[go: up one dir, main page]

WO2025101484A1 - Traitement de cancers endométriaux avec des thérapies lymphocytaires infiltrant les tumeurs - Google Patents

Traitement de cancers endométriaux avec des thérapies lymphocytaires infiltrant les tumeurs Download PDF

Info

Publication number
WO2025101484A1
WO2025101484A1 PCT/US2024/054513 US2024054513W WO2025101484A1 WO 2025101484 A1 WO2025101484 A1 WO 2025101484A1 US 2024054513 W US2024054513 W US 2024054513W WO 2025101484 A1 WO2025101484 A1 WO 2025101484A1
Authority
WO
WIPO (PCT)
Prior art keywords
tils
expansion
days
population
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/054513
Other languages
English (en)
Inventor
Frederick G. Vogt
Friedrich Graf Finck VON FINCKENSTEIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Iovance Biotherapeutics Inc
Original Assignee
Iovance Biotherapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Iovance Biotherapeutics Inc filed Critical Iovance Biotherapeutics Inc
Publication of WO2025101484A1 publication Critical patent/WO2025101484A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex

Definitions

  • TIL tumor-infiltrating lymphocytes
  • metastatic melanoma Sudik AA et al., J Clin Oncol 2021
  • cervical carcinoma Jazaeri AA et al., ASCO 2019 #182
  • non-small-cell lung cancer and other epithelial malignancies.
  • overexpression of different cytokines can modify the tumor microenvironment, that can further promote survival, activity and/or longevity of administered TILs.
  • overexpression of IL-15 can improve homeostatic maintenance of long-lived CD8+ memory T-cells, inhibits AICD and reverses T-cell energy.
  • overexpression of IL-18 induces production of IFN ⁇ and acute Th1 response.
  • overexpression of IL21 supports T-cell survival in absence of IL-2, favoring CM/EM phenotype and decreasing PD1 expression.
  • constitutive co-expression of IL-15 and IL-21 may support high expansion levels in in vivo (and probably in vitro), upon administration of the TILs, and should restrict signaling to TILs and allow them to persist, decrease toxicity. The decrease in toxicity may, for example, enable adoptive cell therapy without nonmyeloablative lymphodepletion. 1 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO IV.
  • the invention provides methods and compositions for the treatment of endometrial cancer using tumor infiltrating lymphocyte therapies. In an embodiment, the invention provides methods and compositions for the treatment of uterine cancer using tumor infiltrating lymphocyte therapies. [0006] In an embodiment, the invention provides methods and compositions for the treatment of endometrial cancer using tumor infiltrating lymphocyte therapies, wherein the endometrial cancer is resected and processed to prepare tumor infiltrating lymphocytes.
  • Embodiments disclosed herein provide a method of treating a subject with endometrial cancer, the method comprising administering expanded tumor infiltrating lymphocytes (TILs) comprising: (a) adding a tumor digest or tumor fragments from an endometrial cancer into a closed system, wherein the tumor digest or tumor fragments comprise a first population of TILs and are obtained from a tumor that was resected from the subject; (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, 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 population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the system; (c) performing a second expansion by supplementing additional cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs),
  • the endometrial cancer is metastatic or unresectable.
  • the subject was refractory to or progressed following prior treatment with a PD-1 or 2 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO PD-L1 inhibitor.
  • the subject was refractory to or progressed following prior treatment with chemotherapy.
  • the chemotherapy is a platinum chemotherapy and a taxane.
  • the platinum chemotherapy is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin.
  • the taxane is selected from the group consisting of paclitaxel, albumin-bound paclitaxel, and docetaxel.
  • the endometrial cancer is microsatellite instability high (MSI-H).
  • the endometrial cancer is mismatch repair deficient (dMMR).
  • the endometrial cancer is microsatellite stable (MSS).
  • the endometrial cancer is mismatch repair proficient (pMMR).
  • the subject is treatment na ⁇ ve.
  • the method comprises the additional step of administering a therapeutically effective dosage of a PD-1 or PD-L1 inhibitor.
  • the anti-PD-1 or anti-PD-L1 antibody is selected from the group consisting of dostarlimab, nivolumab, pembrolizumab, JS001, TSR-042, pidilizumab, BGB- A317, SHR-1210, REGN2810, MDX-1106, PDR001, anti-PD-l from clone: RMP1-14, an anti-PD-l antibodies disclosed in U.S. Patent No.8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, as well as biosimilars thereof.
  • the method further comprises the step of treating the subject with a non-myeloablative lymphodepletion regimen prior to administering the therapeutic population of TILs to the subject.
  • the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg/m 2 /day for two days followed by administration of fludarabine at a dose of 25 mg/m 2 /day for five days.
  • the method further comprises the step of treating the subject with an IL-2 regimen starting on the day after administration of the therapeutic population of TILs to the subject.
  • 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 tolerance.
  • the first expansion is performed over a period of 12 days or less.
  • the second expansion is performed over a period of 12 days or less.
  • steps (a) through (d) are performed in about 10 days to about 22 days.
  • steps (a) through (d) are performed in about 15 days to about 22 days.
  • steps (a) through (d) are performed in about 20 days to about 22 days.
  • the IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL in the first cell culture medium. In some embodiments, the IL-2 is present at an initial concentration of 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 the second cell culture medium.
  • the first expansion is performed using a gas permeable container. In some embodiments, the second expansion is performed using a gas permeable container.
  • TILs tumor infiltrating lymphocytes
  • Some embodiments disclosed herein provide a method of expanding tumor infiltrating lymphocytes (TILs) comprising: (a) adding a tumor digest or tumor fragments from an endometrial cancer into a closed system, wherein the tumor digest or tumor fragments comprise a first population of TILs and are obtained from a tumor that was resected from a subject; (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, 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 population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the system; (c) performing a second expansion by supplementing additional cell culture medium comprising IL-2, OKT-3, and antigen
  • the tumor digest in step (a) was prepared by incubating a sample of the tumor that was resected from the subject in an enzymatic media.
  • the method further comprises disrupting the tumor sample mechanically so as to dissociate the tumor sample.
  • the method further comprises purifying the disassociated tumor sample using a density gradient separation.
  • the enzymatic media comprises DNase.
  • the enzymatic media comprises 30 units/mL of DNase.
  • the enzymatic media comprises collagenase.
  • the enzymatic media comprises 1.0 mg/mL of collagenase.
  • the medium in the first expansion and/or the second expansion is free of human serum.
  • the first expansion is performed over a period of 12 days or less.
  • the second expansion is performed over a period of 12 days or less.
  • steps (a) through (d) are performed in about 10 days to about 22 days.
  • steps (a) through (d) are performed in about 15 days to about 22 days.
  • steps (a) through (d) are performed in about 20 days to about 22 days.
  • the IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL in the first cell culture medium. In some embodiments, the IL-2 is present at an initial concentration of 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 the second cell culture medium.
  • the first expansion is performed using a gas permeable container. In some embodiments, the second expansion is performed using a gas permeable container.
  • the APCs are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are supplemented at a ratio of about 1:25 TIL:PBMCs.
  • TILs tumor infiltrating lymphocytes
  • a method comprising: (a) adding a tumor digest or tumor fragments into a closed system, wherein the tumor digest or tumor fragments comprise a first population of TILs and are obtained from a tumor that was resected from a subject having endometrial cancer; (b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for a first period of about 3 to 11 days to obtain the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion by supplementing the cell culture medium with additional IL-2, OKT-3, and antigen presenting cells (APCs) to
  • the tumor digest in step (a) was prepared by incubating a sample of the tumor that was resected from the subject in an enzymatic media.
  • the method further comprises disrupting the tumor sample mechanically so as to dissociate the tumor sample.
  • the method further comprises purifying the disassociated tumor sample using a density gradient separation.
  • the enzymatic media comprises DNase.
  • the enzymatic media comprises 30 units/mL of DNase.
  • the enzymatic media comprises collagenase.
  • the enzymatic media comprises 1.0 mg/mL of collagenase.
  • the medium in the first expansion and/or the second expansion is free of human serum.
  • the therapeutic population of TILs harvested in step (d) comprises sufficient TILs for use in administering a therapeutically effective dosage to a subject.
  • the number of TILs sufficient for administering a therapeutically effective dosage is from about 1 ⁇ 10 9 to about 5 ⁇ 10 9 .
  • the number of TILs sufficient for administering a therapeutically effective dosage is from about 1 ⁇ 10 9 to about 9 ⁇ 10 10 .
  • the number of TILs sufficient for administering a therapeutically effective dosage is from about 5 ⁇ 10 9 to about 1 ⁇ 10 10 . In some embodiments, the number of TILs sufficient for administering a therapeutically effective dosage is from about 1 ⁇ 10 10 to about 5 ⁇ 10 10 .
  • the therapeutic population of TILs harvested in step (d) exhibits an increased subpopulation of CD8+ cells relative to the first and/or second population of TILs [0016] In some embodiments, the first expansion is performed over a period of 12 days or less. In some embodiments, the second expansion is performed over a period of 12 days or less. In some embodiments, steps (a) through (d) are performed in about 10 days to about 22 days.
  • steps (a) through (d) are performed in about 15 days to about 22 days. In some embodiments, steps (a) through (d) are performed in about 20 days to about 22 days.
  • the IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL in the first cell culture medium. In some embodiments, the IL-2 is present at an initial concentration of 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 the second cell culture medium.
  • the first expansion is performed using a gas permeable container. In some embodiments, the second expansion is performed using a gas permeable container.
  • the APCs are peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs are supplemented at a ratio of about 1:25 TIL:PBMCs.
  • the tumor digest is a cryopreserved tumor digest.
  • Figure 3 Shows a diagram of an embodiment of a cryopreserved TIL exemplary manufacturing process ( ⁇ 22 days).
  • Figure 4 Shows a diagram of an embodiment of Gen 2 (process 2A), a 22-day process for TIL manufacturing.
  • Figure 5 Comparison table of Steps A through F from exemplary embodiments of process 1C and Gen 2 (process 2A) for TIL manufacturing.
  • Figure 6 Detailed comparison of an embodiment of process 1C and an embodiment of Gen 2 (process 2A) for TIL manufacturing.
  • Figure 7 Provides the structures I-A and I-B. The cylinders refer to individual polypeptide binding domains.
  • Structures I-A and I-B comprise three linearly-linked TNFRSF binding domains derived from e.g., 4-1BBL or an antibody that binds 4-1BB, which fold to form a trivalent protein, which is then linked to a second trivalent protein through IgG1-Fc (including CH3 and CH2 domains) is then used to link two of the trivalent proteins together through disulfide bonds (small elongated ovals), stabilizing the structure and providing an agonists capable of bringing together the intracellular signaling domains of the six receptors and signaling proteins to form a signaling complex.
  • IgG1-Fc including CH3 and CH2 domains
  • the TNFRSF binding domains denoted as cylinders may be scFv domains comprising, e.g., a VH and a VL chain connected by a linker that may comprise hydrophilic residues and Gly and Ser sequences for flexibility, as well as Glu and Lys for solubility.
  • Figures 8A-8B A) shows an example of structure and synthesis of lipid click molecule having a BCN group.
  • B) shows an example of structure and synthesis of an anchoring molecule having a PEG-azide molecule tagged thereto.
  • Figures 9A-9B Phenotype of endometrial TIL.
  • Figures 10A-10C NeoTCR8 analysis of endometrial TIL.
  • Figures 11A-11B Enhanced TIL cell activation after co-culture with autologous tumor digest.
  • Figures 12A-12B Increased cytokine production after co-culture with autologous tumor digest.
  • Figure 13 TIL-mediated autologous anti-tumor killing using MOS. Abbreviations: ANOVA, analysis of variance; MOS, micro-organospheres; NIR, near-infrared spectroscopy. ****, p ⁇ 0.0001.
  • SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.
  • SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.
  • SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2 protein.
  • SEQ ID NO:4 is the amino acid sequence of aldesleukin.
  • SEQ ID NO:5 is an IL-2 form.
  • SEQ ID NO:6 is the amino acid sequence of nemvaleukin alfa.
  • SEQ ID NO:7 is an IL-2 form.
  • SEQ ID NO:8 is a mucin domain polypeptide.
  • SEQ ID NO:9 is the amino acid sequence of a recombinant human IL-4 protein.
  • SEQ ID NO:10 is the amino acid sequence of a recombinant human IL-7 protein.
  • SEQ ID NO:11 is the amino acid sequence of a recombinant human IL-15 protein.
  • SEQ ID NO:12 is the amino acid sequence of a recombinant human IL-21 protein.
  • SEQ ID NO:13 is an IL-2 sequence.
  • SEQ ID NO:14 is an IL-2 mutein sequence.
  • SEQ ID NO:15 is an IL-2 mutein sequence.
  • SEQ ID NO:16 is the HCDR1_IL-2 for IgG.IL2R67A.H1.
  • SEQ ID NO:17 is the HCDR2 for IgG.IL2R67A.H1.
  • SEQ ID NO:18 is the HCDR3 for IgG.IL2R67A.H1.
  • SEQ ID NO:19 is the HCDR1_IL-2 kabat for IgG.IL2R67A.H1.
  • SEQ ID NO:20 is the HCDR2 kabat for IgG.IL2R67A.H1.
  • SEQ ID NO:21 is the HCDR3 kabat for IgG.IL2R67A.H1.
  • SEQ ID NO:22 is the HCDR1_IL-2 clothia for IgG.IL2R67A.H1.
  • SEQ ID NO:23 is the HCDR2 clothia for IgG.IL2R67A.H1.
  • SEQ ID NO:24 is the HCDR3 clothia for IgG.IL2R67A.H1.
  • SEQ ID NO:25 is the HCDR1_IL-2 IMGT for IgG.IL2R67A.H1.
  • SEQ ID NO:26 is the HCDR2 IMGT for IgG.IL2R67A.H1.
  • SEQ ID NO:27 is the HCDR3 IMGT for IgG.IL2R67A.H1.
  • SEQ ID NO:28 is the VH chain for IgG.IL2R67A.H1.
  • SEQ ID NO:29 is the heavy chain for IgG.IL2R67A.H1.
  • SEQ ID NO:30 is the LCDR1 kabat for IgG.IL2R67A.H1.
  • SEQ ID NO:31 is the LCDR2 kabat for IgG.IL2R67A.H1.
  • SEQ ID NO:32 is the LCDR3 kabat for IgG.IL2R67A.H1.
  • SEQ ID NO:33 is the LCDR1 chothia for IgG.IL2R67A.H1.
  • SEQ ID NO:34 is the LCDR2 chothia for IgG.IL2R67A.H1.
  • SEQ ID NO:35 is the LCDR3 chothia for IgG.IL2R67A.H1.
  • SEQ ID NO:36 is a VL chain.
  • SEQ ID NO:37 is a light chain.
  • SEQ ID NO:38 is a light chain.
  • SEQ ID NO:39 is a light chain.
  • SEQ ID NO:40 is the amino acid sequence of human 4-1BB.
  • SEQ ID NO:41 is the amino acid sequence of murine 4-1BB.
  • SEQ ID NO:42 is the heavy chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:43 is the light chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:44 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:45 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:46 is the heavy chain CDR1 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:47 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:48 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:49 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:50 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:51 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
  • SEQ ID NO:52 is the heavy chain for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:53 is the light chain for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:54 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:55 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:56 is the heavy chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:57 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:58 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:59 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:60 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:61 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
  • SEQ ID NO:62 is an Fc domain for a TNFRSF agonist fusion protein.
  • SEQ ID NO:63 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:64 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:65 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:66 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:67 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:68 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:69 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:70 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:71 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:72 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:73 is an Fc domain for a TNFRSF agonist fusion protein.
  • SEQ ID NO:74 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:75 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:76 is a linker for a TNFRSF agonist fusion protein.
  • SEQ ID NO:77 is a 4-1BB ligand (4-1BBL) amino acid sequence.
  • SEQ ID NO:78 is a soluble portion of 4-1BBL polypeptide.
  • SEQ ID NO:79 is a heavy chain variable region (VH) for the 4-1BB agonist antibody 4B4- 1-1 version 1.
  • SEQ ID NO:80 is a light chain variable region (VL) for the 4-1BB agonist antibody 4B4-1- 1 version 1.
  • SEQ ID NO:81 is a heavy chain variable region (VH) for the 4-1BB agonist antibody 4B4- 1-1 version 2.
  • SEQ ID NO:82 is a light chain variable region (VL) for the 4-1BB agonist antibody 4B4-1- 1 version 2.
  • SEQ ID NO:83 is a heavy chain variable region (VH) for the 4-1BB agonist antibody H39E3-2.
  • SEQ ID NO:84 is a light chain variable region (VL) for the 4-1BB agonist antibody H39E3- 2.
  • SEQ ID NO:85 is the amino acid sequence of human OX40.
  • SEQ ID NO:86 is the amino acid sequence of murine OX40.
  • SEQ ID NO:87 is the heavy chain for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
  • SEQ ID NO:88 is the light chain for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
  • SEQ ID NO:89 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
  • SEQ ID NO:90 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
  • SEQ ID NO:91 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
  • SEQ ID NO:92 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
  • SEQ ID NO:93 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562). 12 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • SEQ ID NO:94 is the light chain CDR1 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
  • SEQ ID NO:95 is the light chain CDR2 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
  • SEQ ID NO:96 is the light chain CDR3 for the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
  • SEQ ID NO:97 is the heavy chain for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:98 is the light chain for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:99 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:100 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:101 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:102 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:103 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:104 is the light chain CDR1 for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:105 is the light chain CDR2 for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:106 is the light chain CDR3 for the OX40 agonist monoclonal antibody 11D4.
  • SEQ ID NO:107 is the heavy chain for the OX40 agonist monoclonal antibody 18D8.
  • SEQ ID NO:108 is the light chain for the OX40 agonist monoclonal antibody 18D8.
  • SEQ ID NO:109 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 18D8.
  • SEQ ID NO:110 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 18D8. 13 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • SEQ ID NO:111 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody 18D8.
  • SEQ ID NO:112 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody 18D8.
  • SEQ ID NO:113 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody 18D8.
  • SEQ ID NO:114 is the light chain CDR1 for the OX40 agonist monoclonal antibody 18D8.
  • SEQ ID NO:115 is the light chain CDR2 for the OX40 agonist monoclonal antibody 18D8.
  • SEQ ID NO:116 is the light chain CDR3 for the OX40 agonist monoclonal antibody 18D8.
  • SEQ ID NO:117 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody Hu119-122.
  • SEQ ID NO:118 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody Hu119-122.
  • SEQ ID NO:119 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody Hu119-122.
  • SEQ ID NO:120 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody Hu119-122.
  • SEQ ID NO:121 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody Hu119-122.
  • SEQ ID NO:122 is the light chain CDR1 for the OX40 agonist monoclonal antibody Hu119-122.
  • SEQ ID NO:123 is the light chain CDR2 for the OX40 agonist monoclonal antibody Hu119-122.
  • SEQ ID NO:124 is the light chain CDR3 for the OX40 agonist monoclonal antibody Hu119-122.
  • SEQ ID NO:125 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody Hu106-222.
  • SEQ ID NO:126 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody Hu106-222. 14 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • SEQ ID NO:127 is the heavy chain CDR1 for the OX40 agonist monoclonal antibody Hu106-222.
  • SEQ ID NO:128 is the heavy chain CDR2 for the OX40 agonist monoclonal antibody Hu106-222.
  • SEQ ID NO:129 is the heavy chain CDR3 for the OX40 agonist monoclonal antibody Hu106-222.
  • SEQ ID NO:130 is the light chain CDR1 for the OX40 agonist monoclonal antibody Hu106-222.
  • SEQ ID NO:131 is the light chain CDR2 for the OX40 agonist monoclonal antibody Hu106-222.
  • SEQ ID NO:132 is the light chain CDR3 for the OX40 agonist monoclonal antibody Hu106-222.
  • SEQ ID NO:133 is an OX40 ligand (OX40L) amino acid sequence.
  • SEQ ID NO:134 is a soluble portion of OX40L polypeptide.
  • SEQ ID NO:135 is an alternative soluble portion of OX40L polypeptide.
  • SEQ ID NO:136 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 008.
  • SEQ ID NO:137 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 008.
  • SEQ ID NO:138 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 011.
  • SEQ ID NO:139 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 011.
  • SEQ ID NO:140 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 021.
  • SEQ ID NO:141 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 021.
  • SEQ ID NO:142 is the heavy chain variable region (VH) for the OX40 agonist monoclonal antibody 023.
  • SEQ ID NO:143 is the light chain variable region (VL) for the OX40 agonist monoclonal antibody 023.
  • SEQ ID NO:144 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.
  • SEQ ID NO:145 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.
  • SEQ ID NO:146 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.
  • SEQ ID NO:147 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.
  • SEQ ID NO:148 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.
  • SEQ ID NO:149 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.
  • SEQ ID NO:150 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.
  • SEQ ID NO:151 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.
  • SEQ ID NO:152 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.
  • SEQ ID NO:153 is the heavy chain variable region (VH) for a humanized OX40 agonist monoclonal antibody.
  • SEQ ID NO:154 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.
  • SEQ ID NO:155 is the light chain variable region (VL) for a humanized OX40 agonist monoclonal antibody.
  • SEQ ID NO:156 is the heavy chain variable region (VH) for an OX40 agonist monoclonal antibody.
  • SEQ ID NO:157 is the light chain variable region (VL) for an OX40 agonist monoclonal antibody.
  • SEQ ID NO:158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:160 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:161 is the light chain variable region (VL) amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.
  • SEQ ID NO:168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.
  • SEQ ID NO:169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.
  • SEQ ID NO:170 is the heavy chain variable region (VH) amino acid sequence of the PD-1 inhibitor pembrolizumab.
  • SEQ ID NO:171 is the light chain variable region (VL) amino acid sequence of the PD-1 inhibitor pembrolizumab. 17 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • SEQ ID NO:172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.
  • SEQ ID NO:173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.
  • SEQ ID NO:174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.
  • SEQ ID NO:175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.
  • SEQ ID NO:176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.
  • SEQ ID NO:177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.
  • SEQ ID NO:178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.
  • SEQ ID NO:179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.
  • SEQ ID NO:180 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor durvalumab.
  • SEQ ID NO:181 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor durvalumab.
  • SEQ ID NO:182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.
  • SEQ ID NO:183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.
  • SEQ ID NO:184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.
  • SEQ ID NO:185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.
  • SEQ ID NO:186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab. 18 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • SEQ ID NO:187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.
  • SEQ ID NO:188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:190 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:191 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.
  • SEQ ID NO:198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.
  • SEQ ID NO:199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.
  • SEQ ID NO:200 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor atezolizumab.
  • SEQ ID NO:201 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor atezolizumab. 19 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • SEQ ID NO:202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.
  • SEQ ID NO:203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.
  • SEQ ID NO:204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.
  • SEQ ID NO:205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.
  • SEQ ID NO:206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.
  • SEQ ID NO:207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.
  • SEQ ID NO:208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.
  • SEQ ID NO:209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.
  • SEQ ID NO:210 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor ipilimumab.
  • SEQ ID NO:211 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor ipilimumab.
  • SEQ ID NO:212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.
  • SEQ ID NO:213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.
  • SEQ ID NO:214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.
  • SEQ ID NO:215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab. 20 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • SEQ ID NO:216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.
  • SEQ ID NO:217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.
  • SEQ ID NO:218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:220 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:221 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.
  • SEQ ID NO:228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.
  • SEQ ID NO:229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab. 21 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • SEQ ID NO:230 is the heavy chain variable region (VH) amino acid sequence of the CTLA-4 inhibitor zalifrelimab.
  • SEQ ID NO:231 is the light chain variable region (VL) amino acid sequence of the CTLA-4 inhibitor zalifrelimab.
  • SEQ ID NO:232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.
  • SEQ ID NO:233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.
  • SEQ ID NO:234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.
  • SEQ ID NO:235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.
  • SEQ ID NO:236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.
  • SEQ ID NO:237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.
  • SEQ ID NO:238 is a CD8a transmembrane domain.
  • SEQ ID NO:239 is a B7-1 transmembrane-intracellular domain
  • SEQ ID NOs:240-245 are exemplary glycine-serine linkers that are useful in the immunomodulatory fusion proteins described herein.
  • SEQ ID NO:246 is an exemplary linker that is useful in the immunomodulatory fusion proteins described herein.
  • SEQ ID NO:247 is a 2A peptide C-terminus sequence.
  • SEQ ID NO:248 is a porcine teschovirus-12A peptide.
  • SEQ ID NO:249 is an equine rhinitis A virus 2A peptide.
  • SEQ ID NO:250 is a foot-and-mouth disease virus 2A peptide.
  • SEQ ID NO:251 is an exemplary furin-cleavable 2A peptide.
  • SEQ ID NOs:252 and 253 are human IgE signal peptide sequences. 22 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • SEQ ID NO:254 is a human IL-2 signal peptide sequence.
  • SEQ ID NO:255 is a 6X NFAT IL-2 minimal promoter.
  • SEQ ID NO:256 is an NFAT responsive element.
  • SEQ ID NO:557 is a human IL-2 promoter sequence.
  • SEQ ID NO:258 is human IL-15 (N72D mutant).
  • SEQ ID NO:259 is human IL-15R-alpha-Su/Fc domain.
  • SEQ ID NO:260 is human IL-15R-alpha-Su (65aa truncated extracellular domain).
  • SEQ ID NO:261 is human IL-15 isoform 2.
  • SEQ ID NO:262 is human IL-15 isoform 1.
  • SEQ ID NO:263 is human IL-15 (without signal peptide).
  • SEQ ID NO:264 is human IL-15R-alpha (85 aa truncated extracellular domain).
  • SEQ ID NO:265 is human IL-15R-alpha (182aa truncated extracellular domain).
  • SEQ ID NO:266 is human IL-15R-alpha.
  • SEQ ID NO:267 is human IL-12 p35 subunit.
  • SEQ ID NO:268 is human IL-12 p40 subunit.
  • SEQ ID NO:269 is human IL-18.
  • SEQ ID NO:270 is a human IL-18 variant.
  • SEQ ID NO:271 is human IL-21.
  • SEQ ID NO: 272 is human IL-2.
  • SEQ ID NO:273 is human CD40L.
  • SEQ ID NO:274 is agonistic anti-human CD40 VH (Sotigalimab).
  • SEQ ID NO:275 is agonistic anti-human CD40 VL (Sotigalimab).
  • SEQ ID NO:276 is agonistic anti-human CD40 scFv (Sotigalimab).
  • SEQ ID NO:277 is agonistic anti-human CD40 VH (Dacetuzumab).
  • SEQ ID NO:278 is agonistic anti-human CD40 VL (Dacetuzumab).
  • SEQ ID NO:279 is agonistic anti-human CD40 scFv (Dacetuzumab).
  • SEQ ID NO:280 is agonistic anti-human CD40 VH (Lucatutuzumab).
  • SEQ ID NO:281 is agonistic anti-human CD40 VL (Lucatutuzumab).
  • SEQ ID NO:282 is agonistic anti-human CD40 scFv (Lucatutuzumab).
  • SEQ ID NO:283 is agonistic anti-human CD40 VH (Selicrelumab).
  • SEQ ID NO:284 is agonistic anti-human CD40 VL (Selicrelumab).
  • SEQ ID NO:285 is agonistic anti-human CD40 scFv (Selicrelumab).
  • SEQ ID NO:286 is a target PD-1 sequence.
  • SEQ ID NO:287 is a target PD-1 sequence.
  • SEQ ID NO:288 is a repeat PD-1 left repeat sequence.
  • SEQ ID NO:289 is a repeat PD-1 right repeat sequence.
  • SEQ ID NO:290 is a repeat PD-1 left repeat sequence.
  • SEQ ID NO:291 is a repeat PD-1 right repeat sequence.
  • SEQ ID NO:292 is a PD-1 left TALEN nuclease sequence.
  • SEQ ID NO:293 is a PD-1 right TALEN nuclease sequence.
  • SEQ ID NO:294 is a PD-1 left TALEN nuclease sequence.
  • SEQ ID NO:295 is a PD-1 right TALEN nuclease sequence.
  • SEQ ID NO:296 is an exemplary piggyBac (PB) transposase enzyme amino acid sequence.
  • SEQ ID NO:297 is an exemplary Sleeping Beauty transposase enzyme amino acid sequence.
  • SEQ ID NO:298 is an exemplary hyperactive Sleeping Beauty (SB100X) transposase amino acid sequence.
  • SEQ ID NO:299 is an exemplary Clo05 l nuclease domain amino acid sequence.
  • SEQ ID NO:300 is an exemplary the Myc polypeptide sequence.
  • SEQ ID NO:301 is an exemplary MYC fusion protein amino acid sequence.
  • SEQ ID NO:302 is HHHHHH.
  • SEQ ID NO:303 is an exemplary V5 tag amino acid sequence: GKPIPNPLLGLDST.
  • SEQ ID NO:304 is an exemplary V5 tag amino acid sequence: IPNPLLGLD.
  • co-administration encompass administration of two or more active pharmaceutical ingredients (in a preferred 24 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO embodiment of the present invention, for example, a plurality of TILs) to a subject so that both active pharmaceutical ingredients and/or their metabolites are present in the subject at the same time.
  • Co- administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present.
  • in vivo refers to an event that takes place in a subject's body.
  • in vitro refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.
  • ex vivo refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject’s body.
  • the cell, tissue and/or organ may be returned to the subject’s body in a method of surgery or treatment.
  • rapid expansion means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week.
  • a number of rapid expansion protocols are described herein.
  • TILs tumor infiltrating lymphocytes
  • cytotoxic T cells lymphocytes
  • Th1 and Th17 CD4+ T cells natural killer cells
  • dendritic cells dendritic cells
  • M1 macrophages include both primary and secondary TILs.
  • Primary TILs are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations can include genetically modified TILs. [00336] By “population of cells” (including TILs) herein is meant a number of cells that share common traits. In general, populations generally range from 1 X 10 6 to 1 X 10 10 in number, with different TIL populations comprising different numbers.
  • cryopreserved TILs herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored in the range of about -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples.
  • cryopreserved TILs are distinguishable from frozen tissue samples which may be used as a source of primary TILs.
  • thawed cryopreserved TILs herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient.
  • TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment.
  • TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR ⁇ , 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 upon reintroduction into a patient.
  • the term “cryopreservation media” or “cryopreservation medium” refers to any medium that can be used for cryopreservation of cells. Such media can include media comprising 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, as well as combinations thereof.
  • CS10 refers to a cryopreservation medium which is obtained from Stemcell Technologies or from Biolife Solutions.
  • the CS10 medium may be referred to by the trade name “CryoStor® CS10”.
  • the CS10 medium is a serum-free, animal component-free medium which comprises DMSO.
  • central memory T cell refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi).
  • the surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R.
  • central memory T cells Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1.
  • Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCR triggering.
  • Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.
  • effector memory T cell refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7lo) and are heterogeneous or low for CD62L expression (CD62Llo).
  • the surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R.
  • Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon- ⁇ , IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are 26 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin. [00343]
  • the term “closed system” refers to a system that is closed to the outside environment.
  • Closed systems include, for example, but are not limited to, closed G-containers. Once a tumor segment is added to the closed system, the system is no opened to the outside environment until the TILs are ready to be administered to the patient.
  • fragmenting includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue.
  • peripheral blood mononuclear cells and “PBMCs” refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes.
  • T cells lymphocytes
  • B cells lymphocytes
  • monocytes When used as an antigen presenting cell (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.
  • peripheral blood lymphocytes and “PBLs” refer to T cells expanded from peripheral blood. In some embodiments, PBLs are separated from whole blood or apheresis product from a donor.
  • PBLs are separated from whole blood or apheresis product from a donor by positive or negative selection of a T cell phenotype, such as the T cell phenotype of CD3+ CD45+.
  • a T cell phenotype such as the T cell phenotype of CD3+ CD45+.
  • the term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells.
  • Anti-CD3 antibodies include OKT-3, also known as muromonab.
  • Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3 ⁇ .
  • OKT-3 refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof.
  • the amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2).
  • a hybridoma capable 27 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001.
  • a hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No.86022706. TABLE 1.
  • Amino acid sequences of muromonab exemplary OKT-3 antibody).
  • interleukin-2 Identifier Sequence (One-Letter Amino Acid Symbols) known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof.
  • IL-2 is described, e.g., in Nelson, J. Immunol.2004, 172, 3983-88 and Malek, Annu. Rev. Immunol.2008, 26, 453-79, the disclosures of which are incorporated by reference herein.
  • the amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3).
  • IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors.
  • Aldesleukin (des-alanyl-1, serine- 125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa.
  • IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug bempegaldesleukin (NKTR-214, pegylated human recombinant IL-2 as in SEQ ID NO:4 in which an average of 6 lysine residues are N6 substituted with [(2,7-bis ⁇ [methylpoly(oxyethylene)]carbamoyl ⁇ -9H-fluoren-9-yl)methoxy]carbonyl), 28 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO which is available from Nektar Therapeutics, South San Francisco, CA, USA, or which may be prepared by methods known in the art, such as the methods described in Example 19 of International Patent Application Publication No.
  • NKTR-214 pegylated human recombinant IL-2 as in SEQ ID NO:4 in which an average of 6 lysine residues are N6 substituted with [(2,7
  • WO 2018/132496 A1 or the method described in Example 1 of U.S. Patent Application Publication No. US 2019/0275133 A1, the disclosures of which are incorporated by reference herein.
  • Bempegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the 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 by reference herein.
  • Alternative forms of conjugated IL-2 suitable for use in the 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 by reference herein.
  • an IL-2 form suitable for use in the present invention is THOR-707, available from Synthorx, Inc.
  • the preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the 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 by reference herein.
  • IL-2 form suitable for use in the invention is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5.
  • IL-2 interleukin 2
  • 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.
  • 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 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine.
  • the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to an unnatural amino acid.
  • the unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L- lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8- 29 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L- phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine,
  • the IL-2 conjugate has a decreased affinity to IL-2 receptor ⁇ (IL-2R ⁇ ) subunit relative to a wild-type IL-2 polypeptide.
  • the decreased affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease in binding affinity to IL-2R ⁇ relative to a wild-type IL- 2 polypeptide.
  • the decreased 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 more relative to a wild-type IL-2 polypeptide.
  • the conjugating moiety impairs or blocks the binding of IL-2 with IL-2R ⁇ .
  • the conjugating moiety comprises a water-soluble polymer.
  • the additional conjugating moiety comprises a water-soluble polymer.
  • each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ -hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.
  • each of the water-soluble polymers independently comprises PEG.
  • the PEG is a linear PEG or a branched PEG.
  • each of the water-soluble polymers independently comprises a polysaccharide.
  • the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl- starch (HES).
  • each of the water-soluble polymers independently comprises a glycan.
  • each of the water-soluble polymers independently comprises polyamine.
  • the conjugating moiety comprises a protein.
  • the additional conjugating moiety comprises a protein. In some embodiments, each of the proteins independently comprises an albumin, a transferrin, or a transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of IgG. In some embodiments, the conjugating moiety comprises a polypeptide. In some embodiments, the additional conjugating moiety comprises a polypeptide.
  • each of the polypeptides independently comprises a XTEN 30 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.
  • HAP glycine-rich homoamino acid polymer
  • PAS polypeptide glycine-rich homoamino acid polymer
  • ELP elastin-like polypeptide
  • CTP CTP peptide
  • GLK gelatin-like protein
  • the isolated and purified IL-2 polypeptide is modified by glutamylation.
  • the conjugating moiety is directly bound to the isolated and purified IL-2 polypeptide.
  • the conjugating moiety is indirectly bound to the isolated and purified IL-2 polypeptide through a linker.
  • the linker comprises a homobifunctional linker.
  • the homobifunctional linker comprises Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pi
  • the linker comprises a heterobifunctional linker.
  • the heterobifunctional linker comprises N-succinimidyl 3-(2- pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC- sPDP), water-soluble-long-chain N-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl- ⁇ -methyl- ⁇ -(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[ ⁇ - methyl- ⁇ -(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N- maleimidomethyl)cycl
  • the linker comprises a cleavable linker, optionally comprising a dipeptide linker.
  • the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.
  • the linker comprises a non-cleavable linker.
  • the linker comprises a maleimide group, optionally comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo- sMCC).
  • the linker further comprises a spacer.
  • the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analog thereof.
  • the conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.
  • the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.
  • the IL-2 form suitable for use in the invention is a fragment of any of the IL-2 forms described herein.
  • the IL-2 form suitable for use in the 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.
  • the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a 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 the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5.
  • AzK N6-azidoethoxy-L-lysine
  • the 32 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:5.
  • the IL-2 form suitable for use in the invention lacks IL-2R alpha chain engagement but retains normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.
  • the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a 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 the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5.
  • AzK N6-azidoethoxy-L-lysine
  • the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6- azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a 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 the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5.
  • AzK N6- azidoethoxy-L-lysine
  • the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a 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 the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5.
  • AzK N6-azidoethoxy-L-lysine
  • an IL-2 form suitable for use in the invention is nemvaleukin alfa, also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes, Inc.
  • Nemvaleukin alfa is also known as human interleukin 2 fragment (1-59), variant (Cys125>Ser51), fused via peptidyl linker (60GG61) to human interleukin 2 fragment (62-132), fused via peptidyl linker (133GSGGGS138) to human interleukin 2 receptor ⁇ -chain fragment (139-303), produced in Chinese hamster ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133)-peptide [Cys125(51)>Ser]-mutant (1-59), fused via a G2 peptide linker (60-61) to human interleukin 2 (IL-2) (4-74)-peptide (62-132) and via a GSG3S peptid
  • nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at 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 positions: N187, N206, T212 using the numbering in SEQ ID NO:6.
  • an IL-2 form suitable for use in the invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6.
  • an IL-2 form suitable for use in the invention has the amino acid sequence given in SEQ ID NO:6 or conservative amino acid substitutions thereof.
  • an IL-2 form suitable for use in the invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or variants, fragments, or derivatives thereof.
  • an IL-2 form suitable for use in the 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 to amino acids 24-452 of SEQ ID NO:7, or variants, fragments, or derivatives thereof.
  • Other IL-2 forms suitable for use in the present invention are described in U.S. Patent No.
  • an IL-2 form suitable for use in the invention is a fusion protein comprising a first fusion partner that is linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1R ⁇ or a protein having at least 98% amino acid sequence identity to IL- 1R ⁇ and having the receptor antagonist activity of IL-R ⁇ , 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 as compared to a fusion of the first fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker.
  • antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL, wherein the antibody cytokine engrafted protein preferentially expands T effector cells
  • the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL, wherein the 35 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO IL-2 molecule is a mutein, and wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells.
  • the IL-2 regimen comprises administration of an antibody described in U.S.
  • the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL, wherein the IL-2 molecule is a mutein, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over 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: a IgG class light chain comprising SEQ ID NO:39 and a IgG class heavy chain comprising SEQ ID NO:38; a IgG class light chain comprising SEQ ID NO:37 and a Ig
  • an IL-2 molecule or a fragment thereof is engrafted into HCDR1 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR2 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR3 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR1 of the VL, wherein the IL-2 molecule is a mutein.
  • an IL-2 molecule or a fragment thereof is engrafted into LCDR2 of the VL, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR3 of the VL, wherein the IL-2 molecule is a mutein. [00354]
  • the insertion of the IL-2 molecule 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.
  • the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL2 sequence does not frameshift the CDR sequence.
  • the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL-2 sequence replaces all or part of a CDR sequence.
  • the replacement by the IL-2 molecule can be the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region the CDR.
  • a replacement by the IL-2 molecule can be as few as one or two amino acids of a CDR sequence, or the entire CDR sequences.
  • an IL-2 molecule is engrafted directly into a CDR without a peptide linker, with no additional amino acids between the CDR sequence and the IL-2 sequence.
  • an IL-2 molecule is engrafted indirectly into a CDR with a peptide linker, with one or more additional amino acids between the CDR sequence and the IL-2 sequence.
  • the IL-2 molecule described herein is an IL-2 mutein.
  • the IL-2 mutein comprising an R67A substitution.
  • the IL-2 mutein comprises the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15.
  • the IL- 2 mutein comprises an amino acid sequence in Table 1 in U.S. Patent Application Publication No.
  • the antibody cytokine engrafted protein comprises an 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 engrafted protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13 and SEQ ID NO:16.
  • the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of HCDR2 selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26.
  • the antibody cytokine engrafted protein comprises an HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27.
  • the antibody cytokine engrafted protein comprises a VH region comprising the amino acid sequence of SEQ ID NO:28.
  • the antibody cytokine engrafted protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody cytokine engrafted protein comprises a VL region comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine engrafted protein comprises a light chain comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted 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.
  • the antibody cytokine engrafted 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 engrafted 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 engrafted 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.
  • the antibody cytokine engrafted 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.
  • the antibody cytokine engrafted protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No.2020/0270334 A1, or variants, 37 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO derivatives, or fragments thereof, or conservative amino acid substitutions thereof, or proteins with at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto.
  • the antibody components of the antibody cytokine engrafted protein described herein comprise immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab.
  • the antibody cytokine engrafted protein described herein has a longer serum half- life than a wild-type IL-2 molecule such as, but not limited to, aldesleukin or a comparable molecule.
  • the antibody cytokine engrafted protein described herein has a sequence as set forth in Table 3.
  • IL-4 regulates the differentiation of na ⁇ ve helper T cells (Th0 cells) to Th2 T cells. Steinke and Borish, Respir. Res.2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL- 4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching to IgE and IgG1 expression from B cells. Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec- Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No.
  • IL-7 refers to a glycosylated tissue- derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from 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 consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell 40 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO development within the thymus and survival within the periphery.
  • Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071).
  • IL-15 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, glycoforms, biosimilars, and variants thereof.
  • IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein.
  • IL-15 shares ⁇ and ⁇ signaling receptor subunits with IL-2.
  • Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa.
  • Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No.34-8159-82).
  • the amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:11).
  • IL-18 refers to an interleukin that is a heterodimeric cytokine encoded by the IL-18 gene (e.g., Genbank Accession numbers: NM_001243211, NM_001562 and NM_001386420).
  • IL-18 structurally similar to IL-1 ⁇ , is a member of IL-1 superfamily of cytokines. This cytokine, which is expressed by many human lymphoid and nonlymphoid cells, has an important role in inflammatory processes.
  • IL-18 in combination with IL- 12 can activate cytotoxic T cells (CTLs), as well as natural killer (NK) cells, to produce IFN- ⁇ and, therefore, contributes to tumor immunity.
  • CTLs cytotoxic T cells
  • NK natural killer cells
  • IL-18 can enhance the anti-tumor effects of the TIL compositions provided herein.
  • the IL-18 associated with the modified TIL is a full length IL- 18, a fragment or a variant of IL-18.
  • the IL-18 is a human IL-18 or a variant human IL-18.
  • the IL-18 is a biological active human IL-18 variant.
  • the IL-18 includes 1, 2, 3,4 ,5 ,67, 8, 9, or 10 mutations as compared to a wild- type IL-18.
  • the variant IL-18 has the amino acid sequence in Table 21. 41 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO [00363]
  • the TIL compositions provided herein includes a nucleic acid encoding an immunomodulatory fusion protein that includes an IL-18, wherein the nucleic acid is operably linked to a NFAT promoter, as described herein.
  • IL-21 refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc.2014, 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4+ T cells.
  • an anti-tumor effective amount “a tumor-inhibiting effective amount”, or “therapeutic amount”
  • the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the tumor infiltrating lymphocytes (e.g.
  • secondary TILs or genetically modified cytotoxic lymphocytes described herein may be administered at a dosage of 104 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.
  • TILs (including in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these dosages.
  • the TILs can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg, et al., New Eng. J. of Med.1988, 319, 1676).
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the term “hematological malignancy”, “hematologic malignancy” or terms of correlative meaning refer to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system.
  • Hematological malignancies are also referred to as “liquid tumors.” Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic 42 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin’s lymphoma, and non-Hodgkin’s lymphomas.
  • ALL acute lymphoblastic leukemia
  • CLL small lymphocytic lymphoma
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • AoL acute monocytic leukemia
  • Hodgkin’s lymphoma and non-Hodgkin’s lymphomas
  • B cell hematological malignancy refers to hematological malignancies that affect B cells.
  • liquid tumor refers to an abnormal mass of cells that is fluid in nature.
  • Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies.
  • TILs obtained from liquid tumors may also be referred to herein as marrow infiltrating lymphocytes (MILs).
  • MILs marrow infiltrating lymphocytes
  • TILs obtained from liquid tumors, including liquid tumors circulating in peripheral blood may also be referred to herein as PBLs.
  • microenvironment may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within the microenvironment.
  • the tumor microenvironment refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive,” as described in Swartz, et al., Cancer Res., 2012, 72, 2473.
  • the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the invention.
  • the population of TILs may be provided wherein a patient is pre-treated with nonmyeloablative chemotherapy prior to an infusion of TILs according to the present invention.
  • the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m 2 /d for 5 days (days 27 to 23 prior to TIL infusion).
  • the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.
  • lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as 43 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO “immunosuppressive conditioning”) on the patient prior to the introduction of the TILs of the invention.
  • the term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment.
  • a therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, or the manner of administration.
  • the term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and/or cell migration).
  • the specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition.
  • treatment encompasses 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.
  • heterologous when used with reference to portions of a 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.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources.
  • 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).
  • sequence identity refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity can be measured using sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government’s National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. 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 additional publicly available software programs that can be used to align sequences.
  • the term “variant” encompasses but is not limited to antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody by way of one or more substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody.
  • the variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.
  • the variant retains the ability to specifically bind to the antigen of the reference antibody.
  • TILs tumor infiltrating lymphocytes
  • lymphocytes cytotoxic T cells
  • Th1 and Th17 CD4+ T cells natural killer cells
  • dendritic cells dendritic cells
  • M1 macrophages include both primary and secondary TILs.
  • Primary TILs are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussed herein.
  • reREP TILs can include for example second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D, including TILs referred to as reREP TILs).
  • TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment.
  • TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR ⁇ , 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 upon reintroduction into a patient.
  • TILs may further be characterized by potency – for example, TILs may 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.
  • IFN interferon
  • TILs may 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, 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.
  • IFN ⁇ interferon
  • RNA defines a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide defines a nucleotide with a hydroxyl group at the 2' position of a b-D- ribofuranose moiety.
  • RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Nucleotides of the RNA molecules described herein may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients.
  • pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.
  • the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art.
  • a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.
  • compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.” [00383]
