As shown in Figure 42B, 4-1BB expression was found to increase with TS-TIL conditions relative to control conditions (IL-2 only). This experiment highlights the importance of adding IFNγ to the culture medium to enhance the recovery of tumor digest-reactive TILs via increased 4-1BB expression. In an independent experiment, post-REP TILs from five patients were plated at 2e6 cells in 24-well plates in culture medium supplemented with IL-2 (with or without IFNγ) for 24 hours. Notably, IFNγ did not cause a non-specific increase in the percentage of 4-1BB+ CD8 T cells, suggesting that the upregulation may be a response to increased TCR triggering as a result of IFNγ-mediated enhancement of antigen presentation in tumor digests (data shown). Example 15 : Comparison of the ability of several methods to enrich tumor-reactive CD8 TILs

Several methods were analyzed for their ability to enrich for tumor-reactive CD8 TILs, as summarized in Figure 43. In all cases, tumor digests were generated as described previously, and tumor digests were initially plated at 550k cells in 96-well plates and expanded in CM2 medium containing the indicated interleukins. IL2 control: Pre-REP = 6000 IU/mL IL-2 and REP = 3000 IU/mL IL-2 CD103+ enrichment: Pre-REP = 6000 IU/mL IL-2 and REP = 3000 IU/mL IL2 or IL-2 (1000 IU/mL) + IL-15 (10 ng/mL) + IL-21 (10 ng/mL) 4-1BB+ enrichment: Pre-REP = IL-2 (1000 IU/mL) + IL-15 (10 ng/mL) + IL-21 (10 ng/mL) and REP = IL-2 (1000 IU/mL) + IL-15 (10 ng/mL) + IL-21 (10 ng/mL) (these are called "TS-TIL" interleukins).

Media was changed every 2-3 days and cells were split as needed to maintain viable cell populations of less than 400k and more than 50k 3 days after initial tumor digest plating at the pre-REP expansion stage. All pre-REP populations were rapidly expanded (REP) in 24-well GREX plates in the presence of 5e5 irradiated donor PBMCs from Biolegend and 30 ng/mL OKT3 by transferring 25k cells from the pre-REP population into REP.

A bulk TIL control arm was used as shown on the left side of the process flow diagram in Figure 43. For the control arm, TILs from the digest were expanded for 10 days during the pre-REP period and then rapidly expanded for 10 days during the REP period.

For CD103 enrichment, the same day 10 pre-REP cell population was used as a control population. However, on day 10, these cells were flow sorted into four different populations: CD103+CD31+, CD103+CD31-, CD103-CD31+, and CD103-CD31-. These populations were then subjected to individual REP.

For the TS-TIL process, cells were plated in TS-TIL interleukin plus IFNγ (200 ng/mL) for the first 24 hours. They were then sorted using 4-1BB+ magnetic beads (Miltenyia kit) at 24 hours. Sorted cells were then expanded for another 9 days in 6e6 T cell depleted feeders (iPBMC) in the presence of TS-TIL interleukin in 24-well GREX without additional IFNγ (1XREP) during the pre-REP period in 24-well GREX, or alternatively, sorted cells were immediately REPed. On day 10, the two populations were then REPed in TS-TIL cytokines, resulting in two different populations: 1X REP and 2X REP 4-1BB sorted cells. See Figure 43, right.

On process day 20, after REP was completed, all populations were counted, phenotyped for surface marker expression, and autologous tumor responsiveness was analyzed using intracellular interleukin staining as follows:

Autologous tumor reactivity: 200k TIL were plated for seven hours in the presence of 200k digest cells in the presence of monensin (Biolegend), brefeldin A (Biolegend), and anti-CD107a BUV395 antibody (BD Biosciences). After 7 hours, cells were surface stained for CD8, CD4, CD3, and CD45, then fixed, permeabilized, and stained for IFNγ (PE) and TNFα (BV650) and analyzed on a Bio-Rad ZE5 cytometer. For appropriate controls, each TIL population was evaluated under the following conditions: 1) TIL + tumor digest 2) TIL + tumor digest + anti-MHC class I antibody (W6/32) 100 ug/mL to account for non-TCR-mediated (background) CD107a, IFNγ and/or TNFα expression 3) TIL alone (negative control) 4) TIL + TransActCD3/CD28 stimulator (positive control)

The flow data were then gated, processed and analyzed in FlowJo and then plotted in GraphPad Prism. Even with fewer cells driven by a high percentage of tumor-reactive TILs resulting from the CD137 (4-1BB) sorting process, an approximately 10-fold increase in product potency was observed. Note that the CD103-CD31- group had minimal tumor reactivity, representing a bystander group. See Figure 44. Example 16 : Bivariate Flow Cytometric Data from Intracellular Interleukin Tumor Digests (IFNγ and CD107a )

As previously described, bivariate flow cytometry data (IFNγ and CD107a) were analyzed from intracellular interleukin tumor digests. The graphs were gated on live CD8+CD3+ T cells "CD8 TIL". Results are GEN2 (left) and 4-1BB Sort/1x REP "TS-TIL" (right). See Figure 45. The top graph shows TIL co-cultured with autologous tumor digests. The bottom graph shows the same experimental setup, except that an MHC class I blocking antibody (W6/32) was added to account for any non-TCR-mediated (background) expression of the effector interleukin IFNγ or the degranulation marker CD107a. Minimal reactivity was observed in the figure below due to the presence of MHC class I blocking antibodies, which would block CD8 TCR stimulation by tumor antigens as expected. Little tumor-specific reactivity was found in CD8 TIL generated under the control (GEN2/host TIL) process, with only 0.58% of CD8 TIL co-expressing CD107a+ IFNγ+. Notably, reactivity was much stronger in the TS-TIL (4-1BB sorted/1x REP) process, where 11.7% of cells were found to co-express CD107a and IFNγ, an increase of nearly 20-fold. Single positive populations (IFNγ+ or CD107a+) were also increased in the TS-TIL process. See Figure 45. This indicates that the TS-TIL (4-1BB sorting/1x REP) process is enriched for tumor-reactive TIL. Example 17 : Analysis of tumors from 3 additional NSCLC patients

Tumors from three additional NSCLC patients, L4373, L4375, and L4397, were analyzed. Various REP conditions (3000 IU/mL of IL2, CTRL) and a test condition called "Invigo-T" (IL-15 (10 ng/mL) + IL-21 (10 ng/mL) + AKTi (AZD4673) 1 µM) were tested to see how this would affect the expansion of the final (post-REP) TIL production and tumor responsiveness. The 4-1BB sorting/1x REP conditions described previously were used.

GEN2 TIL control conditions vs. TS-TIL4-1BB sorting/1x REP "TS-TIL" conditions vs. CD103 pre-REP post-sorting were tested in three NSCLC patients. For 4-1BB sorting, the effects of the above REP conditions on CD8 tumor-specific reactivity were tested. As before, CD8 tumor digest reactivity was assessed by co-culturing ICS with autologous digests. The results are shown in Figure 46, varying with sorting conditions and REP conditions. Both CD137 (4-1BB) sorting conditions provided optimal enrichment of tumor-reactive CD8 (IFNγ+CD107a+) TILs in the TIL product. CD103 sorting was also improved over the GEN2 main TIL control.

To test the effect of these process changes on the enrichment of tumor-responsive TILs, bivariate flow cytometry of L4397 was analyzed. See Figure 47. In the GEN2 master TIL condition, only 0.24% of CD8 TILs were IFNγ+CD107+, while in the 4-1BB sorting/IL2 3000 IU/mL REP condition, 15.3% of CD8 TILs were IFNγ+CD107a+.

As another example, a primary NSCLC tumor from patient L4463 was digested and a TIL product was generated from only 550k viable digest cells using either the TS-TIL process "TS-TIL" (4-1BB selection) or the control process "CTRL" (host, unselected TIL, expanded with IL-2 only). As shown in Figure 48, the total processing time to produce the final TIL product was 20 days. To compare the tumor-specific reactivity of each day 20 product, 200k TILs were co-cultured with 200k autologous tumor digest cells in 250 uL of AIM V medium supplemented with 300 IU/mL IL-2, Brefeldin A, and Monensin in a 96-well plate for 7 hours. Cells were then stained for CD3, CD45, CD8, CD4, CD107a (exfoliated marker), and IFNγ (effector interleukin) using intracellular interleukin staining, and expression was measured using a Bio-Rad ZE5 flow cytometer. Tumor-specific reactivity was measured in the CD8 population by defining tumor-reactive TILs as those expressing MHC class I-dependent CD107a co-expressing IFNγ. Specifically, live IFNγ+CD107a+CD8+ TIL were measured under the following five conditions for complete analysis with appropriate controls: A) 200k TIL+ 200k autologous digest B) 200k TIL+ 200k autologous digest+ anti-MHC class I blocking antibody (pure line W6/32, Biologend) 100 ug/mL (background subtraction) C) 200k TIL alone (negative control) D) 200k TIL+ CD3/CD8 agonist TransAct (Miltenyi Biosciences, 130-111-160) 1:20 dilution (positive control) E) 200k TIL+CEF peptide mixture (1:100) (Miltenyi Biosciences, 130-098-426) (Used to assess viral reactivity in TILs).

Condition B was used to measure non-specific (i.e., non-peptide:MHC class I-mediated) expression of CD107a and IFNγ. These results are shown in FIG49 .

Specific tumor reactivity was calculated as the percentage of digest-reactive TIL in condition A minus the percentage of digest-reactive TIL in condition B. Because TransAct is a potent T cell stimulatory compound known to trigger T cell receptors (TCR) and co-stimulatory receptors (CD28) and induce secretion of IFNγ and CD107a, TIL alone was used as a negative control (absence of TCR stimulation) and TIL + TransAct was used as a positive control. In addition, TIL were cultured with a CEF peptide cocktail containing multiple known MHC class I epitopes from viral antigens (CMV, EBV, influenza) known to be recognized by T cells across multiple HLA types.

The data showed a significant increase in the percentage of specific tumor-responsive TILs in the TS-TIL product (approximately 7%) versus the CTRL product (approximately 1%). See Figure 49. As expected, both products showed minimal responsiveness when left alone in the absence of TCR stimulation, and were highly responsive when exposed to the potent TCR stimulation TransAct. Notably, the CTRL TIL product showed a higher percentage of TILs that responded to the MHC class I peptide mixture, consistent with the hypothesis that the unselected CTRL TILs contained a higher percentage of "bystander" virus-specific TILs, which were not expected to mediate tumor regression due to the inability to specifically recognize tumor cells.

The same process was repeated for five additional NSCLC patients and the results are shown in Figure 50. To calculate the total number of CD8 tumor-reactive TILs generated by 550k digest cells in each process, the following formula was used:

Total CD8 tumor-responsive production = (total number of cells produced during the pre-REP phase (days 1-10)) × tumor-specific responsiveness (AB) × expansion fold during the REP period.

The total number of CD8 tumor-reactive TILs generated by the TS-TIL process was, on average, 7 times greater than the total number of CD8 tumor-reactive TILs generated by the CTRL process. See Figure 50. Example 18 : Selection of conditions that allow for maximum expansion of tumor-specific CD8 TILs during the REP phase

To select conditions that allowed for maximal expansion of tumor-specific CD8 TILs during the REP phase, multiple REP conditions were tested. Results for total viable cells and fold expansion are shown as a function of REP condition. See Figure 51. Expansion was more robust with GEN2 (host TIL) control cells. However, in both 4-1BB and CD103 sorted populations, REP conditions using 3000 IU/mL IL-2 resulted in the best expansion of tumor-specific TILs. In contrast, REP conditions using Invigo-T (IL-15 + 1L-21 both at 10 ng/mL, L-arginine 5 mM) resulted in poorer expansion. Example 19 : Isolation of CD137 using HD IL-2 REP

Figure 52 shows the total yield of CD8+ tumor-reactive TILs (assessed by autologous digest co-culture ICS) as a function of sorting and REP conditions. Sorting for CD137 (4-1BB) followed by REP with high-dose (HD) IL-2 (3000 ng/mL) was identified as the optimal procedure for expanding tumor-specific TILs. In three patients (L4373, L4397, and L4375), increases in TS-TIL yields of 6.3, 23.7, and 4.7 fold, respectively, were observed.

