WO2025106513A1

WO2025106513A1 - Methods for producing edited tumor infiltrating lymphocytes

Info

Publication number: WO2025106513A1
Application number: PCT/US2024/055677
Authority: WO
Inventors: Chantale Bernatchez; Jason Bock; Micah BENSON; Karrie WONG
Original assignee: KSQ Therapeutics Inc
Current assignee: KSQ Therapeutics Inc
Priority date: 2023-11-13
Filing date: 2024-11-13
Publication date: 2025-05-22
Anticipated expiration: 2026-05-13
Also published as: WO2025106513A8

Abstract

Methods for producing edited tumor infiltrating lymphocytes include producing tumor infiltrating lymphocytes from cell culture media comprising tumor fragments; and modifying the endogenous SOCS1 or Regnase-1 gene in the lymphocytes.

Description

METHODS FOR PRODUCING EDITED TUMOR INFILTRATING

EYMPHOCYTES

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/598,497, filed November 13, 2023, and U.S. provisional application number 63/574,593, filed April 4, 2024, each of which is incorporated by reference herein in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (K071370022WO00-SEQ-EAS.xml; Size: 19,889 bytes; and Date of Creation: November 12, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

Gene-edited tumor infiltrating lymphocyte cell therapy products have been shown to exhibit transformational tumor-killing abilities in preclinical tumor models.

SUMMARY

The disclosure relates, in some aspects to methods for producing (e.g., activating, editing, and expanding) tumor infiltrating lymphocyte (TIL) cell therapy products for treating cancer, for example. The cancer may be selected from melanoma, non-small cell lung cancer (NSCLC), colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), pancreatic ductal adenocarcinoma (PDAC), and uveal melanoma, for example.

In some aspects, a method of the disclosure comprises producing tumor infiltrating lymphocytes from cell culture media comprising tumor fragments, recombinant human interleukin-2 (IL-2), anti-CD3 antibody, and anti-41BB antibody. In some embodiments, a method comprises genetically modifying one or more endogenous gene in tumor infiltrating lymphocytes to produce edited tumor infiltrating lymphocytes (eTIL). In some embodiments, a method comprises genetically modifying the endogenous SOCS1 gene and/or the endogenous REGNASE-1 gene in tumor infiltrating lymphocytes to produce eTIL. In some embodiments, a method comprises expanding eTIL in cell culture media comprising recombinant human IL-2. Some aspects of the disclosure relate to a method comprising: (a) producing tumor infiltrating lymphocytes from cell culture media comprising tumor fragments, recombinant human interleukin-2 (IL-2), anti-CD3 antibody, and anti-4 IBB antibody; (b) genetically modifying an endogenous gene in the tumor infiltrating lymphocytes to produce eTIL; and (c) expanding the eTIL in cell culture media comprising recombinant human IL-2.

Some aspects of the disclosure relate to a method comprising: (a) producing tumor infiltrating lymphocytes from cell culture media comprising tumor fragments, recombinant human IL-2, anti-CD3 antibody, and anti-4 IBB antibody; (b) genetically modifying the endogenous SOCS1 gene and/or the endogenous REGNASE-1 gene in the tumor infiltrating lymphocytes to produce eTIL; and (c) expanding the eTIL in cell culture media comprising recombinant human IL-2.

In other aspects, a method of the disclosure comprises culturing tumor fragments in culture media comprising recombinant human IL-2, anti-CD3 antibody, and anti-4 IBB antibody to produce a first population of cells comprising tumor infiltrating lymphocytes. In some embodiments, a method comprises electroporating a first population of cells with Cas nuclease and one or more guide RNA(s) targeting an endogenous gene to produce a second population of cells comprising edited tumor infiltrating lymphocytes. In some embodiments, a method comprises electroporating a first population of cells with Cas nuclease and one or more guide RNA(s) targeting endogenous SOCS1 and/or endogenous REGNASE-1 to produce a second population of cells comprising edited tumor infiltrating lymphocytes. In some embodiments, a method comprises culturing a second population of cells in culture media comprising recombinant human IL-2 to produce an expanded population of edited tumor infiltrating lymphocytes.

Other aspects of the disclosure relate to a method comprising: (a) culturing tumor fragments in culture media comprising recombinant human IL-2, anti-CD3 antibody, and anti-41BB antibody to produce a first population of cells comprising tumor infiltrating lymphocytes; (b) electroporating the first population of cells with Cas nuclease and one or more guide RNA(s) targeting an endogenous gene to produce a second population of cells comprising edited tumor infdtrating lymphocytes; and (c) culturing the second population of cells in culture media comprising recombinant human IL-2 to produce an expanded population of edited tumor infiltrating lymphocytes.

Other aspects of the disclosure relate to a method comprising: (a) culturing tumor fragments in culture media comprising recombinant human IL-2, anti-CD3 antibody, and anti-41BB antibody to produce a first population of cells comprising tumor infiltrating lymphocytes; (b) electroporating the first population of cells with Cas nuclease and one or more guide RNA(s) targeting endogenous SOCS1 and/or endogenous REGNASE-1 to produce a second population of cells comprising edited tumor infdtrating lymphocytes; and (c) culturing the second population of cells in culture media comprising recombinant human IL-2 to produce an expanded population of edited tumor infiltrating lymphocytes.

Some aspects of the disclosure further comprise administering a therapeutically effective amount of the edited tumor infiltrating lymphocytes to a subject in need thereof, for example, a subject diagnosed with melanoma, NSCLC, CRC, HNSCC, PDAC, or uveal melanoma. In some embodiments, the therapeutically effective amount is about IxlO⁹ edited TIL to about 10xl0⁹ edited TIL. In some embodiments, the therapeutically effective amount is administered with recombinant IL-2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram depicting a process for producing edited tumor infiltrating lymphocytes. This exemplary process starts with processing of autologous tumor material and ends with cry opreservation of the cells. The cells are genome edited during manufacture using CRISPR ribonucleic protein (RNP) complexes composed of Cas9 and single guide RNA (sgRNA or gRNA) to inactivate the SOCS1 gene and/or the REGNASE-1 gene.

FIGs. 2A-2C show that the exemplary process for producing SOCS1 -edited tumor infiltrating lymphocytes (KSQ-001EX), as provided herein, successfully yielded clinical- scale doses from six different solid tumor types (melanoma, uveal melanoma (“uveal”), lung (“NSCLC”), colorectal (“CRC”), head and neck ("HNSCC”), and pancreatic (“PDAC”) cancer). FIG. 2A depicts pre-electroporation (pre-EP) cell yield across the six solid tumor types. Full scale and extrapolated (scaled down) pre-EP yield is shown. All melanoma and colorectal cancer patients were pre-treated with prior therapies prior to tumor harvest. The dotted line indicates the minimum number of TIL (16 xlO⁶) required to proceed to the electroporation (gene editing) step. The line in each bar shows the mean cell yield from the indication. FIG. 2B depicts final cell yield post electroporation (post-EP) of the tumor infiltrating lymphocytes with CRISPR RNP complexes comprising Cas9 and sgRNA targeting endogenous SOCS1 (KSQ-001EX). Full scale and extrapolated (scaled up or down) post-EP yield of freshly harvested end product, prior to cell washing, concentration, and cryopreservation is shown. All melanoma and colorectal cancer patients were pretreated prior to tumor harvest. The dotted lines delineate the lower (1 xlO⁹) and upper (10 xlO⁹) infusion dose. The line in each bar shows the mean cell yield from the indication. Overall Manufacture success rate = 94%. FIG. 2C depicts average of Critical Quality Attributes (viability, percentage of CD3+ cells, and percentage of on-target editing efficiency) for KSQ- 001EX determined by drug product release assays for all indications.