  • antibody and its plural form “antibodies” refer to whole immunoglobulins and any antigen-binding fragment (“antigen-binding portion”) or single chains thereof.
  • VH and VL regions of an antibody may be further subdivided into regions of hypervariability, which are referred to as complementarity determining regions (CDR) or hypervariable regions (HVR), and which can be interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • FR framework regions 47 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen epitope or epitopes.
  • the constant regions of the antibodies may 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.
  • an antigen is a molecule capable of being bound by an antibody or a TCR if presented by major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the term “antigen”, as used herein, also encompasses T cell epitopes.
  • An antigen is additionally capable of being recognized by the immune system.
  • an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the 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 epitope.
  • An antigen can also have one or more epitopes (e.g., B- and T-epitopes).
  • an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which 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 of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • Monoclonal antibodies specific to certain receptors can be made using knowledge and skill in the art of injecting test subjects with suitable antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional characteristics.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.
  • the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a 48 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO 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 an antibody, (v) a domain antibody (dAb) fragment (Ward, et al., Nature, 1989, 341, 544-546), which may consist of a VH or a VL domain; and (vi) an isolated complementarity
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as 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).
  • scFv antibodies are also intended to be encompassed within the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody.
  • a scFv protein domain comprises a VH portion and a VL portion.
  • a scFv molecule is denoted as either VL-L-VH if the VL domain is the N-terminal part of the scFv molecule, or as VH-L-VL if the VH domain is the N-terminal part of the scFv molecule.
  • Methods for making scFv molecules and designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No.4,946,778, R.
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a 49 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • a hybridoma which includes a B cell obtained from a 49 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (such as a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve 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.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) 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 repertoire in vivo.
  • isotype refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.
  • immunoglobulin e.g., IgM or IgG1
  • the phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”
  • human antibody derivatives refers to any modified form of the human antibody, including a conjugate of the antibody and another active pharmaceutical ingredient or antibody.
  • conjugates refers to an antibody, or a fragment thereof, conjugated to another therapeutic moiety, which can be conjugated to antibodies described herein using methods available in the art.
  • humanized antibody “humanized antibodies,” and “humanized” are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • Humanized forms of non-human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the 50 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO recipient are replaced by residues from a 15 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the antibodies described herein may also be modified to employ any Fc variant which is known to impart an improvement (e.g., reduction) in effector function and/or FcR binding.
  • the Fc variants may include, for example, any one of the amino acid substitutions disclosed in International Patent Application Publication Nos.
  • chimeric antibody is intended to refer 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 an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • a “diabody” is a small antibody fragment with two antigen-binding sites.
  • the fragments comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH-VL or VL-VH linker that is too short to allow pairing between the two domains on the same chain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, e.g., European Patent No. EP 404,097, International Patent Publication No. WO 93/11161; and Bolliger, et al., Proc. Natl. Acad. Sci.
  • glycosylation refers to a modified derivative of an antibody.
  • An aglycoslated antibody lacks glycosylation.
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation 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 to thereby eliminate glycosylation at that site.
  • Aglycosylation may increase the affinity of the antibody for antigen, as described in U.S.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • Such altered glycosylation patterns have been demonstrated to increase the ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation.
  • the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.
  • the Ms704, Ms705, and Ms709 FUT8 ⁇ / ⁇ cell lines were created by the targeted disruption of the FUT8 gene in 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).
  • EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme, and also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662).
  • WO 99/54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana, et al., Nat. Biotech.1999, 17, 176-180).
  • GnTIII glycoprotein-modifying glycosyl transferases
  • the fucose residues of the antibody may be cleaved off using a fucosidase enzyme.
  • the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem.1975, 14, 5516-5523. 52 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO [00397]
  • “Pegylation” refers to a modified antibody, or a fragment thereof, that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody.
  • PEG polyethylene glycol
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • a reactive PEG molecule or an analogous reactive water-soluble polymer.
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol- maleimide.
  • the antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, as described for example in European Patent Nos. EP 0154316 and EP 0401384 and U.S.
  • biosimilar means a biological product, including a monoclonal antibody or protein, that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product.
  • a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency.
  • biosimilar is also used synonymously by other national and regional regulatory agencies.
  • Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies.
  • a biological source such as a bacterium or yeast.
  • They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies.
  • the reference IL-2 protein is aldesleukin (PROLEUKIN)
  • a protein approved by drug regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof” of aldesleukin.
  • EMA European Medicines Agency
  • a biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and/or efficacy.
  • the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product.
  • a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product.
  • a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product.
  • a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product.
  • a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product.
  • a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA.
  • the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized “comparator”) in certain studies. Such studies include for example certain clinical and in vivo non- clinical studies.
  • biosimilar also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator.
  • Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins.
  • a protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and/or substitutions of amino acids) which do not significantly affect the function of the polypeptide.
  • the biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%.
  • the biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and/or truncation which is/are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and/or efficacy of the medicinal product.
  • the biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product.
  • the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and/or presentation, providing safety and efficacy of the medicinal product is not compromised.
  • the biosimilar may comprise differences in for example pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization.
  • PK pharmacokinetic
  • PD pharmacodynamic
  • the biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are 54 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product.
  • Gen 2 TIL Manufacturing Processes An exemplary family of TIL processes known as Gen 2 (also known as process 2A) containing some of these features is depicted in Figures 1 and 2. An embodiment of Gen 2 is shown in Figure 2.
  • the present invention can include a step relating to the restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplant into a patient, and methods of testing said metabolic health.
  • TILs are generally taken from a patient sample and manipulated to expand their number prior to transplant into a patient.
  • the TILs may be optionally genetically manipulated as discussed below.
  • the TILs may be cryopreserved. Once thawed, they may also be restimulated to increase their metabolism prior to infusion into a patient.
  • the first expansion (including processes referred to as the pre- REP as well as processes shown in Figure 1 as Step A) is shortened to 3 to 14 days and the second expansion (including processes referred to as the REP as well as processes shown in Figure 1 as Step B) is shorted to 7 to 14 days, as discussed in detail below as well as in the examples and figures.
  • the first expansion (for example, an expansion described as Step B in Figure 1) is shortened to 11 days and the second expansion (for example, an expansion as described in Step D in Figure 1) is shortened to 11 days.
  • the combination of the first expansion and second expansion (for example, expansions described as Step B and Step D in Figure 1) is shortened to 22 days, as discussed in detail below and in the examples and figures.
  • TILs are initially obtained from a patient tumor sample and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, 55 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO restimulated as outlined herein and optionally evaluated for phenotype and metabolic parameters as an indication of TIL health.
  • a patient tumor sample may be obtained using methods known in the art, generally via surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells.
  • multilesional sampling is used.
  • surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells includes multilesional sampling (i.e., obtaining samples from one or more tumor sites and/or locations in the patient, as well as one or more tumors in the same location or in close proximity).
  • the tumor sample may be 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 hematological malignancy.
  • the solid tumor may be of lung tissue.
  • useful TILs are obtained from endometrial cancer.
  • the solid tumor may be of skin tissue.
  • useful TILs are obtained from a melanoma.
  • the tumor sample is generally fragmented using sharp dissection into small pieces of between 1 to about 8 mm 3 , with from about 2-3 mm 3 being particularly useful.
  • the TILs are cultured from these fragments using enzymatic tumor digests.
  • Such tumor digests may be produced by incubation in enzymatic media (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator).
  • enzymatic media e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase
  • mechanical dissociation e.g., using a tissue dissociator
  • a density gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells.
  • Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133 A1, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer.
  • Tumor dissociating enzyme mixtures can include one or more dissociating (digesting) enzymes such as, but not limited to, collagenase (including any blend or type of collagenase), AccutaseTM, AccumaxTM, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseinase, elastase, papain, protease type XIV (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociating or proteolytic enzyme, and any combination thereof.
  • dissociating enzymes such as, but not limited to, collagenase (including any blend or type of collagenase), AccutaseTM, AccumaxTM, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain, trypsin, caseina
  • the dissociating enzymes are reconstituted from lyophilized enzymes.
  • lyophilized enzymes are reconstituted in an amount of sterile buffer such as HBSS.
  • collagenase (such as animal free-type 1 collagenase) is reconstituted in 10 mL of sterile HBSS or another buffer.
  • the lyophilized stock enzyme may be at a concentration of 289.2 PZ U/vial.
  • collagenase is reconstituted in 5 mL to 15 mL buffer.
  • the collagenase stock ranges from about 100 PZ U/mL-about 400 PZ U/mL, e.g., about 100 PZ U/mL-about 400 PZ U/mL, about 100 PZ U/mL-about 350 PZ U/mL, about 100 PZ U/mL-about 300 PZ U/mL, about 150 PZ U/mL-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 289.2 PZ U/mL, about 300 PZ U/mL, about 350 PZ U/mL, or about 400 PZ U/mL
  • neutral protease is reconstituted in 1 mL of sterile HBSS or another buffer.
  • the lyophilized stock enzyme may be at a concentration of 175 DMC U/vial.
  • the neutral protease stock ranges from about 100 DMC/mL-about 400 DMC/mL, e.g., about 100 DMC/mL-about 400 DMC/mL, about 100 DMC/mL-about 350 DMC/mL, about 100 DMC/mL-about 300 DMC/mL, about 150 DMC/mL-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 D
  • DNAse I is reconstituted in 1 mL of sterile HBSS or another buffer.
  • the lyophilized stock enzyme was at a concentration of 4 KU/vial.
  • the DNase I stock ranges from about 1 KU/mL-10 KU/mL, e.g., 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.
  • the stock of enzymes is variable and the concentrations may need to be determined. In some embodiments, the concentration of the lyophilized stock can be verified. In some embodiments, the final amount of enzyme added to the digest cocktail is adjusted based on the determined stock concentration. [00413] In some embodiment, the enzyme mixture includes about 10.2-ul of neutral protease (0.36 DMC U/mL), 21.3 ⁇ L of collagenase (1.2 PZ/mL) and 250-ul of DNAse I (200 U/mL) in about 4.7 mL of sterile HBSS.
  • the TILs are derived from solid tumors.
  • the solid tumors are not fragmented.
  • the solid tumors are not fragmented and are subjected to enzymatic digestion as whole tumors.
  • the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase.
  • the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours.
  • the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C, 5% CO 2 .
  • the tumors are digested in in an enzyme mixture comprising collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C, 5% CO 2 with rotation.
  • the tumors are digested overnight with constant rotation.
  • the tumors are digested overnight at 37°C, 5% CO 2 with constant rotation.
  • the whole tumor is combined with the enzymes to form a tumor digest reaction mixture.
  • the tumors are digested in an enzyme mixture comprising collagenase, DNase, and neutral protease. In some embodiments, the tumors are digested in an enzyme mixture comprising collagenase, DNase, and neutral protease for 1-2 hours. In some embodiments, the tumors are digested in an enzyme mixture comprising collagenase, DNase, and neutral protease for 1-2 hours at 37°C, 5% CO 2. In some embodiments, the tumors are digested in an enzyme mixture comprising collagenase, DNase, and neutral protease for 1-2 hours at 37°C, 5% CO 2 with rotation. In some embodiments, the tumors are digested overnight with constant rotation.
  • the tumors are digested overnight at 37°C, 5% CO 2 with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digest reaction mixture.
  • the tumor is reconstituted with the lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.
  • the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock for the collagenase is a 100 mg/mL 10X working stock. [00418] In some embodiments, the enzyme mixture comprises DNAse.
  • the working stock for the DNAse is a 10,000 IU/mL 10X working stock.
  • the enzyme mixture comprises hyaluronidase.
  • the working stock for the hyaluronidase is a 10 mg/mL 10X working stock.
  • the enzyme mixture comprises 10 mg/mL collagenase, 1000 IU/mL DNAse, and 1 mg/mL hyaluronidase.
  • the enzyme mixture comprises 10 mg/mL collagenase, 500 IU/mL DNAse, and 1 mg/mL hyaluronidase.
  • the harvested cell suspension is called a “primary cell population” or a “freshly harvested” cell population.
  • fragmentation includes physical fragmentation, including for example, dissection as well as digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is dissection. In some embodiments, the fragmentation is by digestion.
  • TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from digesting or fragmenting a tumor sample obtained from a patient.
  • the tumor undergoes physical fragmentation after the tumor sample is obtained in, for example, Step A (as provided in Figure 1).
  • the fragmentation occurs before cryopreservation.
  • the fragmentation occurs after cryopreservation.
  • the fragmentation occurs after obtaining the tumor and in the absence of any cryopreservation.
  • the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first expansion.
  • 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 multiple fragments comprise about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm 3 . In some embodiments, the multiple fragments comprise about 30 to about 60 fragments with a total volume of about 1300 mm 3 to about 1500 mm 3 . In some embodiments, the multiple fragments comprise about 50 fragments with a total volume of about 1350 mm 3 . In some embodiments, the multiple fragments comprise about 50 fragments with a total mass of about 1 gram to about 1.5 grams.
  • the multiple fragments comprise about 4 fragments.
  • the TILs are obtained from tumor fragments.
  • the tumor fragment is obtained by sharp dissection.
  • the tumor fragment is between about 1 mm 3 and 10 mm 3 .
  • the tumor fragment is between about 1 mm 3 and 8 mm 3 .
  • the tumor fragment is about 1 mm 3 .
  • the tumor fragment is about 2 mm 3 .
  • the tumor fragment is about 3 mm 3 .
  • the tumor fragment is about 4 mm 3 .
  • the tumor fragment is about 5 mm 3 .
  • the tumor fragment is about 6 mm 3 .
  • the tumor fragment is about 7 mm 3 . In some embodiments, the tumor fragment is about 59 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO 8 mm 3 . In some embodiments, the tumor fragment is about 9 mm 3 . In some embodiments, the tumor fragment is about 10 mm 3 . In some embodiments, the tumors are 1-4 mm ⁇ 1-4 mm ⁇ 1-4 mm. In some embodiments, the tumors are 1 mm ⁇ 1 mm ⁇ 1 mm. In some embodiments, the tumors are 2 mm ⁇ 2 mm ⁇ 2 mm. In some embodiments, the tumors are 3 mm ⁇ 3 mm ⁇ 3 mm.
  • the tumors are 4 mm ⁇ 4 mm ⁇ 4 mm. [00426] In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic, necrotic, and/or fatty tissues on each piece. In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of necrotic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of fatty tissue on each piece. [00427] In some embodiments, the tumor fragmentation is performed in order to maintain the tumor internal structure.
  • the tumor fragmentation is performed without performing a sawing motion with a scalpel.
  • the TILs are obtained from tumor digests.
  • tumor digests were generated by incubation in enzyme media, for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in enzyme media, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated for 30 minutes at 37 °C in 5% CO 2 and it then mechanically disrupted again for approximately 1 minute.
  • the tumor can be mechanically disrupted a third time for approximately 1 minute.
  • 1 or 2 additional mechanical dissociations were applied to the sample, with or without 30 additional minutes of incubation at 37 °C in 5% CO 2 .
  • a density gradient separation using Ficoll can be performed to remove these cells.
  • the harvested cell suspension prior to the first expansion step is called a “primary cell population” or a “freshly harvested” cell population.
  • cells can be optionally frozen after sample harvest and stored frozen prior to entry into the expansion described in Step B, which is described in further detail below, as well as exemplified in Figure 1.
  • Pleural effusion T-cells, ascites fluid T-cells, and TILs [00430]
  • the sample is a pleural fluid sample.
  • the source of the T-cells or TILs for expansion according to the processes described herein is a pleural 60 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO fluid sample.
  • the sample is a pleural effusion derived sample.
  • the source of the T-cells or TILs for expansion according to the processes described herein is a pleural effusion derived sample. See, for example, methods described in U.S. Patent Publication US 2014/0295426, incorporated herein by reference in its entirety for all purposes.
  • the sample is an ascites fluid sample.
  • the source of the T-cells or TILs for expansion according to the processes described herein is an ascites fluid sample.
  • the sample is an ascites derived sample. In any of the foregoing embodiments, the ascites fluid sample or ascites derived sample is obtained from an endometrial cancer patient.
  • ascites fluid from the abdomen of an endometrial cancer patient may be used to obtain TILs for expansion according to the processes described herein and for treatment of the endometrial cancer patient using TILs and optional co-therapies described herein.
  • any pleural fluid or pleural effusion suspected of and/or containing TILs can be employed.
  • Such a sample may be derived from a primary or metastatic lung cancer, such as NSCLC or SCLC.
  • the sample may be derived from secondary metastatic cancer cells which originated from another organ, e.g., breast, ovary, colon or prostate.
  • the sample for use in the expansion methods described herein is a pleural exudate.
  • the sample for use in the expansion methods described herein is a pleural transudate.
  • Other biological samples may include other serous fluids containing TILs, including, e.g., ascites fluid from the abdomen or pancreatic cyst fluid. Ascites fluid and pleural fluids involve very similar chemical systems; both the abdomen and lung have mesothelial lines and fluid forms in the pleural space and abdominal spaces in the same matter in malignancies and such fluids in some embodiments contain TILs.
  • the disclosed methods utilize pleural fluid, the same methods may be performed with similar results using ascites or other cyst fluids containing TILs.
  • the pleural or ascites fluid is in unprocessed form, directly as removed from the patient.
  • the unprocessed pleural or ascites fluid is placed in a standard blood collection tube, such as an EDTA or Heparin tube, prior to further processing steps.
  • the unprocessed pleural or ascites fluid is placed in a standard CellSave® tube (Veridex) prior to further processing steps.
  • the sample is placed in the CellSave tube immediately after collection from the patient to avoid a decrease in the number of viable TILs.
  • the number of viable TILs can decrease to a significant extent within 24 hours, if left in the untreated pleural or ascites fluid, even at 4°C.
  • the sample is placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the 61 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO patient.
  • the sample is placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after removal from the patient at 4°C.
  • the pleural or ascites fluid sample from the chosen subject may be diluted.
  • 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.
  • diluents include saline, phosphate buffered saline, another buffer or a physiologically acceptable diluent.
  • the sample is placed in the CellSave tube immediately after collection from the patient and dilution to avoid a decrease in the viable TILs, which may occur to a significant extent within 24-48 hours, if left in the untreated pleural fluid, even at 4°C.
  • the pleural or ascites fluid sample is placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient, and dilution.
  • the pleural or ascites fluid sample is placed in the appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after removal from the patient, and dilution at 4°C.
  • pleural or ascites fluid samples are concentrated by conventional means prior to further processing steps. In some embodiments, this pre-treatment of the pleural or ascites fluid is preferable in circumstances in which the pleural or ascites fluid must be cryopreserved for shipment to a laboratory performing the method or for later analysis (e.g., later than 24-48 hours post-collection).
  • the pleural or ascites fluid sample is prepared by centrifuging the pleural or ascites fluid sample after its withdrawal from the subject and resuspending the centrifugate or pellet in buffer.
  • the pleural or ascites fluid sample is subjected to multiple centrifugations and resuspensions, before it is cryopreserved for transport or later analysis and/or processing.
  • pleural or ascites fluid samples are concentrated prior to further processing steps by using a filtration method.
  • the pleural or ascites fluid sample used in further processing is prepared by filtering the fluid through a filter containing a known and essentially uniform pore size that allows for passage of the pleural or ascites fluid through the membrane but retains the tumor cells.
  • the diameter of the pores in the membrane may be at least 4 ⁇ M. In other embodiments the pore diameter may be 5 ⁇ M or more, and in other embodiment, any of 6, 7, 8, 9, or 10 ⁇ M.
  • the cells, including TILs, retained by the membrane may be rinsed off the membrane into a suitable physiologically acceptable buffer.
  • the pleural or ascites fluid sample (including, for example, the untreated pleural or ascites fluid), the diluted pleural or ascites fluid, or the resuspended cell pellet, is contacted with a lytic reagent that differentially lyses non-nucleated red blood cells present in the sample.
  • this step is performed prior to further processing steps in circumstances in which the pleural or ascites fluid contains substantial numbers of RBCs.
  • Suitable lysing reagents include a single lytic reagent or a lytic reagent and a quench reagent, or a lytic agent, a quench reagent and a fixation reagent.
  • Suitable lytic systems are marketed commercially and include the BD Pharm LyseTM system (Becton Dickenson). Other lytic systems include the VersalyseTM system, the FACSlyseTM system (Becton Dickenson), the ImmunoprepTM system or Erythrolyse II system (Beckman Coulter, Inc.), or an ammonium chloride system.
  • the lytic reagent can vary with the primary requirements being efficient lysis of the red blood cells, and the conservation of the TILs and phenotypic properties of the TILs in the pleural or ascites fluid.
  • the lytic systems useful in methods described herein can include a second reagent, e.g., one that quenches or retards the effect of the lytic reagent during the remaining steps of the method, e.g., StabilyseTM reagent (Beckman Coulter, Inc.).
  • a conventional fixation reagent may also be employed depending upon the choice of lytic reagents or the preferred implementation of the method.
  • the pleural or ascites fluid sample, unprocessed, diluted or multiply centrifuged or processed as described herein above is cryopreserved at a temperature of about ⁇ 140°C prior to being further processed and/or expanded as provided herein.
  • the present methods provide for obtaining young TILs, which are capable of increased replication cycles upon administration to a subject/patient and as such may provide additional therapeutic benefits over older TILs (i.e., TILs which have further undergone more rounds of replication prior to administration to a subject/patient).
  • TILs which have further undergone more rounds of replication prior to administration to a subject/patient.
  • the diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited, but large number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T-cell receptors (TCRs).
  • the present invention provides a method for generating TILs which exhibit and increase the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity.
  • the TILs obtained by the present method exhibit an increase in the T- cell repertoire diversity as compared to freshly harvested TILs and/or TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
  • the TILs obtained by the present method exhibit an increase in the T- cell repertoire diversity as compared to freshly harvested TILs and/or TILs prepared using methods referred to as process 1C, as exemplified in Figure 5 and/or Figure 6.
  • the TILs obtained in the first expansion exhibit an increase in the T-cell repertoire diversity.
  • the increase in diversity is an increase in the immunoglobulin diversity and/or the T-cell receptor diversity.
  • the diversity is in the immunoglobulin is in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin light chain. In some embodiments, the diversity is in the T-cell receptor. In some embodiments, the diversity is in one of the T-cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha and/or beta. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) beta.
  • TCRab i.e., TCR ⁇ / ⁇ .
  • the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells.
  • the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum with 6000 IU/mL of IL-2. This primary cell population is cultured for a period of days, generally from 3 to 14 days, resulting in a bulk TIL population, generally about 1 ⁇ 10 8 bulk TIL cells.
  • this primary cell population is cultured for a period of 7 to 14 days, resulting in a bulk TIL population, generally about 1 ⁇ 10 8 bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of 10 to 14 days, resulting in a bulk TIL population, generally about 1 ⁇ 10 8 bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about 1 ⁇ 10 8 bulk TIL cells.
  • expansion of TILs may be performed using an initial bulk TIL expansion step (for example such as those described in Step B of Figure 1, which can include processes referred to as pre-REP) as described below and herein, followed by a second expansion (Step D, including processes referred to as rapid expansion protocol (REP) steps) as described below under Step D and herein, followed by optional cryopreservation, and followed by a second Step D (including processes referred to as restimulation REP steps) as described below and herein.
  • the TILs obtained from this process may be optionally characterized for phenotypic characteristics and metabolic parameters as described herein.
  • 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 Corp., Emeryville, CA).
  • CM complete medium
  • IL-2 6000 IU/mL
  • the tumor fragment is between about 1 mm 3 and 10 mm 3 .
  • the first expansion culture medium is referred to as “CM”, an abbreviation for culture media.
  • CM for Step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin.
  • G-REX10 Wilson Wolf Manufacturing, New Brighton, MN
  • each flask was loaded with 10–40 ⁇ 10 6 viable tumor digest cells or 5–30 tumor fragments in 10–40 mL of CM with IL-2.
  • the culture medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium.
  • the serum-free or defined medium comprises a basal cell medium and a serum supplement and/or a serum replacement.
  • the serum-free or defined medium is used to prevent and/or decrease experimental variation due in part to the lot-to-lot variation of serum-containing media.
  • the serum-free or defined medium comprises a basal cell medium and a serum supplement and/or serum replacement.
  • the basal cell medium includes, but is not limited to CTSTM OpTmizerTM T-cell Expansion Basal Medium , CTSTM OpTmizerTM T-Cell Expansion SFM, CTSTM AIM-V Medium, CTSTM AIM-V SFM, LymphoONETM T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential 65 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal
  • the serum supplement or serum replacement includes, but is not limited to one or more of CTSTM OpTmizer T-Cell Expansion Serum Supplement, CTSTM Immune Cell Serum Replacement, 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 precursors, one or more antibiotics, and one or more trace elements.
  • the defined medium comprises albumin and one or more ingredients 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 saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, Al3+, Ba2+, Cd2+, CO 2 +, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+ and Zr4+.
  • glycine L- histidine, L-isoleucine, L- methionine, L-phenylalanine, L-proline, L- hydroxyproline
  • the defined medium further comprises L-glutamine, sodium bicarbonate and/or 2- mercaptoethanol.
  • the CTSTMOpTmizerTM T-cell Immune Cell Serum Replacement is used with conventional growth media, including but not limited to CTSTM OpTmizerTM T-cell Expansion Basal Medium, CTSTM OpTmizerTM T-cell Expansion SFM, CTSTM AIM-V Medium, CSTTM AIM-V SFM, LymphoONETM T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium
  • the total serum replacement concentration (vol%) in the serum-free or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or defined medium.
  • the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium.
  • the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium.
  • the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.
  • the serum-free or defined medium is CTSTM OpTmizerTM T- cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTSTM OpTmizerTM is useful in the present invention.
  • CTSTM OpTmizerTM T-cell Expansion SFM is a combination of 1L CTSTM OpTmizerTM T-cell Expansion Basal Medium and 26 mL CTSTM OpTmizerTM T-Cell Expansion 66 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO Supplement, which are mixed together prior to use.
  • the CTSTM OpTmizerTM T- cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, the CTSTM OpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2-mercaptoethanol at 55mM. In some embodiments, the CTSTM OpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55 ⁇ M.
  • SR Immune Cell Serum Replacement
  • the defined medium is CTSTM OpTmizerTM T-cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTSTM OpTmizerTM is useful in the present invention.
  • CTSTM OpTmizerTM T-cell Expansion SFM is a combination of 1L CTSTM OpTmizerTM T- cell Expansion Basal Medium and 26 mL CTSTM OpTmizerTM T-Cell Expansion Supplement, which are mixed together prior to use.
  • the CTSTM OpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2-mercaptoethanol at 55mM.
  • the CTSTMOpTmizerTM T- cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine.
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2- mercaptoethanol, and 2mM of L-glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2.
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further comprises about 3000 IU/mL of IL-2.
  • SR Immune Cell Serum Replacement
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further comprises about 6000 IU/mL of IL-2.
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2- mercaptoethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2.
  • SR Immune Cell Serum Replacement
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2- mercaptoethanol, and further comprises about 3000 IU/mL of IL-2. In some embodiments, the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL of IL-2.
  • SR Immune Cell Serum Replacement
  • the 67 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2.
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 3000 IU/mL of IL-2.
  • SR Immune Cell Serum Replacement
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 6000 IU/mL of IL- 2.
  • the CTSTM OpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55 ⁇ M.
  • the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of from about 0.1mM to about 10mM, 0.5mM to about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to about 5 mM.
  • glutamine i.e., GlutaMAX®
  • the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2mM.
  • the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of from about 5mM to about 150mM, 10mM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about 100mM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55mM.
  • the final concentration of 2-mercaptoethanol in the media is 55 ⁇ M.
  • the defined media described in International PCT Publication No. WO/1998/030679, which is herein incorporated by reference, are useful in the present invention.
  • serum-free eukaryotic cell culture media are described.
  • the serum-free, eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting the growth of cells in serum- free culture.
  • the serum-free eukaryotic cell culture medium supplement comprises or is obtained by combining one or more ingredients 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 precursors, one or more trace elements, and one or more antibiotics.
  • the defined medium further comprises L-glutamine, sodium bicarbonate and/or beta-mercaptoethanol.
  • the defined 68 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO medium comprises an albumin or an albumin substitute and one or more ingredients selected from group consisting of 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 precursors, and one or more trace elements.
  • the defined medium comprises albumin and one or more ingredients 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 saturated transferrin, insulin, and compounds containing the trace element moieties Ag+, Al3+, Ba2+, Cd2+, CO 2 +, Cr3+, Ge4+, Se4+, Br, T, Mn2+, P, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+ and Zr4+.
  • glycine L- histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L- hydroxypro
  • the basal cell media is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium Eagle
  • RPMI 1640 F-10, F-12
  • ⁇ MEM Minimal Essential Medium
  • G-MEM Glasgow's Minimal Essential Medium
  • RPMI growth medium RPMI growth medium
  • Iscove's Modified Dulbecco's Medium Iscove's Modified Dulbecco's Medium.
  • the concentration of glycine in the defined medium is in the range of from 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, the concentration of L-tryptophan is about 2-110 mg/L, the concentration of L-tyrosine is about 3-175 mg/L, the concentration of L-valine is about 5-500 mg/L, the concentration of thiamine is about 1-20 mg/L, the concentration of reduced glutathione is about 1-20 mg/L, the concentration of L-ascor
  • the non-trace element moiety ingredients in the defined medium are present in the concentration ranges listed in the column under the heading “Concentration Range in 1X Medium” in Table 4 below. In other embodiments, the non-trace element moiety ingredients in the defined medium are present in the final concentrations listed in the column under the heading “A Preferred Embodiment of the 1X Medium” in Table 4.
  • the defined medium is a basal cell medium comprising a serum free supplement. In some of these embodiments, the serum free supplement comprises non-trace moiety ingredients of the type and in the concentrations listed in the column under the heading “A Preferred Embodiment in Supplement” in Table 4 below.
  • the osmolarity of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmolarity 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 can be further supplemented with L-glutamine (final 70 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO concentration of about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100 ⁇ M), 2-mercaptoethanol (final concentration of about 100 ⁇ M).
  • L-glutamine final 70 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO concentration of about 2 mM
  • NEAA non-essential amino acids
  • 2-mercaptoethanol final concentration of about 100 ⁇ M
  • the cell medium in the first and/or second gas permeable container is unfiltered.
  • the use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells.
  • the cell medium in the first and/or second gas permeable container lacks beta-mercaptoethanol (BME or ⁇ ME; also known as 2-mercaptoethanol, CAS 60-24-2).
  • the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells.
  • the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum (or, in some cases, as outlined herein, in the presence of an APC cell population) with 6000 IU/mL of IL-2.
  • This primary cell population is cultured for a period of days, generally from 10 to 14 days, resulting in a bulk TIL population, generally about 1 ⁇ 10 8 bulk TIL cells.
  • the growth media during the first expansion comprises IL-2 or a variant thereof.
  • the IL is recombinant human IL-2 (rhIL-2).
  • the IL-2 stock solution has a specific activity of 20-30 ⁇ 10 6 IU/mg for a 1 mg vial.
  • the IL-2 stock solution has a specific activity of 20 ⁇ 10 6 IU/mg for a 1 mg vial.
  • the IL-2 stock solution has a specific activity of 25 ⁇ 10 6 IU/mg for a 1 mg vial.
  • the IL-2 stock solution has a specific activity of 30 ⁇ 10 6 IU/mg for a 1 mg vial.
  • the IL- 2 stock solution has a final concentration of 4-8 ⁇ 10 6 IU/mg of IL-2.
  • the IL- 2 stock solution has a final concentration of 5-7 ⁇ 10 6 IU/mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 6 ⁇ 10 6 IU/mg of IL-2. In some embodiments, the IL-2 stock solution is prepare as described in Example 5. In some embodiments, the first expansion culture media comprises about 10,000 IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL- 2, about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mL of IL-2.
  • the first expansion culture media comprises about 9,000 IU/mL of IL-2 to about 5,000 IU/mL of IL-2. In some embodiments, the first expansion culture media comprises about 8,000 IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments, the first expansion culture media comprises about 7,000 IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments, the first expansion culture media comprises about 6,000 IU/mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2.
  • the cell culture medium comprises about 3000 71 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO IU/mL of 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 of IL-2.
  • 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 of IL-2.
  • 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.
  • first expansion culture media comprises about 500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100 IU/mL of IL-15.
  • the first expansion culture media comprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15.
  • the first expansion culture media comprises about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the first expansion culture media comprises about 300 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the first expansion culture media comprises about 200 IU/mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL of 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 of IL-15.
  • first expansion culture media comprises about 20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, or about 0.5 IU/mL of IL-21.
  • the first expansion culture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL of IL-21.
  • the first expansion culture media comprises about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the first expansion culture media comprises about 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the first expansion culture media comprises about 10 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the first expansion culture media comprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In some embodiments, the first expansion culture media comprises about 2 IU/mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL of IL-21.
  • the cell culture medium comprises about 0.5 IU/mL of 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 of IL-21. 72 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO [00463] In some embodiments, the cell culture medium comprises an anti-CD3 agonist antibody, e.g. OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3 antibody.
  • 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 ⁇ g/mL of OKT-3 antibody.
  • 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.
  • the cell culture medium does not comprise OKT-3 antibody.
  • the OKT-3 antibody is muromonab. See, for example, Table 1.
  • the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium.
  • the TNFRSF agonist comprises a 4-1BB agonist.
  • the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, and combinations thereof.
  • the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 ⁇ g/mL and 100 ⁇ g/mL.
  • the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 ⁇ g/mL and 40 ⁇ g/mL.
  • the cell culture medium in addition to one or more TNFRSF agonists, further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.
  • the first expansion culture medium is referred to as “CM”, an abbreviation for culture media. In some embodiments, it is referred to as CM1 (culture medium 1).
  • CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin.
  • G-REX10 Wilson Wolf Manufacturing, New Brighton, MN
  • each flask was loaded with 10–40x10 6 viable tumor digest cells or 5–30 tumor fragments in 10–40mL of CM with IL-2.
  • the G-REX10 and 24-well plates were incubated in a humidified incubator at 37°C in 5% CO 2 and 5 days 73 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO after culture initiation, half the media was removed and replaced with fresh CM and IL-2 and after day 5, half the media was changed every 2–3 days.
  • the CM is the CM1 described in the Examples, see, Example 1.
  • the first expansion occurs in an initial cell culture medium or a first cell culture medium.
  • the initial cell culture medium or the first cell culture medium comprises IL-2.
  • the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre-REP) process is shortened to 3-14 days, as discussed in the examples and figures.
  • the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre-REP) is shortened to 7 to 14 days, as discussed in the Examples and shown in Figures 4 and 5, as well as including for example, an expansion as described in Step B of Figure 1.
  • the first expansion of Step B is shortened to 10-14 days.
  • the first expansion is shortened to 11 days, as discussed in, for example, an expansion as described in Step B of Figure 1.
  • the first TIL expansion can proceed 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 can proceed for 1 day to 14 days. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some embodiments, the first TIL expansion can proceed for 3 days to 14 days. In some embodiments, the first TIL expansion can proceed for 4 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days.
  • the first TIL expansion can proceed for 7 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 9 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 11 days to 14 days. In some embodiments, the first TIL expansion can proceed for 12 days to 14 days. In some embodiments, the first TIL expansion can proceed for 13 days to 14 days. In some embodiments, the first TIL expansion can proceed for 14 days. In some embodiments, the first TIL expansion can proceed for 1 day to 11 days. In some embodiments, the first TIL expansion can proceed for 2 days to 11 days.
  • the first TIL expansion can proceed for 3 days to 11 days. In some embodiments, the first TIL expansion can proceed for 4 days to 11 days. In some embodiments, the first TIL expansion can proceed for 5 days to 11 days. In some embodiments, the first TIL expansion can proceed for 6 days to 11 days. In some embodiments, the first TIL expansion can proceed for 7 days to 11 days. In some embodiments, the first TIL expansion can proceed for 8 days to 11 days. In some embodiments, the first TIL expansion can proceed for 9 days to 11 days. In some embodiments, the first TIL 74 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO expansion can proceed for 10 days to 11 days.
  • the first TIL expansion can proceed for 11 days.
  • a combination of IL-2, IL-7, IL-15, and/or IL-21 are employed as a combination during the first expansion.
  • IL-2, IL-7, IL-15, and/or IL-21 as well as any combinations thereof can be included during the first expansion, including for example during a Step B processes according to Figure 1, as well as described herein.
  • a combination of IL-2, IL-15, and IL-21 are employed as a combination during the first expansion.
  • IL-2, IL-15, and IL-21 as well as any combinations thereof can be included during Step B processes according to Figure 1 and as described herein.
  • the first expansion (including processes referred to as the pre- REP; for example, Step B according to Figure 1) process is shortened to 3 to 14 days, as discussed in the examples and figures.
  • the first expansion of Step B is shortened to 7 to 14 days.
  • the first expansion of Step B is shortened to 10 to 14 days.
  • the first expansion is shortened to 11 days.
  • the first expansion, for example, Step B according to Figure 1 is performed in a closed system bioreactor.
  • a closed system is employed for the TIL expansion, as described herein.
  • a single bioreactor is employed.
  • the single bioreactor employed is for example a G-REX-10 or a G-REX-100.
  • the closed system bioreactor is a single bioreactor.
  • Cytokines and Other Additives generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.
  • cytokine in particular IL-2
  • Step B may also include the addition of OKT-3 antibody or muromonab to the culture media, as described elsewhere herein. In some embodiments, Step B may also include the addition of a 4-1BB agonist to the culture media, as described elsewhere herein.
  • Step B may also include the addition of an OX-40 agonist to the culture media, as 75 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO described elsewhere herein.
  • additives such as peroxisome proliferator- activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as a thiazolidinedione compound, may be used in the culture media during Step B, as described in U.S. Patent Application Publication No. US 2019/0307796 A1, the disclosure of which is incorporated by reference herein.
  • PPAR proliferator-activated receptor
  • the bulk TIL population obtained from the first expansion can be cryopreserved immediately, using the protocols discussed herein below.
  • the TIL population obtained from the first expansion referred to as the second TIL population
  • a second expansion which can include expansions sometimes referred to as REP
  • the first TIL population (sometimes referred to as the bulk TIL population) or the second TIL population (which can in some embodiments include populations referred to as the REP TIL populations) can be subjected to genetic modifications for suitable treatments prior to expansion or after the first expansion and prior to the second expansion.
  • the TILs obtained from the first expansion (for example, from Step B as indicated in Figure 1) are stored until phenotyped for selection.
  • the TILs obtained from the first expansion are not stored and proceed directly to the second expansion.
  • the TILs obtained from the first expansion are not cryopreserved after the first expansion and prior to the second expansion.
  • the transition from the first expansion to the second expansion occurs at 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 from when fragmentation occurs.
  • the transition from the first expansion to the second expansion occurs at about 3 days to 14 days from when fragmentation occurs.
  • the transition from the first expansion to the second expansion occurs at about 4 days to 14 days from when fragmentation occurs.
  • the transition from the first expansion to the second expansion occurs at about 4 days to 10 days from when fragmentation occurs.
  • the transition from the first expansion to the second expansion occurs at about 7 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 14 days from when fragmentation occurs. [00476] In some embodiments, the transition from the first expansion to the second expansion occurs at 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 from when fragmentation occurs. In some embodiments, the transition from 76 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO the first expansion to the second expansion occurs 1 day to 14 days from when fragmentation occurs.
  • the first TIL expansion can proceed 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 from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 14 days from when fragmentation occurs.
  • the transition from the first expansion to the second expansion occurs 9 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 1 day to 11 days from when fragmentation occurs.
  • the transition from the first expansion to the second expansion occurs 2 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 4 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 7 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 8 days to 11 days from when fragmentation occurs.
  • the transition from the first expansion to the second expansion occurs 9 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days from when fragmentation occurs. 77 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO [00477] In some embodiments, the TILs are not stored after the first expansion and prior to the second expansion, and the TILs proceed directly to the second expansion (for example, in some embodiments, there is no storage during the transition from Step B to Step D as shown in Figure 1). In some embodiments, the transition occurs in closed system, as described herein.
  • the TILs from the first expansion, the second population of TILs proceeds directly into the second expansion with no transition period.
  • the transition from the first expansion to the second expansion is performed in a closed system bioreactor.
  • a closed system is employed for the TIL expansion, as described herein.
  • a single bioreactor is employed.
  • the single bioreactor employed is for example a G-REX-10 or a G-REX-100 bioreactor.
  • the closed system bioreactor is a single bioreactor. D.
  • the TIL cell population is expanded in number after harvest and initial bulk processing for example, after Step A and Step B, and the transition referred to as Step C, as indicated in Figure 1).
  • This further expansion is referred to herein as the second expansion, which can include expansion processes generally referred to in the art as a rapid expansion process (REP); as well as processes as indicated in Step D of Figure 1.
  • the second expansion is generally accomplished using a culture media comprising a number of components, including feeder cells, a cytokine source, and an anti-CD3 antibody, in a gas-permeable container.
  • the second expansion or second TIL expansion (which can include expansions sometimes referred to as REP; as well as processes as indicated in Step D of Figure 1) of TIL can be performed using any TIL flasks or containers known by those of skill in the art.
  • the second TIL expansion can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.
  • the second TIL expansion can proceed for about 7 days to about 14 days.
  • the second TIL expansion can proceed for about 8 days to about 14 days.
  • the second TIL expansion can proceed for about 9 days to about 14 days.
  • the second TIL expansion can proceed for about 10 days to about 14 days.
  • the second TIL expansion can proceed for about 11 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 13 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days. 78 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO [00481] In some embodiments, the second expansion can be performed in a gas permeable container using the methods of the present disclosure (including for example, expansions referred to as REP; as well as processes as indicated in Step D of Figure 1).
  • 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).
  • the non-specific T-cell receptor stimulus can include, for example, an anti- CD3 antibody, such as about 30 ng/mL of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA).
  • TILs can be expanded to induce further stimulation of the TILs in vitro by including one or more antigens during the second expansion, including antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 ⁇ MART-1 :26-35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell growth factor, such as 300 IU/mL IL-2 or IL-15.
  • HLA-A2 human leukocyte antigen A2
  • TIL may include, e.g., NY-ESO- 1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof.
  • TIL may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.
  • the TILs can be further re- stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.
  • the re-stimulation occurs as part of the second expansion.
  • the second expansion occurs in the presence of irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.
  • the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL of IL-2.
  • 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 of IL-2.
  • 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 between 8000 IU/mL of IL-2.
  • the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3 antibody.
  • 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, 79 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO about 500 ng/mL, and about 1 ⁇ g/mL of OKT-3 antibody.
  • 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.
  • the cell culture medium does not comprise OKT-3 antibody.
  • the OKT-3 antibody is muromonab.
  • the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium.
  • the TNFRSF agonist comprises a 4-1BB agonist.
  • the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, and combinations thereof.
  • the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 ⁇ g/mL and 100 ⁇ g/mL.
  • the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 ⁇ g/mL and 40 ⁇ g/mL.
  • the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.
  • a combination of IL-2, IL-7, IL-15, and/or IL-21 are employed as a combination during the second expansion.
  • IL-2, IL-7, IL-15, and/or IL-21 as well as any combinations thereof can be included during the second expansion, including for example during a Step D processes according to Figure 1, as well as described herein.
  • a combination of IL-2, IL-15, and IL-21 are employed as a combination during the second expansion.
  • IL-2, IL-15, and IL-21 as well as any combinations thereof can be included during Step D processes according to Figure 1 and as described herein.
  • the second expansion can be conducted in a supplemented cell culture medium comprising IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist.
  • the second expansion occurs in a supplemented cell culture medium.
  • the supplemented cell culture medium comprises IL-2, OKT-3, and antigen- presenting feeder cells.
  • the second cell culture medium comprises IL-2, OKT- 3, and antigen-presenting cells (APCs; also referred to as antigen-presenting feeder cells).
  • the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen presenting cells).
  • the second expansion culture media comprises about 500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100 IU/mL of IL-15.
  • the second expansion culture media comprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15.
  • the second expansion culture media comprises about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the second expansion culture media comprises about 300 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the second expansion culture media comprises about 200 IU/mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL of 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 of IL-15.
