Combination of tumor infiltrating lymphocytes (TIL) and low dose radiation

Abstract

Provided herein are combination therapy methods, compositions and uses involving adoptive T cell therapies, such as tumor infiltrating lymphocyte (TIL) therapies and low dose radiation therapy (LDRT). In some embodiments, the combination therapy methods, composition and uses also involve a chemotherapeutic regimen and / or a checkpoint inhibitor.

Description

COMBINATION OF TUMOR INFILTRATING LYMPHOCYTES (TIL) AND LOW DOSE RADIATIONCross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional patent application No. 63 / 543,720 filed October 11, 2023, entitled “Combination of Tumor Infiltrating Lymphocytes (TIL) and Low Dose Radiation”, the content of which is incorporated by reference in its entirety.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence listing in electronic format. The sequence listing is provided as a filed entitled 220612001740. xml, created October 4, 2024, which is 3,774 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure relates in some aspects to combination therapy methods, compositions and uses involving adoptive T cell therapies, such as tumor infiltrating lymphocyte (TIL) therapies, and low dose radiation. In some embodiments, the combination therapy methods, composition and uses also involve a chemotherapeutic regimen and / or a checkpoint inhibitor.Background

[0004] In most patients with metastatic cancers, systemic chemotherapy is not curative. The last decade has seen significant advances in the treatment of a segment of these patients with developments in the field of immunotherapy, namely checkpoint inhibitors, however these advances still are only effective as monotherapies in a minority of patients (Marra et al., 2019, BMC medicine 17:90; Morse et al., 2019, The oncologist, 24:1453-1461). Thus, a need for novel therapeutic options remains. Adoptive cell therapies hold promise for treating certain cancers, however, their ability to treat solid tumors has remained elusive. The therapeutic success of many adoptive cell therapies (ACTs) for solid tumors including, but not limited to, tumor infiltrating lymphocyte (TIL) based therapies, require ACT cells to migrate into primary or metastatic solid tumor tissues following intravenous administration of the ACT. In some cases, the tumor tissue can represent a hostile and immunosuppressive environment which can limit the ability of ACT cells to migrate into the tumor tissue and / or can compromise the therapeuticbenefit of ACT. Thus, improved methods for ACT particularly involving TIL therapies are needed.Provided herein are embodiments that meet such needs.Summary of the Invention

[0005] Provided herein is a method for treating a solid tumor malignancy in a subject, the method comprising: a. administering a radiation therapy to a subject that has a solid tumor malignancy and b. administering a T lymphocyte infiltrating (TIL) composition enriched in tumor reactive T cells to the subject.

[0006] In some of any of the provided embodiments, the radiation therapy is a low dose radiation therapy. In some of any of the provided embodiments, the radiation therapy is administered as a total dose of no more than about 30 Gy. In some of any of the provided embodiments, the radiation therapy is administered as a total dose of from 0.5 Gy to 20 Gy. In some of any of the provided embodiments, the radiation therapy is administered as a total dose of from 0.5 Gy to 10 Gy. In some of any of the provided embodiments, the radiation therapy is administered as a total dose of about 4 Gy. In some of any of the provided embodiments, the radiation therapy is administered as a total dose to induce infiltration of T cells into the tumor of the subject.

[0007] In some of any of the provided embodiments, the radiation therapy is administered as a total dose that does not damage DNA to reduce or prevent cell division of cancer cells. In some of any of the provided embodiments, the radiation therapy is administered as a total dose that does not kill cancer cells. In some of any of the provided embodiments, the total dose of the radiation therapy is administered prior to and / or after administering the TIL composition to the subject. In some of any of the provided embodiments, the total dose of the radiation therapy is administered prior to administering the TIL composition to the subject. In some of any of the provided embodiments, the total dose of the radiation therapy is administered after administering the TIL composition to the subject.

[0008] In some of any of the provided embodiments, the total dose of the radiation therapy is administered as a divided dose given as a plurality of fraction doses. In some of any of the provided embodiments, the plurality of fraction doses are 2, 3, 4, 5 or 6 doses. In some of any of the provided embodiments, the plurality of fraction doses are 4 doses. In some of any of the provided embodiments, the plurality of fraction doses are administered prior to and / or after administering the TIL composition to the subject. In some of any of the provided embodiments, each of the plurality of fraction doses are administered prior to administering the TIL composition to the subject. In some of any of the provided embodiments, each of the plurality of fraction doses are administered after administering the TIL composition to the subject. In some of any of the provided embodiments, at least one of the plurality of fraction doses is administered prior to administration of the TIL composition and at least one of the plurality of fraction doses is administered after administration of the TIL composition. In some of any ofthe provided embodiments, 2 fraction doses are administered prior to administration of the TIL composition and 2 fraction disease are administered after administration of the TIL composition. In some of any of the provided embodiments, each of the plurality of fraction doses is administered at a dose of no more than 6 Gy per lesion per day. In some of any of the provided embodiments, each of the plurality of fraction doses is administered at a dose of from 0.5 Gy to 4 Gy. In some of any of the provided embodiments, each of the plurality of fraction doses is administered at a dose of from 0.5 Gy to 2 Gy. In some of any of the provided embodiments, each of the plurality of fraction doses is administered at a dose of about 1 Gy.

[0009] In some of any of the provided embodiments, administration of the radiation therapy prior to administration of the TIL composition is initiated no more than 30 days prior to the harvesting of tumor to derive the TIL composition. In some of any of the provided embodiments, the administration of the radiation therapy prior to administration of the TIL composition is initiated no more than 2 days prior to the harvesting of tumor to derive the TIL composition.

[0010] In some of any of the provided embodiments, the administration of the radiation therapy prior to administration of the TIL composition is initiated no more than 10 days prior to the administration of the TIL composition. In some of any of the provided embodiments, the administration of the radiation therapy prior to administration of the TIL composition is initiated no more than 2 days prior to the administration of the TIL composition. In some of any of the provided embodiments, the radiation therapy comprises administering a first total dose of radiation and a second total dose of radiation. In some of any of the provided embodiments, the first total dose of radiation is initiated no more than 30 days prior to the harvesting of tumor to derive the TIL composition and the second total dose of radiation is initiated no more than 10 days prior to the administration of the TIL composition.

[0011] In some of any of the provided embodiments, the radiation therapy is an external beam radiation therapy, a brachytherapy or a systemic radioisotope therapy. In some of any of the provided embodiments, the TIL composition comprises a population of T cells that is enriched in at least 10 different T cell receptor (TCR) clonotypes each with a frequency in the population of at least 2.0%. In some of any of the provided embodiments, at least 11 different TCR clonotypes have a frequency in the population of at least 2.0%. In some of any of the provided embodiments, at least 12 different TCR clonotypes have a frequency in the population of at least 2.0%. In some of any of the provided embodiments, 8 to 15 different T cell receptor (TCR) clonotypes make up at least 50 % of the TCR frequency in the population. In some of any of the provided embodiments, 9 to 12 different TCR clonotypes make up at least 50% of the TCR frequency in the population. In some of any of the provided embodiments, the TCR clonotypes exhibit reactivity to at least one CD4 antigen and at least one CD8 antigen. In some of any of the provided embodiments, the TCR clonotypes exhibit reactivity for 2 to 6 different peptide antigens. In some of any of the provided embodiments, the TCR clonotypes exhibit reactivity for 2 to 4 peptide antigens. In some of any of the provided embodiments, the TCR clonotypesexhibit reactivity for 2 peptide antigens. In some of any of the provided embodiments, the TCR clonotypes exhibit reactivity for one CD8 antigen and one CD4 antigen. In some of any of the provided embodiments, at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity. In some of any of the provided embodiments, the ratio of CD4+ T cells to CD8+ T cells in the composition is at or about 10:1 to 25:1, optionally at or about 20:1. In some of any of the provided embodiments, greater than 75% of the CD3+ T cells, optionally greater than 75% of the CD4 and CD8+ T cells, express markers of an effector memory phenotype. In some of any of the provided embodiments, greater than 80% of the CD3+ T cells, optionally greater than 80% of the CD4 and CD8+ T cells, express markers of an effector memory phenotype. In some of any of the provided embodiments, greater than 85% of the CD3+ T cells, optionally greater than 85% of the CD4 and CD8+ T cells, express markers of an effector memory phenotype. In some of any of the provided embodiments, greater than 90% of the CD3+ T cells, optionally greater than 90% of the CD4 and CD8+ T cells, express markers of an effector memory phenotype.

[0012] In some of any of the provided embodiments, the effector memory phenotype is characterized by surface marker expression of one or more of CD45RO+, CD40RO-, CD62L-, CCR7-, CD28- and CD27-. In some of any of the provided embodiments, the effector memory phenotype is characterized by surface marker expression CD45RO+, CD40RO-, CD62L-, and CCR7-. In some of any of the provided embodiments, the effector memory phenotype is characterized by surface marker expression CD45RO+, CD40RO-, CD62L-, CCR7-, CD28- and CD27-.

[0013] In some of any of the provided embodiments, greater than 95% of the CD4+ and CD8+ T cells in the composition are PD-1-. In some of any of the provided embodiments, greater than 80% of the CD4+ and CD8+ T cells in the composition LAG3-.

[0014] In some of any of the provided embodiments, the population of T cells of the TIL composition are able to produce IFNgamma at a concentration of greater than at or about 30 pg / mL, optionally greater than at or about 60 pg / mL, following antigen-specific stimulation. In some of any of the provided embodiments, the TIL composition is produced by an ex vivo method comprising ex vivo expansion of tumor-reactive T cells that have been obtained from the subject and co-cultured with autologous antigen presenting cells and peptide neoantigens.

[0015] In some of any of the provided embodiments, the ex vivo method for producing the TIL composition comprises: i. processing a biological sample containing a population of T lymphocyte cells obtained from the subject that has a tumor to produce a first population of T lymphocyte cells; ii. stimulating the first population with one or more first T-cell stimulating agents of lymphocytes under conditions for expansion of T cells in the population to produce a second population of activated T cells; iii. co-culturing the second population of T cells in the presence of antigen presenting cells that present one or more peptides on a major histocompatibility complex (MHC), said one or more e peptides are peptides corresponding to peptide neoantigens associated in the tumor of thesubject, to produce a third population of cells containing T cells comprising endogenous T cell receptors reactive to mutation encoding peptides of the tumor; iv. selecting, from the third population of cells, the population of T cells containing endogenous TCR that are reactive to peptides present on the APCs based on one or more upregulation markers expressed on reactive or activated T cells to produce a fourth population containing the selected T cells; v. expanding the fourth population of selected cells in the presence of one or more second T-cell stimulating agents of lymphocytes under conditions to expand T cells in the population to produce the TIL composition.

[0016] In some of any of the provided embodiments, the antigen presenting cells are dendritic cells. In some of any of the provided embodiments, the antigen presenting cells are autologous to the subject. In some of any of the provided embodiments, the MHC molecule is a class I molecule. In some of any of the provided embodiments, the MHC molecule is a Class II molecule. In some of any of the provided embodiments, the MHC molecule is MHC class I and II.

[0017] In some of any of the provided embodiments, the T cells are CD4+ cells. In some of any of the provided embodiments, the T cells are CD8+ cells. In some of any of the provided embodiments, the T cells are CD4+ cells and CD8+ cells.

[0018] In some of any of the provided embodiments, the one or more first T cell stimulating agents are selected from one or more of an anti-CD3 antibody; an anti-CD28 antibody; or a recombinant cytokine selected from among IL-2, IL-7, IL- 15 and IL-21. In some of any of the provided embodiments, the one or more first T-cell stimulating agents is or comprises recombinant IL-2. In some of any of the provided embodiments, the concentration of recombinant IL-2 is from 100 lU / mL to 6000 lU / mL, optionally at from 1000 lU / mL to 6000 lU / mL. In some of any of the provided embodiments, the concentration of recombinant IL-2 is from 3000 lU / mL to 6000 IIJ / mL, optionally about 3000 lU / mL or 6000 lU / mL.

[0019] In some of any of the provided embodiments, the one or more second T cell stimulating agents are selected from one or more of an anti-CD3 antibody; an anti-CD28 antibody; or a recombinant cytokine selected from among IL-2, IL-7, IL- 15 and IL-21. In some of any of the provided embodiments, the one or more second T-cell stimulating agents is or comprises recombinant IL-2. In some of any of the provided embodiments, the concentration of recombinant IL-2 is from 100 lU / mL to 6000 lU / mL, optionally at from 1000 lU / mL to 6000 lU / mL. In some of any of the provided embodiments, the concentration of recombinant IL-2 is from 3000 lU / mL to 6000 lU / mL, optionally about 3000 IIJ / mL or 6000 lU / mL. In some of any of the provided embodiments, the one or more second T cell stimulating agent further comprises an anti-CD3 antibody, optionally OKT3, optionally wherein the concentration of the anti-CD3 antibody is at or about 50 ng / mL. In some of any of the provided embodiments, the one or more second T cell stimulating agent further comprises feeder cells that are non-dividing peripheral blood mononuclear cells (PBMC), optionally wherein the feeder cells are gamma irradiated.

[0020] In some of any of the provided embodiments, the one or more non-native peptides are loaded on antigen presenting cells by peptide pulse, optionally by electroporation. In some of any of the provided embodiments, the one or more non-native peptide is 5-30 amino acids, optionally 12-25 amino acids, optionally at or about 25 amino acids in length. In some of any of the provided embodiments, the one or more non-native peptides are a pool of peptides and the concentration of peptides in the pool of peptides for the peptide pulse is between at or about 0.00001 pg / mL and about 0.01 pg / mL, 0.0001 pg / mL and at or about 0.001 pg / mL, 0.001 pg / mL and at or about 40 pg / mL, 0.01 pg / mL and at or about 40 pg / mL, at or about 0.1 pg / mL and at or about 40 pg / mL, at or about 1 pg / mL and at or about 40 pg / mL, at or about 0.01 pg / mL and at or about 10 pg / mL or at or about 1 pg / mL and at or about 10 pg / mL.

[0021] In some of any of the provided embodiments, the co-culture ratio of antigen presenting cells to T Cells is between 20:1 and 1:1, between 15:1 and 1:1, between 10:1 and 1:1, between 5:1 and 1:1, between 2.5:1 and 1:1, between 1:20 and 1:1, between 1:15 and 1:1, between 1:10 and 1:1, between 1:5 and 1:1, or between 1:2.5 and 1:1. In some of any of the provided embodiments, the co-culture ratio of dendritic cells to T cells is between 5:1 and 1:5 or is between 3:1 and 1:3, optionally is or is about 1:1.

[0022] In some of any of the provided embodiments, the nonsynonymous somatic mutations associated in the tumor of the subject are identified using Whole exome sequencing (WES) and RNAseq. In some of any of the provided embodiments, the co-culturing is for 2 hours to 24 hours. In some of any of the provided embodiments, the co-culturing is for at or about 6 hours. In some of any of the provided embodiments, the selection is performed using a florescence based cell sorter.