FIG. 3 depicts SOCS1 expression in tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX). A Capillary western blot analysis was used to determine SOCS1 expression at the protein level in non-edited and edited tumor infiltrating lymphocytes. Vinculin was used as loading control.

FIGs. 4A-4B depict phenotypic characteristics of tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX). FIG. 4A depicts the frequency of CD8+ T cells and CD4+ T cells out of total live CD45⁺CD3⁺ population in electroporated tumor infiltrating lymphocytes (KSQ-001EX) and donor-matched TIL control that do not undergo electroporation (No EP). FIG. 4B depicts the frequency of CD45RO⁺CCR7‘ T Effector Memory Cells (Tern) out of total live CD45⁺CD3⁺ population.

FIGs. 5A-5C depict functionality of tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX). FIG. 5A depicts a representative kill curve in an A375-tumor spheroid assay. A nonlinear fit curve for cytotoxicity EC50 calculation from 1 representative donor is shown. Tumor spheroid fluorescence was normalized to To and plotted against different E:T ratios. FIG. 5B depicts EC50 values comparing donor-matched No EP and KSQ-001EX after 120-hour co-culture across multiple donors (n=6). FIG. 5C depicts IFNy expression at 24 hours. Supernatant was collected from TIL-tumor- spheroid co-cultures at 24 hours for IFNy measurement. IFNy levels were plotted for 20:1 E:T ratio. Statistical analysis was done using 1-tailed paired Students’ t test; *p-value < 0.05, **p-value < 0.01.

FIG. 6 depicts GSEA Analysis (RNAseq) on genes downstream of the IL-2 STAT5 pathway from tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX). To generate gene-set scores, singscores were used by ranking the normalized gene expression count data. A paired Wilcox test was performed between the conditions KSQ00-1EX vs No EP and the nominal P-value < 0.05 was deemed significant.

FIG. 7 depicts Simpson’s TCR Diversity Index (SDI) from tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX). TCRseq was performed on KSQ-001EX and No EP samples. SDI was calculated to represent TCR repertoire diversity with high-frequency reads with the closer the index value is to 1, the more diverse the TCR repertoire. FIG. 8 is a schematic depicting two processes for producing edited tumor infiltrating lymphocytes. The first process, referred to as Process 1 (top), starts with processing of autologous tumor material, comprises pre-electroporation expansion in the presence of CD3 agonist, CD28 agonist, and IL-2, and ends with cry opreservation of the cells. The second process, referred to as Process 2 (bottom), starts with processing of autologous tumor material, comprises pre-electroporation expansion in the presence of a 41BB agonist and IL- 2, and ends with cryopreservation of the cells. The cells in both processes are genome edited during manufacture using CRISPR ribonucleic protein (RNP) complexes composed of Cas9 and single guide RNA (sgRNA or gRNA) to inactivate the SOCS1 gene (“KSQ-001” from Process 1 and “KSQ-001EX” from Process 2) and both the SOCS1 gene and the REGNASE-1 gene (“KSQ-004” from Process 1 and “KSQ-004EX” from Process 2).

FIG. 9 depict the frequencies of CD3⁺ T cells in edited tumor infiltrating lymphocytes KSQ-001 and KSQ-001EX.

FIG. 10 depict the frequencies of CD3⁺ T cells in edited tumor infiltrating lymphocytes KSQ-004 and KSQ-004EX.

DETAILED DESCRIPTION

The expeditious production of autologous tumor-infiltrating lymphocyte (TIL) therapies is paramount, as it directly impacts the clinical outcomes for patients battling aggressive forms of cancer. TIL therapies generally involve extracting immune cells from a patient’s own tumor, expanding them in the laboratory, and then infusing them back into the patient to attack cancer cells. The effectiveness of this personalized treatment hinges on a narrow therapeutic window where the patient's disease status and overall health must align with the availability of the TIL product. Delays can be detrimental, considering the rapid progression of many cancers and the potential for a patient’s condition to deteriorate, which may render them ineligible for the therapy. Additionally, the functional capacity of the TILs can diminish over time, emphasizing the need for a swift and streamlined manufacturing process. Ensuring the rapid turnaround of TIL products, from the point of tumor resection to cell reinfusion, is an important component of therapy that can lead to improved survival rates and better quality of life for patients.

Provided herein, in some aspects, is an eTIL manufacturing process, which is a continuous process starting with processing of autologous tumor material and ending with cry opreservation of the cells. The cells are genome-edited during manufacture using, for example, CRISPR ribonucleic protein (RNP) complexes composed of Cas9 and single guide RNA (sgRNA) to inactivate the SOCS1 gene, the REGNASE-1 gene or both genes. The manufacturing process herein, in some aspects, includes three key phases: pre-genome editing expansion, genome editing, and post-genome editing expansion. In some aspects, the three key phases are referred to as: pre-electroporation (pre-EP) expansion, electroporation, and post-EP expansion. An exemplary manufacturing process flow diagram for edited TIL production is provided in Figure 1 and is followed by a description of each step in Example 1 below. Manufacturing is patient-specific, in some aspects, such that tumor material from a single patient is used to manufacture product for autologous use. An exemplary engineered TIL therapy using CRISPR/Cas9 technology to knockout the SOCS1 gene (KSQ-001EX), which can be manufactured at clinical doses from surgical resections or core biopsies within 22 days using a simplified feeder-free manufacturing process from multiple cancer cell types (different indications), is provided in Example 2 below. A comparison of manufacturing processes for edited TIL production is provided in Figure 8 and is followed by a comparison of engineered TIL therapies manufactured by each process in Example 3 below.

Tumor Infiltrating Lymphocytes

Tumor infiltrating lymphocytes (TILs) include white blood cells that have left the bloodstream and migrated into a tumor, for example, in response to the presence of cancer cells. TILs include various types of T cells (such as CD8+ cytotoxic T cells and CD4+ helper T cells, including Thl and Thl7 CD4+ T cells), B cells, natural killer T (NKT) cells, and natural killer (NK) cells.

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 as expressing one or more of the following biomarkers: CD4, CD8, TCR aP, TCRgd, CD27, CD28, CD56, CCR7, CD45RA, CD45RO, CD95, PD-1, and CD25. Additionally, or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient.

In some embodiments, TILs are obtained from a patient sample and expanded in culture prior to transplantation into a patient. In some embodiments, the TILs are genetically manipulated and, thus, are considered engineered TILs. In general, TILs are initially obtained from a patient tumor sample (“primary TILs”) and then expanded into a larger population for further manipulation (e.g., genome editing), optionally cryopreserved and re- stimulated. In some embodiments, the TILs are evaluated for phenotype and metabolic parameters as an indication of TIL health using the in vivo antitumor potency models of the present disclosure. In some embodiments, a population of TILs is monoclonal. In other embodiments, a population of TILs is polyclonal. A monoclonal T cell population has predominance of a single TCR-gene rearrangement pattern. In contrast, a polyclonal T cell population has diverse TCR-gene rearrangement pattern.