  • the second expansion culture media comprises about 20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, or about 0.5 IU/mL of IL-21.
  • the second expansion culture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL of IL-21.
  • the second expansion culture media comprises about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the second expansion culture media comprises about 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the second expansion culture media comprises about 10 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the second expansion culture media comprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In some embodiments, the second expansion culture media comprises about 2 IU/mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL of IL-21.
  • the cell culture medium comprises about 0.5 IU/mL of 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 of IL-21.
  • the antigen-presenting feeder cells are PBMCs.
  • the ratio of TILs to PBMCs and/or antigen-presenting cells in the rapid expansion and/or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500.
  • the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1 to 50 and 1 to 300.
  • the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1 to 100 and 1 to 200.
  • REP and/or the second expansion is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mL IL-2 in 150 mL media.
  • Media replacement is done (generally 2/3 media replacement via respiration with fresh media) until the cells are transferred to an alternative growth chamber.
  • Alternative growth chambers include G-REX flasks and gas permeable containers as more fully discussed below.
  • the second expansion (which can include processes referred to as the REP process) is shortened to 7-14 days, as discussed in the examples and figures. In some embodiments, the second expansion is shortened to 11 days.
  • REP and/or the second expansion may 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 cultureware (G-REX flasks).
  • the second expansion (including expansions referred to as rapid expansions) is performed in T-175 flasks, and about 1 x 10 6 TILs suspended in 150 mL of media may be added to each T-175 flask.
  • the TILs may be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3.
  • the T-175 flasks may be incubated at 37° C in 5% CO2. Half the media may be exchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2.
  • cells from two T-175 flasks may be combined in a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 mL of TIL suspension.
  • the number of cells in each bag was counted every day or two and fresh media was added to keep the cell count between 0.5 and 2.0 x 10 6 cells/mL.
  • the second expansion (which can include expansions referred to as REP, as well as those referred to in Step D of Figure 1) may be performed in 500 mL capacity gas permeable flasks with 100 cm gas-permeable silicon bottoms (G-REX-100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 ⁇ 10 6 or 10 ⁇ 10 6 TIL may be cultured with PBMCs in 400 mL of 50/50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3 (OKT3).
  • G-REX-100 gas-permeable silicon bottoms
  • the G-REX-100 flasks may be incubated at 37°C in 5% CO 2 . On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 ⁇ g) for 10 minutes. The TIL pellets may be re- suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-REX-100 flasks.
  • TIL When TIL are expanded serially in G-REX-100 flasks, on day 7 the TIL in each G-REX-100 may be suspended in the 300 mL of media present in each flask and the cell suspension may be divided into 3100 mL aliquots that may be used to seed 3 G-REX-100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added 82 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO to each flask.
  • the G-REX-100 flasks may be incubated at 37° C in 5% CO 2 and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G-REX-100 flask. The cells may be harvested on day 14 of culture.
  • the second expansion (including expansions referred to as REP) is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mL IL-2 in 150 mL media.
  • media replacement is done until the cells are transferred to an alternative growth chamber.
  • the second expansion (including expansions referred to as REP) is performed and further comprises a step wherein TILs are selected for superior tumor reactivity.
  • REP expansions referred to as REP
  • Any selection method known in the art may be used. For example, the methods described in U.S. Patent Application Publication No.2016/0010058 A1, the disclosures of which are incorporated herein by reference, may be used for selection of TILs for superior tumor reactivity.
  • a cell viability assay can be performed after the second expansion (including expansions referred to as the REP expansion), using standard assays known in the art.
  • a trypan blue exclusion assay can be done on a sample of the bulk TILs, which selectively labels dead cells and allows a viability assessment.
  • TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA).
  • viability is determined according to the standard Cellometer K2 Image Cytometer Automatic Cell Counter protocol.
  • the second expansion (including expansions referred to as REP) of TIL can be performed using T-175 flasks and gas-permeable bags as previously described (Tran, et al., 2008, J Immunother., 31, 742–751, and Dudley, et al.2003, J Immunother., 26, 332–342) or gas-permeable G-REX flasks.
  • the second expansion is performed using flasks.
  • the second expansion is performed using gas-permeable G-REX flasks.
  • the second expansion is performed in T-175 flasks, and about 1 ⁇ 10 6 TIL are suspended in about 150 mL of media and this is added to each T-175 flask.
  • the TIL are cultured with irradiated (50 Gy) allogeneic PBMC as “feeder” cells at a ratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3.
  • the T-175 flasks are incubated at 37°C in 5% CO 2 .
  • half the media is changed on day 5 using 50/50 medium with 3000 IU/mL of IL-2.
  • cells from 2 T-175 flasks are combined in a 3 L bag and 300 83 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 is added to the 300 mL of TIL suspension.
  • the number of cells in each bag can be counted every day or two and fresh media can be added to keep the cell count between about 0.5 and about 2.0 ⁇ 10 6 cells/mL.
  • the second expansion (including expansions referred to as REP) are performed in 500 mL capacity flasks with 100 cm 2 gas-permeable silicon bottoms (G-REX- 100, Wilson Wolf) about 5 ⁇ 10 6 or 10 ⁇ 10 6 TIL are cultured with irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2 and 30 ng/ mL of anti-CD3.
  • the G-REX-100 flasks are incubated at 37°C in 5% CO 2 .
  • TILs are expanded serially in G-REX-100 flasks
  • the TIL in each G-REX-100 are suspended in the 300 mL of media present in each flask and the cell suspension was divided into three 100 mL aliquots that are used to seed 3 G-REX-100 flasks.
  • the present invention provides a method for generating TILs which exhibit and increase the T-cell repertoire diversity.
  • the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity.
  • the TILs obtained in the second expansion exhibit an increase in the T-cell repertoire diversity.
  • the increase in diversity is an increase in the immunoglobulin diversity and/or the T-cell receptor diversity.
  • the diversity is in the immunoglobulin is in the immunoglobulin heavy chain.
  • the diversity is in the immunoglobulin is in the immunoglobulin light chain. In some embodiments, the diversity is in the T-cell receptor. In some embodiments, the diversity is in one of the T-cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha and/or beta. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) beta. In some embodiments, there is an increase in the expression of TCRab (i.e., TCR ⁇ / ⁇ ).
  • the second expansion culture medium (e.g., sometimes referred to as CM 2 or the second cell culture medium), comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells (APCs), as discussed in more detail below.
  • the culture medium used in the expansion processes disclosed herein is a serum-free medium or a defined medium.
  • the serum- free or defined medium comprises a basal cell medium and a serum supplement and/or a serum replacement.
  • the serum-free or defined medium is used to prevent and/or decrease experimental variation due in part to the lot-to-lot variation of serum-containing media.
  • the serum-free or defined medium comprises a basal cell medium and a serum supplement and/or serum replacement.
  • the basal cell medium includes, but is not limited to CTSTM OpTmizerTM T-cell Expansion Basal Medium , CTSTM OpTmizerTM T-Cell Expansion SFM, CTSTM AIM-V Medium, CTSTM AIM-V SFM, LymphoONETM T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium Eagle
  • RPMI 1640 F-10, F-12
  • ⁇ MEM Minimal Essential Medium
  • G-MEM Glasgow's Minimal Essential Medium
  • RPMI growth medium
  • the serum supplement or serum replacement includes, but is not limited to one or more of CTSTM OpTmizer T-Cell Expansion Serum Supplement, CTSTM Immune Cell Serum Replacement, 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 precursors, one or more antibiotics, and one or more trace elements.
  • the defined medium comprises albumin and one or more ingredients 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 saturated transferrin, insulin, and compounds containing the trace element moieties 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+ , Ni 2+ , Rb + , Sn 2+ and Zr 4+ .
  • the trace element moieties Ag + , Al 3+ , Ba 2+ , Cd 2+ , CO 2
  • the defined medium further comprises L-glutamine, sodium bicarbonate and/or 2-mercaptoethanol.
  • the CTSTMOpTmizerTM T-cell Immune Cell Serum Replacement is used with conventional growth media, including but not limited to CTSTM OpTmizerTM T-cell Expansion Basal Medium, CTSTM OpTmizerTM T-cell Expansion SFM, CTSTM AIM-V Medium, CSTTM AIM-V SFM, LymphoONETM T-Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium 85 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco'
  • the total serum replacement concentration (vol%) in the serum-free or defined medium is from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or defined medium.
  • the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium.
  • the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium.
  • the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.
  • the serum-free or defined medium is CTSTM OpTmizerTM T- cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTSTM OpTmizerTM is useful in the present invention.
  • CTSTM OpTmizerTM T-cell Expansion SFM is a combination of 1L CTSTM OpTmizerTM T-cell Expansion Basal Medium and 26 mL CTSTM OpTmizerTM T-Cell Expansion Supplement, which are mixed together prior to use.
  • the CTSTM OpTmizerTM T- cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific).
  • SR Immune Cell Serum Replacement
  • the CTSTM OpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2-mercaptoethanol at 55mM.
  • the CTSTM OpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55 ⁇ M.
  • the defined medium is CTSTM OpTmizerTM T-cell Expansion SFM (ThermoFisher Scientific).
  • CTSTM OpTmizerTM T-cell Expansion SFM is a combination of 1L CTSTM OpTmizerTM T- cell Expansion Basal Medium and 26 mL CTSTM OpTmizerTM T-Cell Expansion Supplement, which are mixed together prior to use.
  • the CTSTM OpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), along with 2-mercaptoethanol at 55mM.
  • SR Immune Cell Serum Replacement
  • the CTSTMOpTmizerTM T- cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine.
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2- mercaptoethanol, and 2mM of L-glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2.
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented 86 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further comprises about 3000 IU/mL of IL-2.
  • SR Immune Cell Serum Replacement
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific), 55mM of 2-mercaptoethanol, and 2mM of L-glutamine, and further comprises about 6000 IU/mL of IL-2.
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2- mercaptoethanol, and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2.
  • SR Immune Cell Serum Replacement
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2- mercaptoethanol, and further comprises about 3000 IU/mL of IL-2. In some embodiments, the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and 55mM of 2-mercaptoethanol, and further comprises about 1000 IU/mL to about 6000 IU/mL of IL-2.
  • SR Immune Cell Serum Replacement
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 1000 IU/mL to about 8000 IU/mL of IL-2. In some embodiments, the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 3000 IU/mL of IL-2.
  • SR Immune Cell Serum Replacement
  • the CTSTMOpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and about 2mM glutamine, and further comprises about 6000 IU/mL of IL- 2.
  • the CTSTM OpTmizerTM T-cell Expansion SFM is supplemented with about 3% of the CTSTM Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the media is 55 ⁇ M.
  • the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of from about 0.1mM to about 10mM, 0.5mM to about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to about 5 mM.
  • glutamine i.e., GlutaMAX®
  • the serum-free medium or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2mM.
  • the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of from about 5mM to about 150mM, 10mM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about 100mM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65mM.
  • the 87 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55mM. In some embodiments, the final concentration of 2-mercaptoethanol in the media is 55 ⁇ M.
  • the defined media described in International PCT Publication No. WO/1998/030679, which is herein incorporated by reference, are useful in the present invention. In that publication, serum-free eukaryotic cell culture media are described.
  • the serum-free, eukaryotic cell culture medium includes a basal cell culture medium supplemented with a serum-free supplement capable of supporting the growth of cells in serum- free culture.
  • the serum-free eukaryotic cell culture medium supplement comprises or is obtained by combining one or more ingredients 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 precursors, one or more trace elements, and one or more antibiotics.
  • the defined medium further comprises L-glutamine, sodium bicarbonate and/or beta-mercaptoethanol.
  • the defined medium comprises an albumin or an albumin substitute and one or more ingredients selected from group consisting of 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 precursors, and one or more trace elements.
  • the defined medium comprises albumin and one or more ingredients 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 saturated transferrin, insulin, and compounds containing the trace element moieties 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+ , Ni 2+ , Rb + , Sn 2+ and Zr 4+ .
  • the trace element moieties Ag + , Al 3+ , Ba 2+ , Cd 2+ , CO
  • the basal cell media is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth medium, and Iscove's Modified Dulbecco's Medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium Eagle
  • RPMI 1640 F-10, F-12
  • ⁇ MEM Minimal Essential Medium
  • G-MEM Glasgow's Minimal Essential Medium
  • RPMI growth medium RPMI growth medium
  • Iscove's Modified Dulbecco's Medium Iscove's Modified Dulbecco's Medium.
  • the concentration of glycine in the defined medium is in the range of from 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, the concentration of L-tryptophan is about 2-110 mg/L, the concentration of L-tyrosine is about 3-175 mg/L, the concentration of L-valine is about 5-500 mg/L, the concentration of thiamine is about 1-20 88 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO mg/L
  • the non-trace element moiety ingredients in the defined medium are present in the concentration ranges listed in the column under the heading “Concentration Range in 1X Medium” in Table 4. In other embodiments, the non-trace element moiety ingredients in the defined medium are present in the final concentrations listed in the column under the heading “A Preferred Embodiment of the 1X Medium” in Table 4. In other embodiments, the defined medium is a basal cell medium comprising a serum free supplement. In some of these embodiments, the serum free supplement comprises non-trace moiety ingredients of the type and in the concentrations listed in the column under the heading “A Preferred Embodiment in Supplement” in Table 4.
  • the osmolarity of the defined medium is between about 260 and 350 mOsmol. In some embodiments, the osmolarity 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 can 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 ⁇ M), 2-mercaptoethanol (final concentration of about 100 ⁇ M).
  • the defined media described in Smith, et al., Clin Transl Immunology, 4(1) 2015 (doi: 10.1038/cti.2014.31) are useful in the present invention. Briefly, RPMI or CTSTM OpTmizerTM was used as the basal cell medium, and supplemented with either 0, 2%, 5%, or 10% CTSTM Immune Cell Serum Replacement.
  • the cell medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells.
  • the cell medium in the first and/or second gas permeable container lacks beta-mercaptoethanol (BME or ⁇ ME; also known as 2-mercaptoethanol, CAS 60-24-2).
  • BME or ⁇ ME also known as 2-mercaptoethanol, CAS 60-24-2.
  • the second expansion is performed in a closed system bioreactor.
  • a closed system is employed for the TIL expansion, as described herein.
  • a single bioreactor is employed.
  • the single bioreactor employed is for example a G-REX -10 or a G- REX -100.
  • the closed system bioreactor is a single bioreactor.
  • the step of rapid or second expansion is split into a plurality of steps to achieve a scaling up of the culture by: (a) performing the rapid or second 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) effecting the transfer of the TILs in the small scale culture to a second container larger than the first container, e.g., a G-REX-500-MCS 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.
  • a first container e.g., a G-REX-100 MCS container
  • a second container larger than the first container e.g., a G-REX-500-MCS container
  • the step of rapid or second expansion is split into a plurality of steps to achieve a scaling out of the culture by: (a) performing the rapid or second expansion by culturing TILs in a first 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) effecting the transfer and apportioning of the TILs from the first small scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers that are equal in size to the first container, wherein in each second container the portion of the TILs from first small scale culture transferred to such second container is cultured in a second small scale culture for a period of about 4 to 7 days.
  • a first container e.g., a G-REX-100 MCS container
  • the first small scale TIL culture is apportioned into a plurality of about 2 to 5 subpopulations of TILs.
  • the step of rapid or second expansion is split into a plurality of steps to achieve a scaling out and scaling up of the culture by: (a) performing the rapid or second 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) effecting the transfer and apportioning of the TILs from the small scale culture into and amongst at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers that are larger in size than the first container, e.g., G-REX- 500MCS containers, wherein in each second container the portion of the TILs from the small scale culture transferred to such second container is cultured in a larger scale culture for a period
  • the step of rapid or second expansion is split into a plurality of steps to achieve a scaling out and scaling up of the culture by: (a) performing the rapid or second 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) effecting the transfer and apportioning of the TILs from the small scale culture into and amongst 2, 3 or 4 second containers that are larger in size than the first container, e.g., G-REX-500 MCS containers, wherein in each second container the portion of the TILs from the small scale culture transferred to such second container is cultured in a larger scale culture for a period of about 6 days.
  • a first container e.g., a G-REX-100 MCS container
  • each second container upon the splitting of the rapid or second expansion, comprises at least 10 8 TILs. In some embodiments, upon the splitting of the rapid or second expansion, each second container comprises at least 10 8 TILs, at least 10 9 TILs, or at least 10 10 TILs. In one exemplary embodiment, each second container comprises at least 10 10 TILs.
  • the first small scale TIL culture is apportioned into a plurality of subpopulations. In some embodiments, the first small scale TIL culture is apportioned into a plurality of about 2 to 5 subpopulations.
  • the first small scale TIL culture is apportioned into a plurality of about 2, 3, 4, or 5 subpopulations.
  • the plurality of subpopulations comprises a therapeutically effective amount of TILs.
  • one or more subpopulations of TILs are pooled together to produce a therapeutically effective amount of TILs.
  • each subpopulation of TILs comprises a therapeutically effective amount of TILs.
  • the rapid or second expansion is performed for a period of about 3 to 7 days before being split into a plurality of steps.
  • the splitting of the rapid or second expansion occurs at about day 3, day 4, day 5, day 6, or day 7 after the initiation of the rapid or second expansion. [00527] In some embodiments, the splitting of the rapid or second expansion occurs at about day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, or day 16 day 17, or day 18 after the initiation of the first expansion (i.e., pre-REP expansion). In one exemplary embodiment, the splitting of the rapid or second expansion occurs at about day 16 after the initiation of the first expansion. [00528] In some embodiments, the rapid or second expansion is further performed for a period of about 7 to 11 days after the splitting.
  • the rapid or second expansion is further performed for a period of about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days after the splitting.
  • the cell culture medium used for the rapid or second expansion before the splitting comprises the same components as the cell culture medium used for the rapid or second expansion after the splitting.
  • the cell culture medium used for the rapid or second expansion before the splitting comprises different components from the cell culture medium used for the rapid or second expansion after the splitting.
  • the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2, optionally OKT-3 and further optionally APCs.
  • the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2, OKT-3, and further optionally APCs.
  • the cell culture medium used for the rapid or second expansion before the splitting comprises IL-2, OKT-3 and APCs.
  • the cell culture medium used for the rapid or second expansion before the splitting is generated by supplementing the cell culture medium in the first expansion with fresh culture medium comprising IL-2, optionally OKT-3 and further optionally APCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by supplementing the cell culture medium in the first expansion with fresh culture medium comprising IL-2, OKT-3 and APCs. In some embodiments, the cell culture medium used for the rapid or second expansion before the splitting is generated by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, optionally OKT-3 and further optionally APCs.
  • the cell culture medium used for the rapid or second expansion before the splitting is generated by replacing the cell culture medium in the first expansion with fresh cell culture medium comprising IL-2, OKT-3 and APCs.
  • the cell culture medium used for the rapid or second expansion after the splitting comprises IL-2, and optionally OKT-3.
  • the cell culture medium used for the rapid or second expansion after the splitting comprises IL-2, and OKT-3.
  • the cell culture medium used for the rapid or second expansion after the splitting is generated by replacing the cell culture medium used for the rapid or second expansion before the splitting with fresh culture medium comprising IL-2 and optionally OKT-3.
  • the cell culture medium used for the rapid or second expansion after the splitting is generated by replacing the cell culture medium used for the rapid or second expansion before the splitting with fresh culture medium comprising IL-2 and OKT-3.
  • the splitting of the rapid expansion occurs in a closed system.
  • the scaling up of the TIL culture during the rapid or second expansion comprises adding fresh cell culture medium to the TIL culture (also referred to as feeding the TILs).
  • the feeding comprises adding fresh cell culture medium to the TIL culture frequently.
  • the feeding comprises adding fresh cell culture medium to the TIL culture at a regular interval.
  • the fresh cell culture medium is supplied to the TILs via a constant flow.
  • an automated cell expansion system such as Xuri W25 is used for the rapid expansion and feeding.
  • Feeder Cells and Antigen Presenting Cells [00535]
  • the second expansion procedures described herein (for example including expansion such as those described in Step D from Figure 1, as well as those referred to as REP) require an excess of feeder cells during REP TIL expansion and/or during the second expansion.
  • the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors.
  • the PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.
  • the allogeneic PBMCs are inactivated, either via irradiation or heat treatment, and used in the REP procedures, as described in the examples, which provides an exemplary protocol for evaluating the replication incompetence of irradiate allogeneic PBMCs.
  • PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells on day 14 is less than the initial viable cell number put into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start day of the second expansion).
  • PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start day of the second expansion).
  • the PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2.
  • PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start day of the second expansion).
  • the PBMCs are cultured in the presence of 5-60 ng/mL OKT3 antibody and 1000-6000 IU/mL IL-2.
  • the PBMCs are cultured in the presence of 10-50 ng/mL OKT3 antibody and 2000-5000 IU/mL IL-2.
  • the PBMCs are cultured in the presence of 20-40 ng/mL OKT3 antibody and 2000-4000 IU/mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 25-35 ng/mL OKT3 antibody and 2500-3500 IU/mL IL-2.
  • the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells.
  • the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1 to 93 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300.
  • the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200.
  • the second expansion procedures described herein require a ratio of about 2.5x10 9 feeder cells to about 100x10 6 TIL. In other embodiments, the second expansion procedures described herein require a ratio of about 2.5x10 9 feeder cells to about 50x10 6 TIL. In yet other embodiments, the second expansion procedures described herein require about 2.5x10 9 feeder cells to about 25x10 6 TIL. [00542] In some embodiments, the second expansion procedures described herein require an excess of feeder cells during the second expansion.
  • the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors.
  • PBMCs peripheral blood mononuclear cells
  • the PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.
  • artificial antigen-presenting (aAPC) cells are used in place of PBMCs.
  • the allogeneic PBMCs are inactivated, either via irradiation or heat treatment, and used in the TIL expansion procedures described herein, including the exemplary procedures described in the figures and examples.
  • artificial antigen presenting cells are used in the second expansion as a replacement for, or in combination with, PBMCs. 2.
  • Cytokines and Other Additives generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.
  • a cytokine in particular IL-2
  • IL-15 and IL-21 are examples of cytokines, with the latter finding particular use in many embodiments.
  • Step D may also include the addition of OKT-3 antibody or muromonab to the culture media, as described elsewhere herein.
  • Step D may also include the addition of a 4-1BB agonist to the culture media, as described elsewhere herein.
  • Step D may also include the addition of an OX-40 agonist to the culture media, as described elsewhere herein.
  • additives such as peroxisome proliferator-activated receptor gamma coactivator I-alpha agonists, including proliferator-activated receptor (PPAR)-gamma agonists such as a thiazolidinedione compound, may be used in the culture media during Step D, as described in U.S. Patent Application Publication No. US 2019/0307796 A1, the disclosure of which is incorporated by reference herein.
  • TILs can be harvested in any appropriate and sterile manner, including for example by centrifugation. Methods for TIL harvesting are well known in the art and any such know methods can be employed with the present process. In some embodiments, TILs are harvested using an automated system.
  • Cell harvesters and/or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell based harvester can be employed with the present methods. In some embodiments, the cell harvester and/or cell processing systems is a membrane- based cell harvester.
  • cell harvesting is via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi).
  • LOVO cell processing system also refers to any instrument or device manufactured by any vendor that can pump a solution comprising cells through a membrane or filter such as a spinning membrane or spinning filter in a sterile and/or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture media without pelletization.
  • 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.
  • the harvest for example, Step E according to Figure 1, is performed from a closed system bioreactor.
  • a closed system is employed for the TIL expansion, as described herein.
  • a single bioreactor is employed.
  • the single bioreactor employed is for example a G-REX-10 or a G-REX-100.
  • the closed system bioreactor is a single bioreactor.
  • the closed system is accessed via syringes under sterile conditions in order to maintain the sterility and closed nature of the system.
  • a closed system as described in the Examples is employed.
  • TILs are harvested according to the methods described in the Examples.
  • TILs between days 1 and 11 are harvested using the methods as described in the steps referred herein, such as in the day 11 TIL harvest in the Examples.
  • TILs between days 12 and 24 are harvested using the methods as described in the steps referred herein, such as in the Day 22 TIL harvest in the Examples.
  • TILs between days 12 and 22 are harvested using the methods as described in the steps referred herein, such as in the Day 22 TIL harvest in the Examples.
  • TILs expanded using APCs of the present disclosure are administered to a patient as a pharmaceutical composition.
  • the pharmaceutical composition is a suspension of TILs in a sterile buffer.
  • TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art.
  • the T-cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes.
  • Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.
  • the culture media used in expansion methods described herein include an anti-CD3 antibody e.g. OKT-3.
  • An anti-CD3 antibody in combination with IL-2 induces T cell activation and cell division in the TIL population. This effect can be seen with full length 96 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO antibodies as well as Fab and F(ab’)2 fragments, with the former being generally preferred; see, e.g., Tsoukas et al., J. Immunol.1985, 135, 1719, hereby incorporated by reference in its entirety.
  • the multiplier (0.64) is the random packing density for equivalent spheres as calculated by Jaeger and Nagel, Science, 1992, 255, 1523-3.
  • the divisor 24 is the number of equivalent spheres that could contact a similar object in 4 -dimensional space or “the Newton number” as described in Musin, Russ. Math. Surv., 2003, 58, 794–795. [00564] In some embodiments, the number of antigen-presenting feeder cells exogenously supplied during the priming first expansion is approximately one-half the number of antigen-presenting feeder cells exogenously supplied during the rapid second expansion.
  • the method comprises performing the priming first expansion in a cell culture medium which comprises approximately 50% fewer antigen presenting cells as compared to the cell culture medium of the rapid second expansion.
  • the number of antigen-presenting feeder cells (APCs) exogenously supplied during the rapid second expansion is greater than the number of APCs exogenously supplied during the priming first expansion.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 20:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 10:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 9:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 8:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 7:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 6:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 5:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 4:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 3:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.9:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.8:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.7:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.6:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.5:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.4:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.3:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.2:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.1:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 10:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 5:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 4:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 3:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.9:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.8:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.7:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.6:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.5:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.4:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.3:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about about 2:1 to at or about 2.2:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.1:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is at or about 2:1.
  • the ratio of the number of APCs exogenously supplied during the rapid second expansion to the number of APCs exogenously supplied during the priming first expansion is at or about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.
  • the number of APCs exogenously supplied during the priming first expansion is at or about 1 ⁇ 10 8 , 1.1 ⁇ 10 8 , 1.2 ⁇ 10 8 , 1.3 ⁇ 10 8 , 1.4 ⁇ 10 8 , 1.5 ⁇ 10 8 , 1.6 ⁇ 10 8 , 1.7 ⁇ 10 8 , 1.8 ⁇ 10 8 , 1.9 ⁇ 10 8 , 2 ⁇ 10 8 , 2.1 ⁇ 10 8 , 2.2 ⁇ 10 8 , 2.3 ⁇ 10 8 , 2.4 ⁇ 10 8 , 2.5 ⁇ 10 8 , 2.6 ⁇ 10 8 , 2.7 ⁇ 10 8 , 2.8 ⁇ 10 8 , 2.9 ⁇ 10 8 , 3 ⁇ 10 8 , 3.1 ⁇ 10 8 , 3.2 ⁇ 10 8 , 3.3 ⁇ 10 8 , 3.4 ⁇ 10 8 or 3.5 ⁇ 10 8 APCs, and the number of APCs exogenously supplied during the rapid second expansion is at or about 3.5 ⁇ 10 8 , 3.6 ⁇ 10 8 , 3.7 ⁇ 10 8 , 3.8 ⁇ 10 8 ,
  • the number of APCs exogenously supplied during the priming first expansion is selected from the range of at or about 1.5 ⁇ 10 8 APCs to at or about 3 ⁇ 10 8 APCs
  • the number of APCs exogenously supplied during the rapid second expansion is selected from the range of at or about 4 ⁇ 10 8 APCs to at or about 7.5 ⁇ 10 8 APCs.
  • the number of APCs exogenously supplied during the priming first expansion is selected from the range of at or about 2 ⁇ 10 8 APCs to at or about 2.5 ⁇ 10 8 APCs, and the number of APCs exogenously supplied during the rapid second expansion is selected from the range of at or about 4.5 ⁇ 10 8 APCs to at or about 5.5 ⁇ 10 8 APCs.
  • the number of APCs exogenously supplied during the priming first expansion is at or about 2.5 ⁇ 10 8 APCs, and the number of APCs exogenously supplied during the rapid second expansion is at or about 5 ⁇ 10 8 APCs.
  • the number of APCs (including, for example, PBMCs) added at day 0 of the priming first expansion is approximately one-half of the number of PBMCs added at day 7 of the priming first expansion (e.g., day 7 of the method).
  • the method comprises adding antigen presenting cells at day 0 of the priming first expansion to the first population of TILs and adding antigen presenting cells at day 7 to the second population of TILs, wherein the number of antigen presenting cells added at day 0 is approximately 50% of the number of antigen presenting cells added at day 7 of the priming first expansion (e.g., day 7 of the method).
  • the number of APCs including, for example, PBMCs
  • exogenously supplied at day 7 of the rapid second expansion is greater than the number of PBMCs exogenously supplied at day 0 of the priming first expansion.
  • the APCs exogenously supplied in the priming first expansion are seeded in the culture flask at a density selected from a range of at or about 1.0 ⁇ 10 6 APCs/cm 2 to at or about 4.5 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the priming first expansion are seeded in the culture flask at a density selected from a range of at or about 1.5 ⁇ 10 6 APCs/cm 2 to at or about 3.5 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the priming first expansion are seeded in the culture flask at a density selected from a range of at or about 2 ⁇ 10 6 APCs/cm 2 to at or about 3 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the priming first expansion are seeded in the culture flask at a density of at or about 2 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the priming first expansion are seeded in the culture flask at a density of at or about 1.0 ⁇ 10 6 , 1.1 ⁇ 10 6 , 1.2 ⁇ 10 6 , 1.3 ⁇ 10 6 , 1.4 ⁇ 10 6 , 1.5 ⁇ 10 6 , 1.6 ⁇ 10 6 , 1.7 ⁇ 10 6 , 1.8 ⁇ 10 6 , 1.9 ⁇ 10 6 , 2 ⁇ 10 6 , 2.1 ⁇ 10 6 , 2.2 ⁇ 10 6 , 2.3 ⁇ 10 6 , 2.4 ⁇ 10 6 , 2.5 ⁇ 10 6 , 2.6 ⁇ 10 6 , 2.7 ⁇ 10 6 , 2.8 ⁇ 10 6 , 2.9 ⁇ 10 6 , 3 ⁇ 10 6 , 3.1 ⁇ 10 6 , 3.2 ⁇ 10 6 , 3.3 ⁇ 10 6 , 3.4 ⁇ 10 6 , 3.5 ⁇ 10 6 , 3.6 ⁇ 10 6 , 3.7 ⁇ 10 6 , 3.8 ⁇ 10 6 , 3.9 ⁇ 10 6 , 4 ⁇ 10 6 , 4.1
  • the APCs exogenously supplied in the rapid second expansion are seeded in the culture flask at a density selected from a range of at or about 2.5 ⁇ 10 6 APCs/cm 2 to at or about 7.5 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the rapid second expansion are seeded in the culture flask at a density selected from a range of at or about 3.5 ⁇ 10 6 APCs/cm 2 to about 6.0 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the rapid second expansion are seeded in the culture flask at a density selected from a range of at or about 4.0 ⁇ 10 6 APCs/cm 2 to about 5.5 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the rapid second expansion are seeded in the culture flask at a density selected from a range of at or about 4.0 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the rapid second expansion are seeded in the culture flask at a density of at or about 2.5 ⁇ 10 6 APCs/cm 2 , 2.6 ⁇ 10 6 APCs/cm 2 , 2.7 ⁇ 10 6 APCs/cm 2 , 2.8 ⁇ 10 6 , 2.9 ⁇ 10 6 , 3 ⁇ 10 6 , 3.1 ⁇ 10 6 , 3.2 ⁇ 10 6 , 3.3 ⁇ 10 6 , 3.4 ⁇ 10 6 , 3.5 ⁇ 10 6 , 3.6 ⁇ 10 6 , 3.7 ⁇ 10 6 , seeded in the culture flask at a density of at or about 1.0 ⁇ 10 6 , 1.1 ⁇ 10 6 , 1.2 ⁇ 10 6 , 1.3 ⁇ 10 6 , 1.4 ⁇ 10 6 , 1.5 ⁇ 10 6 , 1.6 ⁇ 10 6 , 1.7 ⁇ 10 6 , 1.8 ⁇ 10 6 , 1.9 ⁇ 10 6 , 2 ⁇ 10 6 , 2.1 ⁇ 10 6 , 2.2 ⁇ 10 6
  • the APCs exogenously supplied in the priming first expansion are seeded in the culture flask at a density selected from a range of at or about 1.0 ⁇ 10 6 APCs/cm 2 to at or about 4.5 ⁇ 10 6 APCs/cm 2
  • the APCs exogenously supplied in the rapid second expansion are seeded in the culture flask at a density selected from a range of at or about 2.5 ⁇ 10 6 APCs/cm 2 to at or about 7.5 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the priming first expansion are seeded in the culture flask at a density selected from a range of at or about 1.5 ⁇ 10 6 APCs/cm 2 to at or about 3.5 ⁇ 10 6 APCs/cm 2
  • the APCs exogenously supplied in the rapid second expansion are seeded in the culture flask at a density selected from a range of at or about 3.5 ⁇ 10 6 APCs/cm 2 to at or about 6 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the priming first expansion are seeded in the culture flask at a density selected from a range of at or about 2 ⁇ 10 6 APCs/cm 2 to at or about 3 ⁇ 10 6 APCs/cm 2
  • the APCs exogenously supplied in the rapid second expansion are seeded in the culture flask at a density selected from a range of at or about 4 ⁇ 10 6 APCs/cm 2 to at or about 5.5 ⁇ 10 6 APCs/cm 2 .
  • the APCs exogenously supplied in the priming first expansion are seeded in the culture flask at a density at or about 2 ⁇ 10 6 APCs/cm 2 and the APCs exogenously supplied in the rapid second expansion are seeded in the culture flask at a density of at or about 4 ⁇ 10 6 APCs/cm 2 .
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of PBMCs exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 20:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of PBMCs exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 10:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of PBMCs exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 9:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 8:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 7:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 6:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 5:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 4:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 3:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.9:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.8:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.7:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.6:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.5:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.4:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.3:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.2:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2.1:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 1.1:1 to at or about 2:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 10:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 5:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 4:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 3:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.9:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.8:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.7:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.6:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.5:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.4:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.3:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about about 2:1 to at or about 2.2:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from a range of from at or about 2:1 to at or about 2.1:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is at or about 2:1.
  • the ratio of the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion to the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is at or about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, or 5:1.
  • the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is at or about 1 ⁇ 10 8 , 1.1 ⁇ 10 8 , 1.2 ⁇ 10 8 , 1.3 ⁇ 10 8 , 1.4 ⁇ 10 8 , 1.5 ⁇ 10 8 , 1.6 ⁇ 10 8 , 1.7 ⁇ 10 8 , 1.8 ⁇ 10 8 , 1.9 ⁇ 10 8 , 2 ⁇ 10 8 , 2.1 ⁇ 10 8 , 2.2 ⁇ 10 8 , 2.3 ⁇ 10 8 , 2.4 ⁇ 10 8 , 2.5 ⁇ 10 8 , 2.6 ⁇ 10 8 , 2.7 ⁇ 10 8 , 2.8 ⁇ 10 8 , 2.9 ⁇ 10 8 , 3 ⁇ 10 8 , 3.1 ⁇ 10 8 , 3.2 ⁇ 10 8 , 3.3 ⁇ 10 8 , 3.4 ⁇ 10 8 or 3.5 ⁇ 10 8 APCs (including, for example, PBMCs), and the number of APCs (including, for example, PBMCs) exogenously supplied
  • the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from the range of at or about 1 ⁇ 10 8 APCs (including, for example, PBMCs) to at or about 3.5 ⁇ 10 8 APCs (including, for example, PBMCs), and the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid 108 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO second expansion is selected from the range of at or about 3.5 ⁇ 10 8 APCs (including, for example, PBMCs) to at or about 1 ⁇ 10 9 APCs (including, for example, PBMCs).
  • the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from the range of at or about 1.5 ⁇ 10 8 APCs to at or about 3 ⁇ 10 8 APCs (including, for example, PBMCs), and the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion is selected from the range of at or about 4 ⁇ 10 8 APCs (including, for example, PBMCs) to at or about 7.5 ⁇ 10 8 APCs (including, for example, PBMCs).
  • the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is selected from the range of at or about 2 ⁇ 10 8 APCs (including, for example, PBMCs) to at or about 2.5 ⁇ 10 8 APCs (including, for example, PBMCs), and the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion is selected from the range of at or about 4.5 ⁇ 10 8 APCs (including, for example, PBMCs) to at or about 5.5 ⁇ 10 8 APCs (including, for example, PBMCs).
  • the number of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion is at or about 2.5 ⁇ 10 8 APCs (including, for example, PBMCs) and the number of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion is at or about 5 ⁇ 10 8 APCs (including, for example, PBMCs)
  • the number of layers of APCs (including, for example, PBMCs) added at day 0 of the priming first expansion is approximately one-half of the number of layers of APCs (including, for example, PBMCs) added at day 7 of the rapid second expansion.
  • the method comprises adding antigen presenting cell layers at day 0 of the priming first expansion to the first population of TILs and adding antigen presenting cell layers at day 7 to the second population of TILs, wherein the number of antigen presenting cell layer added at day 0 is approximately 50% of the number of antigen presenting cell layers added at day 7.
  • the number of layers of APCs (including, for example, PBMCs) exogenously supplied at day 7 of the rapid second expansion is greater than the number of layers of APCs (including, for example, PBMCs) exogenously supplied at day 0 of the priming first expansion.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 2 cell layers and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 4 cell layers.
  • layered APCs including, for example, PBMCs
  • day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 4 cell layers.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about one cell layer and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 3 cell layers.
  • layered APCs including, for example, PBMCs
  • day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 3 cell layers.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 1.5 cell layers to at or about 2.5 cell layers and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 3 cell layers.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about one cell layer and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 2 cell layers.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1,
  • layered APCs including,
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 1 cell layer to at or about 2 cell layers and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 3 cell layers to at or about 10 cell layers.
  • layered APCs including, for example, PBMCs
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 2 cell layers to at or about 3 cell layers and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 4 cell layers to at or about 8 cell layers.
  • layered APCs including, for example, PBMCs
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 2 cell layers and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 4 cell layers to at or about 8 cell layers.
  • layered APCs including, for example, PBMCs
  • day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 4 cell layers to at or about 8 cell layers.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 1, 2 or 3 cell layers and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.
  • layered APCs including, for example, PBMCs
  • day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness of at or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.1 to at or about 1:10.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.1 to at or about 1:8.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.1 to at or about 1:7.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.1 to at or about 1:6.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number 111 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.1 to at or about 1:5.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.1 to at or about 1:4.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.1 to at or about 1:3.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.1 to at or about 1:2.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.2 to at or about 1:8.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.3 to at or about 1:7.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.4 to at or about 1:6.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.5 to at or about 1:5.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.6 to at or about 1:4.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first 113 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.7 to at or about 1:3.5.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.8 to at or about 1:3.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from the range of at or about 1:1.9 to at or about 1:2.5.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is at or about 1: 2.
  • day 0 of the priming first expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example, PBMCs) and day 7 of the rapid second expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of layers of APCs (including, for example, PBMCs), wherein the ratio of the first number of layers of APCs (including, for example, PBMCs) to the second number of layers of APCs (including, for example, PBMCs) is selected from at or about 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1
  • the number of APCs in the priming first expansion is selected from the range of about 1.0 ⁇ 10 6 APCs/cm 2 to about 4.5 ⁇ 10 6 APCs/cm 2
  • the number of APCs in the rapid second expansion is selected from the range of about 2.5 ⁇ 10 6 APCs/cm 2 to about 7.5 ⁇ 10 6 APCs/cm 2 .
  • the number of APCs in the priming first expansion is selected from the range of about 1.5 ⁇ 10 6 APCs/cm 2 to about 3.5 ⁇ 10 6 APCs/cm 2
  • the number of APCs in the rapid second expansion is selected from the range of about 3.5 ⁇ 10 6 APCs/cm 2 to about 6.0 ⁇ 10 6 APCs/cm 2 .
  • the number of APCs in the priming first expansion is selected from the range of about 2.0 ⁇ 10 6 APCs/cm 2 to about 3.0 ⁇ 10 6 APCs/cm 2
  • the number of APCs in the rapid second expansion is selected from the range of about 4.0 ⁇ 10 6 APCs/cm 2 to about 5.5 ⁇ 10 6 APCs/cm 2 .
  • Anti-CD3 Antibodies [00692]
  • the culture media used in expansion methods described herein include an anti-CD3 antibody.
  • An anti-CD3 antibody in combination with IL-2 induces T cell activation and cell division in the TIL population.
  • anti-human CD3 antibodies that find use in the invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies.
  • the OKT3 anti-CD3 antibody muromonab is used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). See, Table 1. [00694] As will be appreciated by those in the art, there are a number of suitable anti-human CD3 antibodies that find use in the invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies. In some embodiments, the OKT3 anti-CD3 antibody muromonab is used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA).
  • the cell culture medium of the priming first expansion and/or the rapid second expansion comprises a TNFRSF agonist.
  • the TNFRSF agonist is a 4-1BB (CD137) agonist.
  • the 4-1BB agonist may be any 4-1BB binding molecule known in the art.
  • the 4-1BB binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1BB.
  • the 4-1BB agonists or 4-1BB binding molecules may comprise an immunoglobulin heavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • the 4-1BB agonist or 4-1BB binding molecule may have both a heavy and a light chain.
  • binding molecule also includes antibodies (including full length antibodies), monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human, humanized or chimeric antibodies, and antibody fragments, e.g., Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the above, and engineered forms of antibodies, e.g., scFv molecules, that bind to 4-1BB.
  • the 4-1BB agonist is an antigen binding protein that is a fully human antibody.
  • the 4-1BB agonist is an antigen binding protein that is a humanized antibody.
  • 4-1BB agonists for use in the presently disclosed methods and compositions include anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4- 1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB adnectins, anti-4-1BB domain antibodies, single chain anti-4-1BB fragments, heavy chain anti-4-1BB fragments, light chain anti-4- 1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives, conjugates, variants, or biosimilars thereof.
  • Agonistic anti-4-1BB antibodies are known to induce strong immune responses. Lee, et al., PLOS One 2013, 8, e69677.
  • the 4-1BB agonist is an agonistic, anti- 4-1BB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).
  • the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utomilumab, or urelumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.
  • the 4-1BB agonist is utomilumab or urelumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.
  • the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein.
  • a multimeric 4-1BB agonist such as a trimeric or hexameric 4-1BB agonist (with three or six ligand binding domains), may induce superior receptor (4-1BBL) clustering and internal cellular signaling complex formation compared to an agonistic monoclonal antibody, which typically possesses two ligand binding domains.
  • Trimeric (trivalent) or hexameric (or hexavalent) or greater fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and 116 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO optionally further linking two or more of these fusion proteins are described, e.g., in Gieffers, et al., Mol. Cancer Therapeutics 2013, 12, 2735-47. [00697]
  • Agonistic 4-1BB antibodies and fusion proteins are known to induce strong immune responses.
  • the 4-1BB agonist is a monoclonal antibody or fusion protein that binds specifically to 4-1BB antigen in a manner sufficient to reduce toxicity.
  • the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cellular toxicity (ADCC), for example NK cell cytotoxicity.
  • ADCC antibody-dependent cellular toxicity
  • the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cell phagocytosis (ADCP).
  • the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates complement-dependent cytotoxicity (CDC).
  • the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein which abrogates Fc region functionality.
  • the 4-1BB agonists are characterized by binding to human 4-1BB (SEQ ID NO:40) with high affinity and agonistic activity.
  • the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO:40).
  • the 4-1BB agonist is a binding molecule that binds to murine 4-1BB (SEQ ID NO:41).
  • Table 5 The amino acid sequences of 4-1BB antigen to which a 4-1BB agonist or binding molecule binds are summarized in Table 5. TABLE 5. Amino acid sequences of 4-1BB antigens.
  • compositions, processes and methods described include a 4-1BB agonist that binds human or murine 4-1BB with a KD of about 100 pM or lower, binds human or murine 4-1BB with a KD of about 90 pM or lower, binds human or murine 4-1BB with a KD of about 80 pM or lower, binds human or murine 4-1BB with a KD of about 70 pM or lower, binds human or 117 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO murine 4-1BB with a KD of about 60 pM or lower, binds human or murine 4-1BB with a KD of about 50 pM or lower, binds human or murine 4-1BB with a KD of about 40 pM or lower, or binds human or murine 4-1BB with a KD
  • compositions, processes and methods described include a 4-1BB agonist that binds to human or murine 4-1BB with a kassoc of about 7.5 ⁇ 10 5 1/M ⁇ s or faster, binds to human or murine 4-1BB with a kassoc of about 7.5 ⁇ 10 5 1/M ⁇ s or faster, binds to human or murine 4- 1BB with a kassoc of about 8 ⁇ 10 5 l/M ⁇ s or faster, binds to human or murine 4-1BB with a kassoc of about 8.5 ⁇ 10 5 1/M ⁇ s or faster, binds to human or murine 4-1BB with a kassoc of about 9 ⁇ 10 5 1/M ⁇ s or faster, binds to human or murine 4-1BB with a kassoc of about 9.5 ⁇ 10 5 1/M ⁇ s or faster, or binds to human or murine 4-1BB with a kassoc of about
  • compositions, processes and methods described include a 4-1BB agonist that binds to human or murine 4-1BB with a kdissoc of about 2 ⁇ 10 -5 1/s or slower, binds to human or murine 4-1BB with a kdissoc of about 2.1 ⁇ 10 -5 1/s or slower , binds to human or murine 4- 1BB with a kdissoc of about 2.2 ⁇ 10 -5 1/s or slower, binds to human or murine 4-1BB with a kdissoc of about 2.3 ⁇ 10 -5 1/s or slower, binds to human or murine 4-1BB with a kdissoc of about 2.4 ⁇ 10 -5 1/s or slower, binds to human or murine 4-1BB with a kdissoc of about 2.5 ⁇ 10 -5 1/s or slower, binds to human or murine 4-1BB with a kdissoc of about
  • compositions, processes and methods described include a 4-1BB agonist that binds to human or murine 4-1BB with an IC50 of about 10 nM or lower, binds to human or murine 4-1BB with an IC50 of about 9 nM or lower, binds to human or murine 4-1BB with an IC50 of about 8 nM or lower, binds to human or murine 4-1BB with an IC50 of about 7 nM or lower, binds to human or murine 4-1BB with an IC50 of about 6 nM or lower, binds to human or murine 4-1BB with an IC50 of about 5 nM or lower, binds to human or murine 4-1BB with an IC50 of about 4 nM or lower, binds to human or murine 4-1BB with an IC50 of about 3 nM or lower, binds to human or murine 4-1BB with an IC50 of about 2 nM or lower, or bind
  • the 4-1BB agonist is utomilumab, also known as PF-05082566 or MOR-7480, or a fragment, derivative, variant, or biosimilar thereof.
  • Utomilumab is available from Pfizer, Inc.
  • Utomilumab is an immunoglobulin G2-lambda, anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cell antigen ILA, CD137)], Homo 118 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO sapiens (fully human) monoclonal antibody.
  • Utomilumab comprises glycosylation sites at Asn59 and Asn292; heavy chain intrachain disulfide bridges at positions 22-96 (VH-VL), 143-199 (CH1-CL), 256-316 (CH2) and 362-420 (CH3); light chain intrachain disulfide bridges at positions 22’-87’ (VH-VL) and 136’-195’ (CH1- CL); interchain heavy chain-heavy chain disulfide bridges at IgG2A isoform positions 218-218, 219- 219, 222-222, and 225-225, at IgG2A/B isoform positions 218-130, 219-219, 222-222, and 225-225, and at IgG2B isoform positions 219-130 (2), 222-222, and 225-225; and interchain heavy chain-light chain disulfide bridges at IgG2A isoform positions 130
  • a 4-1BB agonist comprises a heavy chain given by SEQ ID NO:42 and a light chain given by SEQ ID NO:43.
  • a 4-1BB agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
  • a 4-1BB agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, a 4-1BB agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, a 4-1BB agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. In some embodiments, a 4-1BB agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively.