[0023] In some of any of the provided embodiments, the one or more upregulation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD137 (4-1BB), CD59, CD69, CD90, CD38, CD30, CD154, CD252, CD134 (0X40), CD258, CD256, PD-1, TIM-3 and LAG-3. In some of any of the provided embodiments, the one or more upregulation maker is CD134 and / or CD137. In some of any of the provided embodiments, the one or more upregulation maker is CD134 and CD137. In some of any of the provided embodiments, the stimulating the first population is for 7 to 21 days, optionally 7 to 14 days. In some of any of the provided embodiments, the stimulating the first population in a closed system is using a gas permeable culture vessel. In some of any of the provided embodiments, the stimulating the first population in a closed system is performed using a bioreactor. In some of any of the provided embodiments, the expanding the fourth population is for 7 to 21 days, optionally 7 to 14 days. In some of any of the provided embodiments, the expanding the fourth population is performed in a gas permeable culture vessel. In some of any of the provided embodiments, the expanding the fourth population is performed using a bioreactor.

[0024] In some of any of the provided embodiments, the TIL composition is prepared by an ex vivo method comprising selecting cells surface positive for at least two activation markers from the group consisting of PD-1, CD39 and TIGIT from cells obtained from a tumor of a donor subject and expandingthe cells ex vivo. In some of any of the provided embodiments, contacting cells from an input sample comprising T cells with antibodies that bind to at least two activation markers from the group consisting of PD-1, CD39 and TIGIT, wherein the input sample comprises cells from a tumor of a subject, selecting cells surface positive for the at least two activation markers to obtain selected cells from the sample, and performing an expansion by culture of the selected cells with one or more T-cell stimulating agents of lymphocytes under conditions to produce a population of expanded T cells. In some of any of the provided embodiments, the at least two activation markers are PD-1 and CD39. In some of any of the provided embodiments, the input sample comprising T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from a resected tumor. In some of any of the provided embodiments, the one or more T-cell stimulating agents of lymphocytes comprises one or more recombinant cytokines selected from the group consisting of IL-2, IL-7, IL-15 and IL-21, optionally wherein at least one of the one or more T-cell stimulating agents is recombinant IL- 2. In some of any of the provided embodiments, at least one recombinant cytokine is IL-2 and wherein the concentration of recombinant IL-2 is from 100 lU / mL to 6000 lU / mL. In some of any of the provided embodiments, the expansion is carried out in a closed system. In some of any of the provided embodiments, the closed system comprises at least one of a gas permeable culture vessel or a bioreactor. In some of any of the provided embodiments, the expansion to produce the expanded population of T cells is performed for about 7 to 35 days.

[0025] In some of any of the provided embodiments, the method further comprises administration of a lymphodepleting therapy. In some of any of the provided embodiments, the lymphodepleting therapy is or comprises cyclophosphamide, ifosfamide, altretamine, busulfan, carboplatin, carmustine, cisplatin, dacarbazine, lomustine, melphalan, temozolomide, trabectedin, sodium 2-mercaptoethane sulfonate (Mensa), fludarabine, floxuridine, or any combination thereof. In some of any of the provided embodiments, the lymphodepleting therapy comprises administration of cyclophosphamide from about 30 mg / kg to about 200 mg / kg, optionally wherein the cyclophosphamide is administered at or about 60 mg / kg, optionally wherein the cyclophosphamide is administered within about 5 to 9 days prior to administering the TIL composition, more optionally wherein the cyclophosphamide is administered about 7 days prior to administering the TIL composition (Day -7).

[0026] In some of any of the provided embodiments, the lymphodepleting therapy is or comprises cyclophosphamide, sodium 2-mercaptoethane sulfonate (Mensa), fludarabine, or a combination thereof. In some of any of the provided embodiments, the dose of cyclophosphamide is 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg. In some of any of the provided embodiments, the dose of Mensa is 5, 10, 15, 20, or 25 mg / kg / day. In some of any of the provided embodiments, the dose of fludarabine is 5, 10, 15, 20, 25, 30, or 35 mg / m2. In some of any of the provided embodiments, the administration of the lymphodepleting therapy is one, two, three, four, five, six, or seven days prior to the administration of the TIL composition . In some of any of the provided embodiments, the lymphodepleting therapy comprises administration ofdose of Mensa is from about 5 mg / kg / day to 5, 10, 15, 20, or 25 mg / kg / day, optionally wherein the Mensa is administered at or about 15 mg / kg / day, optionally wherein the Mensa is administered within about 4 to 8 days prior to administering the TIL composition, more optionally wherein the Mensa is administered about 6 days prior to administering the TIL composition (Day -6). In some of any of the provided embodiments, the lymphodepleting therapy comprises administration of fludarabine from about 20-40 mg / m2, optionally at or about 25 mg / m2 daily, optionally wherein the fludarabine is administered for three consecutive days, optionally wherein the fludarabine is administered 3 to 5 days prior to administering the TIL composition (Day -5 to Day -3). In some of any of the provided embodiments, the administration of the lymphodepleting therapy is completed within about 2 to 7 days prior to initiation of the administration of the TIL composition, optionally 2 to 5 days prior to initiation of the administration of the TIL composition.

[0027] In some of any of the provided embodiments, the method further comprising administering recombinant IL-2 to the subject. In some of any of the provided embodiments, the initiation of administration of the recombinant IL-2 is on the same day as the TIL composition. In some of any of the provided embodiments, the initiation of administration of the recombinant IL-2 is 2 hour to 24 hours after administration of the TIL composition. In some of any of the provided embodiments, initiation of administration of the recombinant IL-2 is 2-3 hours after administration of the TIL composition. In some of any of the provided embodiments, the recombinant IL-2 is administered as a plurality of doses. In some of any of the provided embodiments, the plurality of doses are 2 to 12 doses, optionally 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 doses. In some of any of the provided embodiments, the plurality of doses is 6 doses. In some of any of the provided embodiments, the IL-2 is administered twice a day, three times a day or four times a day. In some of any of the provided embodiments, the recombinant IL-2 is administered three times a day, optionally every 8 hours. In some of any of the provided embodiments, each dose of the recombinant IL-2 is from 200,000 lU / kg to 800,000 lU / kg. In some of any of the provided embodiments, each dose of the recombinant IL-2 is about 600,000 lU / kg. In some of any of the provided embodiments, the recombinant IL-2 is administered intravenously. In some of any of the provided embodiments, the recombinant IL-2 is Aldesleukin.

[0028] In some of any of the provided embodiments, the method further comprises administration of an anti-PDl antibody. In some of any of the provided embodiments, the anti-PD-1 antibody is administered at a dose between 100 mg and 600 mg. In some of any of the provided embodiments, the anti-PD-1 antibody is administered in a dosing regimen comprising administration of the dose every other week dosing (Q2W), once monthly (Q4W) or once every six weeks (Q6W) or combinations thereof. In some of any of the provided embodiments, a first dose of the anti-PDl antibody is administered every two weeks (Q2W) for 3 months. In some of any of the provided embodiments, the first dose of the anti-PD-1 antibody is administered at Day 14, Day 35 and Day 56. In some of any of the provided embodiments, the first dose of the anti-PDl antibody is about 100 mg to about 300 mg. In someof any of the provided embodiments, the first dose of the anti-PDl antibody is about 200 mg. In some of any of the provided embodiments, further comprising administering a second dose of the anti-PDl antibody every six weeks (Q6W). In some of any of the provided embodiments, the second dose is about 300 mg to about 600 mg. In some of any of the provided embodiments, the second dose is about 400 mg. In some of any of the provided embodiments, the anti-PDl antibody is administered for up to six months, 12 months, 18 months, 24 months, 30 months, or 36 months post administration of the TIL composition . In some of any of the provided embodiments, the anti-PDl antibody is administered for up to about 24 months. In some of any of the provided embodiments, the anti-PDl antibody is administered intravenously. In some of any of the provided embodiments, the anti-PDl antibody is pembrolizumab.

[0029] In some of any of the provided embodiments, the composition of expanded cells are used to treat a cancer patient. In some of any of the provided embodiments, the tumor is a tumor of an epithelial cancer. In some of any of the provided embodiments, the tumor is a tumor of a melanoma, lung squamous, lung adenocarcinoma, bladder cancer, lung small cell cancer, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, stomach cancer or uterine cancer. In some of any of the provided embodiments, the tumor is a tumor of a non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma cancer, endometrial cancer, optionally wherein the breast cancer is HR+ / Her2- breast cancer, triple negative breast cancer (TNBC) or HER2+ breast cancer. In some of any of the provided embodiments, the solid tumor malignancy is selected from the group comprising: breast cancer, colorectal carcinoma (CRC), uveal melanoma, cutaneous carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, or any advanced solid tumor.

[0030] In some of any of the provided embodiments, the subject to be treated is a subject selected as having a solid tumor malignancy that is a breast cancer. In some of any of the provided embodiments, the subject to be treated is a subject selected as having a solid tumor malignancy that is a colorectal carcinoma. In some of any of the provided embodiments, the subject to be treated is a subject selected as having a solid tumor malignancy that is a uveal melanoma. In some of any of the provided embodiments, the subject to be treated is a subject selected as having a solid tumor malignancy that is a cutaneous melanoma. In some of any of the provided embodiments, the subject to be treated is a subject selected as having a solid tumor malignancy that is a non-small cell lung cancer (NSCLC). In some of any of the provided embodiments, the subject to be treated is a subject selected as having a solid tumor malignancy that is a head and neck squamous cell carcinoma (HNSCC). In some of any of the provided embodiments, the solid tumor malignancy is an advanced solid tumor malignancy. In some of any of the provided embodiments, the solid tumor malignancy is a metastatic disease. In some of any of the provided embodiments, the subject has failed, is refractory to, or is unable to tolerate a standard of care therapy for treating the solid tumor malignancy. In some of any of the provided embodiments, the subject has relapsed or is refractory to treatment of one or more prior treatments for the solid tumor malignancy.

[0031] In some of any of the provided embodiments, the subject has pathologically confirmed, unresectable and / or metastatic solid tumor or tumors.

[0032] In some of any of the provided embodiments, the subject is a human subject. In some of any of the provided embodiments, the subject is an adult human of at least 18 years of age. In some of any of the provided embodiments, the subject has colorectal carcinoma (CRC) and has received at least one prior treatment for metastatic disease. In some of any of the provided embodiments, the therapeutically effective dose of the TIL composition is between 1 x 109and 10 x 1012T cells. In some of any of the provided embodiments, the therapeutically effective dose of the TIL composition is between 1 x 10sand 1 x 1012T cells. In some of any of the provided embodiments, the therapeutically effective dose of the TIL composition is between 1 x 108and 1 x 1010T cells, optionally between 1 x 108and 1 x 109T cells.Brief Description of the Drawings

[0033] FIG. 1A and FIG. IB depict process flow charts reflecting an exemplary process for manufacturing a T cell therapeutic composition in accord with the provided methods described herein. FIG. 1C depicts a full process flow chart for the generation of a population of patient specific tumor- derived infiltrating T cells.

[0034] An exemplary process for generating a TIL therapy of Example 1 is depicted in FIG. 2.Detailed Description

[0035] Various embodiments provided herein relate to methods and uses involving combination therapies involving a T lymphocyte infiltrating (TIL) cell therapy and one or more other administration therapies from radiation therapy, a lymphodepleting therapy, a therapy with a checkpoint inhibitor and a cytokine therapy. In some embodiments, the provided methods involve combination therapy methods involving a TIL therapy and a radiation therapy. In such methods, a lymphodepleting therapy may be administered to the subject prior to the TIL therapy. Still further, in some embodiments, a checkpoint inhibitor therapy and / or a cytokine therapy may be administered to the subject after administration of the TIL therapy. In some of any of the provided embodiments, the radiation therapy is a low dose radiation therapy (LDRT).

[0036] Provided herein are methods for improving delivery of adoptive cell therapies, such as tumor infiltrating lymphocyte (TIL) therapies, into the solid tumor. In some embodiments, the provided methods relate to administering a low dose radiation therapy (LDRT) to a subject in combination with a TIL therapy. In some embodiments, low dose radiation of about 0.5 to 2 Gy per lesion per day (total dose of 0.5 to 10 Gy) is administered to a subject to be treated with the TIL therapy. In particular embodiments, the subject has a solid tumor cancer. In some embodiments, the low dose radiation isadministered prior to the TIL therapy. In some embodiments, the low dose radiation is administered to the subject after the TIL therapy. In some embodiments, the low dose radiation is administered to the subject prior to the TIL therapy and after the TIL therapy.

[0037] TIL therapy is an adoptive cell therapy that utilizes the patient’s existing antitumor T cells to specifically recognize and destroy cancer cells. TIL-ACT is a personalized cancer therapy that utilizes ex-vivo expanded autologous TIL obtained from resected tumor. In many cases, harvested TIL are expanded in the presence of the T cell growth factor IL-2, sometimes at low concentrations.

[0038] TIL therapy has most commonly been applied to metastatic melanoma patients as the disease has particularly favorable characteristics for TIL treatment (Besser et al., 2020). The mutagenic role of UV radiation in cutaneous melanomas contributes a large burden of expressed neoantigens that increase tumor immunogenicity (Klemen et al., 2020). Indeed a high mutational and predicted neoantigen load is associated with clinical benefit of unselected [bulk] TIL for melanoma patients (Lauss et al., 2017). A recent meta-analysis of 13 TIL-ACT studies (published 1988 - 2016) identified a pooled overall ORR of 41% (95% confidence interval [CI] 35%-48%) with an overall CR rate of 12% (95% CI 7%-16%) for 410 pretreated patients with advanced cutaneous melanoma (Dafni et al., 2019).

[0039] There is emerging data to suggest that the clinical efficacy of TIL therapy in patients that failed checkpoint inhibitors is not as good as observed in patients who are checkpoint naive. Indeed, in a recent phase II trial of patients who have had prior anti-PD-l / PD-Ll antibody therapy the ORR was 36% (95% CI 25-49) with a CR rate of 3% which is lower than that that has been reported in studies without a prior checkpoint inhibitor therapy. As patients who have failed antiPD-l / PD-Ll antibody immunotherapy likely have evolved resistance mechanisms that subvert anti-tumor immunity it is not surprising that the clinical results are inferior in patients that are checkpoint inhibitor resistant.

[0040] In particular embodiments of provided methods, the methods utilize a selected TIL product in which methods for their isolation and / or expansion enrich for tumor-reactive T cells. It is believed that tumors outside of cutaneous melanoma, such as CRC, breast and uveal melanoma are feasible targets for TIL therapy when selection strategies to enrich neoantigen reactive TIL are employed, as a TIL product reactive to a single neoantigen can achieve objective responses. For instance, in an analysis of 75 patients with common gastrointestinal cancers, including CRC, neoantigen reactive TIL could be identified in 83% of patients, demonstrating that TIL can be isolated and expanded from low mutational burden cancers (Parkhurst et al., 2019). A TIL product enriched for even a single neoantigen was capable of shrinking CRC tumors as demonstrated in (Tran et al., 2016) where a CRC patient administered a TIL product in which 75% of TIL were reactive to a single KRAS mutation led to the regression of 7 lesions. Additionally, in a breast cancer study, neoantigen reactive TIL were identified in 28 of 42 TIL products produced from lesions isolated from patients with metastatic breast cancer (Zacharakis et al., 2022).