A patient tumor sample from which a primary TIL is obtained may be from any solid tumor, including primary tumors, invasive tumors or metastases. The solid tumor may be of any cancer type, including, but not limited to, bladder cancer, brain cancer, breast cancer (including triple negative breast cancer), cervical cancer, colon and rectal cancer, stomach cancer, endometrial cancer, renal cancer, lip and oral cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)) glioblastoma, glioblastoma multiforme, neuroblastoma, liver cancer, mesothelioma, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer), skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, nonmelanoma skin cancer and melanoma), ovarian cancer, uveal cancer, uterine cancer, pancreatic cancer, prostate cancer, sarcoma, and thyroid cancer. In some embodiments, useful TILs are obtained from malignant melanoma tumors, as these have been reported to have particularly high levels of TILs. Primary lung (including non-small cell lung cancer (NSCLC)), bladder, cervical and melanoma tumors or metastases thereof can be used to obtain TILs. Once obtained, the tumor sample is generally fragmented. In some embodiments TILs are cultured from tumor fragments using enzymatic tumor digests. In some embodiments, TILs are cultured from tumor fragments in the absence of enzymatic tumor digests.

An analysis of human TILs or a human TIL population may include an expression analysis for one or more phenotypic markers, including, for example: TCRa/p, CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD45RO, CD8b, CCR7, CD3, CD38, and HLA-DR. The expression of one or more regulatory markers may also be assessed, including, for example: CD137, CD8a, Lag3, CD4, CD3, PD-1, TIM-3, CD69, CD8b, TIGIT, CD3, KLRG1, and CD154. Other examples include TCRa/p, CD56, CD27, CD28, CD57, CD45RA, CD45RO, CD25, CD127, CD95, IL-2R, CCR7, CD62L, KLRG1, and CD122.

In some embodiments, expression of a memory marker, e.g., CCR7 or CD62L, is assessed.

In some embodiments, human TILs are evaluated for cytokine release. In some embodiments, human TILs are evaluated for interferon-gamma (IFN-y) secretion in response to stimulation with 0KT3. In some embodiments, human TILs are evaluated for IL-2, TNFa and/or IL-6 secretion in response to stimulation either with 0KT3 or coculture with autologous tumor digest. Additional effector cytokines that could be measured include, but are not limited to, IL-1, IL- 12, IL- 17, IL- 18, and granulocyte-macrophage colony stimulating factor (GM-CSF).

Pre-Genome Editing Expansion

Tumor Fragmentation

Methods of the disclosure comprise, in some aspects, producing TILs from cell culture media that comprises tumor fragments. Tumor fragments include small pieces of a tumor used as a starting material for methods described herein. Tumor fragments may be produced using, for example, mechanical fragmentation (e.g., cutting, grinding, or tissue homogenization), enzymatic digestion (e.g., collagenase, trypsin/trypsin-EDTA, DNase, or hyaluronidase), chemical dissociation (e.g., chelating agent or hypotonic solution), physical disruption (e.g., sonication or pression homogenization), or a combination thereof.

The length of the tumor fragments, in some embodiments, are about 0.5 mm to about 5 mm, for example, in each of three dimensions. For example, the length of the tumor fragments are about 0.5-4.5 mm, about 0.5-4 mm, about 0.5-3.5 mm, about 0.5-3 mm, about 1-4.5 mm, about 1-4 mm, about 1-3.5 mm, or about 1-3 mm. In some embodiments, the length of the tumor fragments is about 1 mm to about 3 mm. In some embodiments, the length of the tumor fragments is about 1 mm, about 2 mm, or about 3 mm.

Tumor fragments, in some embodiments, are cultured in one or more flasks, such as G-Rex®10 flask(s), for example. In some embodiments, a single flask (or other cell culture vessel) comprises about 4-6 tumor fragments per 20-40 ml of cell culture media. For example, 4, 5, or 6 tumor fragments may be cultured in 20 ml, 30 ml, or 40 ml of cell culture media. In some embodiments, 5 tumor fragments are cultured in 20 ml of cell culture media. In some embodiments, 5 tumor fragments are cultured in 30 ml of cell culture media. In some embodiments, 5 tumor fragments are cultured in 40 ml of cell culture media.

Cell Culture

The cell culture media, in some embodiments, comprises one or more of RPML1640 medium, Human AB serum (e.g., 8-12% Human AB Serum), HEPES buffer (e.g., 5-15 mM HEPES buffer), and 2-mercaptoethanol (e.g., 5xl0'⁵ to 6xl0'⁵ M 2-mercaptoethanol). In some embodiments, the cell culture media, which may be referred to herein as TIL Complete Medium, comprises RPML1640 medium, 10% Human AB Serum, 10 mM HEPES buffer, and 5.5 x 10'⁵ M 2-mercaptoethanol. The cell culture media, in some embodiments, comprises human interleukin-2 (IL-2), for example, recombinant human IL-2. Human IL-2, in some aspects, is included in the cell culture media prior to genome editing the cells (e.g., in the presence of the tumor fragments and antibodies) and in the cell culture media after genome editing (e.g., during the expansion of edited TILs). Prior to genome editing, the concentration of human IL-2 may be, for example, about 1,000 lU/mL to about 10,000 lU/mL. In some embodiments, the concentration of human IL-2 is about 2,000 lU/mL to about 8,000 lU/mL. In some embodiments, the concentration of human IL-2 is about 4,000 lU/mL to about 8,000 lU/mL. In some embodiments, the concentration of human IL-2 is about 4,000 lU/mL to about 6,000 lU/mL. In some embodiments, the concentration of human IL-2 is about 4,000 lU/mL, about 5,000 lU/mL, about 6,000 lU/mL, about 7,000 lU/mL, or about 8,000 lU/mL. In some embodiments, the concentration of human IL-2 prior to genome editing is about 6,000 lU/mL. After genome editing, during eTIL expansion, for example, the concentration of human IL-2 may be, for example, about 300 lU/mL to about 6,000 lU/mL. In some embodiments, the concentration of human IL-2, following genome editing, is about 300 lU/mL to about 3,000 lU/mL. In some embodiments, the concentration of human IL-2 is about 300 lU/mL, about 1,000 lU/mL, about 2,000 lU/mL, or about 3,000 lU/mL. In some embodiments, the concentration of human IL-2 after genome editing is about 3,000 lU/mL.

The cell culture media, in some embodiments, comprises anti-CD3 antibody. CD3 (cluster of differentiation 3) is a T cell co-receptor that helps to activate both the cytotoxic T cell (CD8+ naive T cells) and also T helper cells (CD4+ naive T cells). CD3 is a protein complex composed of six distinct polypeptide chains (2 CD3 zeta chains, 2 CD3 epsilon chains, 1 CD3e gamma chain, and 1 CD3 delta chain). These chains associate with the T cell receptor (TCR) alpha and beta chains (or gamma and delta chains) to generate an activation signal in T lymphocytes. The TCR alpha and beta chains (or gamma and delta chains), and CD3 molecules together constitute the TCR complex.