Surface phenotype analysis of live CD8+ TILs was performed using the markers CD45RA and CD62L, which roughly classify CD8+ T cells into four categories: CD45RA+CD62L+ (Tscm=stem cell memory), CD45RA-CD62+ (Tcm-central memory), CD45RA-CD62L- (Tem=effector memory) and CD45RA+CD62L- (Temra=terminal effector) cells. Cells were phenotyped during ICS staining as described above. See Figure 53. Based on the results, it was found that the majority (90-95%) of cells were Tem "effector memory" cells, with little difference between TILs made using different processes. In contrast, only about 1% of CD8+ TILs were found to be Tscm cells at the end of REP. Example 20 : Expansion and characterization of TILs using metabolic reprogramming

In this study, different strategies were investigated for REP TILs to enhance their expansion and phenotype. The literature suggests that the presence of some AAs can modulate T cell metabolism and enhance anti-tumor activity (Geiger et al., Cell 2016 , 167(3):829-842; Chang et al., Cell 2015 , 162(6):1229-41; the contents of which are incorporated herein by reference in their entirety). Overall, this idea leads to reprogramming metabolism in TILs to achieve two main goals: i) anti-tumor phenotype and functional properties; and ii) survival and function under tumor microenvironment (TME) conditions. Indeed, engineering of arginine metabolism in CAR-T cells has been shown to achieve cell proliferation and therapeutic activity (Fultang et al., Blood 2020 , 136(10):1155-1160; the contents of which are incorporated herein by reference in their entirety). Materials and Methods T cell counts and survival

REP cells were harvested after TIL expansion. Viable cell counts were assessed by AOPI analysis using a Cellometer cell counter (Nexcelom, Lawrence, MA). Pre-REP and REP Process

Human tumor samples were cut into approximately 3 mm pieces and cultured in Grex10 with the recommended medium containing IL-2 (6000 IU/mL). After 11 days of culture, pre-REP cells were then expanded in the REP expansion process using irradiated PBMC, anti-CD3 antibody and Invigo-T cocktail (10 ng/ml IL-15 and 10 ng/ml IL-21) for another 11 days.

Compounds to be incorporated during REP: L-arginine: Optimized to 5 mM. Since L-arginine is a basic amino acid, it needs to be balanced with 25 mM HEPES. NAD+: Dose response curve has been plotted. Small molecule inhibitor of GSK-3α/b (SMI). Flow cytometry analysis technology

To examine T cell activation and differentiation, phenotypic characterization was performed using antibodies from BD Bioscience, Biolegend, and Thermofisher. Data were acquired using a BioRad ZE5 flow cytometer and analyzed using FlowJo software (FlowJo, LLC, Ashland, OR). Results L-arginine enhanced post-thaw expansion and cell recovery during Invigo-T REP

First, the REP process supplemented different amino acids, and L-arginine showed an increase in expansion and survival (Figure 55A-B). In addition, to mimic product standards before delivery to patients, REP TILs were frozen and thawed to analyze recovery and killing capabilities. L-arginine protected TILs from damage (Figure 55C-D).

L-arginine provides better phenotype and killing ability.

Supplementation of L-arginine during REP showed some favorable phenotypic changes analyzed by flow cytometry, such as an increase in CD127, CD62L, CD25, CD28, ICOS, and CD40L (FIG. 56A). In addition, an increase in CD4+CD107+ TILs was observed (FIG. 56B). Therefore, the cytotoxicity of TILs was tested in an allogeneic environment by co-culturing TILs with KILR THP1 cells and performing multiple TxA stimulations in REP. REP TILs expanded with L-arginine showed better killing ability (FIG. 56C).

NAD+ works synergistically with L-arginine to promote metabolism and provide unique TIL characteristics.

TME conditions are challenging for TILs because of limited nutrients and unfavorable conditions. L-arginine may serve as an energy source and help cells enhance their functions. In addition, to promote metabolism, the role of NAD+ enhancers was studied during REP. Six different NAD+ enhancers (L-Trp, NR, NMN, NAD+, NAM, and P7C3 activator) were screened, and NAD+ showed interesting phenotypes in TIL products (data not shown). A dose response of NAD+ was performed, and 50-75 μM was found to be the optimal concentration for implementing the REP process (Figure 57A-B) because it had a smaller effect on expansion but maintained a favorable phenotype. In addition, NAD+ supplementation enhanced the recovery of cells in thawed samples, and this was more pronounced after 24 hours of cell activation with TransAct (Figure 57C). Example 21 : Lentiviral transduction efficiency and phenotype of til products produced using various modified rep conditions

In this study, tumors were fragmented and cultured in CM2 containing IL-2 (6000 IU/mL) for 11 days. At the end of 11 days, cells (pre-REP TIL) were collected and either frozen or processed fresh for further manufacturing. For frozen pre-REP TIL, cells were thawed and placed in CM2 containing IL-2 (300 IU/mL) for 3 days before further manufacturing. See Figure 58.

For lentiviral transduction, 100,000 pre-REP TILs were transduced with BaEVTR enveloped virus containing a nucleic acid sequence encoding tethered IL-12 (TeIL-12) driven by the NFAT promoter, followed by a sequence encoding truncated CD19 (tCD19) or tethered IL-15 (TeIL-15). See Figure 76 for a vector map of pCMV-3Tag-4a-BaEV-TR (SEQ ID NO: 166); see Figure 77 for a vector map of pLenti-IRES-NFAT-TeIL12-EF1a-TeIL-15 (SEQ ID NO: 167). For rotational transduction, TIL and virus were added to retronectin coated plates in medium containing IL-2 (3000 IU/mL) and then centrifuged at 2000×g, 32°C for 45 minutes. After rotational transduction, TIL were cultured for 72 hours before starting REP. See Figure 58. At the same time, TIL were cultured under the same conditions before REP, but without rotational transduction. These cells are referred to as non-transduced GEN2 controls.

After lentiviral transduction, TILs were placed in REP in media containing OKT3 (30 ng/mL), irradiated allogeneic culture cells from 3 individual healthy donors, and the following: 1. GEN2: IL-2 (3000 IU/mL) 2. GEN2+NAD: IL-2 (3000 IU/mL) + NAD (50 µM) 3. ITARG: IL-15 (10 ng/mL) + IL-21 (10 ng/mL) + L-arg (5 mM) + HEPES (25 mM) 4. ITARG+NAD: IL-15 (10 ng/mL) + IL-21 (10 ng/mL) + L-arg (5 mM) + HEPES (25 mM) + NAD (50 µM) 5. TSREP: IL-2 (3000 IU/mL) + IL-21 (10 ng/mL) + L-arg (5 mM) + HEPES (25 mM) + NAD (50 µM)

Five days after the start of REP culture, a 50% medium change was performed. REP TILs were harvested 11 days after the start of REP culture. See Figure 58. The total viable cells (TVC) and cell viability of TILs in the REP product were then counted, and phenotypes were analyzed by flow cytometry.

Figure 59 shows the transduction efficiency of NFAT-TeIL12-EF1α-tCD19 (mean = 30.3%) and NFAT-TeIL12-EF1α-TeIL15 (mean = 23.2%) in CD3+ T cells. Figure 60A shows the transduction efficiency in CD4+ and CD8+ T cell subsets, demonstrating that the expression of tCD19 in TILs transduced with NFAT-TeIL12-EF1α-tCD19 is similar between CD4+ and CD8+ cells. In contrast, Figure 60B shows that the expression of TeIL15 in TILs transduced with NFAT-TeIL12-EF1α-TeIL15 is higher in CD8+ T cells compared to CD4+ T cells.

Figure 61 shows that lentiviral vector transduced TILs exhibit low/minimal skewness in CD4+ or CD8+ cell percentages in REP TIL products. Figure 62 shows that REP cell medium conditions exhibit similar transduction efficiencies in CD3+ T cells. Figure 63 shows that REP cell medium conditions exhibit similar transduction efficiencies in CD4+ and CD8+ T cell subsets.

Figure 64 shows that the expansion rate and survival rate are enhanced under various REP cell culture medium conditions. Figure 65 shows that various REP cell culture medium conditions increase the number of TIL (TVC) in non-transduced TIL. Figure 66 shows that various REP cell culture medium conditions increase the survival rate of non-transduced TIL.

The expression of TeIL-15 on CD3+ T cells and CD4+/CD8+ T cell subsets was measured. The results showed that GEN2+NAD and ITARG+NAD REP conditions improved TeIL-15 expression in REP TILs (Figure 67).

TILs from the REP product were also cultured with autologous tumor digest cell suspensions. Untransduced TILs and NFAT-TeIL-12-EF1α TeIL-15 TILs were thawed and cultured with autologous tumor digests at a 1:1 effector:target ratio and incubated for 18-24 hours in CM2 medium containing IL-2 (300 IU/mL). TILs were cultured in the presence of tumor digests +/- anti-HLA-I (50 µg/mL) and anti-HLA-II (10 µg/mL) to measure HLA-dependent T cell activation. TILs were also cultured alone or stimulated with anti-CD3/anti-CD28 GMP TransAct (1:100 concentration). The supernatant was collected and analyzed for IFN-γ, TNFα, IL-12p70, and IL-15 concentrations using the Bio-Plex multiplex immunoassay system (Bio-Rad).

The results showed that the production of IFNγ and TNFα was stimulated by autologous tumor digests (Figure 68) and was HLA-dependent (Figure 69). This result shows that the production of IFNγ and TNFα depends on the interaction of TIL and HLA molecules.

The results further showed that NFAT-TeIL-12-EF1a-TeIL-15 TIL produced more IFNγ after autologous stimulation than non-transduced TIL, and GEN2+NAD and ITARG+NAD enhanced this effect (Figure 70), and NFAT-TeIL-12-EF1a-TeIL-15 TIL produced more TNFα after autologous stimulation, and GEN2+NAD, ITARG and ITARG+NAD enhanced this effect (Figure 71). Example 22 : TIL transduced with TeIL-12/TeIL-15 exhibited excellent cytotoxicity against autologous melanoma cell lines

In this study, melanoma tumor cells were isolated from patient-derived xenografts and grown in vitro. Melanoma cells were maintained in medium 254 before incubation with autologous TIL. First, 5000 melanoma cells were seeded on black-walled 96-well cell culture plates and incubated overnight in Incucyte S3. Untransduced NFAT-TeIL-12-EF1α-tCD19 and NFAT-TeIL-12-EF1α-TeIL-15 were added at various ET ratios (8:1, 4:1, 2:1, 1:1, and 0.5:1) and incubated with photo-apoptotic proteinase 3/7 dye for 24 hours to quantify tumor cell death.

TIL transduced with TeIL-12/TeIL-15 exhibited excellent cytotoxicity against autologous melanoma cell lines (Figure 72). Example 23 : TIL transduced with TeIL-12/TeIL-15 exhibited enhanced killing of NY-ESO-1 A375 melanoma cells

In this study, NYESO1 REP TILs were harvested and 5e6 cells per condition were saved and cultured over the weekend in CM2 + 3000IU/mL IL-2 in a grex-24-well plate. A375 target cells were detached from their flasks with 3 mL trypLE, washed with 27 mL CM2 medium, centrifuged at 500 g for 4 minutes, and then resuspended in TME medium at 2E5 cells/mL.

TME medium preparation for 50 mL medium: 3.125 mL DMEM + 10% FBS (high glucose); 46.9 mL DMEM + 10% FBS (no glucose); 30 IU/mL IL2 and 0.09 g lactate.

50 uL of TME medium was added to each well of the Xcelligence plate and background measurements were performed for well-to-well normalization. 50 uL of A375 target cells were then seeded at 1E4 cells/well in TME medium on the Xcelligence plate. The cells were then incubated overnight in the Xcelligence incubator. The next day, NYESO1 REP TILs were harvested from the grex-24-well plate, counted and resuspended at 5E4, 1.25E4 cells/mL, and then 100 μL of cells were added to the wells in triplicate for each condition. Cells were co-cultured for 48 hours and analyzed for killing function according to the plate diagram below.