[0041] Yet, improved strategies are needed for treating solid tumors, including advanced solid tumors, particularly those with lower mutational burdens. A variety of factors influence whether apatient will respond to TIL therapy. Potential reasons for failure of TIL therapy include lack of T cell penetration into the tumor and the inhibitory tumor stroma (microenvironment). The tumor stroma is rich in inhibitory cells, such as T regulatory cells (Tregs), myeloid-derived suppressor cells, M2 macrophages, and cancer-associated cytokines, such as transforming growth factor beta (TGF-P), that impede T-effector cell function, activation, and infiltration (Barsoumian et al., 2020).

[0042] High-dose (40-60 Gy) radiation (HDR) is a current mainstay of treatment of certain solid tumors and the metastatic disease associated with these. At high doses, radiation therapy kills cancer cells or slows their growth by damaging their DNA. Cancer cells whose DNA is damaged beyond repair stop dividing or die. HDR is not cell-type selective i.e., it impacts dividing cancerous cells and non-cancerous. Because of this lack of specificity, efforts are often taken to ensure that high-dose radiation is only administered to carefully controlled areas of the tumor so as to spare as much normal tissue as possible. This approach requires specialized instrumentation and skilled physicians / technicians to administer. Additionally, because of the DNA and cellular damage induced by high-dose radiation, it is thought to contribute to the effectiveness of combination immunotherapies by providing enhanced availability of tumor-derived antigens to circulating lymphocytes; however, HDR is known to be detrimental to lymphocytes within the tumor tissue at the time of treatment i.e., HDR would be contraindicated as a combination therapy with TIL therapy.

[0043] In contrast to HDR, low dose radiation (LDR) [about 0.5-2Gy per lesion per day, total dose of 0.5-10 Gy] does not induce significant DNA or cellular damage and does not need specialized equipment or highly trained staff to administer. Although LDR is not believed to result in increase in the availability of tumor antigens to lymphocytes, evidence indicates that LDR may have an immunomodulatory capacity that is believed to be a result of enhancing lymphocyte, including T- and NK-cells, migration into the tumor, reducing tumoral levels of immunosuppressive factors such transforming growth factor beta, and skewing the differentiation of immunosuppressive M2-phenotype macrophages within the tumor to a immunostimulatory Ml -like phenotype. For instance, LDR has been demonstrated to modulate the stroma to elicit high levels of CD4 and CD8 T cell activation and penetration into the tumor (Barsoumian et al., 2020 J Immunother Cancer 8). Moreover, since the radiation is being provided at a much lower dose, embodiments herein provide for its combination with cellular immunotherapies because it is not expected to be deemed to be harmful to the tumoral lymphocytes.

[0044] The provided embodiments relate to methods that allow the successful combination of LDR and a TIL therapy to thereby improve TIL therapy. In some embodiments, the methods relate to particular dose levels, frequencies and timing of dosing of either LDR or TIL in the combination methods to thereby increase the efficacy of the TIL therapy. In particular embodiments, the subjects are administered a low dose radiation of about 0.5 to 10 Gy delivered locally to the lesion. In someembodiments, the dose of radiation can be administered as a divided dose in one or more fractions. In some embodiments, administration of the low dose radiation promotes the tumor infiltration of T cells.

[0045] In some embodiments, the subject is conditioned with the low dose radiation prior to isolation of TILs from the subject, in which the low dose radiation increases T cell infiltration into the tumor and thereby increases the number of TIL harvested from the tumor. In some embodiments, the isolated tumor cells are then subjected to ex vivo enrichment and expansion before autologous administration of enriched tumor reactive T cells back to the subject.

[0046] In some embodiments, low dose radiation is administered to the subject after autologous administration of the enriched tumor reactive T cells back to the subject. A problem with existing TIL therapies is that they are administered by intravenous infusion to the subject and the administered T cells must migrate out of the bloodstream into solid tumors. Migration of T cell that are introduced into a subject’s bloodstream to a cancerous lesion can be inefficient. Without wishing to be bound by theory, a reason for this is the lack of an active inflammatory response in some tumors, which can be necessary to drive the migration cues of immune cells to leave the bloodstream and enter the tissues. The provided embodiments relate to applying a low dose radiation to the tumor to thereby create an inflammatory process in the tumor and increase the migration of immune cells. Thus, in some embodiments, the subsequent administration of low dose radiation to the subject after administering the TIL is expected to promote the tumor infiltration of the enriched tumor reactive T cells to increase penetration of the TIL therapy to the tumor and thereby improve response. In some embodiments, the provided combination methods also can be carried out with a lower dose of the administered TIL therapy, since a higher number of cells is expected to be able to migrate back into the tumor when administered with the low dose radiation.

[0047] In some embodiments, the provided methods involve conditioning a subject bearing one or more solid tumor lesion prior to a TIL therapy, wherein the conditioning involves administering a low dose radiation to the subject prior to administering the TIL therapy.

[0048] Various embodiments of the invention provide compositions of tumor infiltrating lymphocytes (TILs) enriched in tumor reactive cells in which the cells are characterized by diversity in TCR clonality and exhibit robust neoantigen reactivity to neoantigen peptides, particularly compared to TIL compositions with less TCR clonal diversity. Related embodiments provide methods for manufacturing tumor infiltrating lymphocytes (TILs) enriched in such tumor reactive cells.

[0049] The provided compositions and methods relate to producing a T cell therapy reactive to tumor-associated antigens, such as neoantigens. Cancer cells accumulate lots of different DNA mutations as part of the tumorigenic process. These mutations can cause amino acid changes in protein coding regions. For a mutation to be recognized by the immune system the protein needs to be processed intracellularly and presented on the surface with the Major Histocompatibility Complex (MHC). Peptide neoantigens (also referred to herein as neoepitopes or peptide neoepitopes) are the mutant peptidespresented by the MHC complex that can be recognized by a T-cell via TCR binding. In order for the immune system to recognize the mutation, it must be expressed on the surface of the cancer cell via the MHC complex and the T cell must have a TCR that recognizes the mutated peptide. These neoantigens may be presented by MHC class I and MHC class II, and are recognized by CD8+ and CD4+ T cells respectively.

[0050] In particular embodiments of the provided compositions and methods, the tumor reactive cells express a T cell receptor (TCR) able to recognize the neoantigens.

[0051] In some embodiments, a “T cell receptor” or “TCR” is a molecule that contains a variable a and P chains (also known as TCRa and TCR , respectively) or a variable y and 5 chains (also known as TCRyand TCR5, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the a form. Typically, TCRs that exist in aP and y5 forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0052] In some aspects, the reactive T cells are tumor-reactive T cells that recognize a cancer neoantigen. The majority of neoantigens arise from passenger mutations, meaning they do not infer any growth advantage to the cancer cell. A smaller number of mutations actively promote tumor growth, these are known as driver mutations. Passenger mutations are likely to give rise to neoantigens that are unique to each patient and may be present in a subset of all cancer cells. Driver mutations give rise to neoantigens that are likely to be present in all the tumor cells of an individual and potentially shared. In some embodiments of the provided method, the population of T cells contain tumor-reactive T cells that can recognize neoantigens containing passenger and / or driver mutations.

[0053] In some aspects, neoantigens are ideal targets for immunotherapies because they represent disease-specific targets. For example, such antigens generally are not present in the body before the cancer developed and are truly cancer specific, not expressed on normal cells and are not subjected to off target immune toxicity. Thus, the unique repertoire of neoantigens specific to the patient can elicit a strong immune response specific to the cancer cells, avoiding normal cells. This is an advantage over other cell therapy targets that may not be disease-specific targets, since even low levels of target antigen on normal cells can lead to severe fatal autoimmune toxicity in the context of engineered therapies that target common antigens. For example an anti MAGE-A3-TCR program in melanoma patients was halted due to study related deaths attributed to cross reactivity with a similar target MAGE-A12, which is expressed at a low level in the brain. A significant challenge in cancer immunotherapy has been the identification of cancer targets.

[0054] Recent clinical studies have demonstrated that T cells isolated from surgically resected tumors possess TCRs that recognize neoantigens, and expanding these neoantigen reactive TILpopulations and re-infusing them into the patient can in some cases result in a dramatic clinical benefit. This personalized therapy has generated remarkable clinical responses in certain patients with common epithelial tumors.

[0055] Various embodiments of methods of producing tumor infiltrating lymphocytes (TILs) enriched in such tumor reactive cells involve the ex vivo expansion and production of a T cell therapeutic composition. In some embodiments, the method of manufacturing involves the growth and manipulation of patient cells outside of the body. In particular embodiments, the methods relate to methods for expanding T cells containing an endogenous TCR specific to a tumor-associated antigen (hereinafter “tumor reactive T cells”). For purposes of this disclosure, reference to tumor reactive T cells includes T cells that exhibit reactivity to a tumor antigen, as evidenced by the ability of the TIL to produce or secrete IFNy or TNFa or express a factor involved or related to cytotoxic killing activity (e.g., Granzyme B or a degranulation factor such as CD 107) following exposure to APCs presenting neoantigenic peptides. In some aspects, the frequency of these cells can be low and in order to expand these cells to a therapeutic dose ex vivo methods for enrichment and expansion are necessary, as provided by the present embodiments.

[0056] Thus, among embodiments of TIL compositions provided for use in the combination therapy methods are TIL compositions that exhibit substantial tumor reactivity activity, including degranulation and ability to express more IFN-gamma and TNF-alpha, in response to APCs (e.g. DCs or B cells) presenting neoantigenic peptides. This functional activity is highly preserved even after cryopreservation and thawing of the TIL compositions. The marked increases in cytolytic enzymes, as well as more robust activation phenotypes, underpin the enhanced capacity of the expanded TIL compositions to induce apoptosis of tumor targets. The marked tumor reactivity also supports the utility of the provided TIL composition as a therapy. Exemplary TIL compositions for administration as a TIL therapy in accord with provided methods are described in Sections I.C and Section II.

[0057] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0058] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0059] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. METHODS OF COMBINATION THERAPY

[0060] Provided herein is a method for treating a solid tumor malignancy in a subject involving administering a radiation therapy to a subject that has a solid tumor malignancy and administering a T lymphocyte infiltrating (TIL) therapy to the subject. In provided embodiments, the TIL therapy is a T cell composition enriched in tumor reactive T cells autologous to the subject to be treated that have been selected and expanded from cells of a tumor from the subject. In some embodiments, the provided methods further include administration of a lymphodepleting therapy prior to administering the TIL therapy to the subject. In some embodiments, the provided methods further include administering a checkpoint inhibitor, such as an anti-PD-Ll antibody, after administering the TIL therapy to the subject. In still further embodiments, the provided methods further include administering a recombinant cytokine, such as recombinant IL-2, after administering the TIL therapy to the subject.

[0061] Provided herein are compositions and methods relating to the provided therapeutic cell compositions described herein for use in treating diseases or conditions in a subject such as a cancer. Such methods and uses include therapeutic methods and uses, for example, involving administration of the therapeutic cells, or compositions containing the same, to a subject having a disease, condition, or disorder. In some cases, the disease or disorder is a tumor or cancer. In some embodiments, the cells or pharmaceutical composition thereof is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the cells or pharmaceutical compositions thereof in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0062] In some embodiments, the methods of treatment comprise administering an effective amount of a composition containing tumor reactive CD3+ T cells or CD3+ T cells surface, which may include T cells surface positive for one or more activation marker. Such compositions can include any as described herein, including compositions produced by the provided methods.

[0063] In an embodiment, the subject is pre-treated with non-myeloablative chemotherapy as a lymphodepleting therapy prior to an infusion of TILs. In an embodiment, the non-myeloablative chemotherapy includes one or more chemotherapeutic agents. Any of various regimens known to a skilled artisan can be used, including any as described herein. In an embodiment, the non- myeloablative chemotherapy is cyclophosphamide administered at 60 mg / kg given on Day -7 prior to TIL infusion), Mesna administered at 15 mg / kg / day given on Day -6 prior to TIL infusion, and fludarabine 25 mg / m2given for 3 days (Day -5 to Day -3 prior to TIL infusion).

[0064] The lymphodepleting therapy is administered prior to the provided cell compositions. In some embodiments, the lymphodepleting therapy is carried out within a week of the administration of the provided cell compositions, such as 5-7 days prior to the administration of the dose of cells.

[0065] In an embodiment, after non-myeloablative chemotherapy and TIL infusion (at Day 0), the subject is further administered an intravenous infusion of IL-2 (e.g. aldesleukin). An exemplary regimen includes intravenous administration at 600,000 lU / kg every 8 hours to physiologic tolerance. In some embodiments, the IL-2 is administered between 2-3 hours and up to 24 hours following TIL infusion.

[0066] In some embodiments, the provided methods can be carried out with one or more other immunotherapies, such as an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitors, for example, antibodies directed to PD-1 or PD-L1. Exemplary immune checkpoint inhibitors that can be used in accord with provided methods are described below. In some embodiments, an anti-PD-1 antibody (e.g. pembrolizumab) is administered to the subject. In an exemplary regimen, pembrolizumab is administered to the subject at about 200 mg by intravenous administration every other week (Q2W) beginning about two weeks after administration of the TIL composition. In some embodiments, pembrolizumab is administered on Day 14, Day 35 and Day 56 after administering the TIL composition. In some embodiments, administration of the anti-PD-1 antibody, such as pembrolizumab, can be continued at a less frequent dosing. In some embodiments, after about 3 months (e.g., week 12) of Q2W administration, pembrolizumab may be administered at about 400 mg once every six week (Q6W).

[0067] The compositions described herein can be used in a method for treating hyperproliferative disorders. In a preferred embodiment, they are for use in treating cancers. In some aspects, the cancer can be a melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer, and sarcoma. In some embodiments, the cancer is a cancer with a high mutational burden. In some embodiments, the cancer is melanoma, lung squamous, lung adenocarcinoma, bladder cancer, lung small cell cancer, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, stomach cancer or uterine cancer.

[0068] In some embodiments, the subject has a solid tumor cancer. Non-limiting examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladdercarcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). In several examples, a tumor is melanoma, lung cancer, lymphoma breast cancer or colon cancer.

[0069] In some embodiments, the cancer is a skin cancer. In particular embodiments, the cancer is a melanoma, such as a cutaneous melanoma. In some embodiments, the cancer is a merkel cell or metastatic cutaneous squamous cell carcinoma (CSCC).

[0070] In some embodiments, the cancer is an epithelial cancer. In some embodiments, the cancer is selected from non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma cancer, endometrial cancer.

[0071] In some embodiments, the tumor is a carcinoma, which is a cancer that develops from epithelial cells or is a cancer of epithelial origin. In some embodiments, the cancer arises from epithelial cells which include, but are not limited to, breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancer and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer, cervical cancer, lung cancer, breast cancer and skin cancer, such as squamous cell and basal cell cancers, prostate cancer, renal cell carcinoma, and other known cancers that effect epithelial cells throughout the body.