The phrase “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody, and includes human, humanized, chimeric or murine antibodies that specifically binds to the CD3 receptor component of the T cell antigen receptor of mature T cells (see, e.g., International Publication No. WO2013186613A1, incorporated herein by reference). Anti-CD3 antibodies include OKT3, also known as muromonab. Anti-CD3 antibodies also include the UCHT1 clone, also known as T3 and CD3c. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

Muromonab-CD3 light chain QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC (SEQ ID NO: 1)

Muromonab-CD3 heavy chain

QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 2)

The term “OKT3” refers to the anti-CD3 antibody produced by Miltenyi Biotech, Inc., San Diego, Calif., USA) and or biosimilar or variant thereof (e.g., a humanized, chimeric, or affinity matured variant). A hybridoma capable of producing OKT3 is available in the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT3 is available in the European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706.

The concentration of anti-CD3 antibody in a cell culture may be, for example, about 10-50 ng/mL. In some embodiments, the concentration of anti-CD3 antibody is about 20-40 ng/mL, about 20-30 ng/mL or 30-40 ng/mL. In some embodiments, the concentration of anti- CD3 antibody in a cell culture is about 20 ng/mL. In some embodiments, the concentration of anti-CD3 antibody in a cell culture is about 25 ng/mL. In some embodiments, the concentration of anti-CD3 antibody in a cell culture is about 30 ng/mL. In some embodiments, the concentration of anti-CD3 antibody in a cell culture is about 35 ng/mL. In some embodiments, the concentration of anti-CD3 antibody in a cell culture is about 40 ng/mL.

The cell culture media, in some embodiments, comprises anti-41BB antibody. 41BB (also known as CD137 or TNERSE9) is a protein that is found on the surface of T cells. It is a member of the tumor necrosis factor (TNE) receptor family and is involved in the immune system's response to cancer and infections. 41BB plays important roles in the activation of T cells.

The phrase “anti-41BB antibody” refers to an antibody or variant thereof, e.g.. a monoclonal antibody, and includes human, humanized, chimeric or murine antibodies that specifically binds to the 4 IBB ligand component of the T cell antigen receptor of mature T cells. Anti-41BB antibodies include urelumab, which is a recombinant fully human IgG4 monoclonal antibody that targets the CD 137 receptor. Urelumab specifically binds to and activates CD137-expressing immune cells, stimulating an immune response, in particular a cytotoxic T cell response, against tumor cells. Other anti-41BB antibodies are known and described, for example in International Publication No. WO2021167908.

Urelumab light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKRTVAAPS VFIFPPSDEQEKSGTASVVCEENNFYPREAKVQWKVDNAEQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3)

Urelumab heavy chain QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGY VTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWG RGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4)

The concentration of anti-41BB antibody in a cell culture may be, for example, about 1-20 pg/mL. In some embodiments, the concentration of anti-41BB antibody is about 1-10 g/mL or 10-20 pg/mL. In some embodiments, the concentration of anti-41BB antibody in a cell culture is about 5 pg/mL. In some embodiments, the concentration of anti-41BB antibody in a cell culture is about 10 pg/mL. In some embodiments, the concentration of anti-41BB antibody in a cell culture is about 15 pg/mL. In some embodiments, the concentration of anti- 41BB antibody in a cell culture is about 20 pg/mL.

Culture initiation, in some embodiments, involves combining tumor fragments with TIL Complete Media and supplements (e.g., IL-2, anti-CD3 antibody, and anti-41BB antibody). Following culture initiation, the tumor fragments may be cultured (incubated), for example, in flasks, in a humidified incubator at 37 °C with 5% CO2 in air for about 4 to 6 days. Following this 4 to 6 day period, fresh TIL Complete Media supplemented with IL-2 (e.g., 6,000 lU/mL) may be added. Media exchanges may be performed for another 2 to 4 days by removing a portion of the spent medium and replacing it with fresh medium supplemented with IL-2. This culture period may be referred to herein as a preelectroporation (pre-EP) expansion culture.

At the end of the pre-EP expansion culture (e.g., around day 9, 10, 11 or 12 following culture initiation), cells may be harvested and pooled. In some embodiments, the cells are harvested around day 10 or 11. In some embodiments, a pool reaching 10xl0⁶ - 20x10⁶ viable cells, preferably 16xl0⁶ viable cells, is used for genome editing. This pool of cells following the pre-EP expansion culture may be referred to herein as a “first population of cells.” This population of cells comprises tumor infiltrating lymphocytes produced from the tumor fragments.

Genome Editing

Following a pre-EP expansion culture, a first population of cells may be genetically modified. For example, the endogenous SOCS1 gene, the endogenous REGNASE-1 gene, or both the endogenous SOCS1 gene and the endogenous REGNASE-1 gene, may be inactivated to produce edited tumor infiltrating lymphocytes (eTIL).

Suppressor of Cytokine Signaling 1 (SOCS1) is an inhibitor of the JAK/STAT signaling pathway. The JAK/STAT pathway is activated by cytokines and growth factors and is involved in the regulation of a wide range of biological responses including immune regulation, cell division, and apoptosis (programmed cell death).

REGNASE-1, also known as ZC3H12A or MCPIP1 (Monocyte Chemoattractant Protein- 1 Induced Protein 1), is an RNA-binding protein that has several key roles in immune regulation. It is known for its involvement in the degradation of certain mRNAs encoding pro-inflammatory cytokines and is thus a regulator of the immune response.

Gene activation may be achieved using a programmable nuclease genome editing system such as for example, CRISPR/Cas, ZFNs, or TALENs. In some embodiments, a CRISPR/Cas system is used. Thus, in some embodiments, a first population of cells is transfected with a Cas nuclease (e.g., Cas9 or Casl2a nuclease) and one or more guide RNA(s) targeting endogenous SOCS1 and/or endogenous REGNASE-1 to produce a second population of cells comprising edited cells, such as edited tumor infiltrating lymphocytes.

In some embodiments, a guide RNA (gRNA) targeting endogenous SOCS1 is selected from a sequence in Table 1:

Table 1: Guide RNA (gRNA) targeting endogenous SOCS1

In some embodiments, a gRNA targeting endogenous REGNASE-1 is selected from a sequence in Table 2: Table 2: Guide RNA (gRNA) targeting endogenous REGNASE-1

A genome editing system such as a CRISPR/Cas system may be introduced into a population of cells by transfection, transduction, electroporation, or physical disruption of the cell membrane by a microfluidics device. In some embodiments, a genome editing system is introduced as a polynucleotide encoding one or more components of the system, a protein, or a ribonucleoprotein (RNP) complex (e.g., Cas nuclease complexed with one or more gRNA). For example, Cas nuclease (e.g., Cas9 nuclease) and a gRNA (e.g., targeting SOCS1 or REGNASE-1) may be complexed to form CRISPR RNP complexes. In some embodiments, a Cas nuclease and a gRNA is complexed at a 1:1, 1:1.5, or 1:2 molar ratio. In preferred embodiments, electroporation is used. Thus, in some embodiments, a first population of cells (e.g., comprising a minimum of 16xl0⁶ viable cells) are resuspended in electroporation buffer and transfected with RNP complexes using an electroporation technology (e.g., MaxCyte® ExPERT® GTX™ electroporation technology).