Compared with the lentiviral backbone control and the mock process control, NYESO1 TCR-selected T cells transduced with a single NFAT-teIL-12 construct or a dual NFAT-teIL-12-EF1a-TeIL-15 construct showed superior killing efficacy (Figure 73). Example 24 : Optimized medium formulation enhances post-thaw TIL expansion and cell recovery Materials and methods T cell counts and survival

REP cells were harvested after TIL expansion. The number of viable cells was assessed by AOPI analysis using a Cellometer cell counter (Nexcelom, Lawrence, MA). REP TILs were cryo-frozen using CS10 (StemCell Technologies, Vancouver, Canada) and stored in liquid nitrogen until further use. After thawing, the number of viable cells was assessed by AOPI analysis using a Cellometer cell counter. Pre-REP and REP Process

Human tumor samples were collected and cut into approximately 3 mm pieces and cultured in Grex10 with the recommended medium containing IL-2 (6000 IU/mL). After 11 days of culture, pre-REP cells were propagated in the REP expansion process using irradiated PBMC, anti-CD3 antibody and containing IL-2 (6000 IU/ml). At the same time, pre-REP cells were propagated for another 11 days in the REP expansion process using irradiated PBMC, anti-CD3 antibody and containing 10 ng/ml IL-15 and 10 ng/ml IL-21 and 5 mM L-arginine (ITARG) without or with NAD+ (ITARG+NAD+). Gene Editing Process

To genetically modify TILs to express the tethered interleukins IL-12 and IL-15, 100,000 pre-REP TILs were transduced with BaEVTR enveloped virus containing a nucleic acid sequence encoding tethered IL-12 (TeIL-12) driven by the NFAT promoter followed by a sequence encoding tethered IL-15 (TeIL-15) driven by the EF1a promoter. For rotational transduction, TILs and virus were added to retronectin-coated plates in medium containing IL-2 (3000 IU/mL) and then centrifuged at 2000×g, 32°C for 45 minutes. After rotational transduction, TILs were cultured for 72 hours before starting REP. result

First, REP supplementation with IL-15 and IL-21 and L-arginine (ITARG), without or with NAD+ (ITARG+NAD+), increased the expansion and survival of unmodified TILs compared to medium formulations containing IL alone (Figures 74A-C). TILs genetically modified to express the tethered interleukins IL-12 and IL-15 also had a modest increase in expansion, but significantly improved survival (Figures 75A-C). When expanded under conditions containing ITARG but without or with NAD+ (ITARG+NAD+), the recovery of unmodified TILs after thawing was also improved (Figure 74D). In contrast, ITARG or ITARG+NAD+ did not enhance the recovery of genetically modified TILs (Figure 75D). Example 25 : Optimized medium formulation enhances TIL expansion, survival and cytotoxicity Materials and methods T cell counts and survival

REP cells were harvested after TIL expansion. The number of viable cells was assessed by AOPI analysis using a Cellometer cell counter (Nexcelom, Lawrence, MA). REP TILs were cryo-frozen using CS10 (StemCell Technologies, Vancouver, Canada) and stored in liquid nitrogen until further use. After thawing, the number of viable cells was assessed by AOPI analysis using a Cellometer cell counter. Pre-REP and REP Process

Human tumor samples were collected and cut into approximately 3 mm pieces and cultured in Grex10 with the recommended medium containing IL-2 (6000 IU/mL). After 11 days of culture, pre-REP cells were then propagated in the REP expansion process using irradiated PBMC, anti-CD3 antibody and containing IL-2 (6000 IU/ml). At the same time, pre-REP cells were propagated for another 11 days in the REP expansion process using irradiated PBMC, anti-CD3 antibody and containing 10 ng/ml IL-15 and 10 ng/ml IL-21 and 5 mM L-arginine (ITARG) without or with NAD+ (ITARG+NAD+). Gene Editing Process

To genetically modify TILs to express the tethered interleukins IL-12 and IL-15, 100,000 pre-REP TILs were transduced with BaEVTR enveloped virus containing a nucleic acid sequence encoding tethered IL-12 (TeIL-12) driven by the NFAT promoter followed by a sequence encoding tethered IL-15 (TeIL-15) driven by the EF1a promoter. For rotational transduction, TILs and virus were added to retronectin-coated plates in medium containing IL-2 (3000 IU/mL) and then centrifuged at 2000 × g, 32°C for 45 min. After rotational transduction, TILs were incubated for 72 h before starting REP. In vitro cytotoxicity assay

Transgenic TCRs targeting the Kirsten rat sarcoma virus (KRAS) G12D mutation were used to assess the function of TILs without or with cytokine tethering in vitro. Patient-derived neoantigen-specific CD8 TCRs recognized the common KRAS G12D mutation presented by C*08:02. HPAC pancreatic cancer cell lines harbored the KRAS G12D mutation and expressed the C*08:02 allele. Pre-REP TILs generated from tumor fragments were used for tumor-directed TIL generation. CD8+ pre-REP TILs were transduced with KRAS lentivirus alone or co-transduced with KRAS lentivirus and tethered IL-12/IL-15 lentivirus (KRAS.mteth.IL12.IL15) by adding virus to 0.1e6 TILs in complete medium supplemented with 3000 IU/mL IL-2 in Retronectin-coated 48-well non-tissue culture treated plates. Plates were centrifuged at 2000g for 45 minutes at 32°C and then placed in a 37°C incubator for 3 days. These cells were then sorted for CD8+TCRb+ expression prior to the REP process as described above. To mimic the conditions of a real tumor microenvironment (TME), HPAC cells expressing eGFP/mCherry were seeded in 96-well plates in DMEM basal medium (glucose-free, supplemented with 1.5 mM glucose, 20 mM lactate, 100 μM adenosine, 5 ng/mL TGF β1, with or without 30 IU/mL IL-2) that mimics a real tumor microenvironment. Then, KRAS TCR+ TILs or KRAS.mteth.IL12.IL15 TILs were added to HPAC cells in triplicate. TIL-based tumor cell lysis at different effector to tumor (E:T) ratios was recorded over 4 days using a live cell imaging system (IncuCyte, Sartorius). Results Optimized medium formulations enhance TIL expansion and survival

In 41 donors with a variety of tumor types, the medium used during REP supplemented with IL-15 and IL-21 and L-arginine (ITARG) without or with 50 µM NAD+ (ITARG+NAD+) resulted in increased total viable cell counts (TVC), fold expansion, and survival of unmodified TILs compared with medium formulations containing IL-2 or IL-2 and 50 μM NAD+ (Figure 78A-C). Optimized medium formulations enhance antigen-specific REP TIL in vitro cytotoxicity

To evaluate the impact of the medium formulation on TIL function in inhibiting tumor cell growth, REP pre-TIL transduced with a TCR construct targeting KRAS G12D were co-cultured with HPAC tumor cells expressing KRAS G12D under conditions that mimic the actual TME. A modest benefit in controlling tumor growth was observed when antigen-specific TILs were expanded in the optimized medium formulation compared to medium containing IL-2 alone (IL-2/NAD+, 1.4-fold change; ITARG, 1.2-fold change; ITARG/NAD+, 1.7-fold change) (Figure 79A). Removal of IL-2 (30 IU/mL) from the simulated TME resulted in loss of TIL responsiveness, regardless of the medium conditions used to expand antigen-specific TILs (IL-2/NAD+, 1.05-fold change; ITARG, 1.03-fold change; ITARG/NAD+, 1.2-fold change) (Figure 79B). Membrane-tethered IL12/IL15 antigen-specific TILs display enhanced cytotoxicity in vitro

Next, the effects of membrane-tethered interleukins on antigen-directed TILs expanded in optimized media on tumor cell growth were assessed over time when co-cultured at low E:T ratios. KRAS.mteth.IL15.IL21 TILs expanded in ITARG/NAD+ exhibited the most potent anti-tumor activity (5.0-fold change) compared to all experimental groups (Figure 80A). In addition, the enhanced activity of KRAS.mteth.IL15.IL21 TILs expanded in ITARG/NAD+ was maintained in the absence of IL-2 (5.2-fold change) (Figure 80B). The level of mteth.IL15 on the surface of KRAS-transduced TILs detected by flow cytometry was used as a control for transduction efficiency, as shown in Figure 80C. Example 26 : TILs expressing membrane-tethered IL-12/IL-15 show enhanced in vivo persistence Materials and methods

On day -10, 1E6 PDX M1152 tumor cells were inoculated into 7 or 8 NOG mice in each experimental group #1-#7. On day 0, 10E6 REP TILs were infused into each mouse in treatment groups #2-#7 (see Table 25).

Each mouse in treatment groups #5, #6 and #7 was administered a regimen of nine doses of Proleukin (45,000 IU) by intraperitoneal injection; the administration schedule was: Day 0: One dose of Proleukin (45,000 IU) after ACT Day 1: Two doses of Proleukin (45,000 IU) - early morning and evening Day 2: Two doses of Proleukin (45,000 IU) - early morning and evening Day 3: One dose of Proleukin (45,000 IU) Days 4-6: One dose of Proleukin (45,000 IU) daily Starting from day 7, peripheral blood was collected from TIL-infused mice in EDTA-coated tubes as follows: Day 7: Groups #2, #3, #4 Day 8: Group #5, #6, #7 Day 14: Group #2, #3, #4 Day 15: Group #5, #6, #7

TIL persistence in PB was stained with CD2+CD3+ antibodies and quantified by flow cytometry using absolute counting beads.

In the absence of Proleukin, TIL expressing TeIL-12/TeIL-15 showed a trend of increased persistence after infusion (Figure 81). *  *  *

The above examples are provided to provide those skilled in the art with a complete disclosure and description of embodiments of how to make and use the compositions, systems and methods of the present invention, and are not intended to limit the scope of the invention defined by the inventors. Modifications of the above-described modes of the present invention that are obvious to those skilled in the art are intended to be within the scope of the following patent claims. All patents and publications mentioned in this specification are indicative of the skill level of those skilled in the art to which the present invention pertains.

All titles and section names are used for clarity and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects from different titles and sections as needed according to the spirit and scope of the invention described herein.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

As will be apparent to those skilled in the art, various modifications and variations may be made without departing from the spirit and scope of this application. The specific embodiments and examples described herein are provided by way of example only, and this application is to be limited only by the terms of the appended claims and the full scope of equivalents to which the claims are entitled.