[0072] In some embodiments, the subject has a cancer that is a gastrointestinal cancer involving a cancer of the gastrointestinal tract (GI tract), including cancers or the upper or lower digestive tract, or an accessory organ of digestion, such as esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum or anus. In some embodiments, the cancer is an esophageal cancer, stomach (gastric) cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, cancer of the mucosa-associated lymphoid tissue (MALT lymphoma), cancer of the biliary tree, colorectal cancer (including colon cancer, rectum cancer or both), anal cancer, or a gastrointestinal carcinoid tumor. In particular embodiments, the cancer is a colorectal cancer.

[0073] In some embodiments, the cancer is a colorectal cancer. Colorectal cancer (CRC) is a common tumor of increasing incidence, which, in many cases, does not response to checkpoint inhibition or other immunotherapy. This is the case even though such cancers have properties that are associated with response, e.g. a reasonably high mutation rate and well established association of prognosis with level of T cell infiltration.

[0074] In some embodiments, the cancer is an ovarian cancer. In some embodiments, the cancer is a triple-negative breast cancer (TNBC).

[0075] In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a colorectal cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is a merkel cell cancer. In some embodiments, thecancer is a metastatic cutaneous squamous cell carcinoma (CSCC). In some embodiments, the cancer is a melanoma.

[0076] In some embodiments, the subject is one whose cancer is refractory to, and or who has relapsed following treatment with, a checkpoint blockade, such as an anti-PDl or anti-PD-Ll therapy.

[0077] In some embodiments, the subject is the same subject from with the biological sample was obtained for producing the therapeutic cell composition. In some such embodiments, the provided method of treatment is an adoptive cell therapy with a therapeutic composition containing T cells autologous to the subject.

[0078] In some embodiments, the cell compositions provided herein are autologous to the subject to be treated. In such embodiments, the starting cells for expansion are isolated directly from a biological sample from the subject as described herein, in some cases including with enrichment for T cells surface positive for one or more T cell activation marker as described, and cultured under conditions for expansion as provided herein. In some aspects, the biological sample from the subject is or includes a tumor or lymph node sample and such sample tumor and an amount of such tissue is obtained, such as by resection or biopsy (e.g. core needle biopsy or fine-needle aspiration). In some embodiments, following the culturing under conditions for expansion in accord with the provided methods the cells are formulated and optionally cryopreserved for subsequent administration to the same subject for treating the cancer.

[0079] In some embodiments, the cell compositions provided herein are allogenic to the subject to be treated. In some aspects, the subject from which the cells are derived or isolated is a healthy subject or is not known to have a disease or conditions, such as a cancer. In such embodiments, the starting cells for expansion are isolated directly from a biological sample from such a subject as described herein, in some cases including with enrichment for T cells surface positive for one or more T cell activation marker as described, and cultured under conditions for expansion as provided herein. In some aspects, the biological sample from the subject is or includes a tumor or lymph node sample and such sample tumor and an amount of such tissue is obtained, such as by resection or biopsy (e.g. core needle biopsy or fine-needle aspiration). In some embodiments, following the culturing under conditions for expansion the cells are formulated and optionally cryopreserved for subsequent administration to a different subject for treating a cancer in such different subject.A. Solid Tumors

[0080] In some embodiments, the subject has a solid tumor. In some embodiments, the subject has an advanced solid tumor that is metastatic. In some embodiments, the subject must have filed, be refractory to, or unable to tolerate standard care therapy for the solid tumor. Exemplary solid tumors for treatment with the provided combination therapy methods are provided in the following subsections.Breas t Cancer

[0081] In some embodiments, the subject to be treated has a breast cancer. There is heterogeneity to breast cancer and there are several molecular subtypes that are commonly grouped into clinical subtypes based on receptor status (Perou et al., 2000). If breast cancer cells are hormone receptor (HR) positive, they have estrogen receptors (ERs) and / or progesterone receptors (PgRs) which are responsible for fueling the cancer’s growth. HER2 positive breast cancer cells express high levels of HER2 and grow faster than other types of breast cancer. Triple negative breast cancers are those which lack all three receptors. Most breast cancers are HR positive and HER2 negative. HER2 positive cases account for 15-20% of breast cancer cases while triple-negative breast cancers account for 10-15% of cases.

[0082] Certain therapies, such as certain chemotherapeutic regimens and treatment with EHR2- targeted therapies, have been used to treat nonmetastatic breast cancer with varying levels of success, but the prognosis of metastatic cancer remains poor. Only 25 of patients with metastatic breast cancer achieve 10-20 year relapse-free survival (Kobayashi et al, 2012).

[0083] Tumor infiltrating lymphocyte (TIL) therapy has yielded durable objective responses in patients with metastatic melanoma and responses have been observed in patients with metastatic breast cancer. It is postulated that accrual of mutational load leads to genomic instability, cell injury, and development of an immunogenically primed environment suitable for TIL. In a recent study, TIL were grown and isolated from resected lesions in 42 patients with metastatic breast cancer (Zacharakis et al., 2022). Twenty-eight (28) of 42 patients contained TIL that recognized one immunogenic somatic mutation. Eight patients underwent TIL therapy, in combination with pembrolizumab (< 4 doses). Objective tumor regression was observed in 3 patients, with one response being complete (currently > 5.5 years) and two responses were partial lasting 6 and 10 months. While selection of mutation-reactive TIL is challenging in breast cancers with fewer mutations, these interim results are encouraging and indicate that TIL can serve as a highly personalized treatment of metastatic breast cancer. However, improved methods are needed.

[0084] In some embodiments, the subject to be treated by the provided TIL combination therapy methods has a breast cancer that is HR+ / Her2- breast cancer. In some embodiments, the breast cancer is a triple negative breast cancer (TNBC). In some embodiments, the breast cancer is a HER2+ breast cancer. In some of any of the provided embodiments, the subject has a metastatic breast cancer.

[0085] In some embodiments, the subject to be treated by the provided TIL combination therapy methods has a breast cancer that has relapsed on at least one prior treatment for metastatic disease. In some embodiments, the subject with breast cancer has relapsed on 1, 2, 3, 4 or 5 prior therapies for treatment for metastatic disease. In some embodiments, the subject with breast cancer has relapsed on at least one but not more than three prior treatments for metastatic disease. In some embodiments, the subject has a HER2-positive disease and the subject has received a prior HER2-containing regimen fortargeting the HER2-positive disease. In some embodiments, the HER2-containing regimen includes trastuzumab or Pertuzumab or a combination of trastuzumab and pertuzumab. In some embodiments, the HER2-containing regimen may also include chemotherapy, such as one or more of a Taxotere (e.g., docetaxel), a taxane (e.g., paclitaxel), an anthracycline-containing regimen (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone or valrubicin)2. Colorectal Carcinoma

[0086] In some embodiments, the subject to be treated has a colorectal carcinoma (CRC). In some embodiments, CRC occurs when there is unregulated and abnormal growth in the cells within the colon or rectum. CRC can also be classified based on the nature of the genomic instability that facilitated carcinogenesis. CRC pathogenesis can occur through the chromosomal instability (CIN) pathway and the microsatellite instability (MSI) pathway (Gupta et al., 2018). CIN tumors are associated with karyotypic abnormalities and the accumulation of mutations in critical genes that initiate colorectal cancer progression. MSI tumors occur when there are mutations in DNA mismatch repair (MMR) genes and there is an accumulation of mutations in DNA microsatellites due to lack of adequate repair mechanisms. Tumors with intact MMR are known as microsatellite stable (MSS). In general, colorectal cancers with MSI have a better stage-adjusted survival compared to colorectal cancer with MSS (Gupta et al., 2018; Idos et al., 2020). T

[0087] Once CRC is detected, the disease is staged (1-4) according to the degree of spread. Similar to breast cancer, prognosis is strongly influenced by how early the cancer is detected. While 5-year survival rate with localized stage colorectal cancer can be high, almost a quarter of patients are diagnosed with late-stage colorectal cancer which has a 5-year survival rate of 15%.

[0088] Standard of care treatments are similar to those of other cancers and include surgical resection, chemotherapy, radiation, and immunotherapy. Selection of treatment is personalized based on the stage of the cancer. Over the past 10-15 years a limited number of chemotherapies have been approved including oxaliplatin, irinotecan, regorafenib, and for a subset of patients, bevacizumab and cetuximab / panitumumab. These agents have doubled median overall survival in patients to nearly 2 years; however, subsequent disease progression represents a high unmet need with few therapeutic avenues remaining for these patients (Byrne and Saif, 2019).

[0089] Regimens using checkpoint inhibitors such as pembrolizumab has shown therapeutic responses in patients with MSI tumors, but not in patients with MSS tumors. However, patients with MSI account for only 5% of the advanced colorectal cancer cases, rendering traditional immunotherapies ineffective for most patients with colorectal cancer and restricting use to clinical trial settings (Juat et al., 2022). There is currently an unmet need for immunotherapeutic modalities with a more generalized treatment for metastatic colorectal cancer.

[0090] It has been previously demonstrated that TIL can be selected to recognize mutant proteins encoded by de novo somatic mutations in genes correlated to colorectal carcinogenesis, such as KRAS (Parkhurst et al., 2019; Tran et al., 2015; Tran et al., 2016). A systematic review performed by Juat et al. analyzed the results of 7 clinical trials (n = 108) that investigated the efficacy of adoptive T cell therapy at treating metastatic colorectal cancer (Juat et al., 2022). Out of the 5 trials that reported on overall survival (OS), 3 trials reported median OS estimates exceeding 14 months. All 7 clinical trials reported progression-free survival (PFS) estimates exceeding 4.5 months. To contextualize, the median OS and PFS reported with standard approved therapies in the second / third-line setting was 5 and 1.7 months, respectively. These encouraging results imply that adoptive T cell therapy can be a new and effective treatment paradigm for metastatic colorectal cancer that circumvents the limitations of traditional modalities. However, improved methods are needed.

[0091] In some embodiments, the subject to be treated by the provided TIL combination therapy methods has a CRC that is a metastatic cancer.

[0092] In some embodiments, the subject to be treated by the provided TIL combination therapy methods has a CRC that is a MSI-high (MSI-H) CRC (see e.g., Gatalica et al. Fam Cancer, 2016 15:405- 412). In some embodiments, MSI-H includes hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome and sporadic MSI-H CRC, as well as tumors that overexpress miRNAs that can silence the MMR genes. In some embodiments, Lynch syndrome is characterized by germline mutations in one or more MMR genes MHL1, MSH2, MSH6 and / or PMS2. In some embodiments, Lynch syndrome is characterized by a germline deletion of the last exon of EPCAM. In some embodiments, sporadic MSI-H CRC is characterized by somatic methylation of the MLH1 gene promoter.

[0093] In some embodiments, the subject to be treated by the provided TIL combination therapy methods has a MSS metastatic CRC.

[0094] In some embodiments, the subject with CRC to be treated by the provided TIL combination therapy methods is one that has relapsed on at least one prior treatment for metastatic disease. In some embodiments, the subject with CRC has relapsed on 1, 2, 3, 4 or 5 prior therapies for treatment for metastatic disease. In some embodiments, the subject with CRC has relapsed on at least one but not more than three prior treatments for metastatic disease. In some embodiments, the subject has MSS metastatic CRC and has received and progressed on a fluorouracil-based chemotherapy. In some embodiments, the fluorouracil chemotherapy is oxaliplatin or irinotecan. In some embodiments, the fluorouracil-based chemotherapy is one that is also given with a biologic, such as an anti-EGFR antibody or an anti- angiogenic agent. In some embodiments, the subject has MSI-high CRC has and received a regimen that includes an anti-PD-1 antibody, such as any known anti-PD-1 antibody including any as described herein..7 Ureal Melanoma

[0095] In some embodiments, the subject to be treated has a uveal melanoma (UM). In some embodiments, UM is a cancer in the middle layer of the wall of the eye which includes the iris, the ciliary body, and the choroid. UM is characterized by hematogenous spread with the most common site of metastasis being the liver. The overall long-term prognosis of UM is poor with death occurring in 50% of cases. Notably, the median survival of patients with metastatic disease is less than 12 months and can decrease to approximately 6 months in the case of liver metastasis (Singh et al., 2011).

[0096] First-line treatment options for UM include resection, radiation therapy, and enucleation (Kaliki and Shields, 2017). Recently, the US Food and Drug Administration (FDA) approved tebentafusptebn for the treatment of unresectable or metastatic UM in patients that are human leukocyte antigen (HLA)-A 02:01 genotype positive (US Food and Drug Administration, 2022). Tebentafusp is a bispecific gplOO peptide-HLA-directed CD3 T cell engager that recruits and activates polyclonal T cells to release cytokines and cytolytic mediators against target cells (Nathan et al., 2021; US Food and Drug Administration, 2022)). While these therapies are successful with respect to local disease control, the long-term survival of patients remains poor due to the risk of liver metastasis (Kaliki and Shields, 2017).

[0097] While immunotherapy with checkpoint inhibition has shown promising results in cutaneous melanoma treatment, this efficacy did not translate to UM with some clinical trials being stopped early due to futility (Carvajal et al., 2017; Kastelan et al., 2020). This discrepancy in treatment efficacy may be due to the low mutational burden of UM compared to cutaneous melanoma (Furney et al., 2014; Kastelan et al., 2020). The lack of efficacious therapies in metastatic UM requires new therapeutic modalities, including adoptive T cell therapy, in which activated T cells with antitumor activity are mobilized. This therapy has shown promising results in the treatment of metastatic solid cancers and activity in a UM preclinical mouse model (Carvajal et al., 2017). In addition, the results from a clinical trial investigating the efficacy of TIL therapy were encouraging (Chandran et al., 2017). In this trial, 7 of the 20 evaluable patients showed tumor regression following TIL therapy, with 1 patient achieving a complete response (CR) of numerous hepatic metastases and 6 patients achieving partial responses (PRs). Three of the responsive patients were refractory to prior immune checkpoint blockade. However, improved methods are needed.

[0098] In some embodiments, the subject to be treated by the provided TIL combination therapy methods has metastatic UM.

[0099] In some embodiments, the subject to be treated by the provided TIL combination therapy methods are subjects who have received one or more prior therapy for treating the UM. In some embodiments, the subject has UM and has only received local-regional or adjuvant therapies if systemic treatments are contraindicated or unavailable to the patient. In some embodiments, the subject has UM with metastatic disease but has not received more than three prior therapies for metastatic disease. Insome embodiments, the subject with UM is one that has relapsed on at least one prior treatment for metastatic disease. In some embodiments, the subject with UM has relapsed on 1, 2, 3, 4 or 5 prior therapies for treatment for metastatic disease. In some embodiments, the subject with UM has relapsed on at least one but not more than three prior treatments for metastatic disease.4. Cutaneous Melanoma

[0100] In some embodiments, the subject to be treated has a cutaneous melanoma. Cutaneous melanoma is one of the most severe skin cancers, with a higher risk of metastasis which rapidly becomes life-threatening. The primary treatment for cutaneous melanoma is surgery, which may include dissection of the sentinel lymph node identified by lymph node mapping. Although dacarbazine (DTIC) is approved for use in melanoma, chemotherapy is now rarely used in preference for targeted treatments which have shown better efficacy with less toxicity.