Post- Genome Editing Expansion

Cell Expansion

Following genome editing (e.g., electroporation with CRISPR/Cas components), the cells may be recovered and rested in cell culture media (e.g., TIL Complete Medium). The rested cells may then be expanded in cell culture media (e.g., TIL Complete Medium) supplemented with IL-2, for example, about 300 - 3,000 lU/mL of IL-2 (e.g., recombinant human IL-2). In some embodiments, the rested cells are expanded in cell culture media (e.g., TIL Complete Medium) supplemented with about 3,000 lU/mL IL-2. This culture period may be referred to herein as an expansion culture. In an exemplary embodiment, the cells are cultured in an appropriately sized G-Rex flask in a humidified incubator at 37 °C with 5% CO2 in air. Cell expansion may be supported with IL-2 addition regularly during the expansion culture, for example, every day or every other day for about 9-10 days. In some embodiments, the cells are expanded for about 9, 10, 11 or 12 days following genome editing. In some embodiments, the cells are expanded for about 10 or 11 days following genome editing. At the end of expansion culture, an expanded population of edited tumor infiltrating lymphocytes is produced.

In some embodiments, eTILs are expanded without (in the absence of) feeder cells, i.e., “feeder-free” process. Non-limiting examples of feeder cells include, human fibroblasts, peripheral blood mononuclear cells, and bone marrow stromal cells. Thus, eTILs of the disclosure, in some embodiments, are expanded in the absence of human fibroblasts, peripheral blood mononuclear cells, and/or bone marrow stromal cells. In some embodiments, the entire method, from tumor fragmentation to eTIL expansion, is performed without feeder cells.

Harvest, Formulation, and Cryopreservation

Following post-genome editing cell expansion, a cell culture may be visually inspected for evidence of contamination, for example. In some embodiments, samples of supernatant and cells are taken for mycoplasma release testing.

Methods of the disclosure, in some embodiments further comprise cryopreserving cells, such as edited tumor infiltrating lymphocytes, in cryopreservation medium. In some embodiments, a cryopreservation medium comprises one or more of (or all of) DMSO, glycerol, ethylene glycol, propylene glycol, hydroxycellulose, mammal-derived serum albumin, sodium gluconate, a balanced and buffered salt solution, acetate, L-cysteine, coenzyme Q10, and one or more saccharide (e.g., sucrose, maltose, and trehalose).

A formulated drug product may be filled into appropriately sized bags, and samples removed for endotoxin and sterility release testing. The bags are typically visually checked prior to cryopreservation.

The drug product bags, in some embodiments, are cryopreserved using a controlled rate freezer and stored in the vapor phase of LN2 (< -130 °C). Samples may be collected for cell count, viability, % CD3+ cells, and/or genome (e.g., SOCS1 and/or REGNASE-1) editing efficiency for release testing.

Additional Embodiments

The disclosure also relates to the embodiments set forth in the numbered paragraphs below:

1. A method comprising:

(a) producing tumor infiltrating lymphocytes from cell culture media comprising tumor fragments, recombinant human interleukin-2 (IL-2), anti-CD3 antibody, and anti-41BB antibody;

(b) genetically modifying the endogenous SOCS1 gene and/or the endogenous REGNASE-1 gene in the tumor infiltrating lymphocytes to produce edited tumor infiltrating lymphocytes (eTIL); and

(c) expanding the eTIL in cell culture media comprising recombinant human IL-2.

2. A method comprising:

(a) culturing tumor fragments in culture media comprising recombinant human interleukin-2 (IL-2), anti-CD3 antibody, and anti-41BB antibody to produce a first population of cells comprising tumor infdtrating lymphocytes;

(b) electroporating the first population of cells with Cas nuclease and one or more guide RNA(s) targeting endogenous SOCS1 and/or endogenous REGNASE-1 to produce a second population of cells comprising edited tumor infdtrating lymphocytes; and

(c) culturing the second population of cells in culture media comprising recombinant human IL-2 to produce an expanded population of edited tumor infiltrating lymphocytes.

3. The method of paragraph 1 or 2, wherein (c) is without feeder cells. 4. The method of any one of paragraphs 1-3, wherein the culture media comprises RPMI-1640, 10% Human AB Serum, HEPES buffer, and 2-mercaptoethanol, optionally wherein the concentration of HEPES buffer is about lOmM and/or the concentration of 2- mercaptoethanol is about 5.5 x 10'⁵ M.

5. The method of any one of paragraphs 1-4, wherein the tumor fragments comprise 1- 3 mm fragments.

6. The method of any one of paragraphs 1-5, wherein (a) comprises culturing about 5 of the tumor fragments per 20-40 ml of the culture media.

7. The method of any one of paragraphs 1-6, wherein the concentration of the recombinant human IL-2 of (a) is about 4,000-6,000 lU/mL, the concentration of anti-CD3 antibody is about 20-40 ng/mL, and/or the concentration of anti-4 IBB antibody is about 20- 30 pg/mL; and/or wherein the concentration of the recombinant human IL-2 of (b) is about 300 lU/mL to about 3,000 lU/mL.

8. The method of paragraph 7, wherein the concentration of the recombinant human IL- 2 of (a) is about 6,000 lU/mL, the concentration of anti-CD3 antibody is about 30 ng/mL, and/or the concentration of anti-4 IBB antibody is about 10 pg/mL; and/or wherein the concentration of the recombinant human IL-2 of (b) is about 3,000 IU/mL..

9. The method of any one of paragraphs 1-8, wherein the anti-CD3 antibody comprises the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

10. The method of any one of paragraphs 1-9, wherein the anti-41BB antibody comprises the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4.

11. The method of any one of paragraphs 1-10, wherein (a) occurs within about 9-12 days, and (c) occurs within about 9-12 days.

12. The method of any one of paragraphs 1-10, wherein (a) occurs within about 11 days, and (c) occurs within about 11 days.

13. The method of any one of paragraphs 2-12, wherein the first population of cells comprises at least 16xl0⁶ viable cells.

14. The method of any one of paragraphs 2-13, wherein the first population of cells comprises about 10xl0⁶ to about 20x10⁶ viable cells.

15. The method of any one of paragraphs 2-14, wherein the Cas nuclease is a Cas9 nuclease.

16. The method of any one of paragraphs 2-15, wherein the one or more gRNA(s) comprise(s) the sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and/or SEQ ID NO: 15.

17. The method of any one of paragraphs 2-16, wherein the one or more gRNA(s) comprise(s) the sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:

18, SEQ ID NO: 19, and/or SEQ ID NO: 20.

18. The method of any one of paragraphs 2-17, wherein the Cas nuclease and the one or more gRNA(s) are complex at a 1:1.5 molar ratio to form a ribonucleoprotein complex.

19. The method of any one of paragraphs 1-18, wherein (a) comprises culturing the tumor fragments in multiple flasks, each optionally containing about 5 of the tumor fragments per 20-40 ml of the culture media to produce a subpopulation of cells in each of the flasks.

20. The method of paragraph 19 further comprising pooling the subpopulation of cells from each of the flasks to produce the first population of cells.

21. The method of any one of paragraphs 1-20, further comprising cry opreserving the edited tumor infiltrating lymphocytes in cryopreservation medium.

EXAMPLES

Example 1: Process for Producing Edited Tumor Infiltrating Lymphocytes

The manufacturing process includes the following key phases: pre-electroporation (Pre-EP) expansion; electroporation; and post-EP expansion (FIG. 1).