  • a 4-1BB agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively.
  • the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of utomilumab.
  • the 4-1BB agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:44
  • the 4-1BB agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:45, and conservative amino acid substitutions thereof.
  • a 4-1BB agonist comprises VH and VL regions that are 119 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO each at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively.
  • a 4-1BB agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively.
  • a 4-1BB agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively.
  • a 4-1BB agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively.
  • a 4- 1BB agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively.
  • a 4-1BB agonist comprises an scFv antibody comprising VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45.
  • a 4-1BB agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively, and conservative amino acid substitutions thereof.
  • the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to utomilumab.
  • the biosimilar monoclonal antibody comprises an 4-1BB antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is utomilumab.
  • the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
  • the biosimilar is a 4-1BB agonist antibody authorized or submitted for authorization, wherein the 4-1BB agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is utomilumab.
  • the 4-1BB agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is utomilumab.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more 120 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is utomilumab.
  • TABLE 6 Amino acid sequences for 4-1BB agonist antibodies related to utomilumab.
  • the 4-1BB agonist is the monoclonal antibody urelumab, also known as BMS-663513 and 20H4.9.h4a, or a fragment, derivative, variant, or biosimilar thereof.
  • Urelumab is available from Bristol-Myers Squibb, Inc., and Creative Biolabs, Inc.
  • Urelumab is an immunoglobulin G4-kappa, anti-[Homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4- 1BB, T cell antigen ILA, CD137)], Homo sapiens (fully human) monoclonal antibody.
  • the amino acid sequences of urelumab are set forth in Table 7.
  • Urelumab comprises N-glycosylation sites at positions 298 (and 298’’); heavy chain intrachain disulfide bridges at positions 22-95 (VH-VL), 148- 204 (CH1-CL), 262-322 (CH2) and 368-426 (CH3) (and at positions 22’’-95’’, 148’’-204’’, 262’’- 322’’, and 368’’-426’’); light chain intrachain disulfide bridges at positions 23’-88’ (VH-VL) and 136’-196’ (CH1-CL) (and at positions 23’’’’-88’’’ and 136’’’-196’’’); interchain heavy chain-heavy chain disulfide bridges at positions 227-227’’ and 230-230’’; and interchain heavy chain-light chain disulfide bridges at 135-216’ and 135’’-216’’’.
  • VH-VL heavy chain intrachain disulfide bridges at
  • urelumab preparation and properties of urelumab and its variants and fragments are described in U.S. Patent Nos.7,288,638 and 8,962,804, the disclosures of which are incorporated by reference herein.
  • the preclinical and clinical characteristics of urelumab are described in Segal, et al., Clin. Cancer Res.2016, available at http:/dx.doi.org/ 10.1158/1078- 0432.CCR-16-1272.
  • Current clinical trials of urelumab in a variety of hematological and solid tumor indications include U.S. National Institutes of Health clinicaltrials.gov identifiers NCT01775631, NCT02110082, NCT02253992, and NCT01471210.
  • a 4-1BB agonist comprises a heavy chain given by SEQ ID NO:52 and a light chain given by SEQ ID NO:53.
  • a 4-1BB agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
  • a 4-1BB agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively.
  • a 4-1BB agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, a 4-1BB agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, a 4-1BB agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively. In some embodiments, a 4-1BB agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:52 and SEQ ID NO:53, respectively.
  • the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of urelumab.
  • the 4-1BB agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:54
  • the 4-1BB agonist light chain 122 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO variable region (VL) comprises the sequence shown in SEQ ID NO:55, and conservative amino acid substitutions thereof.
  • a 4-1BB agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively.
  • a 4-1BB agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, a 4-1BB agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively. In some embodiments, a 4-1BB agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively.
  • a 4- 1BB agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively.
  • a 4-1BB agonist comprises an scFv antibody comprising VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55.
  • a 4-1BB agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61, respectively, and conservative amino acid substitutions thereof.
  • the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to urelumab.
  • the biosimilar monoclonal antibody comprises an 4-1BB antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is urelumab.
  • the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
  • the biosimilar is a 4-1BB agonist antibody authorized or submitted for authorization, wherein the 4-1BB agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is urelumab.
  • the 4-1BB agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal 123 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO product or reference biological product is urelumab.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is urelumab. TABLE 7.
  • Patent No.6,362,325 (such as 1D8 or BMS-469492; 3H3 or BMS-469497; or 3El), antibodies disclosed in U.S. Patent No.6,974,863 (such as 53A2); antibodies disclosed in U.S. Patent No.6,210,669 (such as 1D8, 3B8, or 3El), antibodies described in U.S. Patent No.5,928,893, antibodies disclosed in U.S. Patent No.6,303,121, antibodies disclosed in U.S. Patent No.6,569,997, antibodies disclosed in International Patent Application Publication Nos.
  • the 4-1BB agonist is a 4-1BB agonistic fusion protein described in International Patent Application Publication Nos. WO 2008/025516 A1, WO 2009/007120 A1, WO 2010/003766 A1, WO 2010/010051 A1, and WO 2010/078966 A1; U.S. Patent Application Publication Nos.
  • the 4-1BB agonist is a 4-1BB agonistic fusion protein as depicted in Structure I-A (C-terminal Fc-antibody fragment fusion protein) or Structure I-B (N-terminal Fc- antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biosimilar thereof (see, Figure 7).
  • the cylinders refer to individual polypeptide binding domains.
  • Structures I-A and I-B comprise three linearly-linked TNFRSF binding domains derived from e.g., 4-1BBL (4-1BB ligand, CD137 ligand (CD137L), or tumor necrosis factor superfamily member 9 (TNFSF9)) or an antibody that binds 4-1BB, which fold to form a trivalent protein, which is then linked to a second triavelent protein through IgG1-Fc (including CH3 and CH2 domains) is then used to link two of the trivalent proteins together through disulfide bonds (small elongated ovals), stabilizing the structure and providing an agonists capable of bringing together the intracellular 125 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO signaling domains of the six receptors and signaling proteins to form a signaling complex.
  • 4-1BBL 4-1BB ligand, CD137 ligand (CD137L), or tumor nec
  • the TNFRSF binding domains denoted as cylinders may be scFv domains comprising, e.g., a VH and a VL chain connected by a linker that may comprise hydrophilic residues and Gly and Ser sequences for flexibility, as well as Glu and Lys for solubility.
  • Any scFv domain design may be used, such as those described in de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad, et al., Clin. & Dev. Immunol. 2012, 980250; Monnier, et al., Antibodies, 2013, 2, 193-208; or in references incorporated elsewhere herein. Fusion protein structures of this form are described in U.S.
  • the Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO:62) the complete hinge domain (amino acids 1-16 of SEQ ID NO:62) or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO:62).
  • Preferred linkers for connecting a C-terminal Fc-antibody may be selected from the embodiments given in SEQ ID NO:63 to SEQ ID NO:72, including linkers suitable for fusion of additional polypeptides. TABLE 8. Amino acid sequences for TNFRSF agonist fusion proteins, including 4-1BB agonist fusion proteins, with C-terminal Fc-antibody fragment fusion protein design (structure I-A).
  • Amino acid sequences for TNFRSF agonist fusion proteins including 4-1BB agonist fusion proteins, with N-terminal Fc-antibody fragment fusion protein design (structure I-B).
  • Identifier Sequence One-Letter Amino Acid Symbols
  • a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a 4-1BBL sequence. In some embodiments, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO:77. In some embodiments, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a soluble 4-1BBL sequence.
  • a 4- 1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO:78. 127 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO [00720]
  • a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:44 and SEQ ID NO:45, respectively, wherein the VH and VL domains are connected by a linker.
  • a 4- 1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:54 and SEQ ID NO:55, respectively, wherein the VH and VL domains are connected by a linker.
  • a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the VH and VL sequences given in Table 10, wherein the VH and VL domains are connected by a linker. TABLE 10. Additional polypeptide domains useful as 4-1BB binding domains in fusion proteins or as scFv 4-1BB agonist antibodies.
  • the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising an additional domain at the N-terminal and/or C-terminal end, wherein the additional domain is a Fab or Fc fragment domain, wherein each of the soluble 4-1BB domains lacks a stalk region (which contributes to trimerization and provides a certain distance to the cell membrane, but is not part of the 4-1BB binding domain) and the first and the second peptide linkers independently have a length of 3-8 amino acids.
  • the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stalk region and the first and the second peptide linkers independently have a length of 3-8 amino acids, and wherein each TNF superfamily cytokine domain is a 4-1BB binding domain.
  • TNF tumor necrosis factor
  • the 4-1BB agonist is a 4-1BB agonistic scFv antibody comprising any of the foregoing VH domains linked to any of the foregoing VL domains.
  • the 4-1BB agonist is BPS Bioscience 4-1BB agonist antibody catalog no.79097-2, commercially available from BPS Bioscience, San Diego, CA, USA.
  • the 4-1BB agonist is Creative Biolabs 4-1BB agonist antibody catalog no. MOM- 18179, commercially available from Creative Biolabs, Shirley, NY, USA. 3.
  • the TNFRSF agonist is an OX40 (CD134) agonist.
  • the OX40 agonist may be any OX40 binding molecule known in the art.
  • the OX40 binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX40.
  • the OX40 agonists or OX40 binding molecules may comprise an immunoglobulin heavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or 129 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO subclass of immunoglobulin molecule.
  • the OX40 agonist or OX40 binding molecule may have both a heavy and a light chain.
  • binding molecule also includes antibodies (including full length antibodies), monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human, humanized or chimeric antibodies, and antibody fragments, e.g., Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the above, and engineered forms of antibodies, e.g., scFv molecules, that bind to OX40.
  • the OX40 agonist is an antigen binding protein that is a fully human antibody.
  • the OX40 agonist is an antigen binding protein that is a humanized antibody.
  • OX40 agonists for use in the presently disclosed methods and compositions include anti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40 antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40 antibodies, anti-OX40 adnectins, anti-OX40 domain antibodies, single chain anti-OX40 fragments, heavy chain anti-OX40 fragments, light chain anti-OX40 fragments, anti-OX40 fusion proteins, and fragments, derivatives, conjugates, variants, or biosimilars thereof.
  • the OX40 agonist is an agonistic, anti-OX40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).
  • the OX40 agonist or OX40 binding molecule may also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun, et al., J. Immunother.2009, 182, 1481-89.
  • a multimeric OX40 agonist such as a trimeric or hexameric OX40 agonist (with three or six ligand binding domains) may induce superior receptor (OX40L) clustering and internal cellular signaling complex formation compared to an agonistic monoclonal antibody, which typically possesses two ligand binding domains.
  • Trimeric (trivalent) or hexameric (or hexavalent) or greater fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linking two or more of these fusion proteins are described, e.g., in Gieffers, et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.
  • the OX40 agonist is a monoclonal antibody or fusion protein that binds specifically to OX40 antigen in a manner sufficient to reduce toxicity.
  • the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that abrogates antibody-dependent cellular toxicity (ADCC), for example NK cell cytotoxicity.
  • the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that abrogates antibody-dependent cell phagocytosis (ADCP).
  • the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that 130 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO abrogates complement-dependent cytotoxicity (CDC).
  • the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein which abrogates Fc region functionality.
  • the OX40 agonists are characterized by binding to human OX40 (SEQ ID NO:85) with high affinity and agonistic activity.
  • the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO:85).
  • the OX40 agonist is a binding molecule that binds to murine OX40 (SEQ ID NO:86).
  • SEQ ID NO:86 The amino acid sequences of OX40 antigen to which an OX40 agonist or binding molecule binds are summarized in Table 11. TABLE 11. Amino acid sequences of OX40 antigens.
  • n s uman or murine OX40 with a KD of about 90 pM or lower binds human or murine OX40 with a KD of about 80 pM or lower, binds human or murine OX40 with a KD of about 70 pM or lower, binds human or murine OX40 with a KD of about 60 pM or lower, binds human or murine OX40 with a KD of about 50 pM or lower, binds human or murine OX40 with a KD of about 40 pM or lower, or binds human or murine OX40 with a KD of about 30 pM or lower.
  • compositions, processes and methods described include a OX40 agonist that binds to human or murine OX40 with a kassoc of about 7.5 ⁇ 10 5 1/M ⁇ s or faster, binds to human or murine OX40 with a kassoc of about 7.5 ⁇ 10 5 1/M ⁇ s or faster, binds to human or murine OX40 with a kassoc of about 8 ⁇ 10 5 1/M ⁇ s or faster, binds to human or murine OX40 with a kassoc of about 8.5 ⁇ 10 5 1/M ⁇ s or faster, binds to human or murine OX40 with a kassoc of about 9 ⁇ 10 5 1/M ⁇ s or faster, binds to human or murine OX40 with a kassoc of about 9.5 ⁇ 10 5 1/M ⁇ s or faster, or binds to human or murine OX40 with a kassoc of about
  • compositions, processes and methods described include a OX40 agonist that binds to human or murine OX40 with a kdissoc of about 2 ⁇ 10 -5 1/s or slower, binds to human or murine OX40 with a kdissoc of about 2.1 ⁇ 10 -5 1/s or slower , binds to human or murine OX40 with a kdissoc of about 2.2 ⁇ 10 -5 1/s or slower, binds to human or murine OX40 with a kdissoc of about 2.3 ⁇ 10 -5 1/s or slower, binds to human or murine OX40 with a kdissoc of about 2.4 ⁇ 10 -5 1/s or slower, binds to human or murine OX40 with a kdissoc of any of 2.4 ⁇ 10 -5 1/s or slower, binds to human or murine OX40 with a kdissoc
  • compositions, processes and methods described include OX40 agonist that binds to human or murine OX40 with an IC50 of about 10 nM or lower, binds to human or murine OX40 with an IC50 of about 9 nM or lower, binds to human or murine OX40 with an IC50 of about 8 nM or lower, binds to human or murine OX40 with an IC50 of about 7 nM or lower, binds to human or murine OX40 with an IC50 of about 6 nM or lower, binds to human or murine OX40 with an IC50 of about 5 nM or lower, binds to human or murine OX40 with an IC50 of about 4 nM or lower, binds to human or murine OX40 with an IC50 of about 3 nM or lower, binds to human or murine OX40 with an IC50 of about 2 nM or lower, or binds to
  • the OX40 agonist is tavolixizumab, also known as MEDI0562 or MEDI-0562.
  • Tavolixizumab is available from the MedImmune subsidiary of AstraZeneca, Inc.
  • Tavolixizumab is immunoglobulin G1-kappa, anti-[Homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)], humanized and chimeric monoclonal antibody.
  • TNFRSF4 tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134
  • Tavolixizumab comprises N-glycosylation sites at positions 301 and 301’’, with fucosylated complex bi-antennary CHO-type glycans; heavy chain intrachain disulfide bridges at positions 22-95 (VH-VL), 148-204 (CH1-CL), 265-325 (CH2) and 371-429 (CH3) (and at positions 22’’-95’’, 148’’-204’’, 265’’-325’’, and 371’’-429’’); light chain intrachain disulfide bridges at positions 23’-88’ (VH-VL) and 134’-194’ (CH1-CL) (and at positions 23’’’-88’’’’ and 134’’’-194’’’); interchain heavy chain-heavy chain disulfide bridges at positions 230-230’’ and 233-233’’; and interchain heavy chain-light chain disulfide bridges at 224-214’ and 224’’-214’
  • a OX40 agonist comprises a heavy chain given by SEQ ID NO:87 and a light chain given by SEQ ID NO:88.
  • a OX40 agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
  • a OX40 agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively.
  • a OX40 agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively.
  • a OX40 agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, a OX40 agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. In some embodiments, a OX40 agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:87 and SEQ ID NO:88, respectively. [00735] In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of tavolixizumab.
  • VRs variable regions
  • the OX40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:89
  • the OX40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:90, and conservative amino acid substitutions thereof.
  • a OX40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively.
  • a OX40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively.
  • a OX40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively.
  • an OX40 agonist comprises an scFv antibody comprising VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90.
  • a OX40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, respectively, and conservative amino acid substitutions thereof.
  • the OX40 agonist is a OX40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to tavolixizumab.
  • the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is tavolixizumab.
  • the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
  • the biosimilar is a OX40 agonist antibody authorized or submitted for authorization, wherein the OX40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is tavolixizumab.
  • the OX40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is tavolixizumab.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is tavolixizumab. TABLE 12. Amino acid sequences for OX40 agonist antibodies related to tavolixizumab.
  • a OX40 agonist comprises a heavy chain given by SEQ ID NO:97 and a light chain given by SEQ ID NO:98.
  • a OX40 agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
  • a OX40 agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, a OX40 agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, a OX40 agonist comprises heavy and light chains that are each at least 97% 135 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively.
  • a OX40 agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. In some embodiments, a OX40 agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively. [00740] In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4.
  • the OX40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:99
  • the OX40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:100, and conservative amino acid substitutions thereof.
  • a OX40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively.
  • a OX40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively.
  • a OX40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively.
  • a OX40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:10 6 , respectively, and conservative amino acid substitutions thereof.
  • the OX40 agonist is a OX40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to 11D4.
  • the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 11D4.
  • the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
  • the biosimilar is a OX40 agonist antibody authorized or submitted for authorization, wherein the OX40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or 136 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO reference biological product, wherein the reference medicinal product or reference biological product is 11D4.
  • the OX40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 11D4.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 11D4. TABLE 13. Amino acid sequences for OX40 agonist antibodies related to 11D4.
  • a OX40 agonist comprises a heavy chain given by SEQ ID NO:10 7 and a light chain given by SEQ ID NO:10 8 .
  • a OX40 agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:10 7 and SEQ ID NO:10 8 , respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
  • a OX40 agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:10 7 and SEQ ID NO:10 8 , respectively.
  • a OX40 agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:10 7 and SEQ ID NO:10 8 , respectively.
  • a OX40 agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO:10 7 and SEQ ID NO:10 8 , respectively. In some embodiments, a OX40 agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:10 7 and SEQ ID NO:10 8 , respectively. In some embodiments, a OX40 agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:10 7 and SEQ ID NO:10 8 , respectively. [00745] In some embodiments, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 18D8.
  • VRs variable regions
  • the OX40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:10 9
  • the OX40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:110, and conservative amino acid substitutions thereof.
  • a OX40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:10 9 and SEQ ID NO:110, respectively.
  • a OX40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:10 9 and SEQ ID NO:110, respectively.
  • a OX40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO:10 9 and SEQ ID NO:110, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 96% identical to 138 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO the sequences shown in SEQ ID NO:10 9 and SEQ ID NO:110, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:10 9 and SEQ ID NO:110, respectively.
  • a OX40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116, respectively, and conservative amino acid substitutions thereof.
  • the OX40 agonist is a OX40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to 18D8.
  • the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 18D8.
  • the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
  • the biosimilar is a OX40 agonist antibody authorized or submitted for authorization, wherein the OX40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 18D8.
  • the OX40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 18D8.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 18D8.
  • the reference medicinal product or reference biological product is 18D8.
  • the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu119-122.
  • the OX40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:117
  • the OX40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:118, and conservative amino acid substitutions thereof.
  • a OX40 agonist comprises VH and VL regions that are 140 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO each at least 99% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively.
  • a OX40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:117 and SEQ ID NO:118, respectively.
  • a OX40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:122, SEQ ID NO:123, and SEQ ID NO:124, respectively, and conservative amino acid substitutions thereof.
  • the OX40 agonist is a OX40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to Hu119-122.
  • the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hu119-122.
  • the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
  • the biosimilar is a OX40 agonist antibody authorized or submitted for authorization, wherein the OX40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hu119-122.
  • the OX40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hu119-122.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or 141 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO reference biological product, wherein the reference medicinal product or reference biological product is Hu119-122.
  • TABLE 15 Amino acid sequences for OX40 agonist antibodies related to Hu119-122. Identifier Sequence (One-Letter Amino Acid Symbols) ava ab e rom G axoSm t K ne p c.
  • Hu106-222 T e preparat on and propert es o Hu106-222 are descr bed n U.S. Patent Nos.9,006,399 and 9,163,085, and in International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated by reference herein.
  • the amino acid sequences of Hu106-222 are set forth in Table 16. [00753]
  • the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu106-222.
  • the OX40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:125
  • the OX40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:126, and conservative amino acid substitutions thereof.
  • a OX40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively.
  • a OX40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively.
  • a OX40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. In some embodiments, a OX40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively.
  • a OX40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:127, SEQ ID NO:128, and SEQ ID NO:129, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:132, respectively, and conservative amino acid substitutions thereof.
  • the OX40 agonist is a OX40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to Hu106-222.
  • the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hu106-222.
  • the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
  • the biosimilar is a OX40 agonist antibody authorized or submitted for authorization, wherein the OX40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hu106-222.
  • the OX40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hu106-222.
  • the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hu106-222. 143 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO TABLE 16.
  • Identifier Sequence One-Letter Amino Acid Symbols
  • the OX40 agonist is an antibody produced by the 9B12 hybridoma, deposited with Biovest Inc. (Malvern, MA, USA), as described in Weinberg, et al., J. Immunother.2006, 29, 575- 585, the disclosure of which is hereby incorporated by reference in its entirety.
  • the antibody comprises the CDR sequences of MEDI6469.
  • the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of MEDI6469.
  • the OX40 agonist is L106 BD (Pharmingen Product #340420).
  • the OX40 agonist comprises the CDRs of antibody L106 (BD Pharmingen Product #340420). In some embodiments, the OX40 agonist comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody L106 (BD Pharmingen Product #340420). In some embodiments, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, Catalog #20073). In some embodiments, the OX40 agonist comprises the CDRs of antibody ACT35 (Santa Cruz Biotechnology, Catalog #20073).
  • the OX40 agonist comprises a heavy 144 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO chain variable region sequence and/or a light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, Catalog #20073).
  • the OX40 agonist is the murine monoclonal antibody anti-mCD134/mOX40 (clone OX86), commercially available from InVivoMAb, BioXcell Inc, West Riverside, NH. [00758]
  • the OX40 agonist is selected from the OX40 agonists described in International Patent Application Publication Nos.
  • the OX40 agonist is an OX40 agonistic fusion protein as depicted in Structure I-A (C-terminal Fc-antibody fragment fusion protein) or Structure I-B (N-terminal Fc- antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biosimilar thereof.
  • Structure I-A and I-B are described above and in U.S. Patent Nos.9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated by reference herein. Amino acid sequences for the polypeptide domains of structure I-A given in Figure 7 are found in Table 9.
  • the Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO:62) the complete hinge domain (amino acids 1-16 of SEQ ID NO:62) or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO:62).
  • Preferred linkers for connecting a C- terminal Fc-antibody may be selected from the embodiments given in SEQ ID NO:63 to SEQ ID NO:72, including linkers suitable for fusion of additional polypeptides.
  • amino acid sequences for the polypeptide domains of structure I-B given in Figure 7 are found in Table 10.
  • the sequence of the Fc module is preferably that shown in SEQ ID NO:73, and the linker sequences are preferably selected from those embodiments set forth in SED ID NO:74 to SEQ ID NO:76.
  • an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains selected from the group consisting of a variable heavy chain and variable light chain of tavolixizumab, a variable heavy chain and variable light chain of 11D4, a variable heavy chain and variable light chain of 18D8, a variable heavy chain and variable light chain of Hu119-122, a variable heavy chain and variable light chain of Hu106-222, a variable heavy chain and variable light chain selected from the variable heavy chains and variable light chains described in Table 17, any combination of a variable heavy chain and variable light chain of the foregoing, and fragments, derivatives, conjugates, variants, and biosimilars thereof.
  • an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains comprising an OX40L sequence. In some embodiments, an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains comprising a sequence according to SEQ ID NO:133. In some embodiments, an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains comprising a soluble OX40L sequence.
  • a OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains comprising a sequence according to SEQ ID NO:134. In some embodiments, a OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains comprising a sequence according to SEQ ID NO:135.
  • an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:89 and SEQ ID NO:90, respectively, wherein the VH and VL domains are connected by a linker.
  • an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively, wherein the VH and VL domains are connected by a linker.
  • an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:10 9 and SEQ ID NO:110, respectively, wherein the VH and VL domains are connected by a linker.
  • an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:127 and SEQ ID NO:128, respectively, wherein the VH and VL domains are connected by a linker.
  • an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:125 and SEQ ID NO:126, respectively, wherein the VH and VL domains are connected by a linker.
  • an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more OX40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the VH and VL sequences given in Table 17, wherein the VH and VL domains are connected by a linker.
  • Identifier Sequence (One-Letter Amino Acid Symbols) 147 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO SEQ ID NO:147 DIVMTQSHKF MSTSLGDRVS ITCKASQDVG AAVAWYQQKP GQSPKLLIYW ASTRHTGVPD 60 light chain variable region RFTGGGSGTD FTLTISNVQS EDLTDYFCQQ YINYPLTFGG GTKLEIKR 108 polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain, further comprising an additional domain at the N-terminal and/or C-terminal end, and wherein the additional domain is a Fab or Fc fragment domain.
  • the OX40 agonist is a OX40 agonistic single-chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain, further comprising an additional domain at the N-terminal and/or C-terminal end, wherein the additional domain is a Fab or Fc fragment 148 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO domain wherein each of the soluble OX40 binding domains lacks a stalk region (which contributes to trimerisation and provides a certain distance to the cell membrane, but is not part of the OX40 binding domain) and the first and the second peptide linkers independently have a length of 3-8 amino acids.
  • the OX40 agonist is an OX40 agonistic single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stalk region and the first and the second peptide linkers independently have a length of 3-8 amino acids, and wherein the TNF superfamily cytokine domain is an OX40 binding domain.
  • TNF tumor necrosis factor
  • the OX40 agonist is MEDI6383.
  • MEDI6383 is an OX40 agonistic fusion protein and can be prepared as described in U.S. Patent No.6,312,700, the disclosure of which is incorporated by reference herein.
  • the OX40 agonist is an OX40 agonistic scFv antibody comprising any of the foregoing VH domains linked to any of the foregoing VL domains.
  • the OX40 agonist is Creative Biolabs OX40 agonist monoclonal antibody MOM-18455, commercially available from Creative Biolabs, Inc., Shirley, NY, USA.
  • the OX40 agonist is OX40 agonistic antibody clone Ber-ACT35 commercially available from BioLegend, Inc., San Diego, CA, USA. 4.
  • Myc Fusion Proteins [00769]
  • the cell culture medium of the first expansion and/or the second expansion comprises a Myc fusion protein.
  • Myc fusion proteins are described in US Patent No. 11,369,678, the content of which is incorporated by reference in its entirety.
  • a MYC fusion protein comprising (a) a transporter peptide sequence; and (b) a MYC sequence.
  • the MYC fusion protein is a peptide of Formula (I): transporter pep ⁇ de sequence —MYC sequence.
  • a MYC fusion protein disclosed herein comprises (a) a transporter peptide sequence; (b) a MYC sequence; and (c) one or more molecules that link the transporter 149 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO peptide sequence and the MYC sequence.
  • the MYC fusion protein is a peptide of Formula (II): transporter pep ⁇ de sequence—X—MYC sequence, wherein —X— is molecule that links the transporter pep ⁇ de sequence and the MYC sequence.
  • —X— is an amino acid.
  • —X— is at least one amino acid.
  • a “transporter peptide” means a peptide sequence that promotes peptide penetration into cells and tissues.
  • a transporter peptide is the N-terminal 9- amino-acid sequence of the TAT protein transduction domain of HIV-1 (RKKRRQRRR) (SEQ ID NO: 306).
  • a transporter peptide is TAT[48-57] . In some embodiments, a transporter peptide is TAT [57-48] .
  • a “MYC sequence” is a MYC amino acid peptide sequence. In some embodiments, the Myc polypeptide is a complete Myc polypeptide sequence. In some embodiments, the Myc polypeptide is a partial Myc polypeptide sequence. In some embodiments, the MYC is c- MYC.
  • the Myc polypeptide sequence comprises SEQ ID NO:300: MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEE NFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVA VTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDD ETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARG HSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSS AFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEID VVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHN YAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEEN VKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKV
  • a MYC fusion protein disclosed herein comprises (a) TAT, and (b) c-MYC. In some embodiments, a MYC fusion protein disclosed herein comprises (a) TAT [48-57] , and (b) c-MYC. In some embodiments, a MYC fusion protein disclosed herein comprises (a) TAT[57- 4 8] , and (b) c-MYC.
  • a MYC fusion protein disclosed herein comprises (a) TAT, (b) a linker amino acid, and (c) c-MYC.
  • a MYC fusion protein disclosed herein comprises (a) TAT [48-57] , (b) a linker amino acid, and (c) c-MYC.
  • a MYC fusion protein disclosed herein comprises (a) TAT [57-48] , (b) a linker amino acid, and (c) c-MYC.
  • a MYC fusion protein disclosed herein further comprises at least one amino acid sequence that facilitates purification of the fusion protein.
  • a MYC fusion protein disclosed herein comprises a protein tag.
  • a MYC fusion protein disclosed herein comprises a polyhistidine tag.
  • a MYC fusion protein disclosed herein comprises an epitope tag.
  • a MYC fusion protein disclosed herein comprises a polyhistidine tag and an epitope tag.
  • a MYC fusion protein disclosed herein comprises a 6-histidine tag and a V5 epitope tag.
  • the histidine tag is a 6-histidine tag.
  • the histidine tag comprises the sequence HHHHHH (SEQ ID NO:302).
  • a histidine tag is added to a MYC fusion protein disclosed herein by any suitable method.
  • a TAT-Myc polypeptide sequence is cloned into an expression vector encoding a polyHis-tag.
  • a polyhistidine tag is added by PCR (i.e., the PCR primers comprise a polyhistidine sequence).
  • a MYC fusion protein disclosed herein further comprises at least one protein tag.
  • a MYC fusion protein disclosed herein comprises an epitope tag. In some embodiments, a MYC fusion protein disclosed herein further comprises a V5 epitope tag. In some embodiments, the V5 tag comprises the amino acids: GKPIPNPLLGLDST (SEQ ID NO:303). In some embodiments, the V5 tag comprises the amino acids: IPNPLLGLD (SEQ ID NO:304). In some embodiments, a V5 tag is added to a MYC fusion protein disclosed herein by any suitable method. In some embodiments, a MYC fusion protein sequence is cloned into an expression vector encoding a V5 tag.
  • a V5 tag is added by PCR (i.e., the PCR primers comprise a V5 sequence).
  • the amino acids are in the D formation.
  • the amino acids are in the L formation.
  • a first plurality of amino acids is in the D formation and a second plurality is in the L formation.
  • a MYC fusion protein comprises SEQ ID NO:301: MRKKRRQRRRMDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSV QPYFYCDEEENFYQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRR SGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMV 151 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO NQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARK DSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSS PKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSE EEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKR CHVSTHQHNYAAPPSTRKDYPAAKRV
  • a cell viability assay can be performed after the priming first expansion (sometimes referred to as the initial bulk expansion), using standard assays known in the art.
  • the method comprises performing a cell viability assay subsequent to the priming first expansion.
  • a trypan blue exclusion assay can be done on a sample of the bulk TILs, which selectively labels dead cells and allows a viability assessment.
  • Other assays for use in testing viability can include but are not limited to the Alamar blue assay; and the MTT assay. 1.
  • Cell Counts, Viability, Flow Cytometry [00782] In some embodiments, cell counts and/or viability are measured.
  • markers such as but not limited CD3, CD4, CD8, and CD56 can be measured by flow cytometry with antibodies, for example but not limited to those commercially available from BD Bio-sciences (BD Biosciences, San Jose, CA) using a FACSCantoTM flow cytometer (BD Biosciences).
  • the cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL) and viability can be assessed using any method known in the art, including but not limited to trypan blue staining.
  • the cell viability can also be assayed based on U.S. Patent Application Publication No.2018/0282694, incorporated by reference herein in its entirety.
  • the bulk TIL population can be cryopreserved immediately, using the protocols discussed below.
  • the bulk TIL population can be subjected to REP and then cryopreserved as discussed below.
  • the bulk or REP TIL populations can be subjected to genetic modifications for suitable treatments. 152 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO 2.
  • a method for expanding TILs may include using about 5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cell medium.
  • the media is a serum free medium.
  • the media in the priming first expansion is serum free.
  • the media in the second expansion is serum free.
  • the media in the priming first expansion and the second expansion also referred to as rapid second expansion
  • expanding the number of TILs uses no more than one type of cell culture medium.
  • any suitable cell culture medium may be used, e.g., AIM-V cell medium (L-glutamine, 50 ⁇ M streptomycin sulfate, and 10 ⁇ M gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA).
  • AIM-V cell medium L-glutamine, 50 ⁇ M streptomycin sulfate, and 10 ⁇ M gentamicin sulfate cell culture medium (Invitrogen, Carlsbad CA).
  • expanding the number of TIL may comprise feeding the cells no more frequently than every third or fourth day. Expanding the number of cells in a gas permeable container simplifies the procedures necessary to expand the number of cells by reducing the feeding frequency necessary to expand the cells.
  • the cell culture medium in the first and/or second gas permeable container is unfiltered.
  • the use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells.
  • the cell medium in the first and/or second gas permeable container lacks beta-mercaptoethanol (BME).
  • the duration of the method comprising obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas permeable container containing cell medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3 for a duration of about 1 to 8 days, e.g., about 7 days as a priming first expansion, or about 8 days as a priming first expansion; transferring the TILs to a second gas permeable container and expanding the number of TILs in the second gas permeable container containing cell medium including IL-2, 2X antigen- presenting feeder cells, and OKT-3 for a duration of about 7 to 9 days, e.g., about 7 days, about 8 days, or about 9 days.
  • the duration of the method comprising obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas permeable container containing cell medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3 for a duration of about 1 to 7 days, e.g., about 7 days as a priming first expansion; transferring the TILs to a second gas permeable container and expanding the number of TILs in the second gas permeable container containing cell medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3 for a duration 153 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO of about 7 to 14 days, or about 7 to 9 days, e.g., about 7 days, about 8 days, or about 9 days, about 10 days, or about 11 days.
  • the duration of the method comprising obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas permeable container containing cell medium including IL-2, 1X antigen-presenting feeder cells, and OKT-3 for a duration of about 1 to 7 days, e.g., about 7 days, as a priming first expansion; transferring the TILs to a second gas permeable container and expanding the number of TILs in the second gas permeable container containing cell medium including IL-2, 2X antigen-presenting feeder cells, and OKT-3 for a duration of about 7 to 11 days, e.g., about 7 days, about 8 days, about 9 days, about 10, or about 11 days.
  • TILs are expanded in gas-permeable containers.
  • Gas-permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No.2005/0106717 A1, the disclosures of which are incorporated herein by reference.
  • TILs are expanded in gas-permeable bags.
  • TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare).
  • TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare).
  • the cell expansion system includes a gas permeable cell bag with a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.
  • TILs can be expanded in G-REX flasks (commercially available from Wilson Wolf Manufacturing). Such embodiments allow for cell populations to expand from about 5 ⁇ 105 cells/cm 2 to between 10 ⁇ 10 6 and 30 ⁇ 10 6 cells/cm 2 . In some embodiments this is without feeding. In some embodiments, this is without feeding so long as medium resides at a height of about 10 cm in the G-REX flask. In some embodiments this is without feeding but with the addition of one or more cytokines. In some embodiments, the cytokine can be added as a bolus without any need to mix the cytokine with the medium.
  • Such containers, devices, and methods are known in the art and have been used to expand TILs, and include those described in U.S. Patent Application Publication No. US 2014/0377739A1, International Publication No. WO 2014/210036 A1, U.S. Patent Application Publication No. us 2013/0115617 A1, International Publication No. WO 2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228 A1, U.S. Patent No. US 8,809,050 B2, International publication No. WO 2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216 A1, U.S. Patent Application Publication No.
  • closed systems allow for preventing and/or reducing microbial contamination, allow for the use of fewer flasks, and allow for cost reductions.
  • 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.
  • STCDs Sterile connecting devices
  • the closed systems include luer lock and heat-sealed systems as described in the Examples.
  • the closed system is accessed via syringes under sterile conditions in order to maintain the sterility and closed nature of the system.
  • a closed system as described in the examples is employed.
  • the TILs are formulated into a final product formulation container according to the methods described herein in the examples. [00794] In some embodiments, the closed system uses one container from the time the tumor fragments are obtained until the TILs are ready for administration to the patient or cryopreserving.
  • the first container when two containers are used, the first container is a closed G-container and the population of TILs is centrifuged and transferred to an infusion bag without opening the first closed G-container.
  • the infusion bag when two containers are used, the infusion bag is a HypoThermosol-containing infusion bag.
  • a closed system or closed TIL cell culture system is characterized in that once the tumor sample and/or tumor fragments have been added, the system is tightly sealed from the outside to form a closed environment free from the invasion of bacteria, fungi, and/or any other microbial contamination. [00795] In some embodiments, the reduction in microbial contamination is between about 5% and about 100%.
  • the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% 155 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%.
  • the reduction in microbial contamination is 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 for TIL growth in the absence and/or with a significant reduction in microbial contamination.
  • pH, carbon dioxide partial pressure and oxygen partial pressure of the TIL cell culture environment each vary as the cells are cultured.
  • the closed environment still needs to be constantly maintained as an optimal environment for TIL proliferation.
  • the physical factors of pH, carbon dioxide partial pressure and oxygen partial pressure within the culture liquid of the closed environment be monitored by means of a sensor, the signal whereof is used to control a gas exchanger installed at the inlet of the culture environment, and the that gas partial pressure of the closed environment be adjusted in real time according to changes in the culture liquid so as to optimize the cell culture environment.
  • the present invention provides a closed cell culture system which incorporates at the inlet to the closed environment a gas exchanger equipped with a monitoring device which measures the pH, carbon dioxide partial pressure and oxygen partial pressure of the closed environment, and optimizes the cell culture environment by automatically adjusting gas concentrations based on signals from the monitoring device.
  • the pressure within the closed environment is continuously or intermittently controlled. That is, the pressure in the closed environment can be varied by means of a pressure maintenance device for example, thus ensuring that the space is suitable for growth of TILs in a positive pressure state, or promoting exudation of fluid in a negative pressure state and thus promoting cell proliferation.
  • the expanded TILs of the present invention are further manipulated before, during, or after an expansion step, including during closed, sterile manufacturing processes, each as provided herein, in order to alter protein expression.
  • the altered protein expression is due to gene editing.
  • the expanded TILs of the present invention are transduced with vectors and/or other molecules capable of altering protein expression in the TILs.
  • the TFs and/or other molecules that are capable of transiently altering protein expression provide for altered expression of tumor antigens and/or an alteration in the number of tumor antigen-specific T cells in a population of TILs.
  • the present invention includes genetic editing through nucleotide insertion, such as through ribonucleic acid (RNA) insertion, including insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA), into a population of TILs for promotion of the expression of one or more proteins or inhibition of the expression of one or more proteins, as well as simultaneous combinations of both promotion of one set of proteins with inhibition of another set of proteins.
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • siRNA small (or short) interfering RNA
  • the expanded TILs of the present invention undergo transient alteration of protein expression.
  • the transient alteration of protein expression occurs in the bulk TIL population prior to first expansion, including for example in the TIL population obtained from for example, Step A.
  • the transient alteration of protein expression occurs during the first expansion, including for example in the TIL population expanded in for example, Step B. In some embodiments, the transient alteration of protein expression occurs after the first expansion, including for example in the TIL population in transition between the first and second expansion, the TIL population obtained from for example, Step B and included in Step C. In some embodiments, the transient alteration of protein expression occurs in the bulk TIL population prior to second expansion, including for example in the TIL population obtained from for example, Step C and prior to its expansion in Step D. In some embodiments, the transient alteration of protein expression occurs during the second expansion, including for example in the TIL population expanded in for example, Step D.
  • the transient alteration of protein expression occurs after the second expansion, including for example in the TIL population obtained from the expansion in for example, Step D.
  • a method of transiently altering protein expression in a population of TILs includes the step of electroporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. J.1991, 60, 297-306, and U.S. Patent Application Publication No. 2014/0227237 A1, the disclosures of each of which are incorporated by reference herein.
  • a method of transiently altering protein expression in population of TILs includes the step of calcium phosphate transfection.
  • Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci.1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol.1987, 7, 2745-2752; and in U.S. Patent No.
  • a method of transiently altering protein expression in a population of TILs includes the step of liposomal transfection.
  • Liposomal transfection methods such as methods that employ a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n- trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc.
  • DOTMA dioleoyl phophotidylethanolamine
  • a method of transiently altering protein expression in a population of TILs includes the step of transfection using methods described in U.S. Patent Nos.5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.
  • TSCMs Stem Memory T cells
  • TSCMs are early progenitors of antigen-experienced central memory T cells.
  • TSCMs generally display the long-term survival, self-renewal, and multipotency abilities that define stem cells, and are generally desirable for the generation of effective TIL products.
  • TSCM have shown enhanced anti-tumor activity compared with other T cell subsets in mouse models of adoptive cell transfer (Gattinoni et al. Nat Med 2009, 2011; Gattinoni, Nature Rev. Cancer, 2012; Cieri et al. Blood 2013).
  • transient alteration of protein expression results in a TIL population with a composition comprising a high proportion of TSCM.
  • transient alteration of protein expression results in an at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% increase in TSCM percentage.
  • transient alteration of protein expression results in an at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in TSCMs in the TIL population.
  • transient alteration of protein expression results in a TIL population with at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCMs.
  • transient alteration of protein expression results in a therapeutic TIL population with at 158 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCMs. [00805] In some embodiments, transient alteration of protein expression results in rejuvenation of antigen-experienced T-cells.
  • rejuvenation includes, for example, increased proliferation, increased T-cell activation, and/or increased antigen recognition.
  • transient alteration of protein expression alters the expression in a large fraction of the T-cells in order to preserve the tumor-derived TCR repertoire. In some embodiments, transient alteration of protein expression does not alter the tumor-derived TCR repertoire. In some embodiments, transient alteration of protein expression maintains the tumor- derived TCR repertoire. [00807] In some embodiments, transient alteration of protein results in altered expression of a particular gene.
  • the transient alteration of protein expression targets a gene including but not limited to PD-1 (also referred to as PDCD1 or CD279), TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGF ⁇ , CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1 ⁇ ), CCL4 (MIP1- ⁇ ), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and/or cAMP protein kinase A (PKA).
  • PD-1 also referred to as PDCD1 or CD279
  • TGFBR2 CCR4/5,
  • the transient alteration of protein expression targets a gene selected from the group consisting of PD-1, TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGF ⁇ , CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1 ⁇ ), CCL4 (MIP1- ⁇ ), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and/or cAMP protein kinase A (PKA).
  • PKA protein kinase A
  • the transient alteration of protein expression targets PD-1. In some embodiments, the transient alteration of protein expression targets TGFBR2. In some embodiments, the transient alteration of protein expression targets CCR4/5. In some embodiments, the transient alteration of protein expression targets CBLB. In some embodiments, the transient alteration of protein expression targets CISH. In some embodiments, the transient alteration of protein expression targets CCRs (chimeric co-stimulatory receptors). In some embodiments, the transient alteration of protein expression targets IL-2. In some embodiments, the transient alteration of protein expression targets IL-7. In some embodiments, the transient alteration of protein expression targets IL-10. In some embodiments, the transient alteration of protein expression targets IL-12.
  • the transient alteration of protein expression targets IL-15. In some embodiments, the transient alteration of protein expression targets IL-18. In some 159 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO embodiments, the transient alteration of protein expression targets IL-21. In some embodiments, the transient alteration of protein expression targets NOTCH 1/2 ICD. [00808] In some embodiments, the transient alteration of protein expression targets the NOTCH signaling pathway, such as through the NOTCH 1/2 ICD and/or through other NOTCH ligand, such as mDLL1 (see, for example Kondo, T.
  • the transient alteration of protein expression targets TIM3.
  • the transient alteration of protein expression targets LAG3.
  • the transient alteration of protein expression targets TIGIT.
  • the transient alteration of protein expression targets TGF ⁇ .
  • the transient alteration of protein expression targets CCR1.
  • the transient alteration of protein expression targets CCR2.
  • the transient alteration of protein expression targets CCR4.
  • the transient alteration of protein expression targets CCR5. In some embodiments, the transient alteration of protein expression targets CXCR1. In some embodiments, the transient alteration of protein expression targets CXCR2. In some embodiments, the transient alteration of protein expression targets CSCR3. In some embodiments, the transient alteration of protein expression targets CCL2 (MCP-1). In some embodiments, the transient alteration of protein expression targets CCL3 (MIP-1 ⁇ ). In some embodiments, the transient alteration of protein expression targets CCL4 (MIP1- ⁇ ). In some embodiments, the transient alteration of protein expression targets CCL5 (RANTES). In some embodiments, the transient alteration of protein expression targets CXCL1.
  • the transient alteration of protein expression targets CXCL8. In some embodiments, the transient alteration of protein expression targets CCL22. In some embodiments, the transient alteration of protein expression targets CCL17. In some embodiments, the transient alteration of protein expression targets VHL. In some embodiments, the transient alteration of protein expression targets CD44. In some embodiments, the transient alteration of protein expression targets PIK3CD. In some embodiments, the transient alteration of protein expression targets SOCS1. In some embodiments, the transient alteration of protein expression targets cAMP protein kinase A (PKA). [00810] In some embodiments, the transient alteration of protein expression results in increased and/or overexpression of a chemokine receptor.
  • PKA protein kinase A
  • the chemokine receptor that is overexpressed by transient protein expression includes a receptor with a ligand that includes but is not limited to CCL2 (MCP-1), CCL3 (MIP-1 ⁇ ), CCL4 (MIP1- ⁇ ), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17.
  • the chemokine receptor that is overexpressed by transient protein expression includes a receptor with a ligand that includes but is not limited to IL-2, IL-7, IL-10, IL-15, IL-18, and IL-21, and also NOTCH 1/2 intracellular domain (ICD).
  • the transient alteration of protein expression targets the NOTCH signaling pathway, such as through the NOTCH 1/2 ICD and/or through other NOTCH ligand, such as mDLL1 (see, for example Kondo, T. et al., NOTCH-mediated conversion of activated T cells into stem cell memory- like T cells for adoptive immunotherapy, Nature Communications, Vol.8, Article number: 15338 (2017), which is incorporated by reference herein in its entirety).