[0101] Targeted therapies, including immune checkpoint inhibitor (ICI) therapy have demonstrated significant gains in outcomes in patients with malignant melanoma. Approved by the FDA in 2011, ipilimumab (targeting CTLA-4) was the first ICI approved for use in melanoma (Robert et al., 2011). Subsequently, the monoclonal antibodies nivolumab and pembrolizumab targeting PD-1 have shown efficacy in melanoma (Luke et al., 2022; Wolchok et al., 2022). Approximately 50% of patients with cutaneous melanoma have activating mutations in BRAF; targeted therapy with BRAF inhibitors vemurafenib and dabrafenib has shown improved efficacy compared against chemotherapy in patients with metastatic BRAF V600E / K mutant melanoma. Furthermore, combination BRAF inhibition with the mitogen-activated protein kinase (MEK) inhibitor trametinib has shown later onset of treatment resistance (Proietti et al., 2020). A number of novel ICI therapies including anti-LAG3, anti-TIGIT, plus other approaches such as adoptive cell therapies, and vaccines are currently in development (Comito et al., 2022). However, approximately 40% to 65% of melanoma patients have no response to ICI, and most relapse following ICI therapy due to primary or acquired resistance (Owen et al., 2020; Schadendorf et al., 2018). Thus, improved methods are needed.

[0102] In some embodiment, the subject to be treated by the provided TIL combination therapy methods has metastatic cutaneous melanoma. In some embodiments, the subjects for treatment are subjects who have received one or more prior therapy for treating the cutaneous melanoma. In some embodiments, the subject has cutaneous melanoma with metastatic disease but has not received more than three prior therapies for metastatic disease. In some embodiments, the subject with cutaneous melanoma is one that has relapsed on at least one prior treatment for metastatic disease. In some embodiments, the subject with cutaneous melanoma has relapsed on 1, 2, 3, 4 or 5 prior therapies for treatment for metastatic disease. In some embodiments, the subject with cutaneous melanoma has relapsed on at least one but not more than three prior treatments for metastatic disease. In some embodiments, the subject has cutaneous melanoma has relapsed after disease progression followingtreatment with an ICI, such as a PD-1 or PD-L1 inhibitor. In some embodiments, the subject has an activating mutation in BRAF, such as a V600E mutation, and has received or has been ineligible for treatment with a BRAF inhibitor or MEK inhibitor therapy.5. Non-small Celt Lung Cancer

[0103] In some embodiments, the subject to be treated has a non-small cell lung cancer (NSCLC). NSCLC is the most common lung cancer. In some embodiments, the majority of patients with NSCLC either present with advanced disease, or develop metastatic disease at some point during their illness and are potentially candidates for systemic therapy. Platinum-based chemotherapy, with or without targeted therapy, depending on the tumor’s mutational status, is applied as first-line treatment of NSCLC patients. The median survival of patients receiving first-line therapy is approximately 1 year (Gerber and Schiller, 2013; Leighl, 2012) and chemotherapy is associated with substantial toxicity that can negatively impact quality-of-life.

[0104] Tyrosine kinase inhibitors (TKI, e.g. Erlotinib, gefitinib, afatinib) have been demonstrated to be effective in specific subsets representing a minority of patients with NSCLC. In patients with NSCLC harboring an oncogenic driver mutation in epidermal growth factor receptor (EGFR), erlotinib was superior in PFS and response rate (RR) vs gemcitabine vs carboplatin (Zhou et al., 2011 ; Zhou et al., 2015). Similar results were observed with the next generation TKI agent afatinib (Rosell et al., 2012; Sequist et al., 2013; Wu et al., 2015; Yang et al., 2013). In subjects with NSCLC, mutations of EGFR act as one of the most frequent driver genes, although gene mutations in ALK and ROS1 resulting in ALK and ROS 1 rearrangements are also common and also effective therapeutic targets of tyrosine kinase inhibitors (TKIs). In patients with advanced lung adenocarcinomas, testing for somatic mutations and rearrangements in EGFR, ALK and ROS1 genes are typical as part of a pretreatment screening. Examples of EFGR-TKIs are erlotinib, gefitinib and afatinib, examples of ALK inhibitors are crizotinib and ceritinib and examples of ROS 1 inhibitors are crizotinib and entrectinib. However, although improvement in PFS and RR have been observed with TKI, overall survival was not improved (Lee et al., 2013). Immune checkpoint inhibitors have also been effective in some patients with NSCLC. The PD-1 inhibitor nivolumab has been approved for the treatment of squamous NSCLC and more recently for all platinum-refractory NSCLC patients (Borghaei et al., 2015; Brahmer et al., 2015). The PD-1 targeted antibody pembrolizumab (Keytruda, Merck) was evaluated in the platinum-failure NSCLC setting (Garon et al., 2015; Herbst et al., 2015) and has been approved for patients whose tumors express programmed death ligand 1 (PD-L1). However, improved methods for treatment of NSCLC are needed.

[0105] In some embodiments, the subject to be treated by the provided TIL combination therapy methods has metastatic NSCLC.

[0106] In some embodiments, the subject to be treated by the provided TIL combination therapy methods has received one or more prior therapy for treating the NSCLC. In some embodiments, thesubject with NSCLC has experienced disease progression following platinum containing chemotherapy and / or treatment with an ICI, such as a PD-1 or PD-L1 inhibitor. In some embodiments, the subject has a NSCLC harboring an oncogenic driver mutation in one or more of EGFR, ALK or ROS1, and has received or been determined to be ineligible for treatment with a therapy targeting the respective mutation(s). In some embodiments, the subject has NSCLC with metastatic disease but has not received more than three prior therapies for metastatic disease. In some embodiments, the subject with NSCLC is one that has relapsed on at least one prior treatment for metastatic disease. In some embodiments, the subject with NSCLC has relapsed on 1, 2, 3, 4 or 5 prior therapies for treatment for metastatic disease. In some embodiments, the subject with NSCLC has relapsed on at least one but not more than three prior treatments for metastatic disease.A Head and Neck Si / uamous Ced Carcinoma

[0107] In some embodiments, the subject to be treated has a head and neck squamous cell carcinoma. Head and neck squamous cell carcinoma (HNSCC) is the most common subtype of head and neck cancer. Treatment of local or regionally invasive disease consists of surgery or radiation alone, or in combination with chemotherapy. Platinum based combinations are the cornerstone of treatment for more advanced, metastatic disease with response rates of 30-40% and median survival of 6-9 months in the first-line setting (Pfister et al., 2013). Thereafter, in second or greater line of therapy, monotherapy with multiple different agents (including taxanes, EGFR-targeted antibodies, and methotrexate) produced response rates of 10-15% with marginal prolongation of OS (Vermorken et al., 2008; Vermorken et al., 2007). In 2016, immunotherapy with PD-1 targeting antibody treatment was approved by the FDA in the second and later lines of therapy following platinum failure based on data demonstrating improvement in objective response rate (ORR), PFS, and OS compared to standard chemotherapy options. Subsequently in 2019, immunotherapy was approved for front-line treatment of inoperable or metastatic cancer (Bauml et al., 2019). Despite this recent progress all treatments are temporizing, with disease eventually progressing following all current treatment options. Thus, improved treatment methods are needed.

[0108] In some embodiments, the subject to be treated by the provided TIL combination therapy methods has received one or more prior therapy for treating the HNSCC. In some embodiments, the subject with HNSCC has experienced disease progression following treatment with a platinum-based combination therapy and / or and ICI, such as a PD-1 or PD-L1 inhibitor. In some embodiments, the subject has HNSCC with metastatic disease but has not received more than three prior therapies for metastatic disease. In some embodiments, the subject with HNSCC is one that has relapsed on at least one prior treatment for metastatic disease. In some embodiments, the subject with HNSCC has relapsed on 1, 2, 3, 4 or 5 prior therapies for treatment for metastatic disease. In some embodiments, the subject with HNSCC has relapsed on at least one but not more than three prior treatments for metastatic disease.B. Low Dose Radiation

[0109] In some embodiments, the provided methods involve administering a low dose radiation therapy to the subject. In some embodiments, the dose of radiation is administered prior to and / or after administering the TIL therapy. In some embodiments, the dose of radiation is administered as a plurality of fraction doses, and at least one fraction dose is administered prior to administration of the TIL therapy and at least one fraction dose is administered after administration of the TIL therapy.

[0110] Radiotherapy is the use of high-energy radiation from x-rays, gamma rays, neutrons, protons, and other sources to kill cancer cells and shrink tumors. Radiotherapy may also be called irradiation and radiation therapy. In some embodiments, a low dose radiotherapy is administered to a subject to modulate the tumor microenvironment, such as to induce infiltration of T cells into the tumor.

[0111] In certain embodiments, the radiation therapy is a low dose radiation therapy. In certain embodiments, the radiation therapy is administered in a total dose of no more than or up to about 100 Gy, about 80 Gy, about 60 Gy, about 40 Gy, or about 30 Gy, about 25 Gy, about 20 Gy, about 19 Gy, about 18 Gy, about 17 Gy, about 16 Gy, about 15 Gy, about 14 Gy, about 13 Gy, about 12 Gy, about 11 Gy, about 10 Gy, about 9 Gy, about 8 Gy, about 7 Gy, about 6 Gy, about 5 Gy, about 4 Gy, about 3 Gy, about 2.5 Gy, about 2 Gy, about 1.5 Gy, about 1 Gy or about 0.5 Gy. In some embodiments, the radiation therapy is administered in a total dose of no more than or up to about 30 Gy. In certain embodiments, the radiation therapy is administered in a total dose of between about 0.01 Gy and about 30 Gy, between about 0.1 Gy and about 20 Gy, between about 0.1 Gy and about 10 Gy, between about 0.5 Gy and about 30 Gy, between about 1.5 Gy and about 20 Gy, between about 0.5 Gy and about 10 Gy, between about 0.5 Gy and about 5 Gy, between about 1 Gy and about 30 Gy, between about 1 Gy and about 20 Gy, between about 1 Gy and about 10 Gy, between about 1 Gy and about 5 Gy, between about 2 Gy and about 30 Gy, between about 2 Gy and about 20 Gy, between about 2 Gy and about 10 Gy, between about 2 Gy and about 5 Gy,. In certain embodiments, the radiation therapy is administered in a total dose of between about 1 Gy and about 20 Gy.

[0112] In some embodiments, the low dose radiotherapy may comprise a total dose of about 1-13, 2- 12, 1-9, 2-9, 2-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 Gy, or any range derivable therein.

[0113] In some embodiments, the low dose radiotherapy may be separated into separate administrations ( e.g. ., 1, 2, 3, 4, or 5 administrations of the radiotherapy over a period of about 1, 2, 3, 4, or 5 days). The low dose radiotherapy may provide a dose of 10-170, 10-160, 20-160, 30-140, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 170 cGy per fraction, or any range derivable therein. In some embodiments, the low dose radiotherapy is a sub-clinical dose of radiation (e.g., 20-190 cGy per fraction, for a total of 2-12 Gy).

[0114] In some embodiments, the low dose radiation is a dosage of 0.1 -1.9 Gy per fraction, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 Gy per fraction, or anyrange derivable therein. In certain embodiments, the total dose of radiation is administered as a plurality of fractions. In some embodiments, the low dose radiotherapy may be delivered in 1, 2, 3, 4, or 5 fractions. In some embodiments, the total dose is administered in about 1 fraction, about 2 fractions, about 3 fractions, about 4 fractions, about 5 fractions, about 6 fractions, about 7 fractions, about 8 fractions, about 9 fractions, about 10 fractions, about 11 fractions, about 12 fractions, about 13 fractions, about 14 fractions, about 15 fractions or more.

[0115] In certain embodiments, the radiation therapy is administered no later than about 50 days, about 40 days, about 30 days, about 20 days, about 19 days, about 18 days, about 17 days, about 16 days, about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day or about 0.5 day prior to the administration of the TIL composition. In certain embodiments, the radiation therapy is administered no later than about 10 days prior to the administration of the TIL composition. In certain embodiments, the radiation therapy is administered between about 0.5 day and about 20 days prior to the administration of the TIL composition, between about 1 day and about 20 days prior to the administration of the TIL composition, between about 1 day and about 15 days prior to the administration of the TIL composition, between about 2 day and about 20 days prior to the administration of the TIL composition, between about 3 day and about 20 days prior to the administration of the TIL composition, between about 2 day to about 15 days prior and the administration of the TIL composition, or between about 2 day to about 10 days prior to the administration of the TIL composition.

[0116] In certain embodiments, the radiation therapy is administered no more than about 50 days, about 40 days, about 30 days, about 20 days, about 19 days, about 18 days, about 17 days, about 16 days, about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day or about 0.5 day after the administration of the TIL composition. In certain embodiments, the radiation therapy is administered no more than about 10 days after the administration of the TIL composition. In certain embodiments, the radiation therapy is administered between about 0.5 day and about 20 days after the administration of the TIL composition, between about 1 day and about 20 days after the administration of the TIL composition, between about 1 day and about 15 days after the administration of the TIL composition, between about 2 day and about 20 days after the administration of the TIL composition, between about 3 day and about 20 days after the administration of the TIL composition, between about 2 day to about 15 days after the administration of the TIL composition, or between about 2 day to about 10 days after the administration of the TIL composition.

[0117] Suitable types of radiotherapy include conventional external beam radiotherapy, stereotactic radiation therapy ( e.g. ., Axesse, Cyberknife, Gamma Knife, Novalis, Primatom, Synergy, X- Knife, TomoTherapy or Trilogy), Intensity -Modulated Radiation Therapy, particle therapy (e.g., proton therapy), brachytherapy, delivery of radioisotopes, intraoperative radiotherapy, Auger therapy,Volumetric modulated arc therapy (VMAT), Virtual simulation, 3-dimensional conformal radiation therapy, and intensity-modulated radiation therapy, etc.

[0118] In certain embodiments, the radiation therapy is an external beam radiation therapy, a brachytherapy or a systemic radioisotope therapy. X-rays, gamma rays, and charged particles are examples of types of radiation that can be used. The radiation may be delivered by a machine outside the body (external-beam radiation therapy (XRT)), or it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachytherapy). Types of external-beam radiation therapy include: Intensity-modulated radiation therapy (IMRT), Image-guided radiation therapy (IGRT), tomotherapy, stereotactic radiosurgery, stereotactic body radiation therapy, proton therapy, and other charged particle beams. Systemic radiation therapy uses radioactive substances, such as radioactive iodine or a radiolabeled monoclonal antibody, that travel in the blood and / or to tissues throughout the body to kill cancer cells.

[0119] In some embodiments, the radiation therapy is by a stereotactic body radiation therapy (SBRT; also referred to as stereotactic ablative radiotherapy (SABR) involving hypofractionation of the radiation.C. Enriched tumor reactive TIL compositions

[0120] In some embodiments, the provided methods involving administering to a subject a TIL composition that is enriched for tumor reactive T cells.