Tumor fragmentation and culture initiation are the first step in the manufacturing process. Harvested autologous tumor material from the patient was dissected into approximately 1 - 3 mm fragments in each of 3 dimensions. If cryopreserved, the tumor fragments were thawed at 37°C and rinsed in TIL Complete Medium (RPMI-1640, 10% Human AB Serum, HEPES 10 mM, 2-mercaptoethanol 5.5 x 10'⁵ M). Tumor fragments were cultured in G-Rex®10 flask(s) containing 5 fragments per flask in 20 mL of TIL Complete Medium supplemented with 6,000 lU/mL of recombinant human interleukin-2 (IL-2), anti- CD3 antibody (clone OKT3, 30 ng/mL) and anti-4- IBB (urelumab, 10 pg/mL). Each flask was incubated in a humidified incubator at 37°C with 5% CO2 in air.

TIL culture conditions follow tumor fragmentation and culture initiation. Four to six days after culture initiation, fresh culture medium was added to bring the final volume to 40 mL per flask using TIL Complete Medium supplemented with 6,000 lU/mL of IL-2. Media exchanges were performed by removing 20 mL of spent medium and replacing with fresh medium containing IL-2. In-process tests for sterility and cell count were performed as needed during the culture. At the end of the pre-EP expansion culture, cells were harvested and pooled. A pool reaching 16 x 10⁶ viable cells or more advanced to the next steps. A pool that has not reached 16 x 10⁶ viable cells was terminated.

Following TIL culture conditions described above, the TILs were subject to electroporation. Cas9 and sgRNA targeting endogenous SOCS1 and/or endogenous REGNASE-1 were complexed at a 1:1.5 molar ratio to form CRISPR RNP complexes. A minimum of 16 x 10⁶ harvested cells were resuspended in electroporation buffer and transfected with RNP complexes using the MaxCyte® ExPERT® GTx™ electroporation technology.

Following electroporation, the cells were recovered and rested in TIL Complete Medium. The rested cells were expanded in TIL Complete Medium supplemented with 300 - 3,000 lU/mL of IL-2 in an appropriately sized G-Rex flask in a humidified incubator at 37°C with 5% CO2 in air. Cell expansion was supported with IL-2 addition regularly during the culture.

At the end of the cell expansion, the cell culture was visually inspected for evidence of contamination. Samples of supernatant and cells were taken for mycoplasma release testing. The cells were washed and formulated into cryopreservation medium.

The formulated drug product was filled into appropriately sized bags and samples were removed for endotoxin and sterility release testing. The bags were visually checked prior to cryopreservation.

The drug product bags were cryopreserved using a controlled rate freezer and stored in the vapor phase of LN2 (< -130°C). Samples collected for cell count, viability, % CD3⁺ cells, and SOCS1 and/or REGNASE-1 editing efficiency were cryopreserved for release testing.

Table 3 provides data for an exemplary batch of edited TILs using the process disclosed herein.

Table 3: Results for Batch 00157-NCP

Example 2: KSQ-001EX, an Engineered TIL Therapy, Manufactured from a Clinical- Scale, Feeder-Free Process for the Treatment of Solid Tumor Indications

The manufacturing process of Example 1 (pre-electroporation (Pre-EP) expansion; electroporation; and post-EP expansion) was used to produce an engineered TIL therapy using CRISPR/Cas9 technology to knockout the SOCS1 gene (KSQ-001EX). KSQ-001EX characteristics and anti-tumor functionality were subsequently evaluated.

Pre-EP TIL Expansion

Fresh or frozen tumors from melanoma, uveal melanoma (“uveal”), lung (“NSCLC”), colorectal (“CRC”), head and neck ("HNSCC”), and pancreatic (“PDAC”) cancer were cut in 1-3 mm per side fragments and put in culture in complete media supplemented with 10% human AB serum, agonistic antibodies against CD3 (OKT3) and 4- IBB (urelumab) and high dose of IL-2. Tumor infiltrating lymphocytes (“TIL”) were expanded for 11 days following media addition on Days 4-6 and one media change three to four days later. At Day 11, yield and viability was determined using a NucleoCounter NC-200 system. If viable cell count was >16xl0⁶ TIL were washed and concentrated for electroporation. Cell yields from the various tumors are presented in FIG 2A.

Electroporation

For SOCS1 editing, washed and concentrated TIL (between 16xl0⁶ and 480xl0⁶ TIL) were electroporated using the MaxCyte technology in the OC-100, 400 or CL1.1 device with RNP complexes containing Cas9 and the SOCS1 -targeting sgRNA at a 1:1.5 molar ratio.

Post-EP TIL Expansion Edited TIL were expanded post electroporation in a G-rex 100M (< 70x10⁶ TIL) or 500M (> 70xl0⁶ TIL) for 10 to 12 days, for a total process length of 21 to 23 days. Small scale expansions were plated in a G-Rex 10M (6 xlO⁶ TIL). During the post-electroporation expansion, IL-2 (high dose) was added every two to three days. On the final day, for large scale expansions, edited TIL were harvested, concentrated and washed using Lovo cell processing instrument and formulated for cryopreservation at a concentration between 5xl0⁶ to 50xl0⁶ cells/mL. Einal cell yields with an overall manufacture success rate are presented in FIG. 2B.

Post-Thaw Viability

Post-thaw viability of the cryopreserved edited TILs was determined using a NucleoCounter NC-200 system. Cryopreserved final drug product was tested following thaw at 37°C. Cells were diluted to the concentration range of the NC-200 then loaded into singleuse cassettes pre-loaded with Acridine Orange (AO) to stain total cells and 4’,6-Diaminidino- 2-Phenylindole, dihydrochloride (DAPI) to stain non- viable cells. These were subsequently automatically quantified using image cytometry.

Flow Cytometry

The percentage of viable CD3+ cells was measured by flow cytometry using the BD LACS Lyric. Cryopreserved final drug product was tested following thaw at 37°C. A viability stain was used to distinguish between live and dead cells. The % CD3+ of viable cells was determined by first using standard procedures to select live single cells then gating for CD3+.

Editing Efficiency

SOCS1 editing efficiency was determined by ddPCR. gDNA was extracted from cells for testing. A drop-off assay design was used with primer and probes specific to a Reference site and the Target site. The Reference site is upstream of the u728 cut site and the Target site spans the u728 cut site. Droplets that contain edited gDNA were identified as positive for the Reference and negative for the Target. Droplets that contain non-edited gDNA template were identified as positive for both the Reference and Target. SOCS1 editing efficiency (%gDNA) was calculated as the ratio of Edited droplets to the sum of the Edited and Non-Edited droplets. Figure 2C is graph of the viability, %CD3+ cells, and editing efficiency of the edited TIL samples.

SOCS1 Western Blot Analysis

SOCS1 expression levels in tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX) were evaluated as follows. Cryopreserved No EP or KSQ-001EX cells were thawed and stimulated overnight with ImmunoCult Human CD3/CD28/CD2 T cell activator at a concentration of 6.25 mL/ml in REP media (1:1 ratio of RPMI 1640 and AIM V supplemented with 5% heat- inactivated human serum, type AB, and 6000 U/mL IL-2). Cell pellets were collected and then lysed with RIPA buffer containing protease and phosphatase inhibitor and centrifuged at 21,000 x g for 10 minutes at 4°C; concentration of protein lysates in supernatant was quantified by BCA assay following manufacturer protocol. Equal amounts of protein per sample were loaded and detected on a Wes instrument following manufacturer protocol. Vinculin was included as a loading control. SOCS1 protein levels from No EP or KSQ-001EX TIL are presented in FIG. 3.