[ Figure 1A ] - [ Figure 1C ] : Overview of studies used to evaluate the expression and signaling of membrane-bound IL-15/IL-21-transduced pre-REP TILs. [ Figure 2A ] - [ Figure 2B ] : Overview of studies used to evaluate the expression of mIL-15/IL21 and CD8 and CD4 T cell subsets in mIL-15/IL-21-transduced REP TILs. [ Figure 3A ] - [ Figure 3C ] : Overview of studies used to evaluate the phenotype of CD8+ REP TILs transduced with mIL-15/IL-21. [ Figure 4A ] - [ Figure 4C ] : Overview of studies used to evaluate the phenotype of CD4+ REP TILs transduced with mIL-15/IL-21. [ FIG. 5 ] Describes exemplary nucleic acids that allow expression of member-anchored IL-12 (TeIL-12) and PD-1 shRNA in embodiments of the subject TILs provided herein. [ FIG. 6 ] Describes an exemplary workflow for preparing TILs expressing TeIL-12 and/or NFAT-TeIL-12 for administration to an individual. [ FIG. 7A ] - [ FIG. 7B ] : Overview of studies used to evaluate TeIL-12 and/or NFAT-TeIL-12 expression in REP TILs. (A) After REP harvest, surface expression of TeIL-12 on TeIL12 TILs was studied by flow cytometry using an IL-12P70 flow antibody (B). NFAT-TeIL-12-transduced REP-TILs were stimulated with TransACT or PMA at specified dilutions. Surface TeIL-12 expression was investigated 48 hours after stimulation. [ Figure 8 ] : Overview of studies used to evaluate IL-12 activity in TIL expressing TeIL-12. [ Figure 9A ] - [ Figure 9B ] : Overview of studies used to evaluate (A) expansion and (B) survival of TIL after REP transduced to express TeIL-12 or NFAT-TeIL-12. [ Figure 10A ] - [ Figure 10B ] : Overview of studies used to evaluate (A) frequency and (B) viral genome copy number (VCN) per cell of TeIL-12 and/or NFAT-TeIL-12 in REP TIL populations from different tissues including two lung, one head and neck, one breast, and one ovarian tumor samples. [ Figure 11 A ] - [ Figure 11 D ] : Overview of studies used to evaluate (A) and (B) cytotoxicity in THP-1-based allogeneic cytotoxicity assays, (C) and (D) IFN-γ production by TeIL-12 REP-TIL and NFAT-driven induced TeIL-12 REP-TIL. [ Figure 12 A ] - [ Figure 12 B ] : Overview of studies used to evaluate cytotoxicity of TIL expressing TeIL-12 also evaluated by xCelligence RTCA assay using two target cell populations (A) and (B). [ Figure 13A ] - [ Figure 13C ] : Overview of studies used to evaluate killing efficacy of TIL. (A) Schematic diagram of experimental design. (B) KILR® THP-1 cytotoxicity assay and IFN-g quantification, and (C) Xcellgene RTCA killing assay. [ Figure 14 ] Depicts an overview of studies used to assess the distribution of CD8+, CD4+, and CD4+/FoxP3- T cells within REP TIL populations transduced to express TeIL-12 or NFAT -TeIL-12. [ Figure 15A ] - [ Figure 15B ] : An overview of studies used to assess T cell differentiation as measured by various cell markers of (A) CD8+ and (B) CD4+ T cells within REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [ FIG. 16 A ] - [ FIG. 16 B ] : Overview of studies used to assess T cell depletion as measured by various cell markers of (A) CD8+ and (B) CD4+ T cells in REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [ FIG. 17 A ] - [ FIG. 17 B ] : Overview of studies used to assess T cell activation as measured by various cell markers of (A) CD8+ and (B) CD4+ T cells in REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [ FIG. 18 A ] - [ FIG. 18 B ] : Overview of studies used to assess (A) CD8+ and (B) CD4+ T cells as measured by various cell markers in REP TIL populations transduced to express TeIL-12 or NFAT-TeIL-12. [ FIG. 19 A ] - [ FIG. 19 B ] : Shows (A) cell expansion and (B) surface expression of TeIL-15 following the following procedures: Following TeIL-15 lentiviral gene transduction, pre-REP TILs were treated with feeder cells, 3000 IU/ml IL-2, and aCD3 Ab OKT3 or HIT3a for REP expansion. OKT3 (30 ng/ml) or HIT3a (30 ng/ml) was added to REP medium at different days after setting up the REP process (day 0, day 2, and day 4). After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [ Fig. 20A ] - [ Fig. 20B ] : Shows (A) cell expansion and (B) surface expression of TeIL-15/TeIL-21 after the following procedures: After TeIL-15/TeIL-21 lentiviral gene transduction, pre-REP TILs were treated with feeder cells, 3000IU/ml IL-2, and aCD3 Ab OKT3 or HIT3a for REP expansion. OKT3 (30 ng/ml) or HIT3a (30 ng/ml) was added to REP medium at different days after setting up the REP process (day 0, day 2, and day 4). After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [ Fig. 21A ] - [ Fig. 21B ] : Shows (A) cell expansion and (B) surface expression of TeIL-15 after the following process: After TeIL-15 lentiviral gene transduction, pre-REP TILs were treated with feeder cells, 3000IU/ml IL-2, and the indicated concentrations of OKT3 for 11 days of REP expansion. After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [ Fig. 22A ] - [ Fig. 22B ] : Shows (A) cell expansion and (B) surface expression of TeIL-15/TeIL-21 after the following procedures: After TeIL-15/TeIL-21 lentiviral gene transduction, pre-REP TILs were treated with feeder cells, 3000IU/ml IL-2, and the indicated concentrations of OKT3 for REP expansion. After 11 days of REP expansion, post-REP TILs were harvested and analyzed. [ Fig. 23 ] : Shows the surface expression of TeIL-12 after transduction with or without TransAct. [ Fig. 24 ] : Shows the surface expression of TeIL-12 after transduction with Retronection or Vectofusin coated plates with or without centrifugation. [ Figure 25 ] : Surface expression of TeIL-12 after transduction with TransACT or PMA-myomycin stimulation and with or without Lentiboost. [ Figure 26 ] : Surface expression of TeIL-12 after transduction with BaEVTR, RD114 or VSV-G Env plasmids. [ Figures 27A-27D ] : Results of (A) KILR® THP-1 cytotoxicity assay, (B) IFN production, (C) xCelligence RTCA assay and (D) xCelligence RTCA assay of TIL expressing NFAT-TeIL-12 after continuous TransACT stimulation. [ Figure 28 ] : Exemplary expression vectors encoding NFAT-driven TeIL-12 and EF1α-driven bicistronic tCD19 and component X are shown. [ Figure 29A-29B ] : Shows (A) expansion fold and (B) survival rate of TILs transduced with NFAT-TeIL-12 and component X. [ Figure 30A-30B ] : Shows (A) transduction efficiency and (B) viral copy number per cell (VCN) of TILs transduced with NFAT-TeIL-12 and component X. [ Figure 31 ] : Shows the expression of tCD19, TeIL-2, TeIL-15, IL-2, IL-15, GFP shRNA and PD-1 shRNA after different levels of TCR stimulation. [ Figure 32 ] : Shows the results of xCELLgene RTCA cytotoxicity assay of TILs expressing tCD19, TeIL-2, TeIL-15, IL-2 or IL-15. [ Figure 33 ] : p-STAT activation induced by TILs expressing TeIL-2, TeIL-15, IL-2 or IL-15 is shown. [ Figure 34 ] : TILs expressing TeIL-2, TeIL-15, IL-2 or IL-15 are shown to be skewed by cis-proliferation. [ Figures 35A-35B ] : EF-1α-driven IL-2/IL-15 improves T cell proliferation in the absence of IL-2. [ Figures 36A-36B ] : PD-1 shRNA effectively reduces PD-1 positivity in TILs as measured by flow cytometry (A) or gMFI (B). [ Figures 37A-37D ] : TeIL-15 promotes TIL survival in vitro in the absence of IL-2. [ Figure 38A-38B ] : PDX data showing the in vivo efficacy of NFAT-IL-12 engineered TILs using Gen 2 or Invigo-T + I-Arg process. [ Figure 39A-39B ] : Dynamic changes in body weight (A) and % body weight relative to baseline (B) of NFAT-IL-12 engineered TILs using Gen 2 or Invigo-T + L-Arg process. [ Figure 40 ] : Plasma levels of IFN-γ, TNFa, CCL4, and IL-12 p70 in mice infused with Gen 2 or NFAT-IL-12 engineered TILs are shown. [ Figure 41 ] : Enhanced tumor responsiveness is shown when TILs are cultured with ITIL interleukins in the presence of IFNγ. [ Figures 42A-42B ] : Shows the finding that 4-1BB expression was maximal 24 hours after tumor digest application (A) and increased with provision of IFNγ to the cell culture medium (TS-TIL condition) (B). [ Figure 43 ] : Outlines several processes analyzed for the ability to enrich for tumor-reactive CD8 TIL. [ Figure 44 ] : Shows approximately 10-fold increase in product potency for TIL produced by the CD137 sorting process. [ Figure 45 ] : Shows enrichment for tumor-reactive TIL produced by the TS-TIL (4-1BB sorting/1x REP) process. [ Figure 46 ] : Shows that two CD137 (4-1BB) sorting conditions provide optimal enrichment for tumor-reactive CD8 (IFNγ +CD107a+) TIL. [ Figure 47 ] : Demonstrates that CD137 (4-1BB) sorting conditions improve tumor-responsive CD8 (IFNγ +CD107a+) TILs by 60-fold. [ Figure 48 ] : Overview of the "TS-TIL" (4-1BB selection) process used to generate tumor-responsive TILs. [ Figure 49 ] : Demonstrates that the percentage of specific tumor-responsive TILs in the TS-TIL product (approximately 7%) was significantly increased compared to the CTRL product (approximately 1%). [ Figure 50 ] : Demonstrates that the total number of CD8 tumor-responsive TILs generated from the TS-TIL process was, on average, 7-fold greater than the total number of CD8 tumor-responsive TILs generated from the CTRL process. [ Figure 51 ] : Demonstrates the results of total viable cells and expansion fold as a function of REP conditions. [ Figure 52 ] : Shows the total yield of CD8+ tumor-reactive TILs as a function of sorting and REP conditions (assessed by co-culture ICS with autologous digests). [ Figure 53 ] : Shows that the majority of cells were found to be TEM "effector memory" cells, with minimal differences between TILs prepared by different processes. [ Figure 54 ] : Shows that TeIL-15 activates T cells only in a cis-like manner. [ Figures 55A-55D ] : Shows that L-arginine enhances post-thaw expansion and cell recovery during Invigo-T REP. [ Figures 56A-56C ] : Shows that L-arginine provides better phenotype and killing capacity. [ Figure 57A-57C ] : Demonstrates that NAD+ and L-arginine work synergistically to promote metabolism and provide unique T cell characteristics. [ Figure 58 ] Demonstrates the TIL production process from stored and fresh pre-REP TIL. [ Figure 59 ] Demonstrates the transduction efficiency of NFAT-TeIL12-EF1α-tCD19 (mean = 30.3%) and NFAT-TeIL12-EF1α-TeIL15 (mean = 23.2%) in CD3+ T cells. [ Figure 60A ] Demonstrates the transduction efficiency in CD4+ and CD8+ T cell subsets, demonstrating that the expression of tCD19 in TIL transduced with NFAT-TeIL12-EF1α-tCD19 is similar between CD4+ and CD8+ cells. [ FIG. 60B ] shows that the expression of TeIL15 in TILs transduced with NFAT-TeIL12-EF1α-TeIL15 is higher in CD8+ T cells compared to CD4+ T cells. [ FIG. 61 ] shows that TILs transduced with lentiviral vectors show low/minimal skewness in the percentage of CD4+ or CD8+ cells in REP TIL products. [ FIG. 62 ] shows that REP cell medium conditions show similar transduction efficiency in CD3+ T cells. [ FIG. 63A-63B ] shows that REP cell medium conditions show similar transduction efficiency in CD4+ and CD8+ T cell subsets. [ FIG. 64 ] shows that the expansion rate and survival rate are enhanced under various REP cell medium conditions. [ Figure 65 ] Various REP cell culture medium conditions increase the number of TIL (TVC) in non-transduced TIL. [ Figure 66 ] Various REP cell culture medium conditions increase the survival rate of non-transduced TIL. [ Figure 67 ] The expression level of TeIL-15 in REP TIL is enhanced by GEN2+NAD and ITARG+NAD in CD3+ cells ( A ), CD4+ T cells ( B ), and CD8+ T cells ( C ). [ Figure 68 ] shows that untransduced TIL ( A , C ) and NFAT-TeIL-12-EF1α-TeIL-15 TIL ( B , D) exhibited increased production of IFNγ (A , B) and TNFα ( C , D ) when cultured with autologous tumor digest (TIL+digest) compared to TIL that did not receive any stimulation (unstimulated). [ Figure 69 ] shows that untransduced TIL ( A , C ) and NFAT-TeIL12-EF1α-TeIL15 TIL (B, D ) exhibited decreased production of IFNγ ( A , B ) and TNFα ( C , D ) when cultured with autologous tumor digest (TIL +digest) compared to co-cultures incubated with anti-HLA-I and anti-HLA-II (TIL+digest+ HLA block ) . [ Figure 70 ] shows that NFAT-TeIL12-EF1α-TeIL15 TILs produced more IFNγ when cultured with autologous tumor digest (TIL+digest) compared to non-transduced TILs, and this effect was enhanced when TILs underwent GEN2+NAD or ITARG+NAD REP ( A ). NFAT-TeIL12-EF1α-TeIL15 TILs expanded under GEN2 conditions showed a 2.8-fold increase in IFNγ, while NFAT-TeIL12-EF1α-TeIL15 GEN2+NAD TILs and NFAT-TeIL12-EF1α-TeIL15 ITARG+NAD TILs showed a 6.7-fold and 5.1-fold increase in IFNγ, respectively ( B ). [ Figure 7 1 ] shows that NFAT-TeIL12-EF1α-TeIL15 TIL produced more TNFα when cultured with autologous tumor digest (TIL+digest) compared to non-transduced TIL, and this effect was enhanced when TIL underwent GEN2+NAD or ITARG+NAD REP ( A ). NFAT-TeIL12-EF1α-TeIL15 TIL expanded under GEN2 conditions showed a 1.3-fold increase in TNFα compared to NFAT-TeIL12-EF1α-TeIL15 GEN2+NAD TIL (1.8-fold), NFAT-TeIL12-EF1α-TeIL15 ITARG TIL (1.7-fold), and NFAT-TeIL12-EF1α-TeIL15 ITARG+NAD TIL (1.7-fold) ( B ). [ Figure 72 ] shows that TIL transduced with NFAT-TeIL12-EF1α-tCD19 or NFAT-TeIL12-EF1α-TeIL15 exhibited superior cytotoxicity against autologous melanoma cell lines compared to untransduced TIL. When tumor cells were cultured with autologous NFAT-TeIL12-EF1α-tCD19 or NFAT-TeIL12-EF1α-TeIL15 TIL, the percentage of caspase 3/7+ cells was significantly higher at 8:1 ET ratio ( A ) and 4:1 ET ratio ( B ) compared to untransduced TIL. [ Figure 73 ] shows that NYESO1 TCR-selected T cells transduced with a single NFAT-teIL12 construct or dual NFAT-teIL12-EF1a-TeIL15 construct exhibited superior killing efficacy compared to lentiviral backbone controls and mock process controls. [ Figure 74 ] shows ( A ) total live cells, ( B ) expansion fold, and ( C ) survival rate of unmodified TILs expanded in various medium formulations at the end of the 22-day REP process, and ( D ) survival rate 1 hour after thawing. Data are shown as mean ± SD. Data from repeated measures mixed model, post-hoc Tukey's multiple comparisons test. * p＜0.01; **p＜0.001, ***p＜0.0001. Abbreviations: ITARG, IL-15, IL-21, L-arginine; NAD+, nicotinamide adenine dinucleotide. [ Figure 75 ] Shows (A) total live cells, ( B ) expansion folds, and ( C ) survival rate of tethered-interleukin TIL (NFAT-TeIL12/TeIL15) expanded under various conditions at the end of the 22-day REP process, and (D ) survival rate 1 hour after thawing. Data are shown as mean ± SD. Data from repeated measures mixed model, post hoc Tukey multiple comparison test. N, not significant; * p＜0.01; **p＜0.001, ***p＜0.0001. Abbreviations: ITARG, IL-15, IL-21, L-arginine; NAD+, nicotinamide adenine dinucleotide. [ Figure 76 ] shows the vector map of pCMV-3Tag-4a-BaEV-TR (SEQ ID NO: 166). [ Figure 77 ] shows the vector map of pLenti-IRES-NFAT-TeIL12-EF1a-TeIL-15 (SEQ ID NO: 167). [ Figure 78 ]. ( A ) Total viable cells, ( B ) expansion fold, and ( C ) survival rate of unmodified TILs from 41 donors of various tumor types expanded in various medium formulations at the end of the 22-day REP process. Data are shown as mean ± SD. Data from repeated measures mixed effects model with post hoc Tukey multiple comparison test. * p <0.01; ** p < 0.001, *** p < 0.0001. Abbreviations: ITARG, IL-15, IL-21, L-arginine; NAD+, nicotinamide adenine dinucleotide. [ Figure 79 ]. In vitro cytotoxicity of tumor-directed TILs. The cytotoxicity of KRAS-TCR TIL products from 5 donors was evaluated against KRAS (HPAC) tumor cells. Antigen-specific TILs were added to corresponding HPAC cells in modified media that mimic conditions commonly observed in the real tumor microenvironment (TME) (with and without IL-2, (A) and (B) respectively). Data are presented as mean ± SD. Data from repeated measures mixed effects model with post hoc Tukey multiple comparison test. *p <0.01; **p < 0.001, ***p < 0.0001. Abbreviations: ITARG, IL-15, IL-21, L-arginine; NAD+, nicotinamide adenine dinucleotide. [ Figure 80 ]. In vitro cytotoxicity of tumor-directed TILs with membrane-tethered cytokines. Cytotoxicity of KRAS.mteth.IL12.IL15 TIL products from five donors against KRAS (HPAC) tumor cells was evaluated in modified media mimicking conditions commonly observed in the real tumor microenvironment (TME) with and without IL-2, (A) and (B), respectively. Data are presented as mean ± SD. Data from repeated measures mixed-effects model with post hoc Tukey multiple comparison test. *p <0.01; **p < 0.001, ***p < 0.0001. Abbreviations: ITARG, IL-15, IL-21, L-arginine; NAD+, nicotinamide adenine dinucleotide. Surface mteth.IL15 levels of KRAS-transduced TILs detected using flow cytometry were used as a control for transduction efficiency (C). [ FIG. 81 ]. TILs expressing TeIL-12/TeIL-15 show a trend of increased persistence after infusion. [ FIG. 82 ]. Exemplary Gen2 (Process 2A) diagram providing an overview of steps A to F. [ FIG. 83A-83C ]. Process flow diagram of a Gen 2 (Process 2A) embodiment for TIL manufacturing.