[0121] In some embodiments, the provided TIL composition is a multiclonal population that exhibits TCR diversity and enrichment of T cell receptors (TCRs) reactive to neoantigens. In some embodiments, the provided TIL composition is a oligoclonal population that exhibits TCR diversity and enrichment of different TCR clonotypes. In some embodiments, the TIL composition contains up to 40 different TCR clonotypes (e.g. Top40 clones) that make up at least 40% of the TCR frequency in the population. In some embodiments, the TIL composition contains up to 40 different TCR clonotypes that make up at least 50% of the TCR frequency in the population. In some embodiments, the TIL composition contains up to 40 different TCR clonotypes that make up at least 60% of the TCR frequency in the population. In some embodiments, the TIL composition contains up to 40 different TCR clonotypes that make up at least 70% of the TCR frequency in the population. In some of any of the above embodiments, the number of TCR clonotypes making up the percentage is 10 to 40 different TCR clonotypes. In some of any of the above embodiments, the number of TCR clonotypes making up the percentage is 20 to 40 different TCR clonotypes. In some of any of the above embodiments, the number of TCR clonotypes making up the percentage is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 different TCR clonotypes. In some embodiments, the TIL composition contains at least 10 different TCR clonotypes that exhibit neoantigenreactivity. In some embodiments, the TIL composition contains at least 11, 12, 12, 14, 15, 16, 17, 18, 19, 20 or more different TCR clonotypes that exhibit neoantigen reactivity.

[0122] In some embodiments, the population is enriched in at least 10 different TCR clonotypes each with a frequency in the population of at least 2.0%. In some embodiments, the population is enriched in at least 11 different TCR clonotypes each with a frequency in the population of at least 2.0%. In some embodiments, the population is enriched in at least 12 different TCR clonotypes each with a frequency in the population of at least 2.0%. In some embodiments, 8 to 15 different T cell receptor (TCR) clonotypes make up at least 50 % of the TCR frequency in the population. In some embodiments, 9 to 12 different TCR clonotypes make up at least 50% of the TCR frequency in the population.

[0123] In some embodiments, the TIL composition contains 20 to 40 different TCR clonotypes that make up at least 40% of the TCR frequency in the population. In some embodiments, the TIL composition contains 20 to 40 different TCR clonotypes that make up at least 50% of the TCR frequency in the population. In some embodiments, the TIL composition contains 20 to 40 different TCR clonotypes that make up at least 60% of the TCR frequency in the population. In some embodiments, the TIL composition contains 20 to 40 different TCR clonotypes that make up at least 70% of the TCR frequency in the population.

[0124] In some embodiments, the neoantigen reactivity of the TCR clonotypes is reactivity to at least one CD4 antigen and at least one CD8 antigen. In some embodiments, the neoantigen reactivity of the TCR clonotypes is for at least 2 peptide antigens, in which at least one peptide antigen is a CD4 antigen and at least one peptide antigen is a CD8 antigen. In some embodiments, the neoantigen reactivity of the TCR clonotypes is for 2 to 6 different peptide antigens. In some embodiments, the neoantigen reactivity of the TCR clonotypes is for 2 to 4 peptide antigens. In some embodiments, the neoantigen reactivity of the TCR clonotypes is for 2 peptide antigens. In some embodiments, the neoantigen reactivity of the TCR clonotypes is for 2 peptide antigens in which one is a CD8 antigen and one is a CD4 antigen.

[0125] Various methods for assessing the TCR repertoire for clonotype identification and TCR repertoire analysis are known (see e.g. Rosati et al. (2017) BMC Biotechnology, 17:61; Friedensohn et al. (2016) Trends in Biotechnology, 35:203-214). In some aspects, the methods involve high-throughput or next-generation sequencing methods. In some embodiments, the frequency and variety of different clones present in the population or composition can be determined. In some embodiments, the compositions can be assessed the clonality, clonal diversity or clonal heterogeneity of the cells in the population of the composition of cells, for example, based on the determined frequency and / or variety of clonotypes present in the population or composition. In some embodiments, single -cell sequencing methods are carried out to identify a clonotype on a particular cell. In certain aspects, paired ap TCR sequencing methods are used (see e.g. W02017053902A1). In some embodiments, sequencing methods are carried out on DNA, such as genomic DNA or complementary DNA. In some embodiments, sequencingmethods are carried out on RNA. In some embodiments, high-throughput or next-generation sequencing of TCR sequences or by sequencing the whole genome or transcriptome (e.g., RNAseq). In some aspects, the methods used are RNAseq-based methods.

[0126] In some embodiments, T cell clonotype assessment and clonality and diversity in various T cell populations or compositions or samples containing T cells, are determined using high-throughput sequencing of all or a portion of the TCR genes or based on sequences obtained from high-throughput whole genome or transcriptome analysis, on the population or composition of cells, and / or in a single cell. In some embodiments, the provided methods can include various features of the methods as described in WO2016 / 044227, WO2016 / 176322, W02012 / 048340, WO2012 / 048341, WO2014 / 144495, W02017 / 053902, W02017 / 053903 or W02017 / 053905, each incorporated by reference in their entirety.

[0127] The clonotypes of a cell or the clonotypes present in a population or composition of cells, in some examples, may be determined by TCR sequencing. In some embodiments, sequencing methods that can be employed include high-throughput or next-generation sequencing as is known in the art. In some aspects, next-generation sequencing methods can be employed, using genomic DNA or cDNA from T cells, to assess the TCR repertoire, including sequences encoding the complementaritydetermining region 3 (CDR3). In some embodiments, whole transcriptome sequencing by RNAseq can be employed. In some aspects, the TCR repertoire information, e.g., TCR sequences and relative frequency, can be constructed or extracted from whole transcriptome sequencing (e.g., by RNAseq). For example, in some aspects, computational methods such as MIXCR (Such as those described in Bolotin et al. Nature Methods 12 (2015) 380-381, Bolotin et al., Nature Biotechnology 35 (2017) 908-911) or IMREP (Mangul et al., bioRxiv (2017) 089235) can be utilized to determine the repertoire TCR sequences or a portion thereof (e.g., CDR3) from whole transcriptome RNAseq results. In some embodiments, single-cell sequencing methods can be used. In some embodiments, clonotypes can be assessed or determined by spectratype analysis (a measure of the TCR Vp, Va, Vy, or V5 chain hypervariable region repertoire). Clonotypes can also be determined by generation and characterization of antigen-specific clones to an antigen of interest.

[0128] In some embodiments, T cell clonotype assessment are determined using high-throughput sequencing of all or a portion of the TCR genes or based on sequences obtained from high-throughput whole genome or transcriptome analysis, on the population or composition of cells, and / or in a single cell. In some embodiments, bulk sequencing of targeted sequences (e.g., TCR chains or portion thereof) or bulk whole genome or transcriptome sequencing (e.g., by RNAseq) can be used to determine the clonotypes present in the cells in the population or composition. In some aspects, T cell clonotype assessment can involve sequencing of a portion of the variable region of one or more native TCR chains, such as the complementarity-determining region 3 (CDR3). In some aspects, single cell sequencing can be employed. In some embodiments, the provided methods can include various features of the methods as described in WO2016 / 044227, WO2016 / 176322, WO2012 / 048340, WO2012 / 048341,WO2014 / 144495, W02017 / 053902, W02017 / 053903 or WO2017 / 053905, each incorporated by reference in their entirety. In some embodiments, for target TCR molecules, the genes encoding chains of a TCR can be obtained from genomic DNA or mRNA of immune cells or T cells.

[0129] In some embodiments, the composition exhibits clonal diversity, i.e. is multiclonal. In some cases, the clonal diversity is determined based on the relative frequency of the one or more clonotypes and / or one or more TCR sequences. In some embodiments, the determining the clonal diversity is represented as clonality, Shannon-adjusted clonality or top 25 clonality of each of the plurality of samples. In some embodiments, the determining the clonal diversity is represented as Shannon-adjusted clonality in each of the plurality of samples.

[0130] In some embodiments, the cells of the provided TIL compositions exhibit one or more phenotypic or functional markers. In some cases, such cells include cells positive or negative for one or more phenotypic marker or functional feature or attribute.

[0131] As used herein, a statement that a cell or population of cells is “positive” for a particular marker, function or attribute refers to the detectable presence on or in the cell of a particular marker, such as a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and / or at a level substantially similar to that for cell known to be positive for the marker, and / or at a level substantially higher than that for a cell known to be negative for the marker.

[0132] As used herein, a statement that a cell or population of cells is “negative” for a particular marker, function or attribute refers to the absence of substantial detectable presence on or in the cell of a particular marker, such as a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that for cell known to be positive for the marker, and / or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0133] Exemplary markers, functions and attributes of provided TIL compositions are described below. In some embodiments, the TIL composition is characterized by any one or more of such features, such as 2, 3, 4, 5 or more of such features. For instance, a provided TIL composition may be characterized by presence or absence of one or more T cell markers, effector memory phenotype markers, exhaustion markers, the ability to produce or secrete cytokines and / or the ability to produce orsecrete a cytotoxic factor, such as described below. Any 2, 3, 4 or 5 of any of such features may be present in a TIL composition as described.

[0134] In some embodiments, a provided TIL composition comprises CD3+ T cells as a percentage of total cells in the population that is greater than or greater than about 85%, such as greater than or greater than about 90%, greater than or greater than about 95%, greater than or greater than about 97% or greater than or greater than about 98%. In some embodiments, the composition contains CD4+ T cells and CD8+ T cells as a percentage of total cells in the population that is greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, greater than or greater than about 97% or greater than or greater than about 98%.

[0135] In some embodiments, the composition contains a ratio of CD4+ T cells to CD8+ T cells that is between at or about 5:1 to 50:1, between 5:1 to 25:1, between 5:1 to 20:1, between 5:1 to 15:1, between 5:1 to 10:1, between 10:1 to 50:1, between 10:1 to 25:1, between 10:1 to 20:1, between 10:1 to 15:1, between 15:1 to 50:1, between 15:1 to 25:1, between 15:1 to 20:1, between 20:1 to 50:1, between 20:1 to 25:1 or between 25:1 to 50:1. In some embodiments, the composition contains a ratio of CD4+ T cells to CD8+ T cells that is at or about 10:1 to 25:1. In some embodiments, the composition contains a ration of CD4+ T cells to CD8+ T cells that is about 20:1.

[0136] In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 50% express a marker of an effector memory phenotype. In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 60% express an effector memory phenotype marker, greater than about 70% express an effector memory phenotype marker, greater than about 80% express an effector memory phenotype marker, or greater than 90% express an effector memory phenotype marker. In some embodiments, the effector memory phenotype is characterized by surface marker expression of one or more of CD45RO+, CD40RO-, CD62L , CCR7-, CD28- and CD27-. In some embodiments, the effector memory phenotype is characterized by surface marker expression CD45RO+, CD40RO , CD62L , and CCR7 . In some embodiments, the effector memory phenotype is characterized by surface marker expression CD45RO+, CD40RO , CD62L , CCR7 , CD28 and CD27 .

[0137] In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, less than 30% of the cells express an exhaustion phenotype. In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, less than about 25% express an exhaustion phenotype, less than about 20% express an exhaustion phenotype, less than about 15% express an exhaustion phenotype, or less than about 10% express an exhaustion phenotype. In some cases, exhaustion can be assessed by monitoring loss of T cell function, such as reduced or decreased neoantigen-specific reactivity, such as a reduced or decreased ability to produce cytokines or to drive cytolytic activity against target antigen. In some cases, exhaustion also can be assessed by monitoring expression of surface markers on T cells (e.g. CD3+ T cells, or a CD4 and / orCD4 T cell subset thereof) that are associated with an exhaustion phenotype. Among exhaustion markers are inhibitory receptors such as PD-1, CTLA-4, LAG-3 and TIM-3. In some embodiments, the exhaustion phenotype is positive expression for 1 , 2, 3 or 4 of such exhaustion markers.

[0138] In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 70% are PD-1 . In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 75% are PD-1 , greater than 80% are PD-1 , greater than 85% are PD-1 , greater than 90% are PD-1 , or greater than 95% are PD-1 .

[0139] In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 70% are LAG3 . In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 75% are LAG3 , greater than 80% are LAG3 , greater than 85% are LAG3 , greater than 90% are LAG3 , or greater than 95% are LAG3 .

[0140] In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 70% are PD-1 and LAG3 . In some embodiments, among CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 75% are PD-1 and LAG3 , greater than 80% are PD-1 and LAG3 , greater than 85% are PD-1 and LAG3 , greater than 90% are PD-1 and LAG3' , or greater than 95% are PD-1 and LAG3 .

[0141] In some embodiments, a provided TIL composition includes about 10-60 % tumor-reactive T cells. In some embodiments, a provided TIL composition includes greater than about 15% tumor reactive T cells, greater than about 20% tumor-reactive T cells, greater than about 25% tumor-reactive T cells, greater than about 30% tumor-reactive T cells, greater than about 40% tumor-reactive T cells or greater than about 50% tumor-reactive T cells, or any value between any of the foregoing.

[0142] The provided TIL compositions enriched in tumor reactive cells exhibit a number of functional or phenotypic activities that evidence their reactivity to neoantigens. In some embodiments, cells can be assessed for any of a number of functional or phenotypic activities, including but not limited to cytotoxic activity, degranulation, ability to produce or secrete cytokines, and expression of one or more intracellular or surface phenotypic markers. Methods to assess such activities are known and are exemplified herein and in working examples.

[0143] In some embodiments, upon recognition of neoantigens presented by APC, TILs can become activated. Upon activation, the TILs produce cytokines, chemokines and other factors abundantly and at the same time exhibit potent cytolytic activity. In some embodiments, activation triggers the release of cytoplasmic granules containing granzymes, leading to target cell death. Assays to measure cytokines, chemokines and other soluble factors are well known in the art, and include but are not limited to, ELISA, intracellular cytokine staining, cytometric bead array, RT-PCR, ELISPOT, flow cytometry and bio-assays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g. proliferation) in the presence of a test sample.

[0144] In some embodiments, the provided TIL compositions display higher effector cytokine responses than bulk expanded TILs or TILs that exhibit lower multiclonality. In some embodiments, among CD8+ T cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce IFN-y, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides. In some embodiments, among CD4+ T cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce IFN-y, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides. In some embodiments, among total T cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce IFN-y, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides. In some embodiments, among total cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce IFN-y, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides.

[0145] In some embodiments, the TIL composition produces greater than about 20-fold more IFN-y following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides, compared to a TIL composition in which TILs are expanded in bulk or exhibit limited multiclonality. In some embodiments, the TIL composition produces greater than about 30-fold more IFN-y, 40-fold more IFN-y, 50-fold more IFN-y, 60-fold more IFN-y, 70-fold more IFN-y, 80-fold more IFN-y, 90-fold more IFN-y or 100-fold more IFN-y, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides, compared to a TIL composition in which TILs are expanded in bulk or exhibit limited multiclonality.