Cell Surface Staining and Flow Cytometry

Phenotypic characteristics of tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX) were evaluated as follows. Cryopreserved No EP or KSQ-001EX cells were thawed. Single cell suspensions were processed in a 96- well plate, washed with cell staining buffer and cell surface markers stained with a master mix containing viability dye, Fc block, CD45 (clone H130), CD3 (clone SK7 or UCHT1), CD4 (clone L200), CD8 (clone RPA-T8), CD20 (clone 2H7) and CD56 (clone NCAM16.2) antibodies. After surface staining, cells were washed with cell staining buffer, and then fixed, according to manufacturer protocol. Cells were resuspended in cell staining buffer and analyzed using the BD-FACS Symphony A3 or LSR-Fortessa. Cells were gated based on FSC-A and SSC-A, singlets were then defined along the SSC-A = SSC-H line. Viable lymphocytes were gated on CD45⁺ and live/dead" cells. These cells were then subset as CD20⁺, CD3" or CD3⁺ cells. CD56⁺ cells were gated on CD3" cells (CD56⁺CD3‘ NK cell population was reported using CD45⁺ as parent gate), while CD4⁺ and CD8⁺ cells were determined on CD3⁺ gate. CD4+ and CD8+ distribution is shown is shown in FIG. 4A. Frequency of CD45RO⁺CCR7‘ T Effector Memory Cells (Tern) out of total live CD45⁺CD3⁺ population is shown in FIG. 4B. Assessment of Cytotoxicity

Cytotoxicity of tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX) were evaluated as follows. A375-OKT3 cells engineered to express red fluorescent protein (RFP) were cultured in DMEM supplemented with 10% heat-inactivated FBS and lx pen/strep solution. Three days before assay initiation, cells were harvested via TrypLE, with 10,000 cells/well plated in 100 pL RPMI supplemented with 10% heat-inactivated FBS and lx pen/strep solution in ultra-low attachment U-bottom plates to allow for spheroid formation. On the day of assay initiation, No EP and KSQ-001EX cells were added at different effector to target (E:T) ratios to the spheroid plate in 100 pL REP media supplemented with 12,000 U/mL IL-2. Images were taken via Incucyte S3 at 4x magnification in the red fluorescence, brightfield, and phase channels every 6 hours for 6 days to monitor spheroid growth or regression. TIL cytotoxic activity was assessed by changes in red fluorescent intensity as a function of tumor spheroid cell growth or death, normalized to the first co-culture time point. KSQ-001EX exhibited increased anti-tumor activity relative to No EP (FIGs. 5A and 5B).

Assessment of Effector Cytokine Production

Cytokine (IFNy) production was evaluated from KSQ-001EX or donor-matched No EP following challenge with A375-OKT3 spheroid cells as described above in the cytotoxicity functional assay method above. Supernatant was collected 24 hours after the addition of TIL to tumor cells, and cytokine (IFNy) concentration was determined by MSD immunoassay following manufacturer protocol. Supernatant was collected from TIL-tumor- spheroid co-cultures at 24hr for IFNy measurement. KSQ-001EX exhibited increased IFNy secretion relative to No EP (FIG. 5C).

Trans criptomics analysis of KSQ-001EX

General bulk RNASeq methods for tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX) were measured as follows. RNA was extracted using either Qiagen or Autogen kits as per the vendor's guidelines. Libraries for RNA sequencing were prepared following the Truseq Stranded mRNA protocol. The final library concentrations were determined using the KAPA Library Quantification qPCR assay. Sequencing was carried out on a Nextseq2000 platform, aiming for a sequencing depth of 15 million reads for each sample. The raw sequence image files from the sequencer were converted to the fastq format and checked for quality to ensure the sequencing quality scores did not deteriorate at the read-ends. Useful metrics such as the per base sequence quality, the per base sequence content, per base N content, sequence length distribution, adapter and k-mer content and sequence duplication levels were collected for each sequencing run using FastQC (bioinformatics.babraham.ac.uk/projects/fastqc). FastQCs were aligned to the human genome (GRCh38, Gencode version 43) using STAR aligner (Dublin et al., STAR: ultrafast universal RNA-seq aligner; available at pubmedcentral. nih.gov/articlerender.fcgi?artid=3530905&tool=pmcentrez&rendertype=abstr act) and viewed on Integrative Genome Viewer (IGV) (Robinson et al., nature.com/doifinder/10.1038/nbt.1754). Only reads that were uniquely mapped were considered for read annotation to gene features. Reads were annotated with the Gencode v43 gene transfer format (gtf) file using RSEM (Li et al., BMC Bioinformatics 12, 323 (2011)). RSEM provides both read counts and transcripts per million reads (TPMs) for each gene feature in the Gencode gtf.

Differential expression using DESeq2

The R package DESeq2 (Love et al., Genome Biol 15, 550 (2014)) was used for differential gene expression analysis. Genes were considered differentially expressed if they were UP-regulated by log2FC > +1 or DOWN-regulated by log2FC < -1 with an adjusted p- value value < 0.05.

Pathway analysis using GSEA

Pathway analysis was performed using Gene Set Enrichment Analysis (Subramanian et al., PNAS (2005) (GSEA preranked mode) using the MSigDB hallmarks pathways and custom T-cell exhaustion pathways compiled from literature. For all genes included in the differential expression analysis using DESeq2 (previously described) a metric was computed as the product of logFC and -log 10(P- value). Genes were ranked from most significantly overexpressed to most significantly under-expressed. A "running sum" statistic was calculated for each gene set in the pathway database, based on the ranks of the members of the set, relative to those of the non-members. The enrichment score (ES) was defined as the maximum sum of the running sum and the genes that make up this maximum ES contribute to the core enrichment in that pathway. All pathways with a FDR < 0.05 were considered to be significant.

Singscores Gene-set scores per sample were derived using singscores (Foroutan et al., BMC Bioinformatics 19, 404 (2018)), which uses the ranks of genes within a given gene-set, normalized relative to the maximum and minimum theoretical scores for a gene set of a given size. Normalized counts were used to rank the gene expression data for a given gene set and the function simpleScore was used from the singscore R package. A paired Wilcox test was performed between the conditions KSQ-001EX vs NoEP and the nominal P-value < 0.05 was deemed significant.

Following GSEA analysis, KSQ-001EX was determined to exhibit a hallmark IL2 STAT5 signature relative to No EP (FIG. 6).

T-cell Repertoire (TCR)

TCR was evaluated for tumor infiltrating lymphocytes without electroporation (No EP) and with electroporation (KSQ-001EX) as follows. RNA was extracted using either Qiagen kits or Autogen Xtract following the vendor's guidelines. TCRb cDNA was generated using TCRb gene specific primer containing a UMI and adapter sequence on the 5’ end using Super Script IV (Thermo Fisher) protocol. Multiplex PCR using a compact primer set was used to amplify TCRb CDR3 sequences. 2^nd- step PCR was performed to add on Illumina adapters and indices. The final library concentrations were determined using the KAPA Library Quantification qPCR assay. Sequencing was carried out on a Nextseq2000 platform, aiming for a sequencing depth of 1 million reads for each sample. Custom analysis pipeline used to adjust for PCR duplication utilizing the UMI barcode and subsequent fastq read pairs were aligned using MiXCR. Custom python script was used to calculate the Simpson Diversity Index utilizing the following equation:

Where: n = number of individual TCRs

N = total number of TCRs

Following analysis of Simpson’s TCR Diversity Index (SDI), KSQ-001EX exhibited a diverse TCR repertoire (FIG. 7).