  • the NOTCH signaling pathway such as through the NOTCH 1/2 ICD and/or through other NOTCH ligand, such as mDLL1
  • the transient alteration of protein expression results in increased and/or overexpression of one or more binding partners that allow binding of exogenously administered cytokine carriers to the TILs via the one or more binding partners.
  • the transient alteration of protein expression results in a decrease and/or reduced expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGF ⁇ R2, and/or TGF ⁇ (including resulting in, for example, TGF ⁇ pathway blockade).
  • the transient alteration of protein expression results in a decrease and/or reduced expression of CBLB (CBL-B).
  • the transient alteration of protein expression results in a decrease and/or reduced expression of CISH.
  • the transient alteration of protein expression results in increased and/or overexpression of chemokine receptors in order to, for example, improve TIL trafficking or movement to the tumor site.
  • the transient alteration of protein expression results in increased and/or overexpression of a CCR (chimeric co-stimulatory receptor).
  • the transient alteration of protein expression results in increased and/or overexpression of a chemokine receptor selected from the group consisting of CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR3.
  • the transient alteration of protein expression results in increased and/or overexpression of an interleukin.
  • the transient alteration of protein expression results in increased and/or overexpression of an interleukin selected from the group consisting of IL-2, IL-12, IL-15, IL-18, and/or IL-21.
  • the transient alteration of protein expression targets the NOTCH signaling pathway, such as through the NOTCH 1/2 ICD and/or through other NOTCH ligand, such as mDLL1 (see, for example Kondo, T.
  • the transient alteration of protein expression results in increased and/or overexpression of NOTCH 1/2 ICD.
  • the transient alteration of protein expression results in increased and/or overexpression of a NOTCH ligand, such as mDLL1.
  • the transient alteration of protein expression results in increased and/or overexpression of VHL.
  • the transient alteration of protein expression results in increased and/or overexpression of CD44. In some embodiments, the transient alteration of protein expression results in increased and/or overexpression of PIK3CD. In some embodiments, the transient alteration of protein expression results in increased and/or overexpression of SOCS1, [00816] In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of cAMP protein kinase A (PKA).
  • PKA cAMP protein kinase A
  • the transient alteration of protein expression results in decreased and/or reduced expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • the transient alteration of protein expression results in decreased and/or reduced expression of two molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and one molecule selected from the group consisting of LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • the transient alteration of protein expression results in decreased and/or reduced expression of PD-1, LAG-3, CISH, CTLA-4, TIGIT, CBLB, TIM3, and combinations thereof.
  • the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and one of LAG3, CISH, CTLA-4, TIGIT, CBLB, TIM3, and combinations thereof.
  • the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and CTLA-4. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and TIGIT. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and LAG3. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and CISH. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and CBLB. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of CTLA-4 and TIGIT.
  • the transient alteration of protein expression results in decreased and/or reduced expression of CTLA-4 and LAG3. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of CTLA-4 and CISH. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of CTLA-4 and CBLB. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of CTLA-4 and TIM3. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of TIGIT and LAG3.
  • the transient alteration of protein expression 162 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO results in decreased and/or reduced expression of TIGIT and CISH.
  • the transient alteration of protein expression results in decreased and/or reduced expression of TIGIT and CBLB.
  • the transient alteration of protein expression results in decreased and/or reduced expression of TIGIT and TIM3.
  • the transient alteration of protein expression results in decreased and/or reduced expression of LAG3 and CISH.
  • the transient alteration of protein expression results in decreased and/or reduced expression of LAG3 and CBLB.
  • the transient alteration of protein expression results in decreased and/or reduced expression of CISH and CBLB. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of TIM3 and PD-1. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of TIM3 and LAG3. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of TIM3 and CISH. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of TIM3 and CBLB.
  • an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof, is inserted by a gammaretroviral or lentiviral method into the first population of TILs, second population of TILs, or harvested population of TILs (e.g., the expression of the adhesion molecule is increased).
  • the transient alteration of protein expression results in decreased and/or reduced expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof, and increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.
  • the transient alteration of protein expression results in decreased and/or reduced expression of a molecule selected from the group consisting of PD-1, CTLA-4, TIGIT, LAG3, TIM3, CISH, CBLB, and combinations thereof, and increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.
  • a reduction in expression of about 5%, about 10%, about 10%, 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%, or about 95%.
  • transient alteration of protein expression is induced by treatment of the TILs with transcription factors (TFs) and/or other molecules capable of transiently altering protein expression in the TILs.
  • the SQZ vector-free microfluidic platform is employed for intracellular delivery of the transcription factors (TFs) and/or other molecules capable of transiently altering protein expression.
  • WO 2013/059343A1, WO 2017/008063A1, or WO 2017/123663A1, or U.S. Patent Application Publication Nos. US 2014/0287509A1, US 2018/0201889A1, or US 2018/0245089A1 can be employed with the present invention in order to expose a population of TILs to transcription factors (TFs) and/or other molecules capable of inducing transient protein expression, wherein the TFs and/or other molecules capable of inducing transient protein expression provide for increased expression of tumor antigens and/or an 164 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO increase in the number of tumor antigen-specific T cells in the population of TILs, thus resulting in reprogramming of the TIL population and an increase in therapeutic efficacy of the reprogrammed TIL population as compared to a non-reprogrammed TIL population.
  • TFs transcription factors
  • 116983-5130-WO increase in the number of tumor anti
  • the reprogramming results in an increased subpopulation of effector T cells and/or central memory T cells relative to the starting or prior population (i.e., prior to reprogramming) population of TILs, as described herein.
  • the transcription factor (TF) includes but is not limited to TCF-1, NOTCH 1/2 ICD, and/or MYB.
  • the transcription factor (TF) is TCF-1.
  • the transcription factor (TF) is NOTCH 1/2 ICD.
  • the transcription factor (TF) is MYB.
  • the transcription factor (TF) is administered with induced pluripotent stem cell culture (iPSC), such as the commercially available KNOCKOUT Serum Replacement (Gibco/ThermoFisher), to induce additional TIL reprogramming.
  • iPSC induced pluripotent stem cell culture
  • the transcription factor (TF) is administered with an iPSC cocktail to induce additional TIL reprogramming.
  • the transcription factor (TF) is administered without an iPSC cocktail.
  • reprogramming results in an increase in the percentage of TSCMs.
  • reprogramming results in an increase in the percentage of TSCMs by about 5%, about 10%, about 10%, 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%, or about 95% TSCMs.
  • the invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: [00825] (i) obtaining a first population of TILs from a tumor resected from a patient by processing a sample from the resected tumor into multiple tumor fragments; [00826] (ii) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2, and optionally OKT-3, to produce a second population of TILs; [00827] (iii) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is a therapeutic population of TILs; and [00828] (iv) exposing the second and/or third population of TILs to transcription factors (
  • the tumor antigens are selected from, but are not limited to, IL-15, IL-21, IL-12 and IL-18.
  • the TFs alter expression of one or more of IL-15, IL-21, IL-12 and IL-18 so as to increase the expression thereof.
  • the exposure to the TFs and/or the molecules capable of altering protein expression is mediated by sterile electroporation or a SQZ microfluidic membrane disruption.
  • the TFs and/or the molecules capable of altering protein expression include, but are not limited to at least one short interfering RNA or one messenger RNA.
  • an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof is inserted into the first population of TILs, second population of TILs, or harvested population of TILs in addition to the TFs and/or the molecules capable of altering protein expression.
  • the adhesion molecule may be inserted by, for example, a retroviral (e.g., a gammaretroviral), or a lentiviral method.
  • a method of transient altering protein expression may be combined with a method of genetically modifying a population of TILs includes the step of stable incorporation of genes for production of one or more proteins.
  • a method of genetically modifying a population of TILs includes the step of retroviral transduction.
  • a method of genetically modifying a population of TILs includes the step of lentiviral transduction.
  • a method of genetically modifying a population of TILs includes the step of gamma-retroviral transduction.
  • a method of genetically modifying a population of TILs includes the step of transposon-mediated gene transfer.
  • Transposon-mediated gene transfer systems include systems wherein the transposase is provided as DNA expression vector or as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, a transposase provided as an mRNA (e.g., an mRNA comprising a cap 166 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO and poly-A tail).
  • mRNA e.g., an mRNA comprising a cap 166 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO and poly-A tail.
  • Suitable transposon-mediated gene transfer systems including the salmonid-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100x, and engineered enzymes with increased enzymatic activity, are described in, e.g., Bushett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No.6,489,458, the disclosures of each of which are incorporated by reference herein.
  • SB or Sleeping Beauty transposase such as SB10, SB11, and SB100x
  • transient alteration of protein expression is a reduction in expression induced by self-delivering RNA interference (sd-RNA), which is a chemically-synthesized asymmetric siRNA duplex with a high percentage of 2’-OH substitutions (typically fluorine or -OCH- 3) which comprises a 20-nucleotide antisense (guide) strand and a 13 to 15 base sense (passenger) strand conjugated to cholesterol at its 3’ end using a tetraethylenglycol (TEG) linker.
  • sd-RNA self-delivering RNA interference
  • delivery of sd-RNA to a TIL population is accomplished without use of electroporation, SQZ, or other methods, instead using a 1 to 3 day period in which a TIL population is exposed to sd-RNA at a concentration of 1 ⁇ M/10,000 TILs in medium.
  • delivery of sd-RNA to a TIL population is accomplished using a 1 to 3 day period in which a TIL population is exposed to sd-RNA at a concentration of 10 ⁇ M/10,000 TILs in medium. In some embodiments, delivery of sd-RNA to a TIL population is accomplished using a 1 to 3 day period in which a TIL population is exposed to sd- RNA at a concentration of 50 ⁇ M/10,000 TILs in medium.
  • delivery of sd-RNA to a TIL population is accomplished using a 1 to 3 day period in which a TIL population is exposed to sd-RNA at a concentration of between 0.1 ⁇ M/10,000 TILs and 50 ⁇ M/10,000 TILs in medium.
  • delivery of sd-RNA to a TIL population is accomplished using a 1 to 3 day period in which a TIL population is exposed to sd-RNA at a concentration of between 0.1 ⁇ M/10,000 TILs and 50 ⁇ M/10,000 TILs in medium, wherein the exposure to sd-RNA is performed two, three, four, or five times by addition of fresh sd-RNA to the media.
  • Other suitable processes are described, for example, in U.S.
  • the sd-RNA encodes RNA that interferes with NOTCH 1/2 ICD, NOTCH ligand mDLL1, PD-1, CTLA-4 TIM-3, LAG-3, TIGIT, TGF ⁇ , TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH, and/or CBLB.
  • PKA cAMP protein kinase A
  • the reduction in expression is determined based on a percentage of gene silencing, for example, as assessed by flow cytometry and/or qPCR. In some embodiments, there is a reduction in expression of about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, 167 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • sd-RNA Methods [00835]
  • the self-deliverable RNAi technology based on the chemical modification of siRNAs can be employed with the methods of the present invention to successfully deliver the sd-RNAs to the TILs as described herein.
  • the combination of backbone modifications with asymmetric siRNA structure and a hydrophobic ligand allow sd-RNAs to penetrate cultured mammalian cells without additional formulations and methods by simple addition to the culture media, capitalizing on the nuclease stability of sd-RNAs.
  • RNAi-mediated reduction of target gene activity simply by maintaining the active concentration of sd-RNA in the media.
  • backbone stabilization of sd-RNA provides for extended reduction in gene expression effects which can last for months in non-dividing cells.
  • over 95% transfection efficiency of TILs and a reduction in expression of the target by various specific sd-RNA occurs.
  • sd-RNAs containing several unmodified ribose residues were replaced with fully modified sequences to increase potency and/or the longevity of RNAi effect.
  • a reduction in expression effect is maintained for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or more. In some embodiments, the reduction in expression effect decreases at 10 days or more post sd-RNA treatment of the TILs. In some embodiments, more than 70% reduction in expression of the target expression is maintained. In some embodiments, more than 70% reduction in expression of the target expression is maintained TILs.
  • a reduction in expression in the PD- 1/PD-L1 pathway allows for the TILs to exhibit a more potent in vivo effect, which is in some 168 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO embodiments, due to the avoidance of the suppressive effects of the PD-1/PD-L1 pathway.
  • a reduction in expression of PD-1 by sd-RNA results in an increase TIL proliferation.
  • RNAi RNA interference
  • Double stranded DNA can be generally used to define any molecule comprising a pair of complementary strands of RNA, generally a sense (passenger) and antisense (guide) strands, and may include single-stranded overhang regions.
  • dsRNA contrasted with siRNA, generally refers to a precursor molecule that includes the sequence of an siRNA molecule which is released from the larger dsRNA molecule by the action of cleavage enzyme systems, including Dicer.
  • sd-RNA self-deliverable RNA
  • sd-RNA are a new class of covalently modified RNAi compounds that do not require a delivery vehicle to enter cells and have improved pharmacology compared to traditional siRNAs.
  • “Self-deliverable RNA” or “sd-RNA” is a hydrophobically modified RNA interfering-antisense hybrid, demonstrated to be highly efficacious in vitro in primary cells and in vivo upon local administration.
  • sd-RNAs are generally asymmetric chemically modified nucleic acid molecules with minimal double stranded regions.
  • sd-RNA molecules typically contain single stranded regions and double stranded regions, and can contain a variety of chemical modifications within both the single stranded and double stranded regions of the molecule.
  • the sd-RNA molecules can be attached to a hydrophobic conjugate such as a conventional and advanced sterol-type molecule, as described herein.
  • sd-RNAs (and/or RNAs capable of being employed in similar manners to sd-RNAs) and associated methods for making such sd-RNAs have also been described extensively in, for example, U.S.
  • Patent Publication No. US 2017/0369882 U.S. Patent No. US 8,501,706, U.S. Patent Publication No. US 2004/0224405, U.S. Patent No. US 8,252,755, U.S. Patent Publication No. US 2007/0031844, U.S. Patent Publication No. US 2007/0039072, U.S. Patent Publication No. US 2007/0207974, U.S. Patent Publication No. US 2007/0213520, U.S. Patent Publication No. US 2007/0213521, U.S. Patent Publication No. US 2007/0219362, U.S. Patent Publication No. US 2007/0238868, U.S. Patent Publication No. US 2014/0148362, U.S. Patent Publication No.
  • one or more sd-RNAs for use in the present invention can be generated from a linear double-stranded DNA template.
  • the linear double- stranded DNA template for generating the one or more sd-RNAs is one as described in U.S. Patent No.
  • a linear double-stranded DNA template obtained by polymerase chain reaction (PCR) and suitable for in vitro transcription of an mRNA comprises from 5' to 3': an RNA polymerase promoter on the coding strand of the double-stranded DNA, a 5' untranslated region less than 3,000 nucleotides in length and effective for translation of the mRNA into a detectable polypeptide after transfection into a eukaryotic cell, an open reading frame that encodes the polypeptide, wherein the polypeptide is heterologous to the cell to be transfected and wherein the polypeptide is selected from the group consisting of a ligand or a receptor of an immune cell, a polypeptide that stimulates or inhibits a function of the immune system, and a polypeptide that 170 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO inhibits the function of an onc
  • the RNA polymerase promoter comprises a consensus binding sequence for an RNA polymerase selected from the group consisting of T7, T3 or SP6 RNA polymerase.
  • the open reading frame encodes a fusion polypeptide.
  • the open reading frame encodes a polypeptide selected from the group consisting of PD-1, TGFBR2, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-12, IL-15, IL-18, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGF ⁇ , CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP- 1 ⁇ ), CCL4 (MIP1- ⁇ ), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP protein kinase A (PKA) , and combinations thereof.
  • PKA cAMP protein kinase A
  • the open reading frame encodes a polypeptide selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • the linear double-stranded further comprises an internal ribosome entry site.
  • the poly(A) stretch is 300-400 nucleotides in length.
  • the linear double-stranded DNA template of claim 1 wherein from 5' to 3' the template consists of an RNA polymerase promoter on the coding strand of the double- stranded DNA, a 5' untranslated region less than 3,000 nucleotides in length and effective for translation of the mRNA into a detectable polypeptide after transfection into a eukaryotic cell, an open reading frame that encodes the polypeptide, wherein the polypeptide is heterologous to the cell to be transfected and wherein the polypeptide is selected from the group consisting of a ligand or a receptor of an immune cell, a polypeptide that stimulates or inhibits a function of the immune system, and a polypeptide that inhibits the function of an oncogenic polypeptide, a 3' untranslated region effective for translation of the mRNA into a detectable polypeptide after transfection into a eukaryotic cell, and a poly(A) stretch of 50-5,000
  • the 3' untranslated region is at least 100 nucleotides in length.
  • the present invention provides a method of generating the linear double-stranded DNA template described above, wherein the method comprises generating forward and reverse primers, wherein the forward primer comprises a plurality of nucleotides that are substantially complementary to the non-coding strand of a target double-stranded DNA of interest, and a plurality of nucleotides that function as a binding site for an RNA polymerase, wherein the 171 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO reverse primer comprises a plurality of nucleotides that are substantially complementary to the coding strand of a target double-stranded DNA of interest, and a plurality of deoxythymidine nucleotides, and performing polymerase chain reaction amplification of the target DNA using the forward and reverse primers to form the linear double-stranded DNA template.
  • the present invention provides a method of generating the linear double-stranded DNA template described above, wherein the method comprises generating forward and reverse primers, wherein the forward primer comprises a plurality of nucleotides that are substantially complementary to a region of nucleotides directly upstream of a target double-stranded DNA of interest, wherein the reverse primer comprises a plurality of nucleotides that are substantially complementary to a region of nucleotides directly downstream of a target double-stranded DNA of interest, and performing polymerase chain reaction amplification of the target DNA using the forward and reverse primers to form the linear double- stranded DNA template.
  • the primers comprise nucleotide sequences that are substantially complementary to stretches of nucleotides in the 5' and 3' untranslated regions of a double-stranded DNA of interest. In some embodiments, the primers comprise nucleotide sequences that are substantially complementary to stretches of nucleotides within the open reading frame of a double-stranded DNA of interest.
  • the primers comprise nucleotide sequences that are substantially complementary to stretches of nucleotides within the open reading frame of a double-stranded DNA of interest, wherein the primers further comprise stretches of nucleotides that comprise 5' and 3' untranslated regions, wherein the stretch of nucleotides in the forward primer that comprise the 5' untranslated region is between the nucleotides that comprise the RNA polymerase promoter and the nucleotides that are substantially complementary to the non-coding strand of a target double-stranded DNA of interest, and wherein the stretch of nucleotides in the reverse primer that comprise the 3' untranslated region is between the plurality of deoxythymidine nucleotides and the nucleotides that are substantially complementary to the coding strand of a target double-stranded DNA of interest.
  • the RNA targets a polypeptide selected from the group consisting of PD-1, TGFBR2, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-12, IL-15, IL-18, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGF ⁇ , CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1 ⁇ ), CCL4 (MIP1- ⁇ ), CCL5 172 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP protein kinase A (PKA), and combinations thereof.
  • PKA cAMP protein kinase A
  • the RNA targets a polypeptide selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • the invention employs the use of one or more isolated RNAs comprising one or more open reading frames, produced from the linear double-stranded DNA template.
  • the invention provides a method for expressing one or more RNAs in a cell comprising contacting cells with one or more RNAs produced from the linear double-stranded DNA template.
  • the RNAs are present in unequal molar amounts to provide separate expression levels of the RNAs in the cells.
  • the one or more RNAs target a polypeptide selected from the group consisting of PD-1, TGFBR2, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-12, IL-15, IL-18, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGF ⁇ , CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP- 1 ⁇ ), CCL4 (MIP1- ⁇ ), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP protein kinase A (PKA), and combinations thereof.
  • PKA cAMP protein kinase A
  • the one or more RNAs target a polypeptide selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • Untranslated Regions Chemical structures with the ability to promote stability and/or translation efficiency may also be used.
  • the RNA preferably has 5' and 3' UTRs. The examples below demonstrate that inclusion of 44 base pairs of 5' UTR into the PCR template enabled greater translation efficiency of transcribed CFP RNA when compared to PCR templates containing only 6 base pairs of 5' UTR.
  • UTR enables greater translation efficiency of transcribed GFP RNA when compared to PCR templates containing only 11 base pairs of 3, UTR.
  • the length of the 3' UTR exceeds 100 nucleotides, and therefore 3' UTR longer than 100 nucleotides is preferred.
  • the 3' UTR sequence is between 100 and 5000 nucleotides.
  • the length of the 5' UTR is not as critical as the length of the 3' UTR and can be shorter. In some embodiments, the 5' UTR is between zero and 3000 nucleotides in length.
  • the length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA. 173 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO [00847]
  • the 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest.
  • UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA.
  • UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA.
  • AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5' UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
  • the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.
  • RNA Polymerase Promoter To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • Bacteriophage RNA polymerase promoter sequences can be attached to the St UTR by different genetic engineering methods, such as DNA ligation, or can be added to the forward primer (5') of the sequence that is substantially complementary to the target DNA.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 polymerase promoter, as described above.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • Poly(A) Tail and 5' Cap [00850]
  • the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
  • phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc. Acids Res., 13:6223- 36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
  • An endogenous termination signal for T7 RNA polymerase encodes an RNA that can fold into a stem- loop structure followed by a track of uridine residues (Dunn and Studier, J. Mol. Biol., 166:477-535 (1983); Arnaud-Barbe et al., 1998 Nuc. Acids Res., 26:3550-54 (1998)).
  • the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
  • Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA.
  • the poly(A) 175 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO tail is between 100 and 5000 adenosines.
  • RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP).
  • E-PAP E. coli polyA polymerase
  • the examples below demonstrate that increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA. Suitable ATP analogs include, but are not limited to, cordiocipin and 8-azaadenosine.
  • 5' caps can also provide stability to RNA molecules.
  • RNAs produced by the methods disclosed herein include a 5' cap.
  • the 5' cap may, for example, be m7G(5')ppp(5')G, m7G(5')ppp(5')A, G(5')ppp(5')G or G(5')ppp(5')A cap analogs, which are all commercially available.
  • the 5' cap can also be an anti-reverse-cap-analog (ARCA) (see, Stepinski, et al., RNA, 7:1468-95 (2001)) or any other suitable analog.
  • the 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958- 966 (2005)).
  • the RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • IRS internal ribosome entry site
  • the IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
  • the sd-RNA sequences used in the invention exhibit a 70% reduction in expression of the target gene. In some embodiments, the sd-RNA sequences used in the invention exhibit a 75% reduction in expression of the target gene. In some embodiments, the sd-RNA sequences used in the invention exhibit an 80% reduction in expression of the target gene.
  • the sd-RNA sequences used in the invention exhibit an 85% reduction in expression of the target gene. In some embodiments, the sd-RNA sequences used in the invention exhibit a 90% reduction in expression of the target gene. In some embodiments, the sd-RNA sequences used in the invention exhibit a 95% reduction in expression of the target gene. In some embodiments, the sd-RNA sequences used in the invention exhibit a 99% reduction in expression of the target gene.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.25 ⁇ M to about 10 ⁇ M, in some embodiments, about 0.25 ⁇ M to about 4 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.25 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.5 ⁇ M.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.75 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.0 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.25 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.5 ⁇ M.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.75 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.0 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.25 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.5 ⁇ M.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.75 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.0 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.25 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.5 ⁇ M.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.75 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 4.0 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 5.0 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 6.0 ⁇ M.
  • the sd- RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 7.0 ⁇ M. In some embodiments, the sd-RNA sequences used in 177 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 8.0 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 9.0 ⁇ M. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 10.0 ⁇ M.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.25 ⁇ M to about 10 ⁇ M/10,000 TILs, or about 0.25 ⁇ M to about 4 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.25 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.5 ⁇ M/10,000 TILs.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.75 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.0 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.25 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.5 ⁇ M/10,000 TILs.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.75 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.0 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.25 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.5 ⁇ M/10,000 TILs.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.75 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.0 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.25 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.5 ⁇ M/10,000 TILs.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.75 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a 178 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO reduction in expression of the target gene when delivered at a concentration of about 4.0 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 5.0 ⁇ M/10,000 TILs.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 6.0 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 7.0 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 8.0 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 9.0 ⁇ M/10,000 TILs.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 10.0 ⁇ M/10,000 TILs. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.25 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.5 ⁇ M/10,000 TILs/100 ⁇ L media.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.75 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.0 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.25 ⁇ M/10,000 TILs/100 ⁇ L media.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.5 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.75 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.0 ⁇ M/10,000 TILs/100 ⁇ L media.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.25 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.5 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.75 ⁇ M/10,000 TILs/100 ⁇ L media.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.0 ⁇ M/10,000 179 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.25 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.5 ⁇ M/10,000 TILs/100 ⁇ L media.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.75 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 4.0 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 5.0 ⁇ M/10,000 TILs/100 ⁇ L media.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 6.0 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 7.0 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 8.0 ⁇ M/10,000 TILs/100 ⁇ L media.
  • the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 9.0 ⁇ M/10,000 TILs/100 ⁇ L media. In some embodiments, the sd-RNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 10.0 ⁇ M/10,000 TILs/100 ⁇ L media.
  • sd-RNA Modifications [00858]
  • the oligonucleotide agents comprise one or more modification to increase stability and/or effectiveness of the therapeutic agent, and to effect efficient delivery of the oligonucleotide to the cells or tissue to be treated.
  • Such modifications can include a 2'-O-methyl modification, a 2'-O-Fluro modification, a diphosphorothioate modification, 2' F modified nucleotide, a 2'-O-methyl modified and/or a 2'deoxy nucleotide.
  • the oligonucleotide is modified to include one or more hydrophobic modifications including for example, sterol, cholesterol, vitamin D, naphtyl, isobutyl, benzyl, indol, tryptophane, and/or phenyl.
  • chemically modified nucleotides are combination of phosphorothioates, 2'-O-methyl, 2'deoxy, hydrophobic modifications and phosphorothioates.
  • the sugar moiety can be a hexose and incorporated into an oligonucleotide as described (Augustyns, K., et al., Nucl. Acids. Res., 18:4711 (1992)).
  • the double-stranded oligonucleotide of the invention is double- stranded over its entire length, i.e., with no overhanging single-stranded sequence at either end of the molecule, i.e., is blunt-ended.
  • the individual nucleic acid molecules can be of different lengths.
  • a double-stranded oligonucleotide of the invention is not double- stranded over its entire length.
  • one of the molecules e.g., the first molecule comprising an antisense sequence
  • the second molecule hybridizing thereto leaving a portion of the molecule single-stranded.
  • a single nucleic acid molecule when used a single nucleic acid molecule is used a portion of the molecule at either end can remain single-stranded.
  • a double-stranded oligonucleotide of the invention contains mismatches and/or loops or bulges, but is double-stranded over at least about 70% of the length of the oligonucleotide. In some embodiments, a double-stranded oligonucleotide of the invention is double- stranded over at least about 80% of the length of the oligonucleotide. In some embodiments, a double- stranded oligonucleotide of the invention is double-stranded over at least about 90%-95% of the length of the oligonucleotide.
  • a double-stranded oligonucleotide of the invention is double-stranded over at least about 96%-98% of the length of the oligonucleotide.
  • the double-stranded oligonucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.
  • the oligonucleotide can be substantially protected from nucleases e.g., by modifying the 3' or 5' linkages (e.g., U.S. Pat. No.5,849,902 and WO 98/13526).
  • oligonucleotides can be made resistant by the inclusion of a "blocking group.”
  • blocking group refers to substituents (e.g., other than OH groups) that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (e.g., FITC, propyl (CH2-CH2-CH3), glycol (-0-CH2-CH2-O-) phosphate (PO32”), hydrogen phosphonate, or phosphoramidite).
  • Blocking groups can also include “end blocking groups” or “exonuclease blocking groups” which protect the 5' and 3' termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.
  • end blocking groups or "exonuclease blocking groups” which protect the 5' and 3' termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.
  • at least a portion of the contiguous polynucleotides within the sd- RNA are linked by a substitute linkage, e.g., a phosphorothioate linkage.
  • chemical modification can lead to at least a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 181 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO 375, 400, 425, 450, 475, 500 enhancements in cellular uptake.
  • At least one of the C or U residues includes a hydrophobic modification.
  • a plurality of Cs and Us contain a hydrophobic modification.
  • at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90% or at least 95% of the Cs and Us can contain a hydrophobic modification.
  • all of the Cs and Us contain a hydrophobic modification.
  • the sd-RNA or sd-rxRNAs exhibit enhanced endosomal release of sd-rxRNA molecules through the incorporation of protonatable amines.
  • protonatable amines are incorporated in the sense strand (in the part of the molecule which is discarded after RISC loading).
  • the sd-RNA compounds of the invention comprise an asymmetric compound comprising a duplex region (required for efficient RISC entry of 10-15 bases long) and single stranded region of 4-12 nucleotides long; with a 13 nucleotide duplex. In some embodiments, a 6 nucleotide single stranded region is employed. In some embodiments, the single stranded region of the sd-RNA comprises 2-12 phosphorothioate intemucleotide linkages (referred to as phosphorothioate modifications).
  • the sd-RNA compounds of the invention also include a unique chemical modification pattern, which provides stability and is compatible with RISC entry.
  • the guide strand may also be modified by any chemical modification which confirms stability without interfering with RISC entry.
  • the chemical modification pattern in the guide strand includes the majority of C and U nucleotides being 2' F modified and the 5 ' end being phosphorylated.
  • at least 30% of the nucleotides in the sd-RNA or sd-rxRNA are modified.
  • the sd-RNA molecules have minimal double stranded regions.
  • the region of the molecule that is double stranded ranges from 8-15 nucleotides long.
  • the region of the molecule that is double stranded is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long.
  • the double stranded region is 13 nucleotides long.
  • the molecule is either blunt-ended or has a one-nucleotide overhang.
  • the single stranded region of the molecule is In some embodiments, between 4-12 nucleotides long. In some embodiments, the single stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides long. In some embodiments, the single stranded region can also be less than 4 or greater than 12 nucleotides long.
  • the single stranded region is 6 or 7 nucleotides long.
  • the sd-RNA molecules have increased stability.
  • a chemically modified sd-RNA or sd-rxRNA molecule has a half-life in media that is longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more than 24 hours, including any intermediate values.
  • the sd-rxRNA has a half-life in media that is longer than 12 hours.
  • the sd-RNA is optimized for increased potency and/or reduced toxicity.
  • nucleotide length of the guide and/or passenger strand, and/or the number of phosphorothioate modifications in the guide and/or passenger strand can in some aspects influence potency of the RNA molecule, while replacing 2'-fluoro (2'F) modifications with 2'-0- methyl (2'OMe) modifications can in some aspects influence toxicity of the molecule.
  • reduction in 2'F content of a molecule is predicted to reduce toxicity of the molecule.
  • the number of phosphorothioate modifications in an RNA molecule can influence the uptake of the molecule into a cell, for example the efficiency of passive uptake of the molecule into a cell.
  • a guide strand is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate modifications.
  • a guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more than 14 nucleotides that are phosphate-modified.
  • the guide strand may contain one or more modifications that confer increased stability without interfering with RISC entry.
  • the phosphate modified nucleotides such as phosphorothioate modified nucleotides, can be at the 3' end, 5' end or spread throughout the guide strand.
  • the 3' terminal 10 nucleotides of the guide strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides.
  • the guide strand can also contain 2'F and/or 2'OMe modifications, which can be located throughout the molecule.
  • the nucleotide in position one of the guide strand is 2'OMe modified and/or phosphorylated.
  • C and U nucleotides within the guide strand can be 2'F modified.
  • C and U nucleotides in positions 2-10 of a 19 nt guide strand can be 2'F modified.
  • C and U nucleotides within the guide strand can also be 2'OMe modified.
  • 183 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO For example, C and U nucleotides in positions 11-18 of a 19 nt guide strand (or corresponding positions in a guide strand of a different length) can be 2'OMe modified.
  • the nucleotide at the most 3' end of the guide strand is unmodified.
  • the majority of Cs and Us within the guide strand are 2'F modified and the 5' end of the guide strand is phosphorylated.
  • position 1 and the Cs or Us in positions 11-18 are 2'OMe modified and the 5' end of the guide strand is phosphorylated.
  • position 1 and the Cs or Us in positions 11-18 are 2'OMe modified, the 5' end of the guide strand is phosphorylated, and the Cs or Us in position 2-10 are 2'F modified.
  • Delivery of sd-RNA [00871] The self-deliverable RNAi technology provides a method of directly transfecting cells with the RNAi agent, without the need for additional formulations or techniques.
  • the ability to transfect hard-to-transfect cell lines, high in vivo activity, and simplicity of use, are characteristics of the compositions and methods that present significant functional advantages over traditional siRNA- based techniques, and as such, the sd-RNA methods are employed in several embodiments related to the methods of reduction in expression of the target gene in the TILs of the present invention.
  • the sd- RNAi methods allows direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues, both ex-vivo and in vivo.
  • the sd-RNAs described In some embodiments, of the invention herein are commercially available from Advirna LLC, Worcester, MA, USA.
  • sd-RNA are formed as hydrophobically-modified siRNA-antisense oligonucleotide hybrid structures, and are disclosed, for example in Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10): 855-864, incorporated by reference herein in its entirety.
  • the sd-RNA oligonucleotides can be delivered to the TILs described herein using sterile electroporation.
  • the oligonucleotides can be delivered to the cells in combination with a transmembrane delivery system.
  • this transmembrane delivery system comprises lipids, viral vectors, and the like.
  • the oligonucleotide agent is a self- delivery RNAi agent, that does not require any delivery agents.
  • the oligonucleotides such as RNAs or sd-RNAs described herein, can be introduced into target cells using different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MA), NeonTM Transfection System (commercially available from ThermoFisher Scientific, Waltham, MA), and/or the Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendort, Hamburg Germany), cationic 184 DB1/ 152908642.1 A orney Docket No.: 11698
  • TILs can be transfected using NUCLEOFECTOR.TM.-II solution V and the set of recommended regimes for electroporation. In some embodiments, TILs can be transfected using solutions V, T and R and different regimes of electroporation. In some embodiments, TILs can be transfected using T cell NUCLEOFECTOR.TM.-II solution and different regimes of electroporation.
  • Alternative methods of nucleic acids delivery can also be employed to transfect the oligonucleotides described herein used: cationic liposome mediated transfection was performed using LIPOFECTIN or LIPOFECTAMIN (Invitrogen).
  • Electroporation was also performed with the ECM 830 (BTX) (Harvard Instruments, Boston, MA), the Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg Germany), and/or the NeonTM Transfection System (commercially available from ThermoFisher Scientific, Waltham, MA).
  • BTX Garved Instruments
  • Gene Pulser II BioRad, Denver, CO
  • Multiporator Eppendorf, Hamburg Germany
  • NeonTM Transfection System commercially available from ThermoFisher Scientific, Waltham, MA.
  • a pmaxGFP plasmid DNA can be employed as the DNA control.
  • the efficiency of transfection can be determined approximately 3, 6, 9, 12, 15, and/or 18 hours after transfection by fluorescence activated cell sorting (FACS). In some experiments transfectants can be further analyzed every 12 hours to 24 hours until GFP could no longer be detected for GFP controls.
  • cell viability can be determined by trypan blue dye exclusion.
  • Oligonucleotides and oligonucleotide compositions are contacted with (e.g., brought into contact with, also referred to herein as administered or delivered to) and taken up by TILs described herein, including through passive uptake by TILs.
  • the sd-RNA can be added to the TILs as described herein during the first expansion, for example Step B, after the first expansion, for example, during Step C, before or during the second expansion, for example before or during Step D, after Step D and before harvest in Step E, during or after harvest in Step F, before or during final formulation and/or transfer to infusion Bag in Step F, as well as before any optional cryopreservation step in Step F.
  • sd-RNA can be added after thawing from any cryopreservation step in Step F.
  • one or more sd-RNAs targeting genes as described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB may be added to cell culture media comprising TILs and other agents at concentrations selected from the group consisting of 100 nM to 20 mM, 200 nM to 10 mM, 500 nm to 1 mM, 1 ⁇ M to 100 ⁇ M, and 1 ⁇ M to 100 ⁇ M.
  • one or more sd-RNAs targeting 185 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO genes as described herein, including PD-1, LAG-3, TIM-3, CISH, CTLA-4, TIGIT, and CBLB may be added to cell culture media comprising TILs and other agents at amounts selected from the group consisting of 0.1 ⁇ M sd-RNA/10,000 TILs/100 ⁇ L media, 0.5 ⁇ M sd-RNA/10,000 TILs /100 ⁇ L media, 0.75 ⁇ M sd-RNA/10,000 TILs /100 ⁇ L media, 1 ⁇ M sd-RNA/10,000 TILs /100 ⁇ L media, 1.25 ⁇ M sd-RNA/10,000 TILs /100 ⁇ L media, 1.5 ⁇ M sd-RNA/10,000 TILs /100 ⁇ L media, 2 ⁇ M sd- RNA/10,000 TILs /100 ⁇ L ⁇
  • one or more sd-RNAs targeting genes as described herein, including PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, CISH, and CBLB may be added to TIL cultures during the pre-REP or REP stages twice a day, once a day, every two days, every three days, every four days, every five days, every six days, or every seven days.
  • one or more sd-RNAs targeting genes as described herein, including PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, CISH, and CBLB may be added to cell culture media comprising TILs and other agents at amounts selected from the group consisting of 0.1 ⁇ M sd-RNA/10,000 TILs, 0.5 ⁇ M sd-RNA/10,000 TILs, 0.75 ⁇ M sd-RNA/10,000 TILs, 1 ⁇ M sd-RNA/10,000 TILs, 1.25 ⁇ M sd-RNA/10,000 TILs, 1.5 ⁇ M sd-RNA/10,000 TILs, 2 ⁇ M sd-RNA/10,000 TILs, 5 ⁇ M sd-RNA/10,000 TILs, or 10 ⁇ M sd- RNA/10,000 TILs.
  • one or more sd-RNAs targeting genes as described herein, including PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, CISH, and CBLB may be added to TIL cultures during the first, second, and or additional expansion stages twice a day, once a day, every two days, every three days, every four days, every five days, every six days, or every seven days.
  • Oligonucleotide compositions of the invention, including sd-RNA can be contacted with TILs as described herein during the expansion process, for example by dissolving sd-RNA at high concentrations in cell culture media and allowing sufficient time for passive uptake to occur.
  • the high concentrations include 0.1 ⁇ M sd-RNA/10,000 TILs, 0.5 ⁇ M sd-RNA/10,000 TILs, 0.75 ⁇ M sd-RNA/10,000 TILs, 1 ⁇ M sd-RNA/10,000 TILs, 1.25 ⁇ M sd-RNA/10,000 TILs, 1.5 ⁇ M sd-RNA/10,000 TILs, 2 ⁇ M sd-RNA/10,000 TILs, 5 ⁇ M sd-RNA/10,000 TILs, or 10 ⁇ M sd- RNA/10,000 TILs.
  • the high concentrations include 2 ⁇ M sd-RNA/10,000 TILs, 5 ⁇ M sd-RNA/10,000 TILs, or 10 ⁇ M sd-RNA/10,000 TILs. In some embodiments, the high concentrations include 5 ⁇ M sd-RNA/10,000 TILs or up to 10 ⁇ M sd-RNA/10,000 TILs.
  • delivery of oligonucleotides into cells can be enhanced by suitable art recognized methods including calcium phosphate, DMSO, glycerol or dextran, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes using methods known in the art (see, e.g., WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No.4,897,355; Bergan et a 1993. Nucleic Acids Research.21 :3567).
  • suitable art recognized methods including calcium phosphate, DMSO, glycerol or dextran, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes using methods known in the art (see, e.g., WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat
  • more than one sd-RNA is used to reduce expression of a target gene.
  • one or more of PD-1, CTLA-4, TIGIT, TIM-3, CBLB, LAG3 and/or CISH targeting sd-RNAs are used together.
  • a PD-1 sd-RNA is used with one or more of CTLA-4, TIGIT, TIM-3, CBLB, LAG3 and/or CISH in order to reduce expression of more than one gene target.
  • a LAG3 sd-RNA is used in combination with a CISH targeting sd-RNA to reduce gene expression of both targets.
  • the sd-RNAs targeting one or more of PD-1, CTLA-4, TIGIT, TIM-3, CBLB, LAG3 and/or CISH herein are commercially available from Advirna LLC, Worcester, MA, USA.
  • the sd-RNA targets a gene selected from the group consisting of PD- 1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • the sd-RNA targets a gene selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • one sd-RNA targets PD-1 and another sd-RNA targets a gene selected from the group consisting of LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • the sd-RNA targets a gene selected from PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof.
  • the sd-RNA targets a gene selected from PD-1 and one of CTLA-4, TIGIT, LAG3, CISH, CBLB, TIM3, and combinations thereof.
  • one sd-RNA targets TIGIT and one sd-RNA targets CBLB. In some embodiments, one sd-RNA targets TIGIT and one sd-RNA targets TIM3. In some embodiments, one sd-RNA targets LAG3 and one sd-RNA targets CISH. In some embodiments, one sd-RNA targets LAG3 and one sd-RNA targets CBLB. In some embodiments, one sd-RNA targets LAG3 and one sd-RNA targets CTLA-4. In some embodiments, one sd-RNA targets LAG3 and one sd-RNA targets TIGIT. In some embodiments, one sd-RNA targets CISH and one sd-RNA targets CBLB.
  • altering the protein expression of TILs during the TIL expansion method can also allow for expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic population of TILs.
  • altering the protein expression may cause the expression of a stimulatory receptor to be enhanced, which means that it is overexpressed as compared to the expression of a stimulatory receptor that has not been genetically modified.
  • Non-limiting examples of immune checkpoint genes that may exhibit enhanced expression by transiently altering the protein expression in TILs of the present invention include certain chemokine receptors and interleukins, such as CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL- 2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2 intracellular domain (ICD), and/or the NOTCH ligand mDLL1.
  • CCRs & CCLs [00883]
  • T cells need to be trafficked properly into tumors by chemokines.
  • altering the protein expression methods of the present invention may be used to increase the expression of certain chemokine receptors in the TILs, such as one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and/or CX3CR1. Over-expression of CCRs may help promote effector function and proliferation of TILs following adoptive transfer.
  • altering the protein expression methods of the present invention may be used to increase the expression of CCL2 (MCP-1), CCL3 (MIP-1 ⁇ ), CCL4 (MIP1- ⁇ ), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17 in the TILs.
  • CCP-1 CCL2
  • MIP-1 ⁇ CCL3
  • MIP1- ⁇ CCL4
  • CCL5 RANTES
  • CXCL1, CXCL8, CCL22, and/or CCL17 in the TILs CXCL1, CXCL8, CCL22, and/or CCL17 in the TILs.
  • CCR2, CCR4, CCR5, CXCR2, CXCR3 and/or CX3CR1 in TILs is enhanced in accordance with compositions and methods of the present invention.
  • a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A), wherein the method comprises gene-editing at least a portion of the TILs by enhancing the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3 and/or CX3CR1.
  • the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand 188 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO break at a chemokine receptor gene.
  • CRISPR method for example, a CRISPR method, a TALE method, or a zinc finger method may be used to enhance the expression of certain chemokine receptors in the TILs.
  • CCR4 and/or CCR5 adhesion molecules are inserted into a TIL population using a gamma-retroviral or lentiviral method as described herein.
  • CXCR2 adhesion molecule are inserted into a TIL population using a gamma-retroviral or lentiviral method as described in Forget, et al., Frontiers Immunology 2017, 8, 908 or Peng, et al., Clin.
  • gene-editing methods of the present invention may be used to increase the expression of certain interleukins, such as one or more of IL-2, IL-4, IL-7, IL- 10, IL-15, IL-18, and IL-21, and also the NOTCH 1/2 intracellular domain (ICD). Certain interleukins have been demonstrated to augment effector functions of T cells and mediate tumor control.
  • Embodiments of the present invention are directed to methods for expanding TIL populations, the methods comprising one or more steps of gene-editing at least a portion of the TILs in order to enhance their therapeutic effect.
  • “gene-editing,” “gene editing,” and “genome editing” refer to a type of genetic modification in which DNA is permanently modified in the genome of a cell, e.g., DNA is inserted, deleted, modified or replaced within the cell’s genome.
  • gene-editing causes the expression of a DNA sequence to be silenced (sometimes referred to as a gene knockout) or inhibited/reduced (sometimes referred to as a gene knockdown).
  • gene-editing causes the expression of a DNA sequence to be enhanced (e.g., by causing over-expression).
  • gene-editing technology is used to enhance the effectiveness of a therapeutic population of TILs.
  • a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., an exemplary TIL expansion method known as process 2A is described below), wherein the method further comprises gene-editing at least a portion of the TILs.
  • a method for expanding TILs into a therapeutic population of TILs is carried out in accordance with any embodiment of the methods described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, which are incorporated by reference herein in their entireties, wherein the method further comprises gene-editing at least a portion of the TILs.
  • an embodiment of the present invention provides a therapeutic population of TILs that has been expanded in accordance 189 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO with any embodiment described herein, wherein at least a portion of the therapeutic population has been gene-edited, e.g., at least a portion of the therapeutic population of TILs that is transferred to the infusion bag is permanently gene-edited.
  • Specific embodiments of the process of gene-editing are described in detail herein as well as in PCT/2019/029286, and PCT/US2019/012729, each of which is incorporated herein by reference in its entirety.
  • the invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining a first population of TILs from a tumor resected from a patient by processing a sample from the resected tumor into multiple tumor fragments; (b) adding the tumor fragments into a closed system; (c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2, and optionally OKT-3 (e.g., OKT-3 may be present in the culture medium beginning on the start date of the expansion process), to produce a second population of TILs, 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 3-14 days to obtain the second population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the
  • the gene-editing results in a genetic modification of the TILs that causes expression of one or more cytokines, under control of an NFAT promoter, that modulate tumor microenvironment to promote expansion of administered population of TILs.
  • the gene-editing comprises transduction of TILs with an NFAT-IL-18 expression cassette.
  • the gene-editing results in a genetic modification that causes the administered TILs to overexpress IL-18.
  • the gene-editing results in a genetic modification that causes the administered TILs to express ortho-IL2 receptor.
  • the gene-editing results in a genetic modification that causes the administered TILs to express a drug responsive domain (DRD)-cytokine construct that modulate tumor microenvironment to promote expansion of administered population of TILs, wherein the expressed DRD-cytokine is activated by an exogenously administered ligand that binds to the DRD of the DRD-cytokine to activate the cytokine.
  • the cytokine may be IL-2.
  • the gene-editing results in a genetic modification that causes the administered TILs to express IL2-fusion protein, wherein the IL-2 fusion protein releases IL-2 in response to a subsequent administration of a drug to the subject.
  • the fusion protein may be an IL-2-DRD, wherein the DRD is selected to separate from IL-2 in response to an administered drug.
  • the gene-editing results in a genetic modification that causes the administered TILs to express IL2-fusion protein, wherein the IL-2 fusion protein suppresses IL-2 activity in response to a subsequent administration of a drug to the subject.
  • the DRD is selected to mask active domains of IL-2 in response to the administered drug.
  • the gene-editing results in a genetic modification that causes the administered TILs to express a dual IL-15-IL-21 protein.
  • the gene-editing results in a genetic modification that causes TILs to express one or more binding partners that allow binding of exogenously administered cytokine carriers to the TILs via the one or more binding partners.
  • the binding partners may extend extracellularly from hydrophobic tails anchored in the cell membranes for the TILs.
  • the binding partners include proteins or peptides having (i) a segment of hydrophobic amino acids at or close to the C-terminus such that the segment is anchored in the cell membrane of the TILs; and (ii) an antigen or epitope binding domain extending extracellularly from the segment.
  • the binding partner includes an antigen-binding fragment of an antibody and the cytokine carrier may include at least a portion of the antigen recognized by the antibody such that the binding partner recognizes the antigen and binds to the 191 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO cytokine carrier.