TW202509219A_113126279_SEQL.xml

Claims (408)

A nucleic acid molecule comprising a nucleotide sequence encoding tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T cells (NFAT) responsive promoter. The nucleic acid molecule of claim 1, wherein the TeIL-12 comprises a membrane anchor and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit. The nucleic acid molecule of claim 2, wherein the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO: 60 and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO: 61. The nucleic acid molecule of any one of claims 1 to 3, wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62. The nucleic acid molecule of any one of claims 1 to 4, wherein the nucleotide sequence encoding the TeIL-12 is shown in SEQ ID NO: 162. The nucleic acid molecule of any one of claims 1 to 5, further comprising a nucleotide sequence encoding an interleukin selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN γ, TNFa, IFN α, IFN β, GM-CSF, GCSF or its variants. A nucleic acid molecule as claimed in claim 6, wherein the cytokine is under the control of a constitutive promoter. The nucleic acid molecule of claim 7, wherein the constitutive promoter is selected from the group consisting of an EF1α promoter, a CMV promoter, a CAG promoter, a MND promoter and a SSFV promoter. The nucleic acid molecule of claim 7, wherein the constitutive promoter is the EF1α promoter. The nucleic acid molecule of any one of claims 6 to 9, wherein the interleukin is IL-15 or a variant thereof. The nucleic acid molecule of claim 10, wherein the IL-15 is human IL-15. The nucleic acid molecule of claim 11, wherein the human IL-15 has the amino acid sequence of SEQ ID NO: 64. A nucleic acid molecule as in any one of claims 10 to 12, wherein the IL-15 is tethered IL-15 (TeIL-15). The nucleic acid molecule of claim 13, wherein the TeIL-15 comprises a membrane anchor and human IL-15. The nucleic acid molecule of claim 14, wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:73. The nucleic acid molecule of any one of claims 6 to 9, wherein the interleukin is IL-2 or a variant thereof. The nucleic acid molecule of claim 16, wherein the IL-2 is human IL-2. The nucleic acid molecule of claim 17, wherein the human IL-2 has the amino acid sequence of SEQ ID NO:138. A nucleic acid molecule as in any one of claims 16 to 18, wherein the IL-2 is tethered IL-2 (TeIL-2). The nucleic acid molecule of claim 19, wherein the TeIL-2 has the amino acid sequence of SEQ ID NO:139. The nucleic acid molecule of any one of claims 1 to 20, comprising the nucleic acid sequence shown in SEQ ID NO: 148-150. The nucleic acid molecule of any one of claims 1 to 21, further comprising a nucleotide sequence encoding shRNA. The nucleic acid molecule of claim 22, wherein the shRNA inhibits the expression of an immune checkpoint gene. The nucleic acid molecule of claim 23, wherein the immune checkpoint gene is selected from the group consisting of: PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4 , CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11 and BCOR. A nucleic acid molecule as claimed in claim 23, wherein the immune checkpoint gene is PD-1. The nucleic acid molecule of claim 25, wherein the PD-1 shRNA comprises the nucleic acid sequence shown in SEQ ID NO: 141-147. The nucleic acid molecule of any one of claims 1 to 26, comprising the nucleic acid sequence shown in SEQ ID NO: 151. A recombinant expression vector comprising the nucleic acid molecule of any one of claims 1 to 27. A recombinant expression vector as claimed in claim 28, wherein the vector is a transfer vector. The recombinant expression vector of claim 29, wherein the transfer vector is derived from human immunodeficiency virus-1 (HIV-1) and is replication-defective. The recombinant expression vector of claim 30, wherein the transfer vector is a pLenti-IRES-EGFP vector. A lentiviral expression system comprising the recombinant expression vector of claim 30 or 31 and one or more helper plasmids encoding Env protein, Gag protein, Pol protein and Rev protein. The lentiviral expression system of claim 32, comprising an auxiliary plasmid encoding the Env protein, the Gag protein, the Pol protein and the Rev protein. The lentiviral expression system of claim 32, comprising an accessory plasmid encoding the Env protein and an accessory plasmid encoding the Gag protein, the Pol protein and the Rev protein. The lentiviral expression system of claim 32, comprising an accessory plasmid encoding the Env protein, the Gag protein and the Pol protein and an accessory plasmid encoding the Rev protein. The lentiviral expression system of claim 32, comprising an accessory plasmid encoding the Env protein and the Rev protein and an accessory plasmid encoding the Gag protein and the Pol protein. The lentiviral expression system of any one of claims 32 to 36, wherein the Env protein is selected from the group consisting of Ba-EVTR, VSV-G and RD114. The lentiviral expression system of claim 37, wherein the Env protein comprises Ba-EVTR protein. A method for producing recombinant lentiviral particles, comprising: a) culturing a packaging cell population in a cell culture medium; b) contacting the packaging cell population with a lentiviral expression system as described in any one of claims 32 to 38; and c) harvesting the supernatant of the cell culture medium, wherein the supernatant contains the recombinant lentiviral particles. A recombinant lentiviral RNA molecule comprising the nucleic acid molecule of any one of claims 1 to 27. A recombinant lentiviral proviral DNA comprises the nucleic acid molecule of any one of claims 1 to 27. A recombinant lentiviral particle comprising the recombinant lentiviral RNA molecule of claim 40. The recombinant lentiviral particle of claim 42, which is produced by the packaging cell line of any one of claims 240 to 284. The recombinant lentiviral particle of claim 42 or 43, comprising an Env protein selected from the group consisting of Ba-EVTR, VSV-G and RD114. The recombinant lentiviral particle of any one of claims 42 to 44, further comprising a reverse transcriptase. The recombinant lentiviral particle of any one of claims 42 to 45, further comprising a shell encapsulating the recombinant lentiviral RNA molecule. A gene-edited tumor-infiltrating lymphocyte (TIL) comprising the recombinant lentiviral proviral DNA of claim 41. The gene-edited TIL of claim 47, wherein the recombinant lentiviral proviral DNA is integrated into the genome of the gene-edited TIL. A gene-edited tumor-infiltrating lymphocyte (TIL) expressing exogenous tethered IL-12 (TeIL-12). The gene-edited TIL of claim 49, wherein the TeIL-12 comprises a membrane anchor and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit. The gene-edited TIL of claim 50, wherein the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60 and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:61. The gene-edited TIL of claim 51, wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62. The gene-edited TIL of any one of claims 49 to 52, further expressing an interleukin selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN γ, TNFa, IFN α, IFN β, GM-CSF, GCSF and variants thereof. The gene-edited TIL of claim 53, wherein the interleukin is IL-15 or a variant thereof. The gene-edited TIL of claim 54, wherein the IL-15 is human IL-15. The gene-edited TIL of claim 55, wherein the human IL-15 has the amino acid sequence of SEQ ID NO:64. The gene-edited TIL of any one of claims 54 to 56, wherein the IL-15 is tethered IL-15 (TeIL-15). The gene-edited TIL of claim 57, wherein the TeIL-15 comprises membrane anchor and human IL-15. The gene-edited TIL of claim 57, wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:73. A method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; b) performing a second expansion by culturing the TIL population in a second cell culture medium; and c) at any time, transducing the TIL population with a recombinant lentiviral particle as described in any one of claims 42 to 46 to produce the gene-edited TIL population, wherein the gene-edited TIL population expresses TeIL-12. The method of claim 60, further comprising activating the TIL population for 1 day or 2 days before transducing the TIL population with the recombinant lentiviral particles. The method of claim 61, wherein the activation step comprises contacting the TIL population with an interleukin selected from the group consisting of IL-2, IL-15, IL-21, IL-7, and combinations thereof. The method of claim 62, wherein the activation step comprises contacting the TIL population with 20 ng/mL IL-15. The method of claim 62, wherein the activation step comprises contacting the TIL population with 10 ng/mL IL-7. The method of claim 62, wherein the activating step comprises contacting the TIL population with TransAct. The method of claim 62, wherein the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. The method of any one of claims 60 to 66, wherein the transduction step is performed at a TIL concentration of 10 ⁵ cells/ml. The method of any one of claims 60 to 67, wherein the transducing step is performed at a multiplicity of infection (MOI) of about 10 to about 40. The method of any one of claims 60 to 68, wherein the transduction step is performed in the presence of RetroNectin or Vectofusin-1. The method of any of claims 60 to 69, wherein the transducing step comprises centrifugation. The method of any one of claims 60 to 70, wherein the transduction step is performed in the presence of a Lentibooster. A method as in any one of claims 60 to 71, wherein the transduction step is performed after the first amplification step and before the second amplification step. A method as in any one of claims 61 to 72, wherein the activation step is performed after the first expansion step and before the second expansion step. The method of any one of claims 60 to 73, further comprising allowing the TIL population to rest for 2 or 3 days after the transduction step. The method of any one of claims 60 to 74, wherein the TIL population is obtained from a tumor digest. The method of any one of claims 60 to 75, further comprising an initiating step. A method as claimed in claim 76, wherein the triggering step is performed before the first expanding step. The method of claim 76 or 77, wherein the priming step is performed in the presence of IFNγ. The method of claim 78, wherein IFNγ is present at a concentration of 200 ng/mL during the priming step. The method of any one of claims 76 to 79, wherein the priming step is performed in the presence of IL-2, IL-15 and/or IL-21. The method of claim 80, wherein the IL-2 concentration during the eliciting step is 3000 IU/mL or less. The method of claim 80 or 81, wherein the IL-15 and/or IL-21 is present at a concentration of about 1 ng/mL to about 100 ng/mL during the eliciting step. The method of claim 80 or 81, wherein the IL-15 and/or IL-21 is present at a concentration of about 10 ng/mL during the eliciting step. The method of any one of claims 76 to 83, wherein the initiating step lasts for 24-48 hours. The method of any of claims 76 to 83, wherein the initiating step lasts for about 30 hours. The method of any one of claims 60 to 85, further comprising an enrichment step. The method of claim 86, wherein the enrichment step is performed after the priming step and before the first expansion step. The method of claim 86 or 87, wherein the TIL population is enriched for CD137+ T cells. The method of claim 86 or 87, wherein the TIL population is enriched for CD137+ and CD39+ T cells. The method of claim 86 or 87, wherein the TIL population is enriched for CD137+ and CD200+ T cells. The method of claim 86 or 87, wherein the TIL population is enriched for CD137+, CD200+, CD39+ and/or OX40+ T cells. The method of any one of claims 60 to 91, wherein the first expansion is performed in the presence of feeder cells. The method of claim 92, wherein the feeder cells are T cell-depleted PBMCs. The method of any one of claims 60 to 93, wherein the first expansion is performed over a period of about 3-11 days. The method of any one of claims 60 to 93, wherein the first expansion is performed over a period of about 9 days. The method of any one of claims 60 to 95, wherein the second cell culture medium comprises IL-15 and IL-21. The method of claim 96, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. The method of any one of claims 60 to 97, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APC) and L-arginine. The method of claim 98, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM. The method of claim 98, wherein the L-arginine is present at a concentration of about 5 mM. The method of any one of claims 60 to 100, wherein the second cell culture medium comprises a NAD+ enhancer. The method of claim 101, wherein the NAD+ enhancer is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. The method of any one of claims 60 to 102, wherein the second cell culture medium comprises a GSK-3α/β inhibitor. The method of claim 103, wherein the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO and 3F8. The method of any one of claims 60 to 104, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. The method of claim 105, wherein the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oridonin, herbacetin, tehranolide, isoliquiritigenin, scutellarin and honokiol. The method of claim 105, wherein the AKT inhibitor is AZD5363. The method of any one of claims 105 to 107, wherein the AKT inhibitor is at a concentration of about 0.1 µM to about 10 µM. The method of any one of claims 105 to 107, wherein the AKT inhibitor is at a concentration of about 1 µM. The method of any one of claims 60 to 109, wherein the second expansion is performed over a period of about 7-11 days. The method of any one of claims 60 to 109, wherein the second expansion is performed over a period of about 10 days. A method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) priming a tumor digest containing a TIL population in the presence of IFNγ; b) enriching the TIL population for CD137+ T cells; c) performing a first expansion by culturing the TIL population enriched for CD137+ T cells in a first cell culture medium; d) transducing the TIL population enriched for CD137+ T cells with a recombinant lentiviral particle containing a nucleic acid sequence encoding TeIL-12 to produce the gene-edited TIL population; and e) performing a second expansion by culturing the gene-edited TIL population in a second cell culture medium. The method of claim 112, wherein the TeIL-12 is under the control of the NFAT promoter. The method of claim 112 or 113, wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62. The method of claim 112 or 113, wherein the nucleotide sequence encoding the TeIL-12 is shown in SEQ ID NO:162. The method of any one of claims 112 to 115, further comprising activating the TIL population for 1 day or 2 days before transducing the TIL population with the recombinant lentiviral particles. The method of claim 116, wherein the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. The method of any one of claims 112 to 117, wherein the transduction step is performed at a TIL concentration of 10 ⁵ cells/ml. The method of any one of claims 112 to 118, wherein the transducing step is performed at a multiplicity of infection (MOI) of about 10 to about 40. The method of any one of claims 112 to 119, wherein the transduction step is performed in the presence of RetroNectin or Vectofusin-1. The method of any of claims 112 to 