KSQ-001EX manufactured from clinical- scale runs were highly viable (>90%) following cryopreservation and thaw, with > 90% SOCS1 on-target editing and complete knockdown of SOCS1 at the protein level. KSQ-001EX across all indications assessed showed high frequency of CD8 (median -80%), an attribute associated with TIL clinical responses. KSQ-001EX also retained high diversity of TCR repertoire. Consistent with the biology of SOCS1 editing, KSQ-001EX exhibited greater cytotoxicity and higher IFNy production against tumor spheroids in comparison to unedited TIL (No EP) and enhanced IL- 2-STAT5 transcriptomic signature, demonstrating heightened sensitivity of KSQ-001EX to cytokines.

Example 3: Comparison of Engineered TIL Therapy Manufacture Processes

The manufacturing process of Example 1 (pre-electroporation (Pre-EP) expansion; electroporation; and post-EP expansion; referred to as “Process 2”) was used to produce an engineered TIL therapy using CRISPR/Cas9 technology to knockout the SOCS1 gene (KSQ- 001EX) and an engineered TIL therapy using CRISPR/Cas9 technology to knockout both the SOCS1 gene and the ZC3H12A gene (KSQ-004EX). Another manufacturing process, “Process 1” was used to produce an engineered TIL therapy using CRISPR/Cas9 technology to knockout the SOCS1 gene (KSQ-001) and an engineered TIL therapy using CRISPR/Cas9 technology to knockout both the SOCS1 gene and the ZC3H12A gene (KSQ-004). A schematic of the two manufacturing processes is depicted in Figure 8.

The frequencies of CD3+ T cells from engineered TIL therapies KSQ-001, KSQ- 001EX, KSQ-004, and KSQ-004EX were subsequently evaluated using cell surface staining and flow cytometry. KSQ-001EX generated from Process 2 process showed significantly higher frequencies of CD3+ T cells relative to KSQ-001 generated from Process 1 (FIG. 9) and KSQ-004EX generated from Process 2 process showed significantly higher frequencies of CD3+ T cells relative to KSQ-004 generated from Process 1 (FIG. 10).

Process 1

Viably cryopreserved tumor fragments (VCTF) were cultured in TransACT (providing CD3 and CD28 stimulation), IL-2 and growth media for 7-11 days to obtain pre- EP (electroporation) TILs; pre-EP TILs were engineered to knock out SOCS1, or SOCS1 and REGNASE-1, and cultured for additional 7-11 days in IL-2 and growth media. For cell engineering, 50~100xl0⁶/mL Pre-REP TILs were mixed with RNP complexes containing Cas9 and SOCS1 -targeting sgRNA or RNP complexes containing Cas9 and SOCS1 -targeting sgRNA and REGNASE-1 -targeting sgRNA electroporated using MaxCyte instrument per manufacturing protocol (the volume ratio of Pre-REP TILs to RNP was maintained at 4:1).

Editing efficiency was determined at harvest by next generation sequencing (NGS).

Cell Surface Staining and Flow Cytometry

To determine the impact of SOCS1 and REGNASE-1 inactivation on TIL phenotype, cryopreserved TILs were thawed and the expression of key cell surface markers was evaluated to determine their cellular composition and memory phenotype by flow cytometry. The assessment of cellular composition included the percentage of total T-cells (CD3+) and T-cell subtypes (CD4+, CD8+).

Flow cytometry staining and analysis was performed for all TILs. Single-cell suspensions were processed and deposited in a 96-well V-bottom plate and washed with cell staining buffer. Cells were stained using a master mix containing viability dye, Fc block, and antibodies. After surface staining, cells were washed with cell staining buffer, and then fixed according to manufacturer protocol. Cells were resuspended in cell staining buffer and analyzed using the BD-FACS Symphony A3 or LSR-Fortessa.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value. Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

What is claimed is: CLAIMS

1. A method comprising:

(c) expanding the eTIL in cell culture media comprising recombinant human IL-2.

2. A method comprising:

3. The method of claim 1 or 2, wherein (c) is without feeder cells.

4. The method of any one of claims 1-3, wherein the culture media comprises RPML 1640, 10% Human AB Serum, HEPES buffer, and 2-mercaptoethanol, optionally wherein the concentration of HEPES buffer is about lOmM and/or the concentration of 2-mercaptoethanol is about 5.5 x 10'⁵ M.

5. The method of any one of claims 1-4, wherein the tumor fragments comprise l-3mm fragments.

6. The method of any one of claims 1-5, wherein (a) comprises culturing about 5 of the tumor fragments per 20-40 ml of the culture media.

7. The method of any one of claims 1-6, wherein the concentration of the recombinant human IL-2 of (a) is about 4,000-6,000 lU/mL, the concentration of anti-CD3 antibody is about 20-40 ng/mL, and/or the concentration of anti-41BB antibody is about 20-30 pg/mL; and/or wherein the concentration of the recombinant human IL-2 of (b) is about 300 lU/mL to about 3,000 lU/mL.

8. The method of claim 7, wherein the concentration of the recombinant human IL-2 of (a) is about 6,000 lU/mL, the concentration of anti-CD3 antibody is about 30 ng/mL, and/or the concentration of anti-41BB antibody is about 10 |jg/mL; and/or wherein the concentration of the recombinant human IL-2 of (b) is about 3,000 lU/mL.

9. The method of any one of claims 1-8, wherein the anti-CD3 antibody comprises the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

10. The method of any one of claims 1-9, wherein the anti-41BB antibody comprises the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4.

11. The method of any one of claims 1-10, wherein (a) occurs within about 9-12 days, and (c) occurs within about 9-12 days.

12. The method of any one of claims 1-10, wherein (a) occurs within about 11 days, and (c) occurs within about 11 days.

13. The method of any one of claims 2-12, wherein the first population of cells comprises at least 16xl0⁶ viable cells.

14. The method of any one of claims 2-13, wherein the first population of cells comprises about 10xl0⁶ to about 20x10⁶ viable cells.

15. The method of any one of claims 2-14, wherein the Cas nuclease is a Cas9 nuclease.

16. The method of any one of claims 2-15, wherein the one or more gRNA(s) comprise(s) the sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and/or SEQ ID NO: 15.

17. The method of any one of claims 2-16, wherein the one or more gRNA(s) comprise(s) the sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and/or SEQ ID NO: 20.

18. The method of any one of claims 2-17, wherein the Cas nuclease and the one or more gRNA(s) are complex at a 1:1.5 molar ratio to form a ribonucleoprotein complex.

19. The method of any one of claims 1-18, wherein (a) comprises culturing the tumor fragments in multiple flasks, each optionally containing about 5 of the tumor fragments per 20-40 ml of the culture media to produce a subpopulation of cells in each of the flasks.

20. The method of claim 19 further comprising pooling the subpopulation of cells from each of the flasks to produce the first population of cells.

21. The method of any one of claims 1-20, further comprising cry opreserving the edited tumor infiltrating lymphocytes in cryopreservation medium.