  • the binding partner can be a variable fragment of an scFv.
  • the cytokine carriers may include an epitope (e.g., a peptide epitope) extending therefrom such that the binding partner recognizes and binds the epitope.
  • the binding partner includes an antigen recognized by an antigen- binding fragment of an antibody
  • the cytokine carrier may include at least the antigen-binding fragment of the antibody such that the antigen-binding fragment recognizes the antigen and binds the cytokine carrier to the binding partner of the TIL cell surface.
  • the antigen- binding fragment can be a variable fragment of an scFv.
  • the TIL cells may include an epitope (e.g., a peptide epitope) extending therefrom such that the variable fragment of the scFv recognizes and binds the epitope.
  • the binding partner may include a glycosyl-phosphatidylinositol (GPI) anchor enabling the binding partner to be anchored extracellularly in the cell membrane of the TILs.
  • the binding partner may include an antigen displayed on the TIL that can be recognized by an antibody provided on the cytokine carriers enabling the binding partner to bind with the cytokine carriers. Brown and Waneck (3(4) J. Am. Soc. Nephr.1992, 895-906) provide a deailed review of GPI-anchored proteins.
  • the gene-editing results in a genetic modification that reduces expression of one or more of the following immune checkpoint genes selected from the group comprising PD-1, TGIT, TET2, TGF ⁇ R2, PRA, BAFF (BR3), CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGF ⁇ , PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD
  • the gene-editing causes a genetic modification that causes the administered TILs to express cytokine carriers on surface of the genetically modified TILs that modulate tumor microenvironment to promote expansion of administered population of TILs, wherein the carriers link to binding partners extending extracellularly from hydrophobic tails anchored in cell membranes of the modified TILs, and release contents thereof by, one or both of: (i) gradual shedding with click chemistry, and (ii) redox reaction or enzymatic cleavage in the tumor microenvironment.
  • the cytokine may be selected from the group of: IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, and combinations thereof.
  • the gene-editing process may be carried out at any time during the TIL expansion method, which means that the gene editing may be carried out on TILs before, during, or after any of the steps in the expansion method; for example, during any of steps (a)-(f) outlined in the method above, or before or after any of steps (a)-(f) outlined in the method above.
  • TILs are collected during the expansion method (e.g., the expansion method is “paused” for at least a portion of the TILs), and the collected TILs are subjected to a gene-editing process, and, in some cases, subsequently reintroduced back into the expansion method (e.g., back into the culture medium) to continue the expansion process, so that at least a portion of the therapeutic population of TILs that are eventually transferred to the infusion bag are permanently gene-edited.
  • the gene-editing process may be carried out before expansion by activating TILs, performing a gene-editing step on the activated TILs, and expanding the gene-edited TILs according to the processes described herein.
  • alternative embodiments of the expansion process may differ from the method shown above; e.g., alternative embodiments may not have the same steps (a)-(g), or may have a different number of steps.
  • the gene-editing process may be carried out at any time during the TIL expansion method.
  • alternative embodiments may include more than two expansions, and it is possible that gene-editing may be conducted on the TILs during a third or fourth expansion, etc.
  • the gene-editing process is carried out on TILs from one or more of the first population, the second population, and the third population.
  • gene- editing may be carried out on the first population of TILs, or on a portion of TILs collected from the first population, and following the gene-editing process those TILs may subsequently be placed back into the expansion process (e.g., back into the culture medium).
  • gene-editing may be carried out on TILs from the second or third population, or on a portion of TILs collected from the second or third population, respectively, and following the gene-editing process those TILs may subsequently be placed back into the expansion process (e.g., back into the culture medium).
  • gene-editing is performed while the TILs are still in the culture medium and while the expansion is being carried out, i.e., they are not necessarily “removed” from the expansion in order to conduct gene-editing.
  • the gene-editing process is carried out on TILs from the first expansion, or TILs from the second expansion, or both.
  • gene-editing may be carried out on TILs that are collected from the culture 193 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO medium, and following the gene-editing process those TILs may subsequently be placed back into the expansion method, e.g., by reintroducing them back into the culture medium.
  • the gene-editing process is carried out on at least a portion of the TILs after the first expansion and before the second expansion.
  • gene-editing may be carried out on TILs that are collected from the culture medium, and following the gene-editing process those TILs may subsequently be placed back into the expansion method, e.g., by reintroducing them back into the culture medium for the second expansion.
  • the gene-editing process is carried out before step (c) (e.g., before, during, or after any of steps (a)-(b)), before step (d) (e.g., before, during, or after any of steps (a)-(c)), before step (e) (e.g., before, during, or after any of steps (a)-(d)), or before step (f) (e.g., before, during, or after any of steps (a)-(e)).
  • step (c) e.g., before, during, or after any of steps (a)-(b)
  • step (d) e.g., before, during, or after any of steps (a)-(c)
  • step (e) e.g., before, during, or after any of steps (a)-(d)
  • step (f) e.g., before, during, or after any of steps (a)-(e)
  • the cell culture medium may comprise OKT-3 beginning on the start day (Day 0), or on Day 1 of the first expansion, such that the gene-editing is carried out on TILs after they have been exposed to OKT-3 in the cell culture medium on Day 0 and/or Day 1.
  • the cell culture medium comprises OKT-3 during the first expansion and/or during the second expansion, and the gene-editing is carried out before the OKT-3 is introduced into the cell culture medium.
  • the cell culture medium may comprise OKT-3 during the first expansion and/or during the second expansion, and the gene-editing is carried out after the OKT-3 is introduced into the cell culture medium.
  • the cell culture medium may comprise a 4-1BB agonist beginning on the start day (Day 0), or on Day 1 of the first expansion, such that the gene-editing is carried out on TILs after they have been exposed to a 4-1BB agonist in the cell culture medium on Day 0 and/or Day 1.
  • the cell culture medium comprises a 4-1BB agonist during the first expansion and/or during the second expansion, and the gene-editing is carried out before the 4-1BB agonist is introduced into the cell culture medium.
  • the cell culture medium may comprise a 4- 1BB agonist during the first expansion and/or during the second expansion, and the gene-editing is carried out after the 4-1BB agonist is introduced into the cell culture medium.
  • the cell culture medium may comprise IL-2 beginning on the start day (Day 0), or on Day 1 of the first expansion, such that the gene-editing is carried out on TILs after they have been exposed to IL-2 in the cell culture medium on Day 0 and/or Day 1.
  • the cell culture 194 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO medium comprises IL-2 during the first expansion and/or during the second expansion, and the gene- editing is carried out before the IL-2 is introduced into the cell culture medium.
  • the cell culture medium may comprise IL-2 during the first expansion and/or during the second expansion, and the gene-editing is carried out after the IL-2 is introduced into the cell culture medium.
  • OKT-3, 4-1BB agonist and IL-2 may be included in the cell culture medium beginning on Day 0 or Day 1 of the first expansion.
  • OKT-3 is included in the cell culture medium beginning on Day 0 or Day 1 of the first expansion
  • a 4-1BB agonist is included in the cell culture medium beginning on Day 0 or Day 1 of the first expansion
  • IL-2 is included in the cell culture medium beginning on Day 0 or Day 1 of the first expansion.
  • the cell culture medium comprises OKT-3 and a 4- 1BB agonist beginning on Day 0 or Day 1 of the first expansion.
  • the cell culture medium comprises OKT-3, a 4-1BB agonist and IL-2 beginning on Day 0 or Day 1 of the first expansion.
  • the invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining a first population of TILs from at least a portion of a tumor resected from a patient by processing a sample from the resected tumor into multiple tumor fragments; (b) adding the tumor fragments into a closed system; (c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and optionally comprising a 4-1BB agonist antibody for about 3 to 11 days to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area; (d) stimulating the second population of TILs by
  • the foregoing method may be used to provide an autologous harvested TIL population for the treatment of a human subject with cancer.
  • sdRNA is inserted into a population of TILs during manufacturing.
  • the sdRNA encodes RNA that interferes with NOTCH 1/2 ICD, PD-1, CTLA- 4 TIM-3, LAG-3, TIGIT, TGF ⁇ , TGFBR2, cAMP protein kinase A (PKA), BAFF BR3, CISH, and/or CBLB.
  • the reduction in expression is determined based on a percentage of gene silencing, for example, as assessed by flow cytometry and/or qPCR.
  • the sdRNA sequences used in the invention exhibit a 70% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 75% reduction in expression of the target gene.
  • the sdRNA sequences used in the invention exhibit an 80% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit an 85% reduction in expression of the target gene. 196 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO In some embodiments, the sdRNA sequences used in the invention exhibit a 90% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 95% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 99% reduction in expression of the target gene.
  • the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.25 ⁇ M to about 4 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.25 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.5 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 0.75 ⁇ M.
  • the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.0 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.25 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.5 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 1.75 ⁇ M.
  • the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.0 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.25 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.5 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 2.75 ⁇ M.
  • the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.0 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.25 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.5 ⁇ M. In some embodiments, the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 3.75 ⁇ M.
  • the sdRNA sequences used in the invention exhibit a reduction in expression of the target gene when delivered at a concentration of about 4.0 ⁇ M.
  • the oligonucleotide agents comprise one or more modification to increase stability and/or effectiveness of the therapeutic agent, and to effect efficient delivery of the oligonucleotide to the cells or tissue to be treated.
  • Such modifications can include a 2'-O-methyl modification, a 2'-O-Fluro modification, a diphosphorothioate modification, 2' F modified nucleotide, a2'-O-methyl modified and/or a 2'deoxy nucleotide.
  • the oligonucleotide is modified to include one or more hydrophobic modifications including, for example, sterol, cholesterol, vitamin D, naphtyl, isobutyl, benzyl, indol, tryptophane, and/or phenyl.
  • chemically modified nucleotides are combination of phosphorothioates, 2'-O- methyl, 2'deoxy, hydrophobic modifications and phosphorothioates.
  • the sugar moiety can be a hexose and incorporated into an oligonucleotide as described (Augustyns, K., et al., Nucl. Acids. Res.18:4711 (1992)).
  • the double-stranded oligonucleotide of the invention is double- stranded over its entire length, i.e., with no overhanging single-stranded sequence at either end of the molecule, i.e., is blunt-ended.
  • the individual nucleic acid molecules can be of different lengths.
  • a double-stranded oligonucleotide of the invention is not double- stranded over its entire length.
  • one of the molecules e.g., the first molecule comprising an antisense sequence
  • the second molecule hybridizing thereto leaving a portion of the molecule single-stranded.
  • a single nucleic acid molecule when used a single nucleic acid molecule is used a portion of the molecule at either end can remain single-stranded.
  • a double-stranded oligonucleotide of the invention contains mismatches and/or loops or bulges, but is double-stranded over at least about 70% of the length of the oligonucleotide. In some embodiments, a double-stranded oligonucleotide of the invention is double- stranded over at least about 80% of the length of the oligonucleotide. In some embodiments, a double- stranded oligonucleotide of the invention is double-stranded over at least about 90%-95% of the length of the oligonucleotide.
  • a double-stranded oligonucleotide of the invention is double-stranded over at least about 96%-98% of the length of the oligonucleotide.
  • the double-stranded oligonucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.
  • the oligonucleotide can be substantially protected from nucleases e.g., by modifying the 3' or 5' linkages (e.g., U.S. Pat. No.5,849,902 and WO 98/13526).
  • oligonucleotides can be made resistant by the inclusion of a "blocking group.”
  • blocking group refers to substituents (e.g., other than OH groups) that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (e.g., FITC, propyl (CH2-CH2-CH3), glycol (-0-CH2-CH2-O-) phosphate (PO32”), hydrogen phosphonate, or phosphoramidite).
  • Blocking groups can also include “end blocking groups” or “exonuclease blocking groups” which protect the 5' and 3' termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.
  • end blocking groups or "exonuclease blocking groups” which protect the 5' and 3' termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.
  • at least a portion of the contiguous polynucleotides within the sdRNA are linked by a substitute linkage, e.g., a phosphorothioate linkage.
  • chemical modification can lead to at least a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 enhancements in cellular uptake.
  • at least one of the C or U residues includes a hydrophobic modification.
  • a plurality of Cs and Us contain a hydrophobic modification. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90% or at least 95% of the Cs and Us can contain a hydrophobic modification. In some embodiments, all of the Cs and Us contain a hydrophobic modification.
  • the sdRNA or sd-rxRNAs exhibit enhanced endosomal release of sd- rxRNA molecules through the incorporation of protonatable amines. In some embodiments, protonatable amines are incorporated in the sense strand (in the part of the molecule which is discarded after RISC loading).
  • the sdRNA compounds of the invention comprise an asymmetric compound comprising a duplex region (required for efficient RISC entry of 10-15 bases long) and single stranded region of 4-12 nucleotides long; with a 13 nucleotide duplex.
  • a 6 nucleotide single stranded region is employed.
  • the single stranded region of the sdRNA comprises 2-12 phosphorothioate intemucleotide linkages (referred to as phosphorothioate modifications).
  • 6-8 phosphorothioate intemucleotide linkages are employed.
  • the sdRNA compounds of the invention also include a unique chemical modification pattern, which provides stability and is compatible with RISC entry.
  • the guide strand may also be modified by any chemical modification which confirms stability without interfering with RISC entry.
  • the chemical modification pattern in the guide strand includes the majority of C and U nucleotides being 2' F modified and the 5 ' end being phosphorylated. 199 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO [00927] In some embodiments, at least 30% of the nucleotides in the sdRNA or sd-rxRNA are modified.
  • the sdRNA molecules have minimal double stranded regions.
  • the region of the molecule that is double stranded ranges from 8-15 nucleotides long.
  • the region of the molecule that is double stranded is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long.
  • the double stranded region is 13 nucleotides long. There can be 100% complementarity between the guide and passenger strands, or there may be one or more mismatches between the guide and passenger strands.
  • the molecule on one end of the double stranded molecule, is either blunt-ended or has a one-nucleotide overhang.
  • the single stranded region of the molecule is In some embodiments, between 4-12 nucleotides long. In some embodiments, the single stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides long. In some embodiments, the single stranded region can also be less than 4 or greater than 12 nucleotides long. In certain embodiments, the single stranded region is 6 or 7 nucleotides long. [00929] In some embodiments, the sdRNA molecules have increased stability.
  • a chemically modified sdRNA or sd-rxRNA molecule has a half-life in media that is longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more than 24 hours, including any intermediate values.
  • the sd-rxRNA has a half-life in media that is longer than 12 hours.
  • the sdRNA is optimized for increased potency and/or reduced toxicity.
  • nucleotide length of the guide and/or passenger strand, and/or the number of phosphorothioate modifications in the guide and/or passenger strand can in some aspects influence potency of the RNA molecule, while replacing 2'-fluoro (2'F) modifications with 2'-0- methyl (2'OMe) modifications can in some aspects influence toxicity of the molecule.
  • reduction in 2'F content of a molecule is predicted to reduce toxicity of the molecule.
  • the number of phosphorothioate modifications in an RNA molecule can influence the uptake of the molecule into a cell, for example the efficiency of passive uptake of the molecule into a cell.
  • sdRNA has no 2'F modification and yet are characterized by equal efficacy in cellular uptake and tissue penetration.
  • a guide strand is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate modifications.
  • a guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more than 14 nucleotides that are phosphate-modified.
  • the guide strand may contain one or more modifications that confer increased stability without interfering with RISC entry.
  • the phosphate modified nucleotides can be at the 3' end, 5' end or spread throughout the guide strand.
  • the 3' terminal 10 nucleotides of the guide strand contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides.
  • the guide strand can also contain 2'F and/or 2'OMe modifications, which can be located throughout the molecule.
  • the nucleotide in position one of the guide strand is 2'OMe modified and/or phosphorylated.
  • C and U nucleotides within the guide strand can be 2'F modified.
  • C and U nucleotides in positions 2-10 of a 19 nt guide strand can be 2'F modified.
  • C and U nucleotides within the guide strand can also be 2'OMe modified.
  • C and U nucleotides in positions 11-18 of a l9 nt guide strand can be 2'OMe modified.
  • the nucleotide at the most 3' end of the guide strand is unmodified.
  • the majority of Cs and Us within the guide strand are 2'F modified and the 5' end of the guide strand is phosphorylated.
  • position 1 and the Cs or Us in positions 11-18 are 2'OMe modified and the 5' end of the guide strand is phosphorylated. In other embodiments, position 1 and the Cs or Us in positions 11-18 are 2'OMe modified, the 5' end of the guide strand is phosphorylated, and the Cs or Us in position 2-10 are 2'F modified.
  • the self-deliverable RNAi technology provides a method of directly transfecting cells with the RNAi agent, without the need for additional formulations or techniques.
  • the ability to transfect hard-to-transfect cell lines, high in vivo activity, and simplicity of use, are characteristics of the compositions and methods that present significant functional advantages over traditional siRNA- based techniques, and as such, the sdRNA methods are employed in several embodiments related to the methods of reduction in expression of the target gene in the TILs of the present invention.
  • the sdRNAi methods allows direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues, both ex-vivo and in vivo.
  • the sdRNAs described In some embodiments, of the invention herein are commercially available from Advirna LLC, Worcester, MA, USA.
  • the sdRNA are formed as hydrophobically-modified siRNA-antisense oligonucleotide hybrid structures, and are disclosed, for example in Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10): 855-864, incorporated by reference herein in its entirety.
  • the sdRNA oligonucleotides can be delivered to the TILs described herein using sterile electroporation.
  • the method comprises sterile electroporation of a population of TILs to deliver sdRNA oligonucleotides.
  • the oligonucleotides can be delivered to the cells in combination with a transmembrane delivery system.
  • this transmembrane delivery system comprises lipids, viral vectors, and the like.
  • the oligonucleotide agent is a self- delivery RNAi agent, that does not require any delivery agents.
  • the method comprises use of a transmembrane delivery system to deliver sdRNA oligonucleotides to a population of TILs.
  • Oligonucleotides and oligonucleotide compositions are contacted with (e.g., brought into contact with, also referred to herein as administered or delivered to) and taken up by TILs described herein, including through passive uptake by TILs.
  • the sdRNA can be added to the TILs as described herein during the first expansion, for example Step B, after the first expansion, for example, during Step C, before or during the second expansion, for example before or during Step D, after Step D and before harvest in Step E, during or after harvest in Step F, before or during final formulation and/or transfer to infusion Bag in Step F, as well as before any optional cryopreservation step in Step F.
  • sdRNA can be added after thawing from any cryopreservation step in Step F.
  • one or more sdRNAs targeting genes as described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB may be added to cell culture media comprising TILs and other agents at concentrations selected from the group consisting of 100 nM to 20 mM, 200 nM to 10 mM, 500 nm to 1 mM, 1 ⁇ M to 100 ⁇ M, and 1 ⁇ M to 100 ⁇ M.
  • one or more sdRNAs targeting genes as described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB may be added to cell culture media comprising TILs and other agents at amounts selected from the group consisting of 0.1 ⁇ M sdRNA/10,000 TILs/100 ⁇ L media, 0.5 ⁇ M sdRNA/10,000 TILs /100 ⁇ L media, 0.75 ⁇ M sdRNA/10,000 TILs /100 ⁇ L media, 1 ⁇ M sdRNA/10,000 TILs /100 ⁇ L media, 1.25 ⁇ M sdRNA/10,000 TILs /100 ⁇ L media, 1.5 ⁇ M sdRNA/10,000 TILs /100 ⁇ L media, 2 ⁇ M sdRNA/10,000 TILs /100 ⁇ L media, 5 ⁇ M sdRNA/10,000 TILs /100 ⁇ L media, or 10 ⁇ M sdRNA/10,000 TILs /100 ⁇
  • one or more sdRNAs targeting genes as described herein, including PD-1, LAG-3, TIM-3, CISH, and CBLB may be added to TIL cultures during the pre-REP or REP stages twice a day, once a day, every two days, every three days, every four days, every five days, every six days, or every seven days.
  • Oligonucleotide compositions of the invention, including sdRNA can be contacted with TILs as described herein during the expansion process, for example by dissolving sdRNA at high concentrations in cell culture media and allowing sufficient time for passive uptake to occur.
  • the method of the present invention comprises contacting a population of TILs with an oligonucleotide composition as described herein.
  • the method comprises dissolving an oligonucleotide e.g. sdRNA in a cell culture media and contacting the cell culture media with a population of TILs.
  • the TILs may be a first population, a second population and/or a third population as described herein.
  • delivery of oligonucleotides into cells can be enhanced by suitable art recognized methods including calcium phosphate, DMSO, glycerol or dextran, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes using methods known in the art (see, e.g., WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No.4,897,355; Bergan et al 1993. Nucleic Acids Research.21 :3567).
  • more than one sdRNA is used to reduce expression of a target gene.
  • one or more of PD-1, TIM-3, CBLB, LAG3 and/or CISH targeting sdRNAs are used together.
  • a PD-1 sdRNA is used with one or more of TIM-3, CBLB, LAG3 and/or CISH in order to reduce expression of more than one gene target.
  • a LAG3 sdRNA is used in combination with a CISH targeting sdRNA to reduce gene expression of both targets.
  • the sdRNAs targeting one or more of PD-1, TIM-3, CBLB, LAG3 and/or CISH herein are commercially available from Advirna LLC, Worcester, MA, USA.
  • the sdRNA targets a gene selected from the group consisting of PD- 1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • the sdRNA targets a gene selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof.
  • a method for expanding TILs into a therapeutic population may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein the method further comprises gene-editing at least a portion of the TILs by a piggyBac method (e.g., piggyBac transposons and transposases or piggyBac-like transposons and transposases).
  • a piggyBac method e.g., piggyBac transposons and transposases or piggyBac-like transposons and transposases.
  • the use of a piggyBac method during the TIL expansion process causes expression of at least one immunomodulatory composition at the cell surface of at least a portion of the therapeutic population of TILs.
  • the use of a piggyBac method during the TIL expansion process causes expression of one or more binding partners that allow binding of exogenously administered cytokine carriers to the TILs via the one or more binding partners.
  • the use of a piggyBac method during the TIL expansion process causes expression of at least one immunomodulatory composition at the cell surface of, and optionally causes expression of one or more immune checkpoint genes to be modified (e.g., suppressed or enhanced) in, at least a portion of the therapeutic population of TILs.
  • the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.
  • the piggyBac transposon is a mobile genetic element that efficiently transposes between the donor vector and host chromosomes. This system has almost no cargo limit, and is fully reversible, leaving no footprint in the genome after excision.
  • the piggyBac transposon/transposase system consists of a transposase that recognizes piggyBac-specific inverted terminal repeat sequences (ITRs) located on both sides of the transposon cassette.
  • the transposase excises the transposable element to integrate it into TT/AA chromosomal sites that are preferentially located in euchromatic regions of mammalian genomes (Ding et al.2005; Cadina ⁇ os and Bradley 2007; Wilson et al.2007; Wang et al.2008; Li et al.2011).
  • Exemplary piggyBac systems include those described in WO2019/046815, the contents of which are incorporated herein by reference in their entirety.
  • the piggyBac system comprises a transposon/transposase system.
  • a piggyBac method comprises delivering to the TILs, (a) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase enzyme and (b) a recombinant and non-naturally occurring DNA sequence comprising a DNA sequence encoding a transposon.
  • the sequence encoding a transposase enzyme is an mRNA sequence.
  • the sequence encoding a transposase enzyme is a DNA sequence.
  • the DNA sequence is a cDNA sequence.
  • the sequence encoding a transposase enzyme is an amino acid sequence.
  • a protein Super piggybac transposase SPB
  • SPB Super piggybac transposase
  • Exemplary transposon/transposase systems that can be used to genetically modify the subject TILs described herein include, but are not limited to, Sleeping Beauty transposons and transposases, Helraiser transposons and transposases and Tol2 transposons and transposases.
  • the piggyBac transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and moves the contents between the ITRs into TTAA chromosomal sites.
  • ITRs inverted terminal repeat sequences
  • the piggyBac transposon system has no payload limit for the genes of interest that can be included between the ITRs.
  • the transposon is a piggyBac transposon or a piggyBac-like transposon.
  • Examples of piggyBac and piggyBac-like transposases and transposons include, for example, those disclosed in WO2019/046815, the contents of which are incorporated herein by reference in their entirety.
  • the piggyBac or piggyBac-like transposase is hyperactive.
  • a hyperactive piggyBac or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived.
  • the hyperactive piggyBac or piggyBac-like transposase enzyme is isolated or derived from Bombyx mori.
  • a list of hyperactive amino acid substitutions can be found in US patent No.10,041,077, the contents of which are incorporated herein by reference in their entirety.
  • the piggyBac or piggyBac-like transposase is integration deficient.
  • an integration deficient piggyBac or piggyBac-like transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding wild-type transposase.
  • a list of integration deficient amino acid substitutions can be found in US patent No. 10,041,077, the contents of which are incorporated by reference in their entirety.
  • the piggyBac or piggyBac-like transposon is capable of insertion by a piggyBac or piggyBac-like transposase at the sequence 5'-TTAT-3 within a target nucleic acid.
  • the transposase is a piggyBac transposase.
  • the transposase is a piggyBac-like transposase.
  • the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase.
  • the sequence encoding the transposase is an mRNA sequence.
  • the sleeping beauty (SB) transposon is transposed into the target genome by the Sleeping Beauty transposase that recognizes ITRs, and moves the contents between the ITRs into TA chromosomal sites.
  • the transposon is a Sleeping Beauty transposon.
  • the transposase enzyme is a Sleeping Beauty transposase enzyme (see, for example, US Patent No.9,228,180, the contents of which are incorporated herein in their entirety).
  • the Sleeping Beauty transposase is a hyperactive Sleeping Beauty (SB100X) transposase.
  • SB100X hyperactive Sleeping Beauty
  • the Helitron transposase does not contain an RNase-H like catalytic domain, but instead comprises a RepHel motif made up of a replication initiator domain (Rep) and a DNA helicase domain.
  • the Rep domain is a nuclease domain of the HUH superfamily of nucleases.
  • the transposon is a Helraiser transposon.
  • the transposase is flanked by left and right terminal sequences termed LTS and RTS. In some embodiments, these sequences terminate with a conserved 5'-TC/CTAG-3' motif.
  • a 19 bp palindromic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and comprises the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG.
  • the transposase enzyme is a Helitron transposase enzyme.
  • Tol2 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family.
  • Exemplary Tol2 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene encoding the Tol2 transposase, which contains four exons.
  • the transposon is a Tol2 transposon.
  • the transposase enzyme is a Tol2 transposase enzyme.
  • a vector comprises the recombinant and non-naturally occurring DNA sequence encoding the transposon.
  • the vector comprises any form of DNA and wherein the vector comprises at least 100 nucleotides (nts), 500 nts, 1000 nts, 1500 nts, 2000 nts, 2500 nts, 3000 nts, 3500 nts, 4000 nts, 4500 nts, 5000 nts, 6500 nts, 7000 nts, 7500 nts, 206 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO 8000 nts, 8500 nts, 9000 nts, 9500 nts, 10,000 nts or any number of nucleotides in between.
  • nts nucleotides
  • the vector comprises single-stranded or double-stranded DNA. In some embodiments, the vector comprises circular DNA. In some embodiments, the vector is a plasmid vector, a nanoplasmid vector, a minicircle. In some embodiments, the vector comprises linear or linearized DNA. In some embodiments, the vector is a double-stranded doggyboneTM DNA sequence. [00954] In some embodiments, the recombinant and non-naturally occurring DNA sequence encoding a transposon further comprises a sequence encoding one or more immune checkpoint genes.
  • the nucleic acid sequence encoding the transposase enzyme is a DNA sequence
  • an amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon is equal to or less than 10.0 ⁇ g per 100 ⁇ L, less than 7.5 ⁇ g per 100 ⁇ L, less than 6.0 ⁇ g per 100 ⁇ L, less than 5.0 ⁇ g per 100 ⁇ L, less than 2.5 ⁇ g per 100 ⁇ L, or less than 1.67 ⁇ g per 100 ⁇ L, less than 0.55 ⁇ g per 100 ⁇ L, less than 0.19 ⁇ g per 100 ⁇ L, less than 0.10 ⁇ g per 100 ⁇ L of an electroporation or nucleofection reaction.
  • a concentration of the amount of the DNA sequence encoding the transposase enzyme and an amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 100 ⁇ g/mL, equal to or less than 75 ⁇ g/mL, equal to or less than 60 ⁇ g/mL, equal to or less than 50 ⁇ g/mL, equal to or less than 25 ⁇ g/mL, equal to or less than 16.7 ⁇ g/mL, equal to or less than 5.5 ⁇ g/mL, equal to or less than 1.9 ⁇ g/mL, equal to or less than 1.0 ⁇ g/mL.
  • the nucleic acid sequence encoding the transposase enzyme is an RNA sequence
  • an amount of the RNA sequence encoding the transposase enzyme and an amount of the RNA sequence encoding the transposon is equal to or less than 10.0 ⁇ g per 100 ⁇ L, less than 7.5 ⁇ g per 100 ⁇ L, less than 6.0 ⁇ g per 100 ⁇ L, less than 5.0 ⁇ g per 100 ⁇ L, less than 2.5 ⁇ g per 100 ⁇ L, or less than 1.67 ⁇ g per 100 ⁇ L, less than 0.55 ⁇ g per 100 ⁇ L, less than 0.19 ⁇ g per 100 ⁇ L, less than 0.10 ⁇ g per 100 ⁇ L of an electroporation or nucleofection reaction.
  • a concentration of the amount of the RNA sequence encoding the transposase enzyme and an amount of the RNA sequence encoding the transposon in the electroporation or nucleofection reaction is equal to or less than 100 ⁇ g/mL, equal to or less than 75 ⁇ g/mL, equal to or less than 60 ⁇ g/mL, equal to or less than 50 ⁇ g/mL, equal to or less than 25 ⁇ g/mL, equal to or less than 16.7 ⁇ g/mL, equal to or less than 5.5 ⁇ g/mL, equal to or less than 1.9 ⁇ g/mL, equal to or less than 1.0 ⁇ g/mL.
  • the TILs are further modified by a second gene editing tool, including, but not limited to those described herein.
  • the second gene editing tool may include an excision-only piggyBac transposase to re-excise the inserted sequences or any 207 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO portion thereof.
  • the excision-only piggyBac transposase may be used to "re-excise" the transposon.
  • a piggyBac system comprises a transposon/transposase system, wherein the transposase recognizes the ITRs located on both sides of the transposon cassette comprising a cargo encoding one or more immune checkpoint genes, and excises the transposable element to integrate it into TT/AA chromosomal sites, resulting in genomic insertion of the transposon cassette and expression of the one or more immune checkpoint genes.
  • the cargo encodes two or more immune checkpoint molecules.
  • Non-limiting examples of genes that may be enhanced by permanently gene-editing TILs via a piggyBac method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-18, IL-21, the NOTCH 1/2 intracellular domain (ICD), and/or the NOTCH ligand mDLL1.
  • a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding the tumor fragments into a closed system; (c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and optionally comprising OKT-3 and/or a 4-1BB agonist antibody for about 3 to 11 days to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area; (d) stimulating the second population of TILs by adding OKT-3 and culturing for about 1 to 3 days, wherein the transition from step (c) to step (d) occurs without opening the system; 208 DB1/ 152908642.1 A orney Docket No.: 116
  • a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprises: (a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments; (b) adding the tumor fragments into a closed system; (c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and optionally comprising OKT-3 and/or a 4-1BB agonist antibody for about 3 to 11 days to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area; (d) stimulating the second population of TILs by adding OKT-3 and culturing for about 1 to 3 days to obtain the second population of TILs, wherein the transition from step (c) to step (d) occurs without opening the system; 209 DB1/ 152908642.1 A or
  • the at least one immunomodulatory composition comprises an immunomodulatory agent fused to a membrane anchor (e.g., a membrane anchored immunomodulatory fusion protein described herein).
  • the immunomodulatory agent is selected from the group consisting of IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist (e.g., a CD40L or an agonistic CD40 binding domain).
  • the immunomodulatory agent is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.
  • the immunomodulatory agent is selected from the group consisting of IL-12, IL-15, IL-18, IL-21, and a CD40 agonist.
  • a method of genetically modigying a polulation of TILs includes the use of a non-viral technique such as a piggyBac method (e.g., piggyBac transposons and transposases or piggyBac-like transposons and transposases).
  • the method comprises delivering to the TILs: (a) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase enzy; and (b) a recombinant and non-naturally occurring DNA sequence comprising a DNA sequence encoding a transposon.
  • the sequence encoding a transposase enzyme is an mRNA sequence.
  • the mRNA sequence encoding a 210 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO transposase enzyme may be produced in vitro.
  • the sequence encoding a transposase enzyme is a DNA sequence.
  • the DNA sequence encoding a transposase enzyme may be produced in vitro.
  • the DNA sequence may be a cDNA sequence.
  • the sequence encoding a transposase enzyme is an amino acid sequence.
  • the amino acid sequence encoding a transposase enzyme may be produced in vitro.
  • a protein Super piggybac transposase (SPB) may be delivered following pre-incubation with transposon DNA.
  • the transposon is a piggyBac transposon or a piggyBac-like transposon.
  • the transposase is a piggyBac transposase. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac- like transposon, the transposase is a piggyBac-like transposase. In certain embodiments, the piggyBac transposase comprises an amino acid sequence comprising SEQ ID NO: 238.
  • the transposase is a piggyBacTM or a Super piggyBacTM (SPB) transposase.
  • the sequence encoding the transposase is an mRNA sequence.
  • the transposase enzyme is a piggyBacTM (PB) transposase enzyme.
  • the piggyBac (PB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQN VIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKL FFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMV YVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRG RCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNI TCDNWFTSIPLAK
  • the transposon is a Sleeping Beauty transposon.
  • the transposase enzyme is a Sleeping Beauty transposase enzyme (see, for example, US Patent No.9,228,180, the contents of which are incorporated herein in their entirety).
  • the Sleeping Beauty transposase is a hyperactive Sleeping Beauty (SB100X) transposase.
  • the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: 211 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO MGKSKEISQDLRKKIVDLHKSGSSLGAISKRLKVPRSSVQTIVRKYKHHGTTQPSYRSGRRRVLSPRD ERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGRSARKKPLLQNRHKKARLRFATA HGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGT GALHKIDGIMRKENYVDILKQHLKTSVRKLKLGRKWVFQMDNDPKHTSKVVAKWLKDNKVKVLEWPS QSPDLNPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKI
  • the Sleeping Beauty transposase is a hyperactive Sleeping Beauty (SB100X) transposase
  • the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to: MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYRSGRRRVLSPRD ERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATA HGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGT GALHKIDGIMDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPS QSPDLNPIENLWA
  • Embodiments of the present invention are directed to methods for expanding TIL populations, the methods comprising one or more steps of gene-editing at least a portion of the TILs in order to enhance their therapeutic effect.
  • gene-editing refers to a type of genetic modification in which DNA is permanently modified in the genome of a cell, e.g., DNA is inserted, deleted, modified or replaced within the cell’s genome.
  • gene-editing causes the expression of a DNA sequence to be silenced (sometimes referred to as a gene knockout) or inhibited/reduced (sometimes referred to as a gene knockdown).
  • gene-editing causes the expression of a DNA sequence to be enhanced (e.g., by causing over-expression).
  • the DNA sequence exhibiting enhanced expression is an endogenous DNA sequence.
  • the DNA sequence exhibiting expression is an exogenous DNA sequence that originates from outside the edited cell.
  • gene-editing technology is used to enhance the effectiveness of a therapeutic population of TILs.
  • a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs may be carried out in accordance with any embodiment of the methods described herein (e.g., an exemplary TIL expansion method known as process 2A is described below), wherein the method further comprises gene-editing at least a portion of the TILs.
  • TILs tumor infiltrating lymphocytes
  • a method for expanding TILs into a therapeutic population of TILs is carried out in accordance with any embodiment of the methods described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, which are incorporated by reference herein in their entireties, wherein the 212 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO method further comprises gene-editing at least a portion of the TILs.
  • an embodiment of the present invention provides a therapeutic population of TILs that has been expanded in accordance with any embodiment described herein, wherein at least a portion of the therapeutic population has been gene-edited, e.g., at least a portion of the therapeutic population of TILs that is transferred to the infusion bag is permanently gene-edited.
  • Specific embodiments of the process of gene-editing are described in detail herein as well as in PCT/2019/029286, and PCT/US2019/012729, each of which is incorporated herein by reference in its entirety.
  • the expanded TILs of the present invention are further manipulated before, during, or after an expansion step, including during closed, sterile manufacturing processes, each as provided herein, in order to alter protein expression in a transient manner.
  • the transiently altered protein expression is due to transient gene editing.
  • the expanded TILs of the present invention are treated with transcription factors (TFs) and/or other molecules capable of transiently altering protein expression in the TILs.
  • TFs and/or other molecules that are capable of transiently altering protein expression provide for altered expression of tumor antigens and/or an alteration in the number of tumor antigen-specific T cells in a population of TILs.
  • the method comprises genetically editing a population of TILs. In certain embodiments, the method comprises genetically editing the first population of TILs, the second population of TILs and/or the third population of TILs. [00971] In some embodiments, the present invention includes genetic editing through nucleotide insertion, such as through ribonucleic acid (RNA) insertion, including insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA), into a population of TILs for promotion of the expression of one or more proteins or inhibition of the expression of one or more proteins, as well as simultaneous combinations of both promotion of one set of proteins with inhibition of another set of proteins.
  • RNA messenger RNA
  • siRNA small interfering RNA
  • the expanded TILs of the present invention undergo transient alteration of protein expression.
  • the transient alteration of protein expression occurs in the bulk TIL population prior to first expansion, including, for example in the TIL population obtained from for example, Step A.
  • the transient alteration of protein expression occurs during the first expansion, including, for example in the TIL population expanded in for example, Step B.
  • the transient alteration of protein expression occurs after the first expansion, including, for example in the TIL population in transition between the first and second expansion (e.g. the second population of TILs as described herein), the TIL population obtained from for example, Step B and included in Step C.
  • the 213 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO transient alteration of protein expression occurs in the bulk TIL population prior to second expansion, including, for example in the TIL population obtained from for example, Step C and prior to its expansion in Step D.
  • the transient alteration of protein expression occurs during the second expansion, including, for example in the TIL population expanded in for example, Step D (e.g. the third population of TILs).
  • the transient alteration of protein expression occurs after the second expansion, including, for example in the TIL population obtained from the expansion in for example, Step D.
  • a method of transiently altering protein expression in a population of TILs includes the step of electroporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. J.1991, 60, 297-306, and U.S. Patent Application Publication No. 2014/0227237 A1, the disclosures of each of which are incorporated by reference herein. In some embodiments, a method of transiently altering protein expression in population of TILs includes the step of calcium phosphate transfection.
  • a method of transiently altering protein expression in a population of TILs includes the step of liposomal transfection.
  • Liposomal transfection methods such as methods that employ a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n- trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and in U.S.
  • DOTMA cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n- trimethylammonium chloride
  • DOPE dioleoyl phophotidylethanolamine
  • a method of transiently altering protein expression in a population of TILs includes the step of transfection using methods described in U.S. Patent Nos.5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.
  • transient alteration of protein expression results in an increase in Stem Memory T cells (TSCMs).
  • TSCMs are early progenitors of antigen-experienced central memory T cells. TSCMs generally display the long-term survival, self-renewal, and multipotency abilities that define stem cells, and are generally desirable for the generation of effective TIL products. TSCM have shown enhanced anti-tumor activity compared with other T cell subsets in mouse models of adoptive cell transfer (Gattinoni et al. Nat Med 2009, 2011; Gattinoni, Nature Rev. Cancer, 2012; Cieri et al. Blood 2013). In some embodiments, transient alteration of protein expression results in a TIL 214 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO population with a composition comprising a high proportion of TSCM.
  • transient alteration of protein expression results in an at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% increase in TSCM percentage.
  • transient alteration of protein expression results in an at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in TSCMs in the TIL population.
  • transient alteration of protein expression results in a TIL population with at least at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCMs.
  • transient alteration of protein expression results in a therapeutic TIL population with at least at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCMs.
  • transient alteration of protein expression results in rejuvenation of antigen-experienced T-cells.
  • rejuvenation includes, for example, increased proliferation, increased T-cell activation, and/or increased antigen recognition.
  • transient alteration of protein expression alters the expression in a large fraction of the T-cells in order to preserve the tumor-derived TCR repertoire. In some embodiments, transient alteration of protein expression does not alter the tumor-derived TCR repertoire. In some embodiments, transient alteration of protein expression maintains the tumor- derived TCR repertoire. [00977] In some embodiments, transient alteration of protein results in altered expression of a particular gene. In some embodiments, transient alteration of protein results in altered expression of an immune checkpoint gene.
  • the transient alteration of protein expression targets a gene including but not limited to PD-1 (also referred to as PDCD1 or CC279), TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-12, IL-15, IL- 21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGF ⁇ , CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1 ⁇ ), CCL4 (MIP1- ⁇ ), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and/or cAMP protein kinase A (PKA).
  • PD-1 also referred to as PDCD1 or CC279
  • TGFBR2 CCR4/5
  • CBLB CBL
  • the transient alteration of protein expression targets PD-1. In some embodiments, the transient alteration of protein expression targets TGFBR2. In some embodiments, the transient alteration of protein expression targets CCR4/5. In some embodiments, the transient alteration of protein expression targets CBLB. In some embodiments, the transient alteration of protein expression targets CISH. In some embodiments, the transient alteration of protein expression targets CCRs 215 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO (chimeric co-stimulatory receptors). In some embodiments, the transient alteration of protein expression targets IL-2. In some embodiments, the transient alteration of protein expression targets IL-12.
  • the transient alteration of protein expression targets IL-15. In some embodiments, the transient alteration of protein expression targets IL-21. In some embodiments, the transient alteration of protein expression targets NOTCH 1/2 ICD. In some embodiments, the transient alteration of protein expression targets TIM3. In some embodiments, the transient alteration of protein expression targets LAG3. In some embodiments, the transient alteration of protein expression targets TIGIT. In some embodiments, the transient alteration of protein expression targets TGF ⁇ . In some embodiments, the transient alteration of protein expression targets CCR1. In some embodiments, the transient alteration of protein expression targets CCR2. In some embodiments, the transient alteration of protein expression targets CCR4.
  • the transient alteration of protein expression targets CCR5. In some embodiments, the transient alteration of protein expression targets CXCR1. In some embodiments, the transient alteration of protein expression targets CXCR2. In some embodiments, the transient alteration of protein expression targets CSCR3. In some embodiments, the transient alteration of protein expression targets CCL2 (MCP-1). In some embodiments, the transient alteration of protein expression targets CCL3 (MIP-1 ⁇ ). In some embodiments, the transient alteration of protein expression targets CCL4 (MIP1- ⁇ ). In some embodiments, the transient alteration of protein expression targets CCL5 (RANTES). In some embodiments, the transient alteration of protein expression targets CXCL1.
  • the transient alteration of protein expression targets CXCL8. In some embodiments, the transient alteration of protein expression targets CCL22. In some embodiments, the transient alteration of protein expression targets CCL17. In some embodiments, the transient alteration of protein expression targets VHL. In some embodiments, the transient alteration of protein expression targets CD44. In some embodiments, the transient alteration of protein expression targets PIK3CD. In some embodiments, the transient alteration of protein expression targets SOCS1. In some embodiments, the transient alteration of protein expression targets cAMP protein kinase A (PKA). [00978] In some embodiments, the transient alteration of protein expression results in increased and/or overexpression of a chemokine receptor.
  • PKA protein kinase A
  • the chemokine receptor that is overexpressed by transient protein expression includes a receptor with a ligand that includes but is not limited to CCL2 (MCP-1), CCL3 (MIP-1 ⁇ ), CCL4 (MIP1- ⁇ ), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17.
  • MCP-1 CCL2
  • MIP-1 ⁇ CCL3
  • MIP1- ⁇ CCL4
  • CCL5 RANTES
  • CXCL1, CXCL8, CCL22 and/or CCL17.
  • the transient alteration of protein expression results in a decrease and/or reduced expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGF ⁇ R2, and/or TGF ⁇ (including resulting in, for example, TGF ⁇ pathway blockade).
  • the transient alteration of protein expression results in a decrease and/or reduced expression of CBLB (CBL-B).
  • CBL-B CBL-B
  • the transient alteration of protein expression results in a decrease and/or reduced expression of CISH.
  • the transient alteration of protein expression results in increased and/or overexpression of chemokine receptors in order to, for example, improve TIL trafficking or movement to the tumor site.
  • the transient alteration of protein expression results in increased and/or overexpression of a CCR (chimeric co-stimulatory receptor).
  • the transient alteration of protein expression results in increased and/or overexpression of a chemokine receptor selected from the group consisting of CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR3.
  • a chemokine receptor selected from the group consisting of CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR3.
  • the transient alteration of protein expression results in increased and/or overexpression of an interleukin.
  • the transient alteration of protein expression results in increased and/or overexpression of an interleukin selected from the group consisting of IL-2, IL-12, IL-15, and/or IL-21.
  • the transient alteration of protein expression results in increased and/or overexpression of NOTCH 1/2 ICD.
  • the transient alteration of protein expression results in increased and/or overexpression of VHL. In some embodiments, the transient alteration of protein expression results in increased and/or overexpression of CD44. In some embodiments, the transient alteration of protein expression results in increased and/or overexpression of PIK3CD. In some embodiments, the transient alteration of protein expression results in increased and/or overexpression of SOCS1, [00983] In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of cAMP protein kinase A (PKA). [00984] In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of an immune checkpoint.
  • PKA cAMP protein kinase A
  • the transient alteration of protein expression results in decreased and/or reduced expression of a molecule selected from the group consisting of PD-1, TGIT, TET2, TGF ⁇ R2, PRA, BAFF (BR3),CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGF ⁇ , PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, 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, PR
  • the transient alteration of protein expression results in decreased and/or reduced expression of two molecules selected from the 217 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO group consisting of PD-1, TGIT, TET2, TGF ⁇ R2, PRA, BAFF (BR3), CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGF ⁇ , PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, 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,
  • the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and one molecule selected from the group consisting of TGIT, TET2, TGF ⁇ R2, PRA, BAFF (BR3), CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGF ⁇ , PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, 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, PRDM
  • the transient alteration of protein expression results in decreased and/or reduced expression of PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and one of LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and LAG3. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and CISH. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of PD-1 and CBLB.
  • the transient alteration of protein expression results in decreased and/or reduced expression of LAG3 and CISH. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of LAG3 and CBLB. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of CISH and CBLB. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of TIM3 and PD-1. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of TIM3 and LAG3. In some embodiments, the transient alteration of protein expression results in decreased and/or reduced expression of TIM3 and CISH.
  • the transient alteration of protein expression results in decreased and/or reduced expression of TIM3 and CBLB. 218 DB1/ 152908642.1 A orney Docket No.: 116983-5130-WO [00985]
  • an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof is inserted by a gammaretroviral or lentiviral method (as described in Forget, et al., Frontiers Immunology 2017, 8, 908 or Peng, et al., Clin.
  • the transient alteration of protein expression results in decreased and/or reduced expression of one or two molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF ⁇ R2, PKA, CBLB, BAFF (BR3), and combinations thereof, and increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.
  • the transient alteration of protein expression results in decreased and/or reduced expression of one or two molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, CBLB, and combinations thereof, and increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des procédés améliorés et/ou raccourcis et des procédés de préparation de TIL afin de préparer des populations thérapeutiques de TIL ayant une efficacité thérapeutique accrue pour le traitement du cancer de l'endomètre. Dans un mode de réalisation, l'invention concerne des procédés et des compositions pour le traitement du cancer de l'utérus à l'aide de thérapies lymphocytaires infiltrant les tumeurs. Dans un mode de réalisation, l'invention concerne des procédés et des compositions pour le traitement du cancer de l'endomètre à l'aide de thérapies lymphocytaires infiltrant les tumeurs, le cancer de l'endomètre étant réséqué et traité pour préparer des lymphocytes infiltrant les tumeurs.
PCT/US2024/054513 2023-11-06 2024-11-05 Traitement de cancers endométriaux avec des thérapies lymphocytaires infiltrant les tumeurs Pending WO2025101484A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202363596588P 2023-11-06 2023-11-06
US63/596,588 2023-11-06
US202463694763P 2024-09-13 2024-09-13
US63/694,763 2024-09-13
US202463699700P 2024-09-26 2024-09-26
US63/699,700 2024-09-26