120, wherein the transducing step comprises centrifugation. The method of any one of claims 112 to 121, wherein the transduction step is performed in the presence of a Lentibooster. The method of any one of claims 112 to 122, further comprising allowing the TIL population to rest for 2 or 3 days after the transduction step. The method of any one of claims 112 to 123, wherein IFNγ is present at a concentration of 200 ng/mL during the priming step. The method of any one of claims 112 to 124, wherein the priming step is performed in the presence of IL-2, IL-15 and/or IL-21. The method of claim 125, wherein the IL-2 concentration during the eliciting step is 3000 IU/mL or less. The method of claim 125 or 126, wherein the IL-15 and/or IL-21 is present at a concentration of about 10 ng/mL during the eliciting step. The method of any of claims 112 to 127, wherein the initiating step lasts for about 30 hours. The method of any one of claims 112 to 128, wherein the first expansion is performed in the presence of a feeder cell. The method of claim 129, wherein the feeder cells are T cell-depleted PBMCs. The method of any one of claims 112 to 130, wherein the first expansion is performed in the presence of IL-2, IL-15 and/or IL-21. The method of claim 131, wherein the IL-2 concentration during the eliciting step is 3000 IU/mL or less. The method of claim 130 or 131, wherein the IL-15 and/or IL-21 is present at a concentration of about 10 ng/mL during the first expansion period. The method of any of claims 112 to 133, wherein the first expansion is performed over a period of about 9 days. The method of any one of claims 112 to 134, wherein the second cell culture medium comprises IL-15 and IL-21. The method of claim 135, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. The method of any one of claims 112 to 136, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs) and L-arginine. The method of claim 137, wherein the L-arginine is present at a concentration of about 5 mM. The method of any one of claims 112 to 138, wherein the second cell culture medium comprises a NAD+ enhancer. The method of claim 139, wherein the NAD+ enhancer is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. The method of any one of claims 112 to 140, wherein the second cell culture medium comprises a GSK-3α/β inhibitor. The method of claim 141, wherein the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO and 3F8. The method of any of claims 112 to 142, wherein the second expansion is performed over a period of about 10 days. A method for producing a gene-edited tumor-infiltrating lymphocyte (TIL) population, comprising: a) priming a tumor digest containing a TIL population in the presence of IFNγ; b) enriching the TIL population for CD137+ T cells; c) performing a first expansion by culturing the TIL population enriched for CD137+ T cells in a first cell culture medium; d) transducing the TIL population enriched for CD137+ T cells with recombinant lentiviral particles containing nucleic acid sequences encoding TeIL-12 and TeIL-15 to produce the gene-edited TIL population; and e) performing a second expansion by culturing the gene-edited TIL population in a second cell culture medium. The method of claim 144, wherein the TeIL-12 is under the control of the NFAT promoter. The method of claim 144 or 145, wherein the TeIL-15 is under the control of a constitutive promoter. The method of claim 146, wherein the constitutive promoter is an EF1α promoter. The method of any one of claims 145 to 147, wherein the NFAT promoter and the constitutive promoter are introduced into the same nucleic acid coding sequence. The method of any one of claims 144 to 148, wherein the TeIL-12 comprises the amino acid sequence shown in SEQ ID NO:62. The method of any one of claims 144 to 148, wherein the nucleotide sequence encoding the TeIL-12 is shown in SEQ ID NO: 162. The method of any one of claims 144 to 150, wherein the TeIL-15 has the amino acid sequence of SEQ ID NO: 73. The method of any one of claims 144 to 151, further comprising activating the TIL population for 1 day or 2 days before transducing the TIL population with the recombinant lentiviral particles. The method of claim 152, wherein the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. The method of any one of claims 144 to 153, further comprising allowing the TIL population to rest for 2 or 3 days after the transduction step. The method of any one of claims 144 to 154, wherein IFNγ is present at a concentration of 200 ng/mL during the priming step. The method of any one of claims 144 to 155, wherein the priming step is performed in the presence of IL-2, IL-15 and/or IL-21. The method of claim 156, wherein the IL-2 concentration during the eliciting step is 3000 IU/mL or less. The method of claim 156 or 157, wherein the IL-15 and/or IL-21 is present at a concentration of about 10 ng/mL during the eliciting step. The method of any of claims 144 to 158, wherein the initiating step lasts for about 30 hours. The method of any one of claims 144 to 159, wherein the first expansion is performed in the presence of feeder cells. The method of claim 160, wherein the feeder cells are T cell-depleted PBMCs. The method of any of claims 144 to 161, wherein the first expansion is performed over a period of about 9 days. The method of any one of claims 144 to 162, wherein the second cell culture medium comprises IL-15 and IL-21. The method of claim 163, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. The method of any one of claims 144 to 164, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs) and L-arginine. The method of claim 165, wherein the L-arginine is present at a concentration of about 5 mM. The method of any one of claims 144 to 166, wherein the second cell culture medium comprises a NAD+ enhancer. The method of claim 167, wherein the NAD+ enhancer is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. The method of any one of claims 144 to 168, wherein the second cell culture medium comprises a GSK-3α/β inhibitor. The method of claim 169, wherein the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO and 3F8. The method of any of claims 144 to 170, wherein the second expansion is performed over a period of about 10 days. A therapeutic gene-edited TIL population produced by the method of any one of claims 60 to 171. A therapeutic gene-edited TIL population as claimed in claim 172, wherein approximately 15-60% of the gene-edited TIL population expresses TeIL-12. A therapeutic gene-edited TIL population as claimed in claim 172, wherein more than 30% of the gene-edited TIL population expresses TeIL-12. A therapeutic gene-edited TIL population as claimed in claim 172, wherein more than 50% of the gene-edited TIL population expresses TeIL-12. A therapeutic gene-edited TIL population as claimed in any of claims 172 to 175, wherein approximately 15-60% of the gene-edited TIL population expresses TeIL-15. A therapeutic gene-edited TIL population as claimed in any of claims 172 to 175, wherein more than 30% of the gene-edited TIL population expresses TeIL-15. A therapeutic gene-edited TIL population as claimed in any one of claims 172 to 175, wherein more than 50% of the gene-edited TIL population expresses TeIL-15. A therapeutic gene-edited TIL population as claimed in any of claims 172 to 178, wherein approximately 15-60% of the gene-edited TIL population expresses TeIL-2. A therapeutic gene-edited TIL population as claimed in any one of claims 172 to 178, wherein more than 30% of the gene-edited TIL population expresses TeIL-2. A therapeutic gene-edited TIL population as claimed in any one of claims 172 to 178, wherein more than 50% of the gene-edited TIL population expresses TeIL-2. A therapeutic gene-edited TIL population as claimed in any of claims 172 to 181, wherein more than 30% of the gene-edited TIL population has reduced PD-1 expression. A therapeutic gene-edited TIL population as claimed in any of claims 172 to 181, wherein more than 40% of the gene-edited TIL population has reduced PD-1 expression. A therapeutic gene-edited TIL population as claimed in any of claims 172 to 181, wherein more than 60% of the gene-edited TIL population has reduced PD-1 expression. A pharmaceutical composition comprising a therapeutic gene-edited TIL population as described in any one of claims 172 to 184. A method for treating cancer in a patient in need thereof, comprising: a) removing a tumor sample from the patient, wherein the tumor sample comprises a TIL population; b) performing a first expansion by culturing the TIL population in a first cell culture medium; c) performing a second expansion by culturing the TIL population in a second cell culture medium; d) at any time, transducing the TIL population with a recombinant lentiviral particle as described in any one of claims 42 to 46 to produce a therapeutic gene-edited TIL population; and e) administering the therapeutic gene-edited TIL population to the patient. The method of claim 186, further comprising activating the TIL population for 1 day or 2 days before transducing the TIL population with the recombinant lentiviral particles. The method of claim 187, wherein the activation step comprises contacting the TIL population with an interleukin selected from the group consisting of IL-2, IL-15, IL-21, IL-7, and combinations thereof. The method of claim 187, wherein the activating step comprises contacting the TIL population with TransAct. The method of any one of claims 186 to 189, wherein the transduction step is performed at a TIL concentration of 10 ⁵ cells/ml. The method of any one of claims 186 to 190, wherein the transducing step is performed at a multiplicity of infection (MOI) of about 10 to about 40. The method of any one of claims 186 to 191, wherein the transduction step is performed in the presence of RetroNectin or Vectofusin-1. The method of any of claims 186 to 192, wherein the transducing step comprises centrifugation. The method of any one of claims 186 to 193, wherein the transduction step is performed in the presence of a Lentibooster. A method as in any of claims 186 to 194, wherein the transduction step is performed after the first amplification step and before the second amplification step. A method as in any of claims 187 to 195, wherein the activation step is performed after the first expansion step and before the second expansion step. The method of any one of claims 186 to 196, further comprising allowing the TIL population to rest for 2 or 3 days after the transduction step. The method of any one of claims 186 to 197, wherein the TIL population is obtained from a tumor digest. The method of claim 198, further comprising an initiation step, wherein the tumor digest is initiated in the presence of IL-2, IL-15, IL-21 and IFNγ. The method of claim 199, wherein IFNγ is present at a concentration of 200 ng/mL during the priming step. The method of claim 199, wherein the IL-2 concentration during the eliciting step is 3000 IU/mL or less. The method of any one of claims 199 to 201, wherein the IL-15 and/or IL-21 is present at a concentration of about 1 ng/mL to about 100 ng/mL during the eliciting step. The method of any one of claims 199 to 201, wherein the IL-15 and/or IL-21 is present at a concentration of about 10 ng/mL during the eliciting step. The method of any of claims 199 to 203, wherein the initiating step lasts 24-48 hours. The method of any of claims 199 to 203, wherein the initiating step lasts for about 30 hours. The method of any one of claims 186 to 205, further comprising an enrichment step, wherein the TIL population is enriched for CD137+ T cells. The method of any one of claims 186 to 205, further comprising an enrichment step, wherein the TIL population is enriched for CD137+ and CD39+ T cells. The method of any one of claims 186 to 205, further comprising an enrichment step, wherein the TIL population is enriched for CD137+ and CD200+ T cells. The method of any one of claims 186 to 205, further comprising an enrichment step, wherein the TIL population is enriched for CD137+, CD200+, CD39+ and/or OX40+ T cells. The method of any one of claims 186 to 209, wherein the first expansion is performed in the presence of feeder cells. The method of claim 210, wherein the feeder cells are T cell-depleted PBMCs. The method of any one of claims 186 to 211, wherein the first expansion is performed over a period of about 3-11 days. The method of any of claims 186 to 211, wherein the first expansion is performed over a period of about 9 days. The method of any one of claims 186 to 213, wherein the second cell culture medium comprises IL-15 and IL-21. The method of claim 214, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. The method of any one of claims 186 to 215, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs) and L-arginine. The method of claim 216, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM. The method of claim 216, wherein the L-arginine is present at a concentration of about 5 mM. The method of any one of claims 186 to 218, wherein the second cell culture medium comprises a NAD+ enhancer. The method of claim 219, wherein the NAD+ enhancer is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. The method of any one of claims 186 to 220, wherein the second cell culture medium comprises a GSK-3α/β inhibitor. The method of claim 221, wherein the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO and 3F8. The method of any one of claims 186 to 222, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. The method of claim 223, wherein the AKT inhibitor is selected from the group consisting of patasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, perifosine, oregano, herbicide, terheiinolide, isoliquiritigenin, baicalein and honsenol. The method of claim 223, wherein the AKT inhibitor is AZD5363. The method of any one of claims 223 to 225, wherein the AKT inhibitor is at a concentration of about 0.1 µM to about 10 µM. The method of any one of claims 223 to 225, wherein the AKT inhibitor is at a concentration of about 1 µM. The method of any one of claims 186 to 227, wherein the second expansion is performed over a period of about 7-11 days. The method of any of claims 186 to 227, wherein the second expansion is performed over a period of about 10 days. The method of any one of claims 186 to 227, wherein the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. The method of any of claims 186 to 230, further comprising the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the TILs to the patient. The method of claim 231, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administering fludarabine at a dose of 25 mg/m2/day for three days. The method of claim 231, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. The method of claim 231, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for one day. The method of any one of claims 232 to 234, wherein the cyclophosphamide is administered with mesna. The method of any one of claims 186 to 235, further comprising the step of treating the patient with an IL-2 regimen starting the day after administering the TIL to the patient. The method of any one of claims 186 to 235, further comprising the step of starting treatment of the patient with an IL-2 regimen on the same day that the TILs are administered to the patient. The method of claim 236 or 237, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg aldesleukin or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerated. The method of any one of claims 186 to 235, which does not include the step of treating the patient with an IL-2 regimen. A packaging cell line comprising a recombinant expression vector as described in any one of claims 28 to 31 and one or more recombinant DNA molecules, each of the one or more recombinant DNA molecules encoding any one protein, any two proteins, any three proteins or all four proteins selected from the group consisting of: encoding Env protein, Gag protein, Pol protein and Rev protein, with the proviso that any protein selected from the group consisting of the Env protein, the Gag protein, the Pol protein and the Rev protein is encoded by at least one recombinant DNA molecule of the one or more recombinant DNA molecules, wherein each of the one or more recombinant DNA molecules is integrated into the genome of the packaging cell line or contained by an auxiliary plasmid. The packaging cell line of claim 240, wherein the one or more recombinant DNA molecules consist of a first recombinant DNA molecule. The packaging cell line of claim 241, wherein the first recombinant DNA molecule is integrated into the genome of the packaging cell line or contained by a helper plasmid. The packaging cell line of claim 241, wherein the one or more recombinant DNA molecules consist of a first recombinant DNA molecule and a second recombinant DNA molecule. The packaging cell line of claim 243, wherein the first recombinant DNA molecule encodes a single protein selected from the group consisting of the Env protein, the Gag protein, the Pol protein and the Rev protein. The packaging cell line of claim 244, wherein the first recombinant DNA molecule encodes the Env protein. The packaging cell line of claim 244, wherein the first recombinant DNA molecule encodes the Gag protein. The packaging cell line of claim 244, wherein the first recombinant DNA molecule encodes the Pol protein. The packaging cell line of claim 244, wherein the first recombinant DNA molecule encodes the Rev protein. The packaging cell line of claim 243, wherein the first recombinant DNA molecule encodes two proteins selected from the group consisting of the Env protein, the Gag protein, the Pol protein and the Rev protein. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Env protein and the Gag protein. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Env protein and the Pol protein. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Env protein and the Rev protein. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Gag protein and the Pol protein. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Gag protein and the Rev protein. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Pol protein and the Rev protein. The packaging cell line of any one of claims 243 to 255, wherein each of the first recombinant DNA molecule and the second recombinant DNA molecule is integrated into the genome of the packaging cell line or contained by a helper plasmid. The packaging cell line of claim 240, wherein the one or more recombinant DNA molecules consist of a first recombinant DNA molecule, a second recombinant DNA molecule, and a third recombinant DNA molecule. A packaging cell line as claimed in claim 257, wherein the first recombinant DNA molecule encodes a first protein and the second recombinant DNA molecule encodes a second protein, wherein the first protein and the second protein are independently selected from the group consisting of the Env protein, the Gag protein, the Pol protein and the Rev protein, with the proviso that the first protein and the second protein are not the same. The packaging cell line of claim 258, wherein the first protein is the Env protein and the second protein is the Gag protein. The packaging cell line of claim 258, wherein the first protein is the Env protein and the second protein is the Pol protein. The packaging cell line of claim 258, wherein the first protein is the Env protein and the second protein is the Rev protein. The packaging cell line of claim 258, wherein the first protein is the Gag protein and the second protein is the Pol protein. The packaging cell line of claim 258, wherein the first protein is the Gag protein and the second protein is the Rev protein. The packaging cell line of claim 258, wherein the first protein is the Pol protein and the second protein is the Rev protein. A packaging cell line as claimed in claim 257, wherein the first recombinant DNA molecule encodes a first protein and the second recombinant DNA molecule encodes two proteins, wherein the first protein and each of the two proteins are independently selected from the group consisting of the Env protein, the Gag protein, the Pol protein and the Rev protein, with the proviso that any protein is different from any other protein in the group consisting of the first protein and the two proteins. The packaging cell line of claim 265, wherein the first protein is the Env protein, and the two proteins are the Gag protein and the Pol protein. The packaging cell line of claim 265, wherein the first protein is the Env protein, and the two proteins are the Gag protein and the Rev protein. The packaging cell line of claim 265, wherein the first protein is the Env protein, and the two proteins are the Pol protein and the Rev protein. The packaging cell line of claim 265, wherein the first protein is the Gag protein, and the two proteins are the Env protein and the Pol protein. The packaging cell line of claim 265, wherein the first protein is the Gag protein, and the two proteins are the Env protein and the Rev protein. The packaging cell line of claim 265, wherein the first protein is the Gag protein, and the two proteins are the Pol protein and the Rev protein. The packaging cell line of claim 265, wherein the first protein is the Pol protein, and the two proteins are the Env protein and the Gag protein. The packaging cell line of claim 265, wherein the first protein is the Pol protein, and the two proteins are the Env protein and the Rev protein. The packaging cell line of claim 265, wherein the first protein is the Pol protein, and the two proteins are the Gag protein and the Rev protein. The packaging cell line of claim 265, wherein the first protein is the Rev protein, and the two proteins are the Env protein and the Gag protein. The packaging cell line of claim 265, wherein the first protein is the Rev protein, and the two proteins are the Env protein and the Pol protein. The packaging cell line of claim 265, wherein the first protein is the Rev protein, and the two proteins are the Gag protein and the Pol protein. The packaging cell line of any one of claims 257 to 277, wherein each of the first recombinant DNA molecule, the second recombinant DNA molecule, and the third recombinant DNA molecule is integrated into the genome of the packaging cell line or contained by a helper plasmid. The packaging cell line of claim 240, wherein the one or more recombinant DNA molecules consist of a first recombinant DNA molecule, a second recombinant DNA molecule, a third recombinant DNA molecule, and a fourth recombinant DNA molecule. The packaging cell line of claim 279, wherein each of the first recombinant DNA molecule, the second recombinant DNA molecule, the third recombinant DNA molecule, and the fourth recombinant DNA molecule is integrated into the genome of the packaging cell line or contained by a helper plasmid. The packaging cell line of any one of claims 240 to 280, wherein the Env protein comprises Ba-EVTR protein. The packaging cell line of any one of claims 240 to 281, wherein the nucleic acid molecule encoding TeIL-12 is integrated into the genome of the packaging cell line. A packaging cell line as in any one of claims 240 to 282, wherein the recombinant DNA molecule encoding one or more of the Env protein, the Gag protein, the Pol protein and the Rev protein is under the control of an inducible promoter. The packaging cell line of any one of claims 240 to 283, comprising a 293T cell line. A method for enriching tumor-responsive tumor-infiltrating lymphocytes (TILs), comprising: a. eliciting a TIL population obtained from a tumor digest in the presence of IFNγ; b. enriching the TIL population for CD137+ TILs; c. performing a first expansion by culturing the TIL population enriched for CD137+ T cells in a first cell culture medium; and d. performing a second expansion by culturing the TIL population enriched for CD137+ TILs in a second cell culture medium to produce a TIL population enriched for tumor-responsive TILs. The method of claim 285, wherein the TIL population enriched for tumor-reactive TILs comprises an increased percentage of tumor-reactive TILs compared to TILs expanded using a reference expansion procedure. The method of claim 285 or 286, wherein step (a) is performed for about 30 hours. The method of any one of claims 285 to 287, wherein step (c) is performed for about 9 days. The method of any one of claims 285 to 288, wherein step (d) is performed for about 10 days. The method of any one of claims 285 to 289, wherein the first cell culture medium comprises IL-2 at a concentration of 3000 IU/mL, IL-15 at a concentration of 10 ng/mL, and IL-21 at a concentration of 10 ng/mL. The method of any one of claims 285 to 289, wherein the first cell culture medium comprises IL-2 at a concentration of 6000 IU/mL. The method of any one of claims 285 to 291, wherein step (c) is performed in the presence of iPBMCs. The method of claim 292, wherein the iPBMCs are depleted of T cells. The method of any one of claims 285 to 293, wherein the IFNγ is present at a concentration of 200 ng/mL. The method of any one of claims 285 to 294, wherein the second cell culture medium comprises IL-2 at a concentration of 3000 IU/mL, IL-21 at a concentration of 10 ng/mL, and IL-15 at a concentration of 10 ng/mL. The method of any one of claims 285 to 295, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs) and L-arginine. The method of claim 296, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM. The method of claim 296, wherein the L-arginine is present at a concentration of about 5 mM. The method of any one of claims 285 to 298, wherein the second cell culture medium comprises a NAD+ enhancer. The method of claim 299, wherein the NAD+ enhancer is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. The method of any one of claims 285 to 300, wherein the second cell culture medium comprises a GSK-3α/β inhibitor. The method of claim 301, wherein the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO and 3F8. The method of any of claims 285 to 302, wherein enriching the TIL population for CD137+ TILs comprises contacting the TIL population with an anti-CD137 antibody immobilized on beads. The method of any one of claims 285 to 303, further comprising contacting the TIL population with an anti-CD39 antibody. The method of any of claims 285 to 304, further comprising contacting the TIL population with an anti-CD200 antibody. A method for enriching tumor-responsive tumor-infiltrating lymphocytes (TILs), comprising: a. eliciting a TIL population obtained from a tumor digest in the presence of IFNγ; b. enriching the TIL population for CD103+ TILs; c. performing a first expansion by culturing the TIL population enriched for CD103+ T cells in a first cell culture medium; and d. performing a second expansion by culturing the TIL population enriched for CD103+ T cells in a second cell culture medium to produce a TIL population enriched for tumor-responsive TILs. The method of claim 306, wherein the population of TILs enriched for tumor-reactive TILs comprises an increased percentage of tumor-reactive TILs compared to TILs expanded using a reference expansion procedure. The method of claim 306 or 307, wherein step (a) is performed for about 24 hours. The method of any of claims 306 to 308, wherein selecting CD103+ TILs comprises sorting the second TIL population using flow cytometry. The method of any one of claims 306 to 309, wherein step (b) comprises selecting CD103+CD31- TILs. The method of any one of claims 306 to 310, wherein step (c) is performed for about 9 days. The method of any one of claims 306 to 311, wherein step (d) is performed for about 10 days. The method of any one of claims 306 to 312, wherein the first cell culture medium comprises IL-2 at a concentration of 3000 IU/mL, IL-15 at a concentration of 10 ng/mL, and IL-21 at a concentration of 10 ng/mL. The method of any one of claims 306 to 313, wherein the first cell culture medium comprises IL-2 at a concentration of 6000 IU/mL. The method of any one of claims 306 to 314, wherein step (c) is performed in the presence of iPBMCs. The method of claim 315, wherein the iPBMCs are depleted of T cells. The method of any one of claims 306 to 316, wherein the IFNγ is present at a concentration of 200 ng/mL. The method of any one of claims 306 to 317, wherein the second cell culture medium comprises IL-2 at a concentration of 3000 IU/mL, IL-21 at a concentration of 10 ng/mL, and IL-15 at a concentration of 10 ng/mL. The method of any one of claims 306 to 318, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs) and L-arginine. The method of claim 319, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM. The method of claim 319, wherein the L-arginine is present at a concentration of about 5 mM. The method of any one of claims 306 to 321, wherein the second cell culture medium comprises a NAD+ enhancer. The method of claim 322, wherein the NAD+ enhancer is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. The method of any one of claims 306 to 323, wherein the second cell culture medium comprises a GSK-3α/β inhibitor. The method of claim 324, wherein the GSK-3α/β inhibitor is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, lithium chloride hydrate, BIO and 3F8. A method for treating cancer in a patient in need thereof, comprising: a. removing a tumor sample from the patient; b. digesting the tumor sample to obtain a tumor digest; c. generating a TIL population enriched for tumor-reactive TILs from the tumor digest using a method as described in any of claims 285 to 325; and d. administering the therapeutic gene-edited TIL population to the patient. The method of claim 326, wherein the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. The method of claim 326 or 327, further comprising the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the TILs to the patient. The method of claim 328, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administering fludarabine at a dose of 25 mg/m2/day for three days. The method of claim 328, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. The method of claim 328, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for one day. The method of any one of claims 329 to 331, wherein the cyclophosphamide is administered with mesnat. The method of any one of claims 326 to 332, further comprising the step of treating the patient with an IL-2 regimen starting the day after administering the TIL to the patient. The method of any one of claims 326 to 332, further comprising the step of starting treatment of the patient with an IL-2 regimen on the same day that the TILs are administered to the patient. The method of claim 333 or 334, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg of aldesleukin or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerated. The method of any one of claims 326 to 332, which does not include the step of treating the patient with an IL-2 regimen. A method for producing a tumor infiltrating lymphocyte (TIL) population, comprising: a) performing a first expansion