[0146] In some embodiments, the provided TIL compositions display higher effector cytokine responses than bulk expanded TILs or TILs that exhibit lower multiclonality. In some embodiments, among CD8+ T cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce TNF-a, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides. In some embodiments, among CD4+ T cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce TNF-a, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides. In some embodiments, among total T cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce TNF-a, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides. In some embodiments, amongtotal cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% produce TNF- a, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides.

[0147] In some embodiments, the TIL composition produces greater than about 50-fold more TNF- a following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides, compared to a TIL composition in which TILs are expanded in bulk or exhibit limited multiclonality. In some embodiments, the TIL composition produces greater than about 75-fold more TNF-a, 100-fold more TNF-a, 150-fold more TNF-a, 200-fold more TNF-a, 250-fold more TNF-a, 300-fold more TNF- a, 350-fold more TNF-a or 400-fold more TNF-a, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides, compared to a TIL composition in which TILs are expanded in bulk or exhibit limited multiclonality

[0148] In some embodiments, the cytotoxic activity can be determined based on the ability to produce or secrete granzyme B. In some embodiments, the TIL composition produces greater than about 10-fold more granzyme B following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides, compared to a TIL composition in which TILs are expanded in bulk or exhibit limited multiclonality. In some embodiments, the TIL composition produces greater than about 20-fold more granzyme B, 30-fold more granzyme B, 40-fold more granzyme B or 50-fold more granzyme B, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides, compared to a TIL composition in which TILs are expanded in bulk or exhibit limited multiclonality.

[0149] In some embodiments, the provided TIL compositions display higher de granulation responses than bulk expanded TILs or TILs that exhibit lower multiclonality. In some embodiments, among CD8+ T cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% exhibit degranulation, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides. In some embodiments, degranulation activity can be measured by CD107a expression. In some embodiments, among CD8+ T cells in a provided TIL composition, greater than at or about 15%, greater than at or about 20%, greater than at or about 30%, greater than at or about 40% or greater than at or about 50% express CD107a, following culture with autologous APCs (e.g. DCs or B cells) presenting neoantigen peptides.

[0150] In certain embodiments, the number of such cells in the composition is a therapeutically effective amount. An effective amount of cells can vary depending on the patient, as well as the type, severity and extent of disease. Thus, a physician can determine what an effective amount is after considering the health of the subject, the extent and severity of disease, and other variables. In some embodiments, the amount is an amount that reduces the severity, the duration and / or the symptoms associated with cancer in an animal. In some embodiments, a therapeutically effective amount is a doseof cells that results in a reduction of the growth or spread of cancer by at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, by at least 90%, at least 95%, or at least 99% in a patient or an animal administered a composition described herein relative to the growth or spread of cancer in a patient (or an animal) or a group of patients (or animals) not administered the composition. In some embodiments, a therapeutically effective amount is an amount to result in cytotoxic activity resulting in activity to inhibit or reduce the growth of cancer cells.

[0151] In some embodiments, the TIL composition provided herein enriched in tumor reactive cells comprises an amount of cells that is from at or about 103and at or about 1012cells. In some embodiments, the TIL composition provided herein enriched in tumor reactive cells comprises an amount of cells from at or about 103to at or about 108cells. In some embodiments, the TIL composition provided herein enriched in tumor reactive cells comprises an amount of cells from at or about 106and at or about 1012. In some embodiments, the TIL composition provided herein enriched in tumor reactive cells comprises an amount of cells from at or about 10sand at or about 10'1cells. In some embodiments, the TIL composition provided herein enriched in tumor reactive cells comprises an amount of cells from at or about 109and at or about 1010cells. In some embodiments, the TIL composition provided herein enriched in tumor reactive cells comprises an amount of greater than or greater than at or about 105cells, greater than or greater than at or about 106cells, greater than or greater than at or about 107cells, greater than or greater than at or about 108cells, greater than or greater than at or about 109cells, greater than or greater than at or aboutlO10cells, greater than or greater than at or about 1011cells, or greater than or greater than at or about 1012cells. In some embodiments, such an amount can be administered to a subject having a disease or condition, such as to a cancer patient.

[0152] In some embodiments, the volume of the composition is at least or at least about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL or 500 mL, such as is from or from about 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL or 200 mL to 500 mL, each inclusive. In some embodiments, the composition has a cell density of at least or at least about 1 x 105cells / mL, 5 x 103cells / mL, 1 x 106cells / mL, 5 x 106cells / mL, 1 x 107cells / mL, 5 x 107cells / mL or 1 x 108cells / mL. In some embodiment, the cell density of the composition is between or between about 1 x 105cells / mL to 1 x 10scells / mL, 1 x 105cells / mL to 1 x 107cells / mL, 1 x 103cells / mL to 1 x 106cells / mL, 1 x 106cells / mL to 1 x 107cells / mL, 1 x 106cells / mL to 1 x 108cells / mL, 1 x 106cells / mL to 1 x 107cells / mL or 1 x 107cells / mL to 1 x 108cells / mL, each inclusive.

[0153] Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. In some embodiments, the cells are formulated with a pharmaceutically acceptable carrier.

[0154] A pharmaceutically acceptable carrier can include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (Gennaro, 2000, Remington: The science and practice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia, PA). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical carrier should be one that is suitable for cells, such as a saline solution, a dextrose solution or a solution comprising human serum albumin.

[0155] In some embodiments, the pharmaceutically acceptable carrier or vehicle for such compositions is any non-toxic aqueous solution in which the cells can be maintained, or remain viable, for a time sufficient to allow administration of live cells. For example, the pharmaceutically acceptable carrier or vehicle can be a saline solution or buffered saline solution. The pharmaceutically acceptable carrier or vehicle can also include various bio materials that may increase the efficiency of cells. Cell vehicles and carriers can, for example, include polysaccharides such as methylcellulose (M. C. Tate, D. A. Shear, S. W. Hoffman, D. G. Stein, M. C. LaPlaca, Biomaterials 22, 1113, 2001, which is incorporated herein by reference in its entirety), chitosan (Suh J K F, Matthew H W T. Biomaterials, 21, 2589, 2000; Lahiji A, Sohrabi A, Hungerford D S, et al., J Biomed Mater Res, 51, 586, 2000, each of which is incorporated herein by reference in its entirety), N-isopropylacrylamide copolymer P(NIPAM- co-AA) (Y. H. Bae, B. Vernon, C. K. Han, S. W. Kim, J. Control. Release 53, 249, 1998; H. Gappa, M. Baudys, J. J. Koh, S. W. Kim, Y. H. Bae, Tissue Eng. 7, 35, 2001, each of which is incorporated herein by reference in its entirety), as well as Poly(oxyethylene) / poly(D,L-lactic acid-co-glycolic acid) (B. Jeong, K. M. Lee, A. Gutowska, Y. H. An, Biomacromolecules 3, 865, 2002, which is incorporated herein by reference in its entirety), P(PF-co-EG) (Suggs L J, Mikos A G. Cell Trans, 8, 345, 1999, which is incorporated herein by reference in its entirety), PEO / PEG (Mann B K, Gobin A S, Tsai A T, Schmedlen R H, West J L., Biomaterials, 22, 3045, 2001; Bryant S J, Anseth K S. Biomaterials, 22, 619, 2001, each of which is incorporated herein by reference in its entirety), PVA (Chih-Ta Lee, Po-Han Kung and Yu-Der Lee, Carbohydrate Polymers, 61, 348, 2005, which is incorporated herein by reference in its entirety), collagen (Lee C R, Grodzinsky A J, Spector M., Biomaterials 22, 3145, 2001, which is incorporated herein by reference in its entirety), alginate (Bouhadir K H, Lee K Y, Alsberg E, Damm K L, Anderson K W, Mooney D J. Biotech Prog 17, 945, 2001; Smidsrd O, Skjak-Braek G., Trends Biotech, 8, 71, 1990, each of which is incorporated herein by reference in its entirety).

[0156] In some embodiments, the composition, including pharmaceutical composition, is sterile. In some embodiments, isolation or enrichment of the cells is carried out in a closed or sterile environment, for example, to minimize error, user handling and / or contamination. In some embodiments, sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0157] Also provided herein are compositions that are suitable for cryopreserving the provided T cells, including tumor-reactive T cells. In some embodiments, the composition comprises a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or s glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, such as ultra low temperatures, for example, storage with temperature ranges from -40 °C to -150 °C, such as or about 80 °C ± 6.0 ° C.

[0158] In some embodiments, the cryopreserved cells are prepared for administration by thawing. In some cases, the cells can be administered to a subject immediately after thawing. In such an embodiment, the composition is ready-to-use without any further processing. In other cases, the cells are further processed after thawing, such as by resuspension with a pharmaceutically acceptable carrier, incubation with an activating or stimulating agent, or are activated washed and resuspended in a pharmaceutically acceptable buffer prior to administration to a subject.

[0159] In some embodiment, a subject (e.g. autologous) is administered from at or about 105to at or about 1012cells of the TIL composition, such as from at or about 105to at or about 10scells, from at or about 106and at or about 1012cells, from at or about 10sand at or about 10” cells, from at or about 109and at or about 1010cells. In some embodiments, the subject is administered 1 x 109and 1 x 1012cells. In some embodiments, the subject is administered 1 x 108and 1 x 1010cells. In some embodiments, the subject is administered 1 x 108and 1 x 109cells. In some embodiments, the therapeutically effective amount for administration comprises greater than or greater than at or about 105cells, greater than at or about 106cells, greater than at or about 107cells, greater than at or about 108cells, greater than at or about 109cells, greater than at or about 1010cells, greater than at or about 10” cells, or greater than at or about 1012cells p. In some embodiments, such an amount can be administered to a subject having a solid tumor malignancy, such as to a cancer patient, in accord with provided methods. In some embodiments, the number of cells are total cells in the T cell composition. In some embodiments, the number of cells are CD3+ T cells in the TIL composition. In some embodiments, the number of cells in a given dose are viable cells, such as viable T cells.

[0160] In some embodiments, the subject is administered 1 x 109and 1 x 1012T cells. In some embodiments, the subject is administered 1 x 108and 1 x 1010T cells. In some embodiments, the subject is administered 1 x 108and 1 x 109T cells. In some embodiments, the number of cells in a given dose are viable T cells.

[0161] In some embodiments, the amount is administered as a flat dose. In other embodiments, the amount is administered per kilogram body weight of the subject.

[0162] In some embodiments, the TIL composition, such as produced by any of the provided methods or containing tumor-reactive T cells, are administered to an individual soon after expansion according to the provided methods. In other embodiments, the expanded T cells, such as expanded tumor-reactive T cells, are cryopreserved prior to administration, such as by methods described above.For example, the T cells, such as tumor-reactive T cells, can be stored for greater than 6, 12, 18, or 24 months prior to administration to the individual. Such cryopreserved cells can be thawed prior to the administration.

[0163] In some embodiments, the provided compositions, such as provided by any of the provided methods or containing tumor-reactive T cells, can be administered to a subject by any convenient route including parenteral routes such as subcutaneous, intramuscular, intravenous, and / or epidural routes of administration.

[0164] In some embodiments, the compositions, such as provided by any of the provided methods or containing tumor-reactive T cells may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, tumor-reactive T cells may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. In some embodiments, a single infusion of cells of the TIL composition is administered. Administration of such compositions and cells may continue as long as necessary.D. Immune Checkpoint Inhibitors (e.g., PD-1 Inhibitors)

[0165] In some embodiments, the provided methods can be carried out with one or more other immunotherapies. In some embodiments, the immunotherapy is an immune modulating agent that is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically binds a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, 0X40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28, TIGIT and VISTA. In some embodiments, the immune checkpoint inhibitor is and antibody or antigen-binding fragment, a small molecule or a polypeptide. In some embodiments, the immune checkpoint inhibitor is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP- 870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C, or is an antigenbinding fragment thereof.

[0166] In some embodiments, the provided methods include combination therapy of a cell therapy as described and PD-1 or PD-L1 inhibitors. A PD-1 or PD-L1 inhibitor can include binding antibodies, antagonists, or inhibitors (i.e., blockers). In some embodiments of the combination therapy methods, combinations, kits and uses provided herein, the combination therapy can be administered in combination with one or more further compositions, e.g., a pharmaceutical composition containing a PD-1 inhibitor (e.g. an anti-PD-1 antibody, such as pembrolizumab).

[0167] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: nivolumab, camrelizumab, cemiplimab,dostarlimab, MEDI-0680, pembrolizumab, spartalizumab, SSI-361, tislelizumab, or a combination thereof. In some embodiments, the anti-PD-1 antibody is nivolumab.

[0168] In an embodiment, the PD-I inhibitor is nivolumab (commercially available as OPDIVO from Bristol-Myers Squibb Co.), or biosimilars, antigen-binding fragments, conjugates, or variants thereof. Nivolumab is a fully human IgG4 antibody blocking the PD-I receptor. In an embodiment, the anti-PD-I antibody is an immunoglobulin G4 kappa, anti-(human CD274) antibody. Nivolumab is assigned Chemical Abstracts Service (CAS) registry number 946414-94-4 and is also known as 5C4, BMS-936558, l\tIDX-l 106, and ONO-4538. The preparation and properties of nivolumab are described in U.S. Patent No. 8,008,449 and International Patent Publication No. WO 2006 / 121168.

[0169] In another embodiment, the PD-1 inhibitor comprises pembrolizumab ( commercially available as KEYTRUDA from Merck & Co., Inc., Kenilworth, NJ, USA), or antigen-binding fragments, conjugates, or variants thereof. Pembrolizumab is assigned CAS registry number 1374853-91-4 and is also known as lambrolizumab, MK-3475, and SCH-900475. The properties, uses, and preparation of pembrolizumab are described in International Patent Publication No. WO 2008 / 156712 Al, U.S. Patent No. 8,354,509 and U.S. Patent Application Publication Nos. US 2010 / 0266617 Al, US 2013 / 0108651 Al, and US 2013 / 0109843 A2.

[0170] In an embodiment, the PD-LI inhibitor is durvalumab, also known as MEDI4736 (which is commercially available from Medimmune, LLC, Gaithersburg, JVIaryland, a subsidiary of AstraZeneca pic.), or antigen-binding fragments, conjugates, or variants thereof. In an embodiment, the PD-LI inhibitor is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013 / 0034559.

[0171] In an embodiment, the PD-LI inhibitor is avelumab, also known as MSB0010718C (commercially available from Merck KGaA / EMD Serono), or antigen-binding fragments, conjugates, or variants thereof. The preparation and properties of avelumab are described in U.S. Patent Application Publication No. US 2014 / 0341917 Al.

[0172] In an embodiment, the PD-LI inhibitor is atezolizumab, also known as MPDL3280A or RG7446 (commercially available as TECENTRIQ from Genentech, Inc., a subsidiary of Roche Holding AG, Basel, Switzerland), or antigen-binding fragments, conjugates, or variants thereof. In an embodiment, the PD-LI inhibitor is an antibody disclosed in U.S. Patent No. 8,217,149, the disclosure of which is specifically incorporated by reference herein. In an embodiment, the PD-LI inhibitor is an antibody disclosed in U.S. Patent Application Publication Nos. 2010 / 0203056 Al, 2013 / 0045200 Al, 2013 / 0045201 Al, 2013 / 0045202 Al, or 2014 / 0065135 Al. The preparation and properties of atezolizumab are described in U.S. Patent No. 8,217,149.