Publications (1)

Publication Number Publication Date
WO2025101484A1 true WO2025101484A1 (fr) 2025-05-15

Family

ID=93590894

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/054513 Pending WO2025101484A1 (fr) 2023-11-06 2024-11-05 Traitement de cancers endométriaux avec des thérapies lymphocytaires infiltrant les tumeurs

Country Status (1)

Country Link
WO (1) WO2025101484A1 (fr)

Citations (203)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0154316A2 (fr) 1984-03-06 1985-09-11 Takeda Chemical Industries, Ltd. Lymphokine chimiquement modifiée et son procédé de préparation
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US4766106A (en) 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
WO1988007089A1 (fr) 1987-03-18 1988-09-22 Medical Research Council Anticorps alteres
US4897355A (en) 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4902502A (en) 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1990014074A1 (fr) 1989-05-22 1990-11-29 Vical, Inc. Formulations liposomiques ameliorees de nucleotides et d'analogues de nucleotides
EP0401384A1 (fr) 1988-12-22 1990-12-12 Kirin-Amgen, Inc. Facteur de stimulation de colonies de granulocytes modifies chimiquement
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
US5019034A (en) 1988-01-21 1991-05-28 Massachusetts Institute Of Technology Control of transport of molecules across tissue using electroporation
WO1991016024A1 (fr) 1990-04-19 1991-10-31 Vical, Inc. Lipides cationiques servant a l'apport intracellulaire de molecules biologiquement actives
WO1991017424A1 (fr) 1990-05-03 1991-11-14 Vical, Inc. Acheminement intracellulaire de substances biologiquement actives effectue a l'aide de complexes de lipides s'auto-assemblant
US5089261A (en) 1989-01-23 1992-02-18 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US5128257A (en) 1987-08-31 1992-07-07 Baer Bradford W Electroporation apparatus and process
US5137817A (en) 1990-10-05 1992-08-11 Amoco Corporation Apparatus and method for electroporation
US5173158A (en) 1991-07-22 1992-12-22 Schmukler Robert E Apparatus and methods for electroporation and electrofusion
US5206344A (en) 1985-06-26 1993-04-27 Cetus Oncology Corporation Interleukin-2 muteins and polymer conjugation thereof
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
US5232856A (en) 1990-06-25 1993-08-03 Firth Kevin L Electroporation device
US5273525A (en) 1992-08-13 1993-12-28 Btx Inc. Injection and electroporation apparatus for drug and gene delivery
US5279833A (en) 1990-04-04 1994-01-18 Yale University Liposomal transfection of nucleic acids into animal cells
US5304120A (en) 1992-07-01 1994-04-19 Btx Inc. Electroporation method and apparatus for insertion of drugs and genes into endothelial cells
US5318514A (en) 1992-08-17 1994-06-07 Btx, Inc. Applicator for the electroporation of drugs and genes into surface cells
WO1995012673A1 (fr) 1993-11-03 1995-05-11 The Board Of Trustees Of The Leland Stanford Junior University Recepteur situe sur la surface de lymphocytes t actives, appele act-4
WO1995021925A1 (fr) 1994-02-14 1995-08-17 American Cyanamid Company Recepteurs heterologues couples a des proteines g et exprimes dans la levure, leur fusion avec des proteines g et leur utilisation dans des dosages biologiques
EP0672141A1 (fr) 1992-10-23 1995-09-20 Immunex Corporation Procede de preparation de proteines oligomeres solubles
WO1996014339A1 (fr) 1994-11-05 1996-05-17 The Wellcome Foundation Limited Anticorps
US5593875A (en) 1994-09-08 1997-01-14 Genentech, Inc. Methods for calcium phosphate transfection
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
WO1998005787A1 (fr) 1996-08-02 1998-02-12 Bristol-Myers Squibb Company Procede servant a inhiber la toxicite provoquee par les immunoglobulines provenant de l'utilisation d'immunoglobulines en therapie et en diagnostic in vivo
WO1998013526A1 (fr) 1996-09-26 1998-04-02 Oligos Etc. Inc. Oligonucleotides antisens chimeres a trois composants
US5739277A (en) 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
WO1998023289A1 (fr) 1996-11-27 1998-06-04 The General Hospital Corporation Modulation de la fixation de l'igg au fcrn
US5766902A (en) 1993-08-20 1998-06-16 Therexsys Limited Transfection process
WO1998030679A1 (fr) 1997-01-10 1998-07-16 Life Technologies, Inc. Substitut de serum pour cellules souches embryonnaires
WO1998040510A1 (fr) 1997-03-11 1998-09-17 Regents Of The University Of Minnesota Systeme transposon a base d'adn permettant d'introduire de l'acide nucleique dans l'adn d'une cellule
US5834250A (en) 1988-10-28 1998-11-10 Genentech, Inc. Method for identifying active domains and amino acid residues in polypeptides and hormone variants
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US5908635A (en) 1994-08-05 1999-06-01 The United States Of America As Represented By The Department Of Health And Human Services Method for the liposomal delivery of nucleic acids
US5928893A (en) 1995-04-08 1999-07-27 Lg Chemical Ltd. Monoclonal antibody specific for human 4-1BB and cell line producing same
WO1999051642A1 (fr) 1998-04-02 1999-10-14 Genentech, Inc. Variants d'anticorps et fragments de ceux-ci
WO1999054342A1 (fr) 1998-04-20 1999-10-28 Pablo Umana Modification par glycosylation d'anticorps aux fins d'amelioration de la cytotoxicite cellulaire dependant des anticorps
WO1999058572A1 (fr) 1998-05-08 1999-11-18 Cambridge University Technical Services Limited Molecules de liaison derivees d'immunoglobulines ne declenchant pas de lyse dependante du complement
US6010613A (en) 1995-12-08 2000-01-04 Cyto Pulse Sciences, Inc. Method of treating materials with pulsed electrical fields
US6025337A (en) 1994-06-27 2000-02-15 Johns Hopkins University Solid microparticles for gene delivery
WO2000009560A2 (fr) 1998-08-17 2000-02-24 Abgenix, Inc. Production de molecules modifiees avec demi-vie serique prolongee
US6056938A (en) 1995-02-21 2000-05-02 Imarx Pharaceutical Corp. Cationic lipids and the use thereof
WO2000032767A1 (fr) 1998-12-03 2000-06-08 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RECEPTEURS SOLUBLES DE RECOMBINAISON DU Fc
WO2000042072A2 (fr) 1999-01-15 2000-07-20 Genentech, Inc. Variants polypeptidiques ayant une fonction effectrice alteree
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
WO2001009312A2 (fr) 1999-08-03 2001-02-08 Sequitur, Inc. Perfectionnements apportes a des oligomeres antisens, administration d'oligomeres antisens, et identification de cibles d'oligomeres antisens
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
US6210669B1 (en) 1996-10-11 2001-04-03 Bristol-Myers Squibb Co. Methods and compositions for immunomodulation
US6242195B1 (en) 1998-04-02 2001-06-05 Genentech, Inc. Methods for determining binding of an analyte to a receptor
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
US6303121B1 (en) 1992-07-30 2001-10-16 Advanced Research And Technology Method of using human receptor protein 4-1BB
WO2001081565A2 (fr) 2000-04-27 2001-11-01 Max-Delbrück-Centrum für Molekulare Medizin Sleeping beauty, un vecteur transposon a large gamme d'hotes pour la transformation genetique chez les vertebres
US6312700B1 (en) 1998-02-24 2001-11-06 Andrew D. Weinberg Method for enhancing an antigen specific immune response with OX-40L
EP1176195A1 (fr) 1999-04-09 2002-01-30 Kyowa Hakko Kogyo Co., Ltd. Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle
US6362325B1 (en) 1988-11-07 2002-03-26 Advanced Research And Technology Institute, Inc. Murine 4-1BB gene
WO2002044215A2 (fr) 2000-12-01 2002-06-06 Cockbain, Julian Produit
WO2002060919A2 (fr) 2000-12-12 2002-08-08 Medimmune, Inc. Molecules a demi-vies longues, compositions et utilisations de celles-ci
US6475994B2 (en) 1998-01-07 2002-11-05 Donald A. Tomalia Method and articles for transfection of genetic material
US6479626B1 (en) 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6534484B1 (en) 1995-06-07 2003-03-18 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
WO2003035835A2 (fr) 2001-10-25 2003-05-01 Genentech, Inc. Compositions de glycoproteine
WO2003074569A2 (fr) 2002-03-01 2003-09-12 Immunomedics, Inc. Mutations ponctuelles dans un anticorps bispecifique, permettant d'augmenter le taux de clairance
US6627442B1 (en) 2000-08-31 2003-09-30 Virxsys Corporation Methods for stable transduction of cells with hiv-derived viral vectors
WO2004016750A2 (fr) 2002-08-14 2004-02-26 Macrogenics, Inc. Anticorps specifiques du recepteur fc$g(g)riib et procedes d'utilisation de ces anticorps
US6706289B2 (en) 2000-10-31 2004-03-16 Pr Pharmaceuticals, Inc. Methods and compositions for enhanced delivery of bioactive molecules
WO2004029207A2 (fr) 2002-09-27 2004-04-08 Xencor Inc. Variants fc optimises et methodes destinees a leur generation
WO2004035752A2 (fr) 2002-10-15 2004-04-29 Protein Design Labs, Inc. Modification d'affinites de liaison pour fcrn ou de demi-vies seriques d'anticorps par mutagenese
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US6746838B1 (en) 1997-05-23 2004-06-08 Gendaq Limited Nucleic acid binding proteins
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
WO2004063351A2 (fr) 2003-01-09 2004-07-29 Macrogenics, Inc. Identification et elaboration d'anticorps avec des regions du variant fc et procedes d'utilisation associes
WO2004074455A2 (fr) 2003-02-20 2004-09-02 Applied Molecular Evolution Variants de la region fc
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
US20040224405A1 (en) 2003-05-06 2004-11-11 Dharmacon Inc. siRNA induced systemic gene silencing in mammalian systems
WO2004099249A2 (fr) 2003-05-02 2004-11-18 Xencor, Inc. Variants fc optimises et leurs procedes de generation
US6887673B2 (en) 2002-07-30 2005-05-03 Bristol-Myers Squibb Company Humanized antibodies against human 4-1BB
US20050095244A1 (en) 2003-10-10 2005-05-05 Maria Jure-Kunkel Fully human antibodies against human 4-1BB
WO2005040217A2 (fr) 2003-10-17 2005-05-06 Cambridge University Technical Services Limited Polypeptides comprenant des regions constantes modifiees
US20050106717A1 (en) 2003-10-08 2005-05-19 Wilson John R. Cell culture methods and devices utilizing gas permeable materials
WO2005070963A1 (fr) 2004-01-12 2005-08-04 Applied Molecular Evolution, Inc Variants de la region fc
WO2005077981A2 (fr) 2003-12-22 2005-08-25 Xencor, Inc. Polypeptides fc a nouveaux sites de liaison de ligands fc
WO2005092925A2 (fr) 2004-03-24 2005-10-06 Xencor, Inc. Variantes d'immunoglobuline a l'exterieur de la region fc
WO2005123780A2 (fr) 2004-04-09 2005-12-29 Protein Design Labs, Inc. Modification des affinites de liaison pour le fcrn ou de la demi-vie serique d'anticorps par mutagenese
WO2006019447A1 (fr) 2004-07-15 2006-02-23 Xencor, Inc. Variantes genetiques de fc optimisees
US7013219B2 (en) 1999-01-12 2006-03-14 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
WO2006047350A2 (fr) 2004-10-21 2006-05-04 Xencor, Inc. Variants d'immunoglobuline igg a fonction effectrice optimisee
WO2006085967A2 (fr) 2004-07-09 2006-08-17 Xencor, Inc. Anticorps monoclonaux optimises anti-cd20 a variants fc
WO2006121810A2 (fr) 2005-05-06 2006-11-16 Providence Health System Proteine de fusion trimere immunoglobulinique ox-40 et procedes d'utilisation
WO2006122442A1 (fr) 2005-05-14 2006-11-23 Fudan University Piggybac utilise comme outil pour la manipulation genetique et l'analyse chez les vertebres
US20060276635A1 (en) 2002-09-05 2006-12-07 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20070031844A1 (en) 2002-11-14 2007-02-08 Anastasia Khvorova Functional and hyperfunctional siRNA
US7189705B2 (en) 2000-04-20 2007-03-13 The University Of British Columbia Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers
US20070213521A1 (en) 2002-11-14 2007-09-13 Dharmacon, Inc. siRNA targeting cell division cycle 25 (CDC25C)
WO2008025516A2 (fr) 2006-08-28 2008-03-06 Apogenix Gmbh Protéines de fusion de superfamille
WO2009007120A2 (fr) 2007-07-10 2009-01-15 Apogenix Gmbh Protéines de fusion collectines de la superfamille des tnf
WO2009045457A2 (fr) 2007-10-02 2009-04-09 Rxi Pharmaceuticals Corp. CONSTRUCTIONS D'ARNi À STRUCTURE TRIPARTITE
US7550140B2 (en) 2002-06-13 2009-06-23 Crucell Holland B.V. Antibody to the human OX40 receptor
WO2009102427A2 (fr) 2008-02-11 2009-08-20 Rxi Pharmaceuticals Corp. Polynucléotides d'arni modifiés et leurs utilisations
US7585849B2 (en) 1999-03-24 2009-09-08 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
WO2010003766A2 (fr) 2008-06-17 2010-01-14 Apogenix Gmbh Récepteurs multimériques tnf
WO2010010051A1 (fr) 2008-07-21 2010-01-28 Apogenix Gmbh Molécules à une seule chaîne
WO2010033247A2 (fr) 2008-09-22 2010-03-25 Rxi Pharmaceuticals Corporation Composés d'arni de taille réduite à auto-délivrance
US7687070B2 (en) 1994-02-11 2010-03-30 Life Technologies Corporation Reagents for intracellular delivery of macromolecules
US7696175B2 (en) 2004-10-29 2010-04-13 University Of Southern California Combination cancer immunotherapy with co-stimulatory molecules
WO2010042433A1 (fr) 2008-10-06 2010-04-15 Bristol-Myers Squibb Company Combinaison d'anticorps cd137 et d'anticorps ctla-4 pour le traitement de maladies prolifératives
US20100136030A1 (en) 2007-02-27 2010-06-03 Lamhamedi-Cherradi Salah-Eddine Antagonist ox40 antibodies and their use in the treatment of inflammatory and autoimmune diseases
WO2010078536A1 (fr) 2009-01-05 2010-07-08 Rxi Pharmaceuticals Corporation Inhibition de pcsk9 par arni
WO2010078966A1 (fr) 2009-01-09 2010-07-15 Apogenix Gmbh Protéines de fusion formant des trimères
WO2010085699A2 (fr) 2009-01-23 2010-07-29 The Johns Hopkins University Transposon piggybac de mammifère et procédés d'utilisation
WO2010090762A1 (fr) 2009-02-04 2010-08-12 Rxi Pharmaceuticals Corporation Duplexes d'arn avec régions de nucléotide phosphorothioate à brin unique pour fonctionnalité supplémentaire
WO2010099296A1 (fr) 2009-02-26 2010-09-02 Transposagen Biopharmaceuticals, Inc. Transposases piggybac hyperactives
WO2010099301A2 (fr) 2009-02-25 2010-09-02 The Johns Hopkins University Variants de transposon piggybac et procédés d'utilisation
US20110136228A1 (en) 2009-12-08 2011-06-09 Vera Juan F Methods of cell culture for adoptive cell therapy
US7961515B2 (en) 2006-07-14 2011-06-14 Semiconductor Energy Laboratory Co., Ltd. Nonvolatile memory
US7960515B2 (en) 2007-12-14 2011-06-14 Bristol-Myers Squibb Company Binding molecules to the human OX40 receptor
US20110201118A1 (en) 2010-06-14 2011-08-18 Iowa State University Research Foundation, Inc. Nuclease activity of tal effector and foki fusion protein
US8008449B2 (en) 2005-05-09 2011-08-30 Medarex, Inc. Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
WO2011109698A1 (fr) 2010-03-04 2011-09-09 Rxi Pharmaceuticals Corporation Formulations et procédés d'administration ciblée à des cellules phagocytaires
WO2011119871A1 (fr) 2010-03-24 2011-09-29 Rxi Phrmaceuticals Corporation Arn interférant dans des indications oculaires
WO2011119887A1 (fr) 2010-03-24 2011-09-29 Rxi Pharmaceuticals Corporation Arn interférant dans des indications dermiques et fibrosiques
WO2011119852A1 (fr) 2010-03-24 2011-09-29 Rxi Pharmaceuticals Corporation Composés d'arni de taille réduite s'auto-administrant
WO2012027328A2 (fr) 2010-08-23 2012-03-01 Board Of Regents, The University Of Texas System Anticorps anti-ox40 et leurs procédés d'utilisation
WO2012032433A1 (fr) 2010-09-09 2012-03-15 Pfizer Inc. Molécules de liaison 4-1bb
WO2012065086A1 (fr) 2010-11-12 2012-05-18 Nektar Therapeutics Conjugués d'une fraction il-2 et d'un polymère
US8252755B2 (en) 2006-09-22 2012-08-28 Dharmacon, Inc. Duplex oligonucleotide complexes and methods for gene silencing by RNA interference
WO2012129201A1 (fr) 2011-03-22 2012-09-27 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés de mise en croissance de lymphocytes infiltrant une tumeur dans contenants perméables au gaz
WO2012177788A1 (fr) 2011-06-20 2012-12-27 La Jolla Institute For Allergy And Immunology Modulateurs de 4-1bb et réponses immunitaires
WO2013028231A1 (fr) 2011-08-23 2013-02-28 Board Of Regents, The University Of Texas System Anticorps anti-ox40 et leurs procédés d'utilisation
WO2013038191A2 (fr) 2011-09-16 2013-03-21 Bioceros B.V. Anticorps anti-cd134 (ox40) et leurs utilisations
WO2013059343A1 (fr) 2011-10-17 2013-04-25 Massachusetts Institute Of Technology Administration intracellulaire
US20130102075A1 (en) 2009-12-08 2013-04-25 Juan F. Vera Methods of cell culture for adoptive cell therapy
US20130115617A1 (en) 2009-12-08 2013-05-09 John R. Wilson Methods of cell culture for adoptive cell therapy
US20130117869A1 (en) 2011-04-05 2013-05-09 Cellectis S.A. Method for the generation of compact tale-nucleases and uses thereof
US8586526B2 (en) 2010-05-17 2013-11-19 Sangamo Biosciences, Inc. DNA-binding proteins and uses thereof
WO2013173835A1 (fr) 2012-05-18 2013-11-21 Wilson Wolf Manufacturing Corporation Procédés de culture cellulaire améliorés pour thérapie cellulaire adoptive
US20130315884A1 (en) 2012-05-25 2013-11-28 Roman Galetto Methods for engineering allogeneic and immunosuppressive resistant t cell for immunotherapy
WO2013188427A1 (fr) 2012-06-11 2013-12-19 Wilson Wolf Manufacturing Corporation Procédés améliorés de culture cellulaire pour une thérapie cellulaire adoptive
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US20140148362A1 (en) 2002-11-14 2014-05-29 Thermo Fisher Scientific Biosciences Inc. Methods and Compositions for Selecting siRNA of Improved Functionality
US8795965B2 (en) 2012-12-12 2014-08-05 The Broad Institute, Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US20140227237A1 (en) 2011-09-16 2014-08-14 The Trustees Of The University Of Pennsylvania Rna engineered t cells for the treatment of cancer
US8815818B2 (en) 2008-07-18 2014-08-26 Rxi Pharmaceuticals Corporation Phagocytic cell delivery of RNAI
WO2014148895A1 (fr) 2013-03-18 2014-09-25 Biocerox Products B.V. Anticorps anti-cd134 (ox40) humanisés et leurs utilisations
US20140295426A1 (en) 2011-07-28 2014-10-02 Veridex Llc Methods for Diagnosing Cancer by Characterization of Tumor Cells Associated with Pleural or Serous Fluids
US8859229B2 (en) 2007-02-02 2014-10-14 Yale University Transient transfection with RNA
US8865406B2 (en) 2012-12-12 2014-10-21 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8889356B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8906616B2 (en) 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US20140377739A1 (en) 2013-06-24 2014-12-25 Wilson Wolf Manufacturing Closed system device and methods for gas permeable cell culture process
WO2015006700A1 (fr) 2013-07-12 2015-01-15 University Of South Alabama Vecteurs piggybac minimaux pour intégration génomique
US8962804B2 (en) 2010-10-08 2015-02-24 City Of Hope Meditopes and meditope-binding antibodies and uses thereof
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US20150190506A1 (en) 2013-12-17 2015-07-09 Genentech, Inc. Combination therapy comprising ox40 binding agonists and pd-1 axis binding antagonists
US20150203871A1 (en) 2012-06-05 2015-07-23 Cellectis Transcription Activator-Like Effector (TALE) Fusion Protein
WO2015119923A1 (fr) 2014-02-04 2015-08-13 Pfizer Inc. Combinaison d'un antagoniste de pd -1 et d'un agoniste de 4-1bb pour le traitement du cancer
US9228180B2 (en) 2007-07-04 2016-01-05 Max-Delbruck-Centrum Fur Molekulare Medizin Polypeptide variants of sleeping beauty transposase
US20160010058A1 (en) 2013-03-01 2016-01-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Methods of producing enriched populations of tumor-reactive t cells from tumor
US20160120906A1 (en) 2013-05-13 2016-05-05 Cellectis Methods for engineering highly active t cell for immunotheraphy
WO2016094845A2 (fr) 2014-12-12 2016-06-16 Woolf Tod M Compositions et procédés d'édition d'acides nucléiques dans des cellules à l'aide d'oligonucléotides
US20160193242A1 (en) 2002-11-14 2016-07-07 Thermo Fisher Scientific Inc. Methods and Compositions for Selecting siRNA of Improved Functionality
US20160194646A1 (en) 2002-11-14 2016-07-07 Thermo Fisher Scientific Inc. Methods and Compositions for Selecting siRNA of Improved Functionality
US20160201065A1 (en) 2002-11-14 2016-07-14 Thermo Fisher Scientific Inc. Methods and Compositions for Selecting siRNA of Improved Functionality
US20160230188A1 (en) 2007-02-02 2016-08-11 Yale University Method of de-differentiating and re-differentiating somatic cells using rna
US20160304873A1 (en) 2013-12-02 2016-10-20 Mirimmune Inc. Immunotherapy of Cancer
US20160319278A1 (en) 2015-04-03 2016-11-03 University Of Massachusetts Fully stabilized asymmetric sirna
WO2017008063A1 (fr) 2015-07-09 2017-01-12 Massachusetts Institute Of Technology Administration de substances à des cellules anucléées
US20170043024A1 (en) 2015-08-14 2017-02-16 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
US20170107490A1 (en) 2014-06-11 2017-04-20 Polybiocept Ab Expansion of lymphocytes with a cytokine composition for active cellular immunotherapy
WO2017070151A1 (fr) 2015-10-19 2017-04-27 Rxi Pharmaceuticals Corporation Composés d'acides nucléiques de taille réduite à auto-administration ciblant des longs arn non codants
WO2017123663A1 (fr) 2016-01-12 2017-07-20 Sqz Biotechnologies Company Administration intracellulaire de complexes
US9790490B2 (en) 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
US20170312367A1 (en) 2016-01-31 2017-11-02 University Of Massachusetts Branched oligonucleotides
WO2017193053A1 (fr) 2016-05-06 2017-11-09 Woolf Tod M Procédés perfectionnés d'édition de génome avec et sans nucléases programmables
WO2018081473A1 (fr) 2016-10-26 2018-05-03 Iovance Biotherapeutics, Inc. Re-stimulation de lymphocytes infiltrant les tumeurs cryoconservés
WO2018132496A1 (fr) 2017-01-10 2018-07-19 Nektar Therapeutics Conjugués polymères à bras multiples de composés agonistes de tlr et méthodes de traitement immunothérapeutiques associées
US10041077B2 (en) 2014-04-09 2018-08-07 Dna2.0, Inc. DNA vectors, transposons and transposases for eukaryotic genome modification
US20180245089A1 (en) 2015-09-04 2018-08-30 Sqz Biotechnologies Company Intracellular delivery of biomolecules to cells comprising a cell wall
US20180282694A1 (en) 2017-03-29 2018-10-04 Iovance Biotherapeutics, Inc. Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy
US10183979B2 (en) 2012-06-08 2019-01-22 Alkermes, Inc. Fusion polypeptides comprising mucin-domain polypeptide linkers
WO2019046815A1 (fr) 2017-08-31 2019-03-07 Poseida Therapeutics, Inc. Système de transposon et procédés d'utilisation
WO2019160829A1 (fr) 2018-02-13 2019-08-22 Iovance Biotherapeutics, Inc. Expansion de lymphocytes infiltrant les tumeurs (til) avec des antagonistes du récepteur a2a de l'adénosine et combinaisons thérapeutiques de til et d'antagonistes du récepteur a2a de l'adénosine
US20190275133A1 (en) 2016-11-10 2019-09-12 Nektar Therapeutics Immunotherapeutic tumor treatment method
US20190307796A1 (en) 2016-06-03 2019-10-10 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Use of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (pgc1alpha) agonists to improve ex vivo expansion of tumor infiltrating lymphocytes (tils)
US20200181220A1 (en) 2017-08-03 2020-06-11 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases
US20200270334A1 (en) 2017-05-24 2020-08-27 Novartis Ag Antibody-cytokine engrafted proteins and methods of use in the treatment of cancer
US20200330601A1 (en) 2019-02-06 2020-10-22 Synthorx, Inc. IL-2 Conjugates and Methods of Use Thereof
US20210038684A1 (en) 2019-06-11 2021-02-11 Alkermes Pharma Ireland Limited Compositions and Methods for Cancer Immunotherapy
WO2022087324A1 (fr) * 2020-10-22 2022-04-28 Iovance Biotherapeutics, Inc. Système culture cellulaire et ses procédés d'utilisation
US11369678B2 (en) 2008-08-28 2022-06-28 Taiga Biotechnologies, Inc. Compositions and methods for modulating immune cells
WO2022165260A1 (fr) * 2021-01-29 2022-08-04 Iovance Biotherapeutics, Inc. Procédés de fabrication de lymphocytes infiltrant les tumeurs modifiés et leur utilisation dans la thérapie cellulaire adoptive
WO2022170219A1 (fr) * 2021-02-05 2022-08-11 Iovance Biotherapeutics, Inc. Traitement adjuvant du cancer
WO2023147486A1 (fr) * 2022-01-28 2023-08-03 Iovance Biotherapeutics, Inc. Lymphocytes infiltrant les tumeurs modifiés pour exprimer des charges utiles
WO2023196877A1 (fr) * 2022-04-06 2023-10-12 Iovance Biotherapeutics, Inc. Traitement de patients souffrant de cpnpc avec des thérapies lymphocytaires infiltrant les tumeurs