by culturing the TIL population in a first cell culture medium; and b) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APC), IL-21, IL-15 and L-arginine. The method of claim 337, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. The method of claim 337 or 338, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM. The method of claim 337 or 338, wherein the L-arginine is present at a concentration of about 5 mM. The method of any one of claims 337 to 340, wherein the second cell culture medium comprises a NAD+ enhancer. The method of claim 341, wherein the NAD+ enhancer is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. The method of claim 341, wherein the NAD+ enhancer is NAD+. The method of claim 343, wherein the NAD+ is present at a concentration of about 50-75 µM. The method of any one of claims 337 to 344, wherein the first expansion is performed over a period of about 3-14 days. The method of any of claims 337 to 344, wherein the first expansion is performed over a period of about 11 days. The method of any of claims 337 to 345, wherein the second expansion is performed over a period of about 7-14 days. The method of any of claims 337 to 345, wherein the second expansion is performed over a period of about 11 days. A therapeutic TIL population produced by the method of any one of claims 337 to 348. A pharmaceutical composition comprising the therapeutic TIL population of claim 349. A method for treating cancer in a patient in need thereof, comprising: a) removing a tumor sample from the patient, wherein the tumor sample comprises a TIL population; b) performing a first expansion by culturing the TIL population in a first cell culture medium; c) performing a second expansion by culturing the TIL population in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, and L-arginine; and d) administering the therapeutic gene-edited TIL population to the patient. The method of claim 351, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. The method of claim 351 or 352, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM. The method of claim 351 or 352, wherein the L-arginine is present at a concentration of about 5 mM. The method of any one of claims 351 to 354, wherein the second cell culture medium comprises a NAD+ enhancer. The method of claim 355, wherein the NAD+ enhancer is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. The method of claim 355, wherein the NAD+ enhancer is NAD+. The method of claim 357, wherein the NAD+ is present at a concentration of about 50-75 µM. The method of any one of claims 351 to 358, wherein the first expansion is performed over a period of about 3-14 days. The method of any of claims 351 to 358, wherein the first expansion is performed over a period of about 11 days. The method of any of claims 351 to 360, wherein the second expansion is performed over a period of about 7-14 days. The method of any of claims 351 to 360, wherein the second expansion is performed over a period of about 11 days. The method of any one of claims 351 to 362, wherein the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. The method of any one of claims 351 to 363, further comprising the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the TILs to the patient. The method of claim 364, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administering fludarabine at a dose of 25 mg/m2/day for three days. The method of claim 364, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. The method of claim 364, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for one day. The method of any one of claims 365 to 367, wherein the cyclophosphamide is administered with mesnat. The method of any one of claims 351 to 368, further comprising the step of treating the patient with an IL-2 regimen starting the day after administering the TIL to the patient. The method of any one of claims 351 to 368, further comprising the step of starting treatment of the patient with an IL-2 regimen on the same day that the TILs are administered to the patient. The method of claim 369 or 370, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg of aldesleukin or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion once every eight hours until tolerated. The method of any one of claims 351 to 368, which does not include the step of treating the patient with an IL-2 regimen. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, comprising: (a) adding tumor fragments processed from a tumor resected from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) The second TIL population is cultured in a cell culture medium containing IL-21, IL-15, L-arginine, OKT-3 and antigen presenting cells (APC) to perform a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (b) to step (c) is performed without opening the system; (d) Harvesting the therapeutic TIL population obtained from step (c), wherein the transfer from step (c) to step (d) is performed without opening the system, and wherein the harvested therapeutic TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (e) transferring the TIL population harvested from step (d) to an infusion bag, wherein the transfer from step (d) to (e) is performed without opening the system, and (f) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process. A method for expanding tumor infiltrating lymphocytes (TIL) into a therapeutic TIL population, comprising: (a) adding tumor fragments processed from a tumor resected from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) The second TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, NAD+, OKT-3 and antigen presenting cells (APC) to perform a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (b) to step (c) is performed without opening the system; (d) Harvesting the therapeutic TIL population obtained from step (c), wherein the transfer from step (c) to step (d) is performed without opening the system, and wherein the harvested therapeutic TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (e) transferring the TIL population harvested from step (d) to an infusion bag, wherein the transfer from step (d) to (e) is performed without opening the system, and (f) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process. A method for producing a therapeutic gene-edited TIL population, comprising: (a) adding tumor fragments processed from a tumor removed from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) Transducing the second TIL population with a recombinant lentiviral particle as described in any one of claims 42 to 46 to produce a gene-edited TIL population, wherein the transformation from step (b) to step (c) is performed without opening the system; (d) The gene-edited TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, OKT-3 and antigen presenting cells (APC) for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is the therapeutic gene-edited TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (c) to step (d) is performed without opening the system; (e) Harvesting the therapeutic gene-edited TIL population obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system, and wherein the harvested therapeutic gene-edited TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (f) transferring the therapeutic gene-edited TIL population harvested from step (e) to an infusion bag, wherein the transfer from step (e) to (f) is performed without opening the system, and (g) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process. A method for producing a therapeutic gene-edited TIL population, comprising: (a) adding tumor fragments processed from a tumor removed from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a closed container providing a first gas permeable surface area, wherein the first expansion is performed for about 3-11 days to obtain the second TIL population, and wherein the transition from step (a) to step (b) is performed without opening the system; (c) Transducing the second TIL population with a recombinant lentiviral particle as described in any one of claims 42 to 46 to produce a gene-edited TIL population, wherein the transformation from step (b) to step (c) is performed without opening the system; (d) The gene-edited TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, NAD+, OKT-3 and antigen presenting cells (APC) for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is the therapeutic gene-edited TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (c) to step (d) is performed without opening the system; (e) Harvesting the therapeutic gene-edited TIL population obtained from step (d), wherein the transition from step (d) to step (e) is performed without opening the system, and wherein the harvested therapeutic gene-edited TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (f) transferring the therapeutic gene-edited TIL population harvested from step (e) to an infusion bag, wherein the transfer from step (e) to (f) is performed without opening the system, and (g) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process. A method for producing a therapeutic gene-edited TIL population, comprising: (a) processing a tumor resected from a patient to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and wherein the first expansion is performed for about 3-11 days to obtain the second TIL population; (c) transducing the second TIL population with a recombinant lentiviral particle as described in any one of claims 42 to 46 to produce a gene-edited TIL population; (d) The gene-edited TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, OKT-3 and antigen presenting cells (APC) for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is the therapeutic gene-edited TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (e) Harvesting the therapeutic gene-edited TIL population obtained from step (d), wherein the harvested therapeutic gene-edited TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (f) transferring the therapeutic gene-edited TIL population harvested from step (e) to an infusion bag, and (g) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process. A method for producing a therapeutic gene-edited TIL population, comprising: (a) processing a tumor resected from a patient to obtain a first TIL population; (b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and wherein the first expansion is performed for about 3-11 days to obtain the second TIL population; (c) transducing the second TIL population with a recombinant lentiviral particle as described in any one of claims 42 to 46 to produce a gene-edited TIL population; (d) The gene-edited TIL population is cultured in a second cell culture medium containing IL-21, IL-15, L-arginine, NAD+, OKT-3 and antigen presenting cells (APC) for a second expansion to produce a third TIL population, wherein the second expansion is performed for about 7-11 days to obtain the third TIL population, wherein the third TIL population is the therapeutic gene-edited TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area; (e) Harvesting the therapeutic gene-edited TIL population obtained from step (d), wherein the harvested therapeutic gene-edited TIL population contains sufficient TILs for a therapeutically effective dose of the TILs; (f) transferring the therapeutic gene-edited TIL population harvested from step (e) to an infusion bag, and (g) cryopreserving the infusion bag containing the harvested TIL population using a cryopreservation process. The method of any one of claims 373 to 378, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng/mL and IL-21 at a concentration of about 10 ng/mL. The method of any one of claims 373 to 379, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM. The method of any one of claims 373 to 379, wherein the L-arginine is present at a concentration of about 5 mM. The method of any one of claims 374, 376, 378 to 381, wherein the NAD+ is present at a concentration of about 50-75 µM. The method of any one of claims 373 to 382, wherein the first expansion is performed over a period of about 3-11 days. The method of any of claims 373 to 382, wherein the first expansion is performed over a period of about 11 days. The method of any of claims 373 to 384, wherein the second expansion is performed over a period of about 7-11 days. The method of any of claims 373 to 384, wherein the second expansion is performed over a period of about 11 days. The method of any one of claims 373 to 386, further comprising activating the TIL population for 1 day or 2 days before transducing the TIL population with the recombinant lentiviral particles. The method of claim 387, wherein the activating step comprises contacting the TIL population with TransAct. The method of claim 387, wherein the activation step comprises contacting the TIL population with TransAct at a ratio of 1:100. The method of any one of claims 373 to 389, wherein the transduction step is performed at a TIL concentration of 10 ⁵ cells/ml. The method of any one of claims 373 to 390, wherein the transducing step is performed at a multiplicity of infection (MOI) of about 10 to about 40. The method of any one of claims 373 to 391, wherein the transduction step is performed in the presence of RetroNectin or Vectofusin-1. The method of any of claims 373 to 392, wherein the transducing step comprises centrifugation. The method of any one of claims 373 to 393, wherein the transducing step is performed in the presence of a Lentibooster. The method of any one of claims 373 to 394, further comprising allowing the TIL population to rest for 2 or 3 days after the transduction step. A therapeutic gene-edited TIL population produced by the method of any one of claims 373 to 395. A pharmaceutical composition comprising a therapeutic gene-edited TIL population as claimed in claim 396. A use of a therapeutic TIL population as claimed in claim 349 or a therapeutic gene-edited TIL population as claimed in any one of claims 172 to 184 and 396 for treating cancer in a patient in need thereof, comprising administering the therapeutic TIL population or the therapeutic gene-edited TIL population to the patient. The use of claim 398, wherein the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer and renal cell carcinoma. The use of claim 398 or 399, further comprising the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the TILs to the patient. The use of claim 400, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. The use of claim 400, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for three days. The use of claim 400, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of administering cyclophosphamide at a dose of 60 mg/m2/day and fludarabine at a dose of 25 mg/m2/day for two days, followed by administering fludarabine at a dose of 25 mg/m2/day for one day. The use of any one of claims 401 to 403, wherein the cyclophosphamide is administered together with mesnat. The use of any one of claims 398 to 404, further comprising the step of treating the patient with an IL-2 regimen starting the day after the TIL is administered to the patient. The use of any one of claims 398 to 404, further comprising the step of starting treatment of the patient with an IL-2 regimen on the same day that the TIL is administered to the patient. The use of claim 405 or 406, wherein the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg of aldesleukin or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion once every eight hours until tolerated. The method of any one of claims 398 to 404, which does not include the step of treating the patient with an IL-2 regimen.