Patent Citations (289)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0154316A2 (fr) 1984-03-06 1985-09-11 Takeda Chemical Industries, Ltd. Lymphokine chimiquement modifiée et son procédé de préparation
US4897355A (en) 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US5206344A (en) 1985-06-26 1993-04-27 Cetus Oncology Corporation Interleukin-2 muteins and polymer conjugation thereof
US4766106A (en) 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US5648260A (en) 1987-03-18 1997-07-15 Scotgen Biopharmaceuticals Incorporated DNA encoding antibodies with altered effector functions
WO1988007089A1 (fr) 1987-03-18 1988-09-22 Medical Research Council Anticorps alteres
US5128257A (en) 1987-08-31 1992-07-07 Baer Bradford W Electroporation apparatus and process
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5019034A (en) 1988-01-21 1991-05-28 Massachusetts Institute Of Technology Control of transport of molecules across tissue using electroporation
US5019034B1 (en) 1988-01-21 1995-08-15 Massachusetts Inst Technology Control of transport of molecules across tissue using electroporation
US5834250A (en) 1988-10-28 1998-11-10 Genentech, Inc. Method for identifying active domains and amino acid residues in polypeptides and hormone variants
US6905685B2 (en) 1988-11-07 2005-06-14 Byoung S. Kwon Methods of using antibodies to human receptor protein 4-1BB
US6362325B1 (en) 1988-11-07 2002-03-26 Advanced Research And Technology Institute, Inc. Murine 4-1BB gene
US6974863B2 (en) 1988-11-07 2005-12-13 Indiana University Research And Technology Corporation Antibody for 4-1BB
EP0401384A1 (fr) 1988-12-22 1990-12-12 Kirin-Amgen, Inc. Facteur de stimulation de colonies de granulocytes modifies chimiquement
US5824778A (en) 1988-12-22 1998-10-20 Kirin-Amgen, Inc. Chemically-modified G-CSF
US5089261A (en) 1989-01-23 1992-02-18 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US4902502A (en) 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
WO1990014074A1 (fr) 1989-05-22 1990-11-29 Vical, Inc. Formulations liposomiques ameliorees de nucleotides et d'analogues de nucleotides
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
US5279833A (en) 1990-04-04 1994-01-18 Yale University Liposomal transfection of nucleic acids into animal cells
WO1991016024A1 (fr) 1990-04-19 1991-10-31 Vical, Inc. Lipides cationiques servant a l'apport intracellulaire de molecules biologiquement actives
WO1991017424A1 (fr) 1990-05-03 1991-11-14 Vical, Inc. Acheminement intracellulaire de substances biologiquement actives effectue a l'aide de complexes de lipides s'auto-assemblant
US5232856A (en) 1990-06-25 1993-08-03 Firth Kevin L Electroporation device
US5137817A (en) 1990-10-05 1992-08-11 Amoco Corporation Apparatus and method for electroporation
US5173158A (en) 1991-07-22 1992-12-22 Schmukler Robert E Apparatus and methods for electroporation and electrofusion
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
US6350861B1 (en) 1992-03-09 2002-02-26 Protein Design Labs, Inc. Antibodies with increased binding affinity
US5304120A (en) 1992-07-01 1994-04-19 Btx Inc. Electroporation method and apparatus for insertion of drugs and genes into endothelial cells
US6303121B1 (en) 1992-07-30 2001-10-16 Advanced Research And Technology Method of using human receptor protein 4-1BB
US5273525A (en) 1992-08-13 1993-12-28 Btx Inc. Injection and electroporation apparatus for drug and gene delivery
US5318514A (en) 1992-08-17 1994-06-07 Btx, Inc. Applicator for the electroporation of drugs and genes into surface cells
EP0672141A1 (fr) 1992-10-23 1995-09-20 Immunex Corporation Procede de preparation de proteines oligomeres solubles
US5766902A (en) 1993-08-20 1998-06-16 Therexsys Limited Transfection process
WO1995012673A1 (fr) 1993-11-03 1995-05-11 The Board Of Trustees Of The Leland Stanford Junior University Recepteur situe sur la surface de lymphocytes t actives, appele act-4
US7687070B2 (en) 1994-02-11 2010-03-30 Life Technologies Corporation Reagents for intracellular delivery of macromolecules
WO1995021925A1 (fr) 1994-02-14 1995-08-17 American Cyanamid Company Recepteurs heterologues couples a des proteines g et exprimes dans la levure, leur fusion avec des proteines g et leur utilisation dans des dosages biologiques
US6025337A (en) 1994-06-27 2000-02-15 Johns Hopkins University Solid microparticles for gene delivery
US6410517B1 (en) 1994-06-27 2002-06-25 Johns Hopkins University Targeted gene delivery system
US5908635A (en) 1994-08-05 1999-06-01 The United States Of America As Represented By The Department Of Health And Human Services Method for the liposomal delivery of nucleic acids
US6110490A (en) 1994-08-05 2000-08-29 The United States Of America As Represented By The Department Of Health And Human Services Liposomal delivery system for biologically active agents
US5593875A (en) 1994-09-08 1997-01-14 Genentech, Inc. Methods for calcium phosphate transfection
WO1996014339A1 (fr) 1994-11-05 1996-05-17 The Wellcome Foundation Limited Anticorps
US6056938A (en) 1995-02-21 2000-05-02 Imarx Pharaceutical Corp. Cationic lipids and the use thereof
US6569997B1 (en) 1995-03-23 2003-05-27 Advanced Research And Technology Institute, Inc. Antibody specific for H4-1BB
US5928893A (en) 1995-04-08 1999-07-27 Lg Chemical Ltd. Monoclonal antibody specific for human 4-1BB and cell line producing same
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US5739277A (en) 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
US6998253B1 (en) 1995-04-14 2006-02-14 Genentech, Inc. Altered polypeptides with increased half-life
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
US6534484B1 (en) 1995-06-07 2003-03-18 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US6078490A (en) 1995-12-08 2000-06-20 Cyto Pulse Sciences, Inc. Method of treating materials with pulsed electrical fields
US6010613A (en) 1995-12-08 2000-01-04 Cyto Pulse Sciences, Inc. Method of treating materials with pulsed electrical fields
WO1998005787A1 (fr) 1996-08-02 1998-02-12 Bristol-Myers Squibb Company Procede servant a inhiber la toxicite provoquee par les immunoglobulines provenant de l'utilisation d'immunoglobulines en therapie et en diagnostic in vivo
WO1998013526A1 (fr) 1996-09-26 1998-04-02 Oligos Etc. Inc. Oligonucleotides antisens chimeres a trois composants
US5849902A (en) 1996-09-26 1998-12-15 Oligos Etc. Inc. Three component chimeric antisense oligonucleotides
US6210669B1 (en) 1996-10-11 2001-04-03 Bristol-Myers Squibb Co. Methods and compositions for immunomodulation
WO1998023289A1 (fr) 1996-11-27 1998-06-04 The General Hospital Corporation Modulation de la fixation de l'igg au fcrn
WO1998030679A1 (fr) 1997-01-10 1998-07-16 Life Technologies, Inc. Substitut de serum pour cellules souches embryonnaires
US6821505B2 (en) 1997-03-03 2004-11-23 Board Of Regents, The University Of Texas System Immunoglobin-like domains with increased half lives
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
WO1998040510A1 (fr) 1997-03-11 1998-09-17 Regents Of The University Of Minnesota Systeme transposon a base d'adn permettant d'introduire de l'acide nucleique dans l'adn d'une cellule
US6489458B2 (en) 1997-03-11 2002-12-03 Regents Of The University Of Minnesota DNA-based transposon system for the introduction of nucleic acid into DNA of a cell
US6746838B1 (en) 1997-05-23 2004-06-08 Gendaq Limited Nucleic acid binding proteins
US7241573B2 (en) 1997-05-23 2007-07-10 Gendaq Ltd. Nucleic acid binding proteins
US6866997B1 (en) 1997-05-23 2005-03-15 Gendaq Limited Nucleic acid binding proteins
US7241574B2 (en) 1997-05-23 2007-07-10 Gendaq Ltd. Nucleic acid binding proteins
US6475994B2 (en) 1998-01-07 2002-11-05 Donald A. Tomalia Method and articles for transfection of genetic material
US6312700B1 (en) 1998-02-24 2001-11-06 Andrew D. Weinberg Method for enhancing an antigen specific immune response with OX-40L
US7504101B2 (en) 1998-02-24 2009-03-17 Sisters Of Providence In Oregon Methods for enhancing antigen-specific immune response using antibodies that bind OX-40
US7622444B2 (en) 1998-02-24 2009-11-24 Sisters Of Providence In Oregon Methods for using OX-40 ligand to enhance an antigen specific immune response
US6903185B2 (en) 1998-03-02 2005-06-07 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6479626B1 (en) 1998-03-02 2002-11-12 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US7595376B2 (en) 1998-03-02 2009-09-29 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
US6538124B1 (en) 1998-04-02 2003-03-25 Genentech, Inc. Polypeptide variants
WO1999051642A1 (fr) 1998-04-02 1999-10-14 Genentech, Inc. Variants d'anticorps et fragments de ceux-ci
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
US6242195B1 (en) 1998-04-02 2001-06-05 Genentech, Inc. Methods for determining binding of an analyte to a receptor
WO1999054342A1 (fr) 1998-04-20 1999-10-28 Pablo Umana Modification par glycosylation d'anticorps aux fins d'amelioration de la cytotoxicite cellulaire dependant des anticorps
WO1999058572A1 (fr) 1998-05-08 1999-11-18 Cambridge University Technical Services Limited Molecules de liaison derivees d'immunoglobulines ne declenchant pas de lyse dependante du complement
WO2000009560A2 (fr) 1998-08-17 2000-02-24 Abgenix, Inc. Production de molecules modifiees avec demi-vie serique prolongee
WO2000032767A1 (fr) 1998-12-03 2000-06-08 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RECEPTEURS SOLUBLES DE RECOMBINAISON DU Fc
US7220719B2 (en) 1999-01-12 2007-05-22 Sangamo Biosciences, Inc. Modulation of endogenous gene expression in cells
US6824978B1 (en) 1999-01-12 2004-11-30 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US7013219B2 (en) 1999-01-12 2006-03-14 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6979539B2 (en) 1999-01-12 2005-12-27 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6607882B1 (en) 1999-01-12 2003-08-19 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6933113B2 (en) 1999-01-12 2005-08-23 Sangamo Biosciences, Inc. Modulation of endogenous gene expression in cells
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
WO2000042072A2 (fr) 1999-01-15 2000-07-20 Genentech, Inc. Variants polypeptidiques ayant une fonction effectrice alteree
US7030215B2 (en) 1999-03-24 2006-04-18 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
US7585849B2 (en) 1999-03-24 2009-09-08 Sangamo Biosciences, Inc. Position dependent recognition of GNN nucleotide triplets by zinc fingers
EP1176195A1 (fr) 1999-04-09 2002-01-30 Kyowa Hakko Kogyo Co., Ltd. Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle
WO2001009312A2 (fr) 1999-08-03 2001-02-08 Sequitur, Inc. Perfectionnements apportes a des oligomeres antisens, administration d'oligomeres antisens, et identification de cibles d'oligomeres antisens
US7189705B2 (en) 2000-04-20 2007-03-13 The University Of British Columbia Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers
WO2001081565A2 (fr) 2000-04-27 2001-11-01 Max-Delbrück-Centrum für Molekulare Medizin Sleeping beauty, un vecteur transposon a large gamme d'hotes pour la transformation genetique chez les vertebres
US6627442B1 (en) 2000-08-31 2003-09-30 Virxsys Corporation Methods for stable transduction of cells with hiv-derived viral vectors
US6706289B2 (en) 2000-10-31 2004-03-16 Pr Pharmaceuticals, Inc. Methods and compositions for enhanced delivery of bioactive molecules
US6794136B1 (en) 2000-11-20 2004-09-21 Sangamo Biosciences, Inc. Iterative optimization in the design of binding proteins
WO2002044215A2 (fr) 2000-12-01 2002-06-06 Cockbain, Julian Produit
WO2002060919A2 (fr) 2000-12-12 2002-08-08 Medimmune, Inc. Molecules a demi-vies longues, compositions et utilisations de celles-ci
US7083784B2 (en) 2000-12-12 2006-08-01 Medimmune, Inc. Molecules with extended half-lives, compositions and uses thereof
WO2003035835A2 (fr) 2001-10-25 2003-05-01 Genentech, Inc. Compositions de glycoproteine
WO2003074569A2 (fr) 2002-03-01 2003-09-12 Immunomedics, Inc. Mutations ponctuelles dans un anticorps bispecifique, permettant d'augmenter le taux de clairance
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
US7550140B2 (en) 2002-06-13 2009-06-23 Crucell Holland B.V. Antibody to the human OX40 receptor
US8133983B2 (en) 2002-06-13 2012-03-13 Crucell Holland B.V. Agonistic binding molecules to the human OX40 receptor
US7214493B2 (en) 2002-07-30 2007-05-08 Bristol-Myers Squibb Company Polynucleotides encoding humanized antibodies against human 4-1BB
US6887673B2 (en) 2002-07-30 2005-05-03 Bristol-Myers Squibb Company Humanized antibodies against human 4-1BB
WO2004016750A2 (fr) 2002-08-14 2004-02-26 Macrogenics, Inc. Anticorps specifiques du recepteur fc$g(g)riib et procedes d'utilisation de ces anticorps
US20060276635A1 (en) 2002-09-05 2006-12-07 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
WO2004029207A2 (fr) 2002-09-27 2004-04-08 Xencor Inc. Variants fc optimises et methodes destinees a leur generation
WO2004035752A2 (fr) 2002-10-15 2004-04-29 Protein Design Labs, Inc. Modification d'affinites de liaison pour fcrn ou de demi-vies seriques d'anticorps par mutagenese
US20160201058A1 (en) 2002-11-14 2016-07-14 Thermo Fisher Scientific Inc. METHODS AND COMPOSITIONS FOR SELECTING siRNA OF IMPROVED FUNCTIONALITY
US20170349904A1 (en) 2002-11-14 2017-12-07 Thermo Fisher Scientific Inc. Methods and Compositions for Selecting siRNA of Improved Functionality
US20120052487A9 (en) 2002-11-14 2012-03-01 Dharmacon, Inc. Methods and compositions for selecting sirna of improved functionality
US20140148362A1 (en) 2002-11-14 2014-05-29 Thermo Fisher Scientific Biosciences Inc. Methods and Compositions for Selecting siRNA of Improved Functionality
US20070031844A1 (en) 2002-11-14 2007-02-08 Anastasia Khvorova Functional and hyperfunctional siRNA
US20070039072A1 (en) 2002-11-14 2007-02-15 Dharmacon Inc. Functional and hyperfunctional siRNA
US7834170B2 (en) 2002-11-14 2010-11-16 Dharmacon, Inc. Functional and hyperfunctional siRNA
US20160193242A1 (en) 2002-11-14 2016-07-07 Thermo Fisher Scientific Inc. Methods and Compositions for Selecting siRNA of Improved Functionality
US8090542B2 (en) 2002-11-14 2012-01-03 Dharmacon Inc. Functional and hyperfunctional siRNA
US20160194646A1 (en) 2002-11-14 2016-07-07 Thermo Fisher Scientific Inc. Methods and Compositions for Selecting siRNA of Improved Functionality
US20160201065A1 (en) 2002-11-14 2016-07-14 Thermo Fisher Scientific Inc. Methods and Compositions for Selecting siRNA of Improved Functionality
US20070207974A1 (en) 2002-11-14 2007-09-06 Dharmacon Inc. Functional and hyperfunctional siRNA
US20070213521A1 (en) 2002-11-14 2007-09-13 Dharmacon, Inc. siRNA targeting cell division cycle 25 (CDC25C)
US20070213520A1 (en) 2002-11-14 2007-09-13 Dharmacon, Inc. siRNA targeting calcium/calmodulin dependent protein kinase IV (CAMK4)
US20070219362A1 (en) 2002-11-14 2007-09-20 Dharmacon, Inc. siRNA targeting azurocidin 1 (Cartionic Antimicrobial protein 37)
US20070238868A1 (en) 2002-11-14 2007-10-11 Dharmacon, Inc. siRNA targeting chemokine (C-X-C motif) receptor 4 (CXCR4)
US20180119144A1 (en) 2002-11-14 2018-05-03 Thermo Fisher Scientific Inc. METHODS AND COMPOSITIONS FOR SELECTING siRNA OF IMPROVED FUNCTIONALITY
WO2004063351A2 (fr) 2003-01-09 2004-07-29 Macrogenics, Inc. Identification et elaboration d'anticorps avec des regions du variant fc et procedes d'utilisation associes
WO2004074455A2 (fr) 2003-02-20 2004-09-02 Applied Molecular Evolution Variants de la region fc
WO2004099249A2 (fr) 2003-05-02 2004-11-18 Xencor, Inc. Variants fc optimises et leurs procedes de generation
US20040224405A1 (en) 2003-05-06 2004-11-11 Dharmacon Inc. siRNA induced systemic gene silencing in mammalian systems
US20050106717A1 (en) 2003-10-08 2005-05-19 Wilson John R. Cell culture methods and devices utilizing gas permeable materials
US20050095244A1 (en) 2003-10-10 2005-05-05 Maria Jure-Kunkel Fully human antibodies against human 4-1BB
US7288638B2 (en) 2003-10-10 2007-10-30 Bristol-Myers Squibb Company Fully human antibodies against human 4-1BB
WO2005040217A2 (fr) 2003-10-17 2005-05-06 Cambridge University Technical Services Limited Polypeptides comprenant des regions constantes modifiees
WO2005077981A2 (fr) 2003-12-22 2005-08-25 Xencor, Inc. Polypeptides fc a nouveaux sites de liaison de ligands fc
WO2005070963A1 (fr) 2004-01-12 2005-08-04 Applied Molecular Evolution, Inc Variants de la region fc
WO2005092925A2 (fr) 2004-03-24 2005-10-06 Xencor, Inc. Variantes d'immunoglobuline a l'exterieur de la region fc
WO2005123780A2 (fr) 2004-04-09 2005-12-29 Protein Design Labs, Inc. Modification des affinites de liaison pour le fcrn ou de la demi-vie serique d'anticorps par mutagenese
WO2006085967A2 (fr) 2004-07-09 2006-08-17 Xencor, Inc. Anticorps monoclonaux optimises anti-cd20 a variants fc
WO2006019447A1 (fr) 2004-07-15 2006-02-23 Xencor, Inc. Variantes genetiques de fc optimisees
WO2006047350A2 (fr) 2004-10-21 2006-05-04 Xencor, Inc. Variants d'immunoglobuline igg a fonction effectrice optimisee
US7696175B2 (en) 2004-10-29 2010-04-13 University Of Southern California Combination cancer immunotherapy with co-stimulatory molecules
WO2006121810A2 (fr) 2005-05-06 2006-11-16 Providence Health System Proteine de fusion trimere immunoglobulinique ox-40 et procedes d'utilisation
US8008449B2 (en) 2005-05-09 2011-08-30 Medarex, Inc. Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
WO2006122442A1 (fr) 2005-05-14 2006-11-23 Fudan University Piggybac utilise comme outil pour la manipulation genetique et l'analyse chez les vertebres
US7961515B2 (en) 2006-07-14 2011-06-14 Semiconductor Energy Laboratory Co., Ltd. Nonvolatile memory
WO2008025516A2 (fr) 2006-08-28 2008-03-06 Apogenix Gmbh Protéines de fusion de superfamille
US20110027218A1 (en) 2006-08-28 2011-02-03 Apogenix Gmbh Tnf superfamily fusion proteins
US8252755B2 (en) 2006-09-22 2012-08-28 Dharmacon, Inc. Duplex oligonucleotide complexes and methods for gene silencing by RNA interference
US8501706B2 (en) 2006-09-22 2013-08-06 Dharmacon, Inc. Duplex oligonucleotide complexes and methods for gene silencing by RNA interference
US20160230188A1 (en) 2007-02-02 2016-08-11 Yale University Method of de-differentiating and re-differentiating somatic cells using rna
US8859229B2 (en) 2007-02-02 2014-10-14 Yale University Transient transfection with RNA
US20100136030A1 (en) 2007-02-27 2010-06-03 Lamhamedi-Cherradi Salah-Eddine Antagonist ox40 antibodies and their use in the treatment of inflammatory and autoimmune diseases
US9228180B2 (en) 2007-07-04 2016-01-05 Max-Delbruck-Centrum Fur Molekulare Medizin Polypeptide variants of sleeping beauty transposase
US20150126709A1 (en) 2007-07-10 2015-05-07 Apogenix Gmbh Trail collectin fusion proteins
WO2009007120A2 (fr) 2007-07-10 2009-01-15 Apogenix Gmbh Protéines de fusion collectines de la superfamille des tnf
WO2009045457A2 (fr) 2007-10-02 2009-04-09 Rxi Pharmaceuticals Corp. CONSTRUCTIONS D'ARNi À STRUCTURE TRIPARTITE
US9028824B2 (en) 2007-12-14 2015-05-12 Pfizer Inc. Binding molecules to the human OX40 receptor
US7960515B2 (en) 2007-12-14 2011-06-14 Bristol-Myers Squibb Company Binding molecules to the human OX40 receptor
US8236930B2 (en) 2007-12-14 2012-08-07 Pfizer Inc. Binding molecules to the human OX40 receptor
US20110039914A1 (en) 2008-02-11 2011-02-17 Rxi Pharmaceuticals Corporation Modified rnai polynucleotides and uses thereof
WO2009102427A2 (fr) 2008-02-11 2009-08-20 Rxi Pharmaceuticals Corp. Polynucléotides d'arni modifiés et leurs utilisations
WO2010003766A2 (fr) 2008-06-17 2010-01-14 Apogenix Gmbh Récepteurs multimériques tnf
US20110111494A1 (en) 2008-06-17 2011-05-12 Oliver Hill Multimeric tnf receptors
US8815818B2 (en) 2008-07-18 2014-08-26 Rxi Pharmaceuticals Corporation Phagocytic cell delivery of RNAI
WO2010010051A1 (fr) 2008-07-21 2010-01-28 Apogenix Gmbh Molécules à une seule chaîne
US9340599B2 (en) 2008-07-21 2016-05-17 Apogenix Ag Single chain CD40L fusion polypeptides
US9359420B2 (en) 2008-07-21 2016-06-07 Apogenix Ag Single chain trail fusion polypeptides and encoding nucleic acids
US8921519B2 (en) 2008-07-21 2014-12-30 Apogenix Gmbh Single chain fusion polypeptides comprising soluble light cytokine domains
US20150110734A1 (en) 2008-07-21 2015-04-23 Apogenix Gmbh Trail single chain molecules
US8450460B2 (en) 2008-07-21 2013-05-28 Apogenix Gmbh Single-chain TNFSF fusion polypeptides
US11369678B2 (en) 2008-08-28 2022-06-28 Taiga Biotechnologies, Inc. Compositions and methods for modulating immune cells
WO2010033247A2 (fr) 2008-09-22 2010-03-25 Rxi Pharmaceuticals Corporation Composés d'arni de taille réduite à auto-délivrance
WO2010033246A1 (fr) 2008-09-22 2010-03-25 Rxi Pharmaceuticals Corporation Emploi d’arni dans des applications dermatologiques
WO2010033248A2 (fr) 2008-09-22 2010-03-25 Rxi Pharmaceuticals Corporation Nanotransporteurs neutres
US20110263680A1 (en) 2008-09-22 2011-10-27 Rxi Pharmaceuticals Corporation Reduced size self-delivering rnai compounds
WO2010042433A1 (fr) 2008-10-06 2010-04-15 Bristol-Myers Squibb Company Combinaison d'anticorps cd137 et d'anticorps ctla-4 pour le traitement de maladies prolifératives
WO2010078536A1 (fr) 2009-01-05 2010-07-08 Rxi Pharmaceuticals Corporation Inhibition de pcsk9 par arni
WO2010078966A1 (fr) 2009-01-09 2010-07-15 Apogenix Gmbh Protéines de fusion formant des trimères
US20150126710A1 (en) 2009-01-09 2015-05-07 Apogenix Gmbh Fusion proteins forming trimers
WO2010085699A2 (fr) 2009-01-23 2010-07-29 The Johns Hopkins University Transposon piggybac de mammifère et procédés d'utilisation
WO2010090762A1 (fr) 2009-02-04 2010-08-12 Rxi Pharmaceuticals Corporation Duplexes d'arn avec régions de nucléotide phosphorothioate à brin unique pour fonctionnalité supplémentaire
WO2010099301A2 (fr) 2009-02-25 2010-09-02 The Johns Hopkins University Variants de transposon piggybac et procédés d'utilisation
WO2010099296A1 (fr) 2009-02-26 2010-09-02 Transposagen Biopharmaceuticals, Inc. Transposases piggybac hyperactives
US8809050B2 (en) 2009-12-08 2014-08-19 Wilson Wolf Manufacturing Methods of cell culture for adoptive cell therapy
US8956860B2 (en) 2009-12-08 2015-02-17 Juan F. Vera Methods of cell culture for adoptive cell therapy
US20110136228A1 (en) 2009-12-08 2011-06-09 Vera Juan F Methods of cell culture for adoptive cell therapy
WO2011072088A2 (fr) 2009-12-08 2011-06-16 Wilson Wolf Manufacturing Corporation Procédés améliorés de culture cellulaire pour thérapie cellulaire adoptive
US20130115617A1 (en) 2009-12-08 2013-05-09 John R. Wilson Methods of cell culture for adoptive cell therapy
US20130102075A1 (en) 2009-12-08 2013-04-25 Juan F. Vera Methods of cell culture for adoptive cell therapy
US20160208216A1 (en) 2009-12-08 2016-07-21 Juan F. Vera Methods of cell culture for adoptive cell therapy
US20150175966A1 (en) 2009-12-08 2015-06-25 Juan F. Vera Methods of cell culture for adoptive cell therapy
WO2011109698A1 (fr) 2010-03-04 2011-09-09 Rxi Pharmaceuticals Corporation Formulations et procédés d'administration ciblée à des cellules phagocytaires
WO2011119852A1 (fr) 2010-03-24 2011-09-29 Rxi Pharmaceuticals Corporation Composés d'arni de taille réduite s'auto-administrant
US20130131141A1 (en) 2010-03-24 2013-05-23 Anastasia Khvorova Reduced size self-delivering rnai compounds
US9080171B2 (en) 2010-03-24 2015-07-14 RXi Parmaceuticals Corporation Reduced size self-delivering RNAi compounds
US20130131142A1 (en) 2010-03-24 2013-05-23 Lyn Libertine Rna interference in ocular indications
WO2011119887A1 (fr) 2010-03-24 2011-09-29 Rxi Pharmaceuticals Corporation Arn interférant dans des indications dermiques et fibrosiques
WO2011119871A1 (fr) 2010-03-24 2011-09-29 Rxi Phrmaceuticals Corporation Arn interférant dans des indications oculaires
US8586526B2 (en) 2010-05-17 2013-11-19 Sangamo Biosciences, Inc. DNA-binding proteins and uses thereof
US20110201118A1 (en) 2010-06-14 2011-08-18 Iowa State University Research Foundation, Inc. Nuclease activity of tal effector and foki fusion protein
US9006399B2 (en) 2010-08-23 2015-04-14 Board Of Regents, The University Of Texas System Anti-OX40 antibodies and methods of using the same
US9163085B2 (en) 2010-08-23 2015-10-20 Board Of Regents, The University Of Texas System Anti-OX40 antibodies and methods of treating cancer
WO2012027328A2 (fr) 2010-08-23 2012-03-01 Board Of Regents, The University Of Texas System Anticorps anti-ox40 et leurs procédés d'utilisation
US8337850B2 (en) 2010-09-09 2012-12-25 Pfizer Inc. 4-1BB binding molecules
US9468678B2 (en) 2010-09-09 2016-10-18 Pfizer Inc. Method of producing 4-1BB binding molecules and associated nucleic acids
WO2012032433A1 (fr) 2010-09-09 2012-03-15 Pfizer Inc. Molécules de liaison 4-1bb
US8821867B2 (en) 2010-09-09 2014-09-02 Pfizer Inc 4-1BB binding molecules
US8962804B2 (en) 2010-10-08 2015-02-24 City Of Hope Meditopes and meditope-binding antibodies and uses thereof
US20140328791A1 (en) 2010-11-12 2014-11-06 Nektar Therapeutics Conjugates of an IL-2 Moiety and a Polymer
WO2012065086A1 (fr) 2010-11-12 2012-05-18 Nektar Therapeutics Conjugués d'une fraction il-2 et d'un polymère
WO2012129201A1 (fr) 2011-03-22 2012-09-27 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés de mise en croissance de lymphocytes infiltrant une tumeur dans contenants perméables au gaz
US20120244133A1 (en) 2011-03-22 2012-09-27 The United States of America, as represented by the Secretary, Department of Health and Methods of growing tumor infiltrating lymphocytes in gas-permeable containers
US20130117869A1 (en) 2011-04-05 2013-05-09 Cellectis S.A. Method for the generation of compact tale-nucleases and uses thereof
WO2012177788A1 (fr) 2011-06-20 2012-12-27 La Jolla Institute For Allergy And Immunology Modulateurs de 4-1bb et réponses immunitaires
US20140295426A1 (en) 2011-07-28 2014-10-02 Veridex Llc Methods for Diagnosing Cancer by Characterization of Tumor Cells Associated with Pleural or Serous Fluids
WO2013028231A1 (fr) 2011-08-23 2013-02-28 Board Of Regents, The University Of Texas System Anticorps anti-ox40 et leurs procédés d'utilisation
WO2013038191A2 (fr) 2011-09-16 2013-03-21 Bioceros B.V. Anticorps anti-cd134 (ox40) et leurs utilisations
US20140227237A1 (en) 2011-09-16 2014-08-14 The Trustees Of The University Of Pennsylvania Rna engineered t cells for the treatment of cancer
US20150132288A1 (en) 2011-09-16 2015-05-14 Biocerox Products B.V. Anti-cd134 (ox40) antibodies and uses thereof
US20140287509A1 (en) 2011-10-17 2014-09-25 Massachusetts Institute Of Technology Intracellular Delivery
WO2013059343A1 (fr) 2011-10-17 2013-04-25 Massachusetts Institute Of Technology Administration intracellulaire
WO2013173835A1 (fr) 2012-05-18 2013-11-21 Wilson Wolf Manufacturing Corporation Procédés de culture cellulaire améliorés pour thérapie cellulaire adoptive
US20130315884A1 (en) 2012-05-25 2013-11-28 Roman Galetto Methods for engineering allogeneic and immunosuppressive resistant t cell for immunotherapy
US20150203871A1 (en) 2012-06-05 2015-07-23 Cellectis Transcription Activator-Like Effector (TALE) Fusion Protein
US10183979B2 (en) 2012-06-08 2019-01-22 Alkermes, Inc. Fusion polypeptides comprising mucin-domain polypeptide linkers
WO2013188427A1 (fr) 2012-06-11 2013-12-19 Wilson Wolf Manufacturing Corporation Procédés améliorés de culture cellulaire pour une thérapie cellulaire adoptive
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8871445B2 (en) 2012-12-12 2014-10-28 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8999641B2 (en) 2012-12-12 2015-04-07 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US8795965B2 (en) 2012-12-12 2014-08-05 The Broad Institute, Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
US8945839B2 (en) 2012-12-12 2015-02-03 The Broad Institute Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8865406B2 (en) 2012-12-12 2014-10-21 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8771945B1 (en) 2012-12-12 2014-07-08 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8889356B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8932814B2 (en) 2012-12-12 2015-01-13 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8895308B1 (en) 2012-12-12 2014-11-25 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8906616B2 (en) 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
US20160010058A1 (en) 2013-03-01 2016-01-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Serv Methods of producing enriched populations of tumor-reactive t cells from tumor
US20140377284A1 (en) 2013-03-18 2014-12-25 Janssen Pharmaceuticals, Inc. Humanized anti-cd134 (ox40) antibodies and uses thereof
WO2014148895A1 (fr) 2013-03-18 2014-09-25 Biocerox Products B.V. Anticorps anti-cd134 (ox40) humanisés et leurs utilisations
US20160120906A1 (en) 2013-05-13 2016-05-05 Cellectis Methods for engineering highly active t cell for immunotheraphy
US20140377739A1 (en) 2013-06-24 2014-12-25 Wilson Wolf Manufacturing Closed system device and methods for gas permeable cell culture process
WO2014210036A1 (fr) 2013-06-24 2014-12-31 Wilson Wolf Manufacturing Corporation Dispositif à système clos et procédés de processus de culture de cellules perméable aux gaz
WO2015006700A1 (fr) 2013-07-12 2015-01-15 University Of South Alabama Vecteurs piggybac minimaux pour intégration génomique
US20160304873A1 (en) 2013-12-02 2016-10-20 Mirimmune Inc. Immunotherapy of Cancer
US20150190506A1 (en) 2013-12-17 2015-07-09 Genentech, Inc. Combination therapy comprising ox40 binding agonists and pd-1 axis binding antagonists
WO2015119923A1 (fr) 2014-02-04 2015-08-13 Pfizer Inc. Combinaison d'un antagoniste de pd -1 et d'un agoniste de 4-1bb pour le traitement du cancer
US10041077B2 (en) 2014-04-09 2018-08-07 Dna2.0, Inc. DNA vectors, transposons and transposases for eukaryotic genome modification
US20170107490A1 (en) 2014-06-11 2017-04-20 Polybiocept Ab Expansion of lymphocytes with a cytokine composition for active cellular immunotherapy
WO2016094845A2 (fr) 2014-12-12 2016-06-16 Woolf Tod M Compositions et procédés d'édition d'acides nucléiques dans des cellules à l'aide d'oligonucléotides
US20170369882A1 (en) 2015-04-03 2017-12-28 University Of Massachusetts Fully stabilized asymmetric sirna
US20160319278A1 (en) 2015-04-03 2016-11-03 University Of Massachusetts Fully stabilized asymmetric sirna
US9790490B2 (en) 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
WO2017008063A1 (fr) 2015-07-09 2017-01-12 Massachusetts Institute Of Technology Administration de substances à des cellules anucléées
US20180201889A1 (en) 2015-07-09 2018-07-19 Massachusetts Institute Of Technology Delivery of materials to anucleate cells
US20170043024A1 (en) 2015-08-14 2017-02-16 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
US20180245089A1 (en) 2015-09-04 2018-08-30 Sqz Biotechnologies Company Intracellular delivery of biomolecules to cells comprising a cell wall
WO2017070151A1 (fr) 2015-10-19 2017-04-27 Rxi Pharmaceuticals Corporation Composés d'acides nucléiques de taille réduite à auto-administration ciblant des longs arn non codants
WO2017123663A1 (fr) 2016-01-12 2017-07-20 Sqz Biotechnologies Company Administration intracellulaire de complexes
US20170312367A1 (en) 2016-01-31 2017-11-02 University Of Massachusetts Branched oligonucleotides
WO2017193053A1 (fr) 2016-05-06 2017-11-09 Woolf Tod M Procédés perfectionnés d'édition de génome avec et sans nucléases programmables
US20190307796A1 (en) 2016-06-03 2019-10-10 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Use of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (pgc1alpha) agonists to improve ex vivo expansion of tumor infiltrating lymphocytes (tils)
WO2018081473A1 (fr) 2016-10-26 2018-05-03 Iovance Biotherapeutics, Inc. Re-stimulation de lymphocytes infiltrant les tumeurs cryoconservés
US20190275133A1 (en) 2016-11-10 2019-09-12 Nektar Therapeutics Immunotherapeutic tumor treatment method
WO2018132496A1 (fr) 2017-01-10 2018-07-19 Nektar Therapeutics Conjugués polymères à bras multiples de composés agonistes de tlr et méthodes de traitement immunothérapeutiques associées
US20180282694A1 (en) 2017-03-29 2018-10-04 Iovance Biotherapeutics, Inc. Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy
US20180280436A1 (en) 2017-03-29 2018-10-04 Iovance Biotherapeutics, Inc. Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy
US20200270334A1 (en) 2017-05-24 2020-08-27 Novartis Ag Antibody-cytokine engrafted proteins and methods of use in the treatment of cancer
US20200181220A1 (en) 2017-08-03 2020-06-11 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases
WO2019046815A1 (fr) 2017-08-31 2019-03-07 Poseida Therapeutics, Inc. Système de transposon et procédés d'utilisation
WO2019160829A1 (fr) 2018-02-13 2019-08-22 Iovance Biotherapeutics, Inc. Expansion de lymphocytes infiltrant les tumeurs (til) avec des antagonistes du récepteur a2a de l'adénosine et combinaisons thérapeutiques de til et d'antagonistes du récepteur a2a de l'adénosine
US20200330601A1 (en) 2019-02-06 2020-10-22 Synthorx, Inc. IL-2 Conjugates and Methods of Use Thereof
US20210038684A1 (en) 2019-06-11 2021-02-11 Alkermes Pharma Ireland Limited Compositions and Methods for Cancer Immunotherapy
WO2022087324A1 (fr) * 2020-10-22 2022-04-28 Iovance Biotherapeutics, Inc. Système culture cellulaire et ses procédés d'utilisation
WO2022165260A1 (fr) * 2021-01-29 2022-08-04 Iovance Biotherapeutics, Inc. Procédés de fabrication de lymphocytes infiltrant les tumeurs modifiés et leur utilisation dans la thérapie cellulaire adoptive
WO2022170219A1 (fr) * 2021-02-05 2022-08-11 Iovance Biotherapeutics, Inc. Traitement adjuvant du cancer
WO2023147486A1 (fr) * 2022-01-28 2023-08-03 Iovance Biotherapeutics, Inc. Lymphocytes infiltrant les tumeurs modifiés pour exprimer des charges utiles
WO2023196877A1 (fr) * 2022-04-06 2023-10-12 Iovance Biotherapeutics, Inc. Traitement de patients souffrant de cpnpc avec des thérapies lymphocytaires infiltrant les tumeurs

Non-Patent Citations (90)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. NM_001386420
ALIMAD ET AL., CLIN. & DEV. IMMUNOL., 2012, pages 980250
ARNAUD-BARBE ET AL., NUC. ACIDS RES., vol. 26, 1998, pages 3550 - 54
AUGUSTYNS, K. ET AL., NUCL. ACIDS. RES., vol. 18, 1992, pages 4711
BEANE ET AL., MOL. THERAPY, vol. 23, 2015, pages 1380 - 1390
BERGAN ET AL., NUCLEIC ACIDS RESEARCH, vol. 21, 1993, pages 3567
BESSER ET AL., CLIN. CANCER RES., vol. 19, 2013
BESSER ET AL., J. IMMUNOTHER., vol. 182, 2009, pages 1481 - 423
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
BOCZKOWSKI ET AL., CANCER RES., vol. 60, 2000, pages 1028 - 34
BOLLIGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
BROWNWANED, J. AM. SOC. NEPHR., vol. 3, no. 4, 1992, pages 895 - 906
BYRNE ET AL., J. OCUL. PHARMACOL. THER., vol. 29, 2013, pages 855 - 864
BYRNE ET AL., J. OCULAR PHARMACOLOGY AND THERAPEUTICS, vol. 29, no. 10, December 2013 (2013-12-01), pages 855 - 864
CEPKOPEAR, CUR. PROT. MOL. BIOL., vol. 9, 1996
CHENOKAYARCA, MOL. CELL. BIOL., vol. 7, 1987, pages 2745 - 2752
CIERI ET AL., BLOOD, 2013
COUGOT ET AL., TRENDS IN BIOCHEM. SCI., vol. 29, 2001, pages 436 - 444
COX ET AL., NATURE MEDICINE, vol. 21, 2015
CURTI ET AL., CANCER RES., vol. 73, 2013, pages 7189 - 98
DONIA ET AL., SCAND. J. IMMUNOL., vol. 75, 2012, pages 157 - 167
DUDLEY ET AL., CLIN. CANCER RES., vol. 16, 2010, pages 5458 - 6131
DUDLEY ET AL., J. IMMUNOTHER., vol. 26, 2003, pages 332 - 42
DULL ET AL., J. VIROLOGY, vol. 72, 1998, pages 8463 - 71
DUNNSTUDIER, J. MOL. BIOL., vol. 166, 1983, pages 477 - 535
ELANGO ET AL., BIOCHEM RIOPHYS RES COMMUN., vol. 330, 2005, pages 958 - 966
ELANGO ET AL., BIOCHIM. BIOPHYS. RES. COMMUN., vol. 330, 2005, pages 958 - 966
FEHNIGERCALIGIURI, BLOOD, vol. 97, 2001, pages 14 - 32
FELGNER. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 84, 1987, pages 7413 - 7417
FISHER ET AL., CANCER IMMUNOLOG. & IMMUNOTHER., vol. 61, 2012, pages 1721 - 33
FORGET ET AL., FRONTIERS IMMUNOLOGY, vol. 8, 2017, pages 908
FRYMACKALL, BLOOD, vol. 99, 2002, pages 3892 - 904
GATTINONI ET AL., NAT MED, vol. 2009, 2011
GATTINONI., NATURE REV. CANCER, 2012
GIEFFERS. ET AL., MOL. CANCER THERAPEUTICS, vol. 12, 2013, pages 2735 - 47
GRAHAMVAN DER EB, VIROLOGY, vol. 52, 1973, pages 456 - 467
GREISBECK ET AL., J. IMMUNOLOGY, vol. 195, 2015
HACKETT. ET AL., MOL. THERAPY, vol. 18, 2010, pages 674 - 83
HUANG ET AL., J. IMMUNOTHER., vol. 28, 2005, pages 258 - 267
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
JAEGERNAGEL, SCIENCE, vol. 255, 1992, pages 1523 - 3
JAZAERI AA ET AL., ASCO, 2019, pages 182
JIN ET AL., J. IMMUNOTHERAPY, vol. 35, 2012, pages 283 - 292
JIN, J. IMMUNOTHER., vol. 35, 2012, pages 283 - 292
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
KHVOROVAWATTS, NAT. BIOTECHNOL., vol. 35, 2017, pages 238 - 248
KIYAMAOISHI, NUC. ACIDS RES., vol. 24, 1996, pages 4577 - 4583
KONDO. T. ET AL.: "NOTCH-mediated conversion of activated T cells into stem cell memory-like T cells for adoptive immunotherapy", NATURE COMMUNICATIONS, vol. 8, 2017, XP055821918, DOI: 10.1038/ncomms15338
LEE ET AL., PLOS ONE, vol. 8, 2013, pages 69677
LEVINE ET AL., PROC. NAT'L ACAD. SCI., vol. 103, no. 173, 2006, pages 17372 - 77
LIGTENBERG ET AL., MOL. THERAPY, 2018
MACDONALD ET AL., J. MOL. BIOL., vol. 232, 1993, pages 1030 - 47
MALEK, ANNU. REV. IMMUNOL., vol. 26, 2008, pages 453 - 79
MARCO, MICROBIAL CELL FACTORIES., vol. 10, 2011, pages 44
MONNIER ET AL., ANTIBODIES, vol. 2, 2013, pages 193 - 208
MUSIN, RUSS. MATH. SURV., vol. 58, 2003, pages 794 - 795
NACHEVABERZAL-HERRANZ, EUR. J. BIOCHEM., vol. 270, 2003, pages 1485 - 65
NAKANO ET AL., BIOTECHNOL. BIOENG., vol. 64, pages 194 - 99
NISHIKAWA ET AL., HUM GENE THER., vol. 12, no. 8, 2001, pages 861 - 70
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
R. E. BIRDB. W. WALKER, SINGLE CHAIN ANTIBODY VARIABLE REGIONS, TIBTECH, vol. 9, 1991, pages 132 - 137
R. RAAGM. WHITLOW: "Single Chain Fvs.", FASEB, vol. 9, 1995, pages 73 - 80
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
ROBBINS ET AL., J. IMMUNOL., vol. 173, 2004, pages 7125 - 7130
ROSE ET AL., BIOTECHNIQUES, vol. 10, 1991, pages 520 - 525
ROSENBERG ET AL., NEW ENG. J. OF MED., vol. 319, 1988, pages 1676
SAEBOE-LARSSEN ET AL., J. IMMUNOL. METH., vol. 259, 2002, pages 191 - 203
SAMAIK AA ET AL., J CLIN ONCOL, 2021
SCHENBORNMIERENDORF, NUC. ACIDS RES., vol. 13, 1985, pages 6223 - 36
SEGAL ET AL., CLIN. CANCER RES., 2016
SHAREI ET AL., PLOS ONE, 2015
SHAREI ET AL., PNAS, 2013
SHEN ET AL., J. IMMUNOTHER., vol. 30, 2007, pages 123 - 129
SHIELDS. ET AL., J. BIOL. CHEM., vol. 277, 2002, pages 26733 - 26740
SMITH. ET AL., CLIN TRANSL IMMUNOLOGY, vol. 4, no. 1, 2015
SPOLSKILEONARD, NAT. REV. DRUG. DISC., vol. 13, 2014, pages 379 - 95
STEINKEBORISH, RESPIR. RES., vol. 2, 2001, pages 66 - 70
STEPINSKI ET AL., RNA, vol. 7, 2001, pages 1468 - 95
SWARTZ ET AL., CANCER RES., vol. 72, 2012, pages 2473
TARENTINO ET AL., BIOCHEM., vol. 14, 1975, pages 5516 - 5523
TRAN ET AL., J. IMMUNOTHER., vol. 31, 2008, pages 742 - 751
TRIANA-ALONSO ET AL., J. BIOL. CHEM., vol. 270, 1995, pages 6298 - 307
TSONG., BIOPHYS. J., vol. 60, 1991, pages 297 - 306
TSOUKAS ET AL., J. IMMUNOL., vol. 135, 1985, pages 1719
UMANA. ET AL., NAT. BIOTECH., vol. 17, 1999, pages 176 - 180
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
WEINBERG ET AL., J. IMMUNOTHER., vol. 29, 2006, pages 575 - 585
WIGLER ET AL., PROC. NATL. ACAD. SCI., vol. 76, 1979, pages 1373 - 1376
YAMANE-OHNUKI. ET AL., BIOTECHNOL. BIOENG., vol. 87, 2004, pages 614 - 622
ZUFFEREY ET AL., NAT. BIOTECHNOL., vol. 15, 1997, pages 871 - 75

Similar Documents

Publication Publication Date Title
EP4284919A1 (fr) Procédés de fabrication de lymphocytes infiltrant les tumeurs modifiés et leur utilisation dans la thérapie cellulaire adoptive
EP4259164A1 (fr) Traitement de patients atteints de cancer par des thérapies de lymphocytes infiltrant les tumeurs en combinaison avec des inhibiteurs de braf et/ou des inhibiteurs de mek
EP4377446A1 (fr) Traitement de patients atteints d'un cancer avec des thérapies de lymphocytes infiltrant les tumeurs en combinaison avec des inhibiteurs de kras
EP4326287A2 (fr) Récepteurs costimulateurs chimériques, récepteurs de chimiokines et leur utilisation dans des immunothérapies cellulaires
US20250099588A1 (en) Cytokine associated tumor infiltrating lymphocytes compositions and methods
WO2022133140A1 (fr) Traitement avec des thérapies de lymphocytes infiltrant les tumeurs en combinaison avec des inhibiteurs de ctla-4 et de pd-1
JP2024519029A (ja) Ｐｄ－１遺伝子編集された腫瘍浸潤リンパ球及び免疫療法におけるその使用
EP4262827A1 (fr) Traitement de cancers à l'aide de lymphocytes infiltrant les tumeurs
WO2023196877A1 (fr) Traitement de patients souffrant de cpnpc avec des thérapies lymphocytaires infiltrant les tumeurs
JP2024535002A (ja) Ｐｄ－１ ｔａｌｅｎノックダウンを使用したｔｉｌ製品を生成するためのプロセス
US20250101380A1 (en) Tumor infiltrating lymphocytes engineered to express payloads
EP4565683A1 (fr) Récepteurs de costimulation chimériques, récepteurs de chimiokines et leur utilisation dans des immunothérapies cellulaires
WO2022187741A2 (fr) Stockage de tumeur et compositions de culture cellulaire
WO2025101484A1 (fr) Traitement de cancers endométriaux avec des thérapies lymphocytaires infiltrant les tumeurs
WO2023220608A1 (fr) Traitement de patients atteints d'un cancer avec des thérapies lymphocytaires infiltrant les tumeurs en combinaison avec un agoniste d'il-15r
EP4373270A2 (fr) Procédé de cryoconservation de fragments de tumeur solide
WO2024098027A1 (fr) Procédés d'expansion de lymphocytes infiltrant les tumeurs (til) liés à la sélection de cd39/cd103
AU2022388729A1 (en) Methods of expansion treatment utilizing cd8 tumor infiltrating lymphocytes
CN116829156A (zh) 使用肿瘤浸润性淋巴细胞疗法与ctla-4及pd-1抑制剂组合的治疗

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24809507

Country of ref document: EP

Kind code of ref document: A1