[0173] In some embodiments, the composition, e.g., a pharmaceutical composition containing a PD- 1 inhibitor, e.g., an anti-PD-1 antibody such as pembrolizumab, can include carriers such as a diluent, adjuvant, excipient, or vehicle with which the inhibitor / s), and / or the cells are administered. Examples ofsuitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of a PD-1 inhibitor, e.g., an anti-PD-1 antibody, generally in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. The pharmaceutical compositions can contain any one or more of a diluents(s), adjuvant(s), antiadherent(s), binder(s), coating(s), filler(s), flavor(s), color(s), lubricant(s), glidant(s), preservative(s), detergent(s), sorbent(s), emulsifying agent(s), pharmaceutical excipient(s), pH buffering agent(s), or sweetener(s) and a combination thereof. In some embodiments, the pharmaceutical composition can be liquid, solid, a lyophilized powder, in gel form, and / or combination thereof. In some aspects, the choice of carrier is determined in part by the particular inhibitor and / or by the method of administration.

[0174] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG), stabilizers and / or preservatives. The compositions containing a PD-1 inhibitor, e.g., an anti-PD-1 antibody such as nivolumab, can also be lyophilized.

[0175] In some embodiments, the pharmaceutical compositions comprising a PD-1 inhibitor to be administered with the combination therapy described herein can be formulated for administration by any route known to those of skill in the art including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, otic, inhalational, buccal (e.g., sublingual), and transdermal administration or any route. In some embodiments, other modes of administration also are contemplated. In some embodiments, the administration is by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntivalinjection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, administration is by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0176] In some embodiments, compositions comprising a PD-1 inhibitor also can be administered with other biologically active agents, either sequentially, intermittently or in the same composition. In some embodiments, administration also can include controlled release systems including controlled release formulations and device controlled release, such as by means of a pump. In some embodiments, the administration is oral.

[0177] In some embodiments, a PD-1 inhibitor, e.g., an anti-PD-1 antibody such as pembrolizumab, is typically formulated and administered in unit dosage forms or multiple dosage forms. Each unit dose contains a predetermined quantity of a therapeutically active PD-1 inhibitor, e.g., pembrolizumab, sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. In some embodiments, unit dosage forms, include, but are not limited to, tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil water emulsions containing suitable quantities of a PD-1 inhibitor, e.g., pembrolizumab. Unit dose forms can be contained ampoules and syringes or individually packaged tablets or capsules. Unit dose forms can be administered in fractions or multiples thereof. In some embodiments, a multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose form. Examples of multiple dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. / . Dosing

[0178] In some embodiments, the provided combination therapy methods involve administering to the subject a checkpoint inhibitor therapy (e.g. a PD-1 inhibitor) and a T cell therapy (e.g. TIL composition). In some embodiments, the provided combination therapy methods involve initiation administration of the checkpoint inhibitor therapy (e.g. a PD-1 inhibitor) subsequent to initiation of the T cell therapy (e.g. TIL composition). In some embodiments, the provided combination therapy methods involve initiating administration of a checkpoint inhibitor therapy (e.g. a PD-1 inhibitor) between about 1 day and about 3 weeks after initiation of administration of the T cell therapy (e.g., TIL composition). In some embodiments, initiation of administration of the checkpoint inhibitor therapy (e.g. a PD-1 inhibitor) is between about one week and about two weeks after initiation of administration of the T cell therapy (e.g. TIL composition).

[0179] In some embodiments, the provided combination therapy methods involve initiating administration of the T cell therapy (e.g. TIL composition) on Day 1. In some embodiments, the provided combination therapy methods involve initiating administration of a checkpoint inhibitor therapy (e.g. aPD-1 inhibitor) between about Day 2 and Day 20. In some embodiments, the provided combination therapy methods involve initiating administration of a checkpoint inhibitor therapy (e.g. a PD-1 inhibitor) on about Day 8 or Day 15. In some embodiments, the provided combination therapy methods involve initiating administration of a checkpoint inhibitor therapy (e.g. a PD-1 inhibitor) on about Day 8. In some embodiments, the provided combination therapy methods involve initiating administration of a checkpoint inhibitor therapy (e.g. a PD-1 inhibitor) on about Day 15.

[0180] In some embodiments, the method involves initiating administration of the checkpoint inhibitor therapy (e.g. a PD-1 inhibitor) after activation-induced cell death (AICD) of the cells of the T cell therapy (e.g. TIL composition) has peaked.

[0181] In some embodiments, the PD-1 inhibitor is administered between Day 8 and Day 15, inclusive. In some embodiments, the PD-1 inhibitor is administered on Day 8. In some embodiments, the PD-1 inhibitor is administered on Day 15. In some embodiments, the PD-1 inhibitor is administered for no longer than about three months. In some embodiments, a final dose of the PD-1 inhibitor is administered between about Day 80 and about Day 90. In some embodiments, the final dose of the PD-1 inhibitor is administered at about Day 85.

[0182] . In some embodiments, the amount of the PD-1 inhibitor is between about 100 mg and 600 mg, between about 100 mg and about 550 mg, between about 100 mg and about 500 mg, between about 100 mg and about 450 mg, between about 100 mg and about 400 mg, between about 100 mg and about 350 mg, between about 100 mg and about 300 mg, between about 100 mg and about 250 mg, between about 100 mg and about 200 mg, between about 100 mg and about 150 mg, between about 150 mg and 600 mg, between about 150 mg and about 550 mg, between about 150 mg and about 500 mg, between about 150 mg and about 450 mg, between about 150 mg and about 400 mg, between about 150 mg and about 350 mg, between about 150 mg and about 300 mg, between about 150 mg and about 250 mg, between about 150 mg and about 200 mg, between about 200 mg and 600 mg, between about 200 mg and about 550 mg, between about 200 mg and about 500 mg, between about 200 mg and about 450 mg, between about 200 mg and about 400 mg, between about 200 mg and about 350 mg, between about 200 mg and about 300 mg, between about 200 mg and about 250 mg, between about 250 mg and 600 mg, between about 250 mg and about 550 mg, between about 250 mg and about 500 mg, between about 250 mg and about 450 mg, between about 250 mg and about 400 mg, between about 250 mg and about 350 mg, between about 250 mg and about 300 mg, between about 300 mg and 600 mg, between about 300 mg and about 550 mg, between about 300 mg and about 500 mg, between about 300 mg and about 450 mg, between about 300 mg and about 400 mg, between about 300 mg and about 350 mg, between about 350 mg and 600 mg, between about 350 mg and about 550 mg, between about 350 mg and about 500 mg, between about 350 mg and about 450 mg, between about 350 mg and about 400 mg, between about 400 mg and 600 mg, between about 400 mg and about 550 mg, between about 400 mg and about 500 mg, between about 400 mg and about 450 mg, between about 450 mg and 600 mg, between about 450 mgand about 550 mg, between about 450 mg and about 500 mg, between about 500 mg and about 600 mg, between about 500 mg and about 550 mg, or between about 550 mg and about 600 mg.

[0183] In some embodiments, the first amount of the PD-1 inhibitor is between about 140 mg and about 580 mg. In some embodiments, the first amount of the PD-1 inhibitor is between about 180 mg and about 540 mg. In some embodiments, the first amount of the PD-1 inhibitor is about 240 mg or about 480 mg. In some embodiments, the first amount of the PD-1 inhibitor is about 240 mg. In some embodiments, the first amount of the PD-1 inhibitor is about 480 mg. In some embodiments, the first amount of the PD-1 inhibitor is about 200 mg. In some embodiments, the first amount of the PD-1 inhibitor is about 400 mg.

[0184] In some embodiments, the PD-1 inhibitor is administered every two weeks (Q2W). In some embodiments, the PD-1 inhibitor is administered every two weeks (Q2W).

[0185] In some embodiments, the amount of the PD-1 inhibitor is between at or about 160 mg and 560 mg. In some embodiments, the amount of the PD-1 inhibitor is at or about 240 mg or at or about 480 mg. In some embodiments, the amount of the PD-1 inhibitor is 240 mg. In some embodiments, the amount of the PD-1 inhibitor is 480 mg. In some embodiments, a dose is administered about every two weeks (Q2W). In some embodiments, a dose is administered about every four weeks (Q4W).

[0186] In some embodiments, an amount of about 240 mg of the PD-1 inhibitor is administered Q2W. In some embodiments, an amount of about 480 mg of the PD-1 inhibitor is administered Q4W.E. Lymphodepleting Therapy

[0187] In some aspects, the provided methods can further include administering one or more lymphodepleting therapies, such as prior to or simultaneous with initiation of administration of the TIL composition.

[0188] The lymphodepleting therapy can include administration of fludarabine and / or cyclophosphamide (the active form being referred to as mafosfamide) and combinations thereof. Such methods are described in Gassner et al. (Cancer Immunol Immunother . 201 1 , 60(l):75-85, Muranski, et al, Nat Clin Pract Oncol, 2006 3(12):668-681 , Dudley, et al., J Clin Oncol 2008, 26:5233-5239, and Dudley, et al., J Clin Oncol. 2005, 23(10):2346-2357, all of which are incorporated by reference herein in their entireties.

[0189] In some embodiments, the lymphodepleting therapy comprises administration of a phosphamide, such as cyclophosphamide. In some embodiments, the lymphodepleting therapy can include administration of fludarabine. In some embodiments, the lymphodepleting therapy can include Mesna (sodium 2-mercaptoethane sulfonate).

[0190] Preconditioning with lymphodepleting therapy can be carried out with the goal of reducing the risk of one or more of various outcomes that could dampen efficacy of the therapy. These include the phenomenon known as “cytokine sink,” by which T cells, B cells, NK cells compete with TILs forhomeostatic and activating cytokines, such as IL-2, IL-7, and / or IL-15; suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system; impact of negative regulators in the tumor microenvironment. Muranski et al., Nat Clin Pract Oncol. December; 3(12): 668-681 (2006).

[0191] Thus in some embodiments, the provided method further involves administering a lymphodepleting therapy to the subject. In some embodiments, the method involves administering the lymphodepleting therapy to the subject prior to the initiation of the administration of the dose of cells (e.g., TIL composition). In some embodiments, the lymphodepleting therapy contains a chemotherapeutic agent such as fludarabine and / or cyclophosphamide and / or Mesna (sodium 2- mercaptoethane sulfonate). In some embodiments, the lymphodepleting therapy contains a chemotherapeutic agent such as fludarabine and a second lymphodepleting therapy that is cyclophosphamide. In some embodiments, the administration of the cells (e.g., TIL composition) and / or the lymphodepleting therapy is carried out via outpatient delivery.

[0192] In some embodiments, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, Mesna (sodium 2- mercaptoethane sulfonate), or combinations thereof, to a subject prior to the initiation of the administration of the dose of cells (e.g., TIL composition). For example, the subject may be administered a preconditioning agent at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the first or subsequent dose. In some embodiments, the subject is administered a preconditioning agent no more than 7 days prior, such as no more than 6, 5, 4, 3, or 2 days prior, to the initiation of administration of the dose of cells (e.g., TIL composition). In some embodiments, the subject is administered a preconditioning agent between 2 and 7, inclusive, such as at 2, 3, 4, 5, 6, or 7, days prior to the initiation of the administration of the dose of cells (e.g., TIL composition). In some embodiments, the subject I administered a first lymphodepleting therapy no more than 7 days prior, such as no more than 6, 5, 4, 3, or 2 days prior, to the initiation of administration of the dose of cells (e.g., TIL composition). In some embodiments, the subject I administered a second lymphodepleting therapy no more than 5 days prior, such as no more than 5, 4, 3, or 2 days prior, to the initiation of administration of the dose of cells (e.g., TIL composition).

[0193] In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg / kg and 100 mg / kg, such as between or between about 40 mg / kg and 80 mg / kg. In some aspects, the subject is preconditioned with or with about 60 mg / kg of cyclophosphamide. In some embodiments, the cyclophosphamide is administered at a dosage of 100 mg / m2 / day, 150 mg / m2 / day, 175 mg / m2 / day, 200 mg / m2 / day, 225 mg / m2 / day, 250 mg / m2 / day, 275 mg / m2 / day, or 300 mg / m2 / day. In some embodiments, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, cyclophosphamide is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some embodiments, the cyclophosphamide is administered intravenously(i.e., i.v.). In some embodiments, the cyclophosphamide treatment is for 2-7 days, such as 3-5 days, at or about 3 days, at or about 4 days or at or about 5 days. In some embodiments, the cyclophosphamide is administered once daily for one or two days. In some instances, the subject is administered about 60 mg / kg of cyclophosphamide within 5 to 9 days prior to administering the TIL composition. In some embodiments, the cyclophosphamide is administered at or about 7 days prior to (Day -7) administering the TIL composition.

[0194] In some embodiments, where the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose between or between about 1 mg / m2and 100 mg / m2, such as between or between about 10 mg / m2and 75 mg / m2, 15 mg / m2and 50 mg / m2, 20 mg / m2and 40 mg / m2" 24 mg / m2and 35 mg / m2, 20 mg / m2and 30 mg / m2, or 24 mg / m2and 26 mg / m2. In some instances, the subject is administered 25 mg / m2of fludarabine. In some embodiments, the subject is administered about 30 mg / m2of fludarabine. In some embodiments, the fludarabine is administered at a dosage of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day, or a dosage amount between a range of any of the foregoing. In some embodiments, the fludarabine is administered for 2-7 days, such as for 3-5 days, such as at or about 3 days, at or about 4 days or at or about 5 days. In some embodiments, the fludarabine treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, fludarabine is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 25 mg / m2of fludarabine, daily for 3 days, prior to administering the TIL composition. In some embodiments, the daily administration involves administration on consecutive days. In some embodiments, the fludarabine is administered 2 to 6 days prior to administering the TIL composition. In some embodiments, the fludarabine is administered daily for 3 consecutive days beginning about 5 days prior to (Day-5) administering the TIL composition. In some embodiments, the fludarabine is administered on Day -5, Day -4 and Day -3.

[0195] In some embodiments, lymphodepletion is performed by administering the fludarabine and the cyclophosphamide are together to a patient (e.g., the lymphodepleting therapy). In some embodiments, fludarabine is administered at 25 mg / m2 / day i.v. and cyclophosphamide is administered at 250 mg / m2 / day i.v. over 4 days. In an embodiment, the lymphodepletion is performed by administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for 3-5 days, such as for about 3 days.

[0196] In some embodiments, where the lymphodepleting agent comprises Mesna (sodium 2- mercaptoethane sulfonate), the subject is administered Mesna (sodium 2-mercaptoethane sulfonate) at a dose between or between about 1 mg / m2and 100 mg / m2, such as between or between about 10 mg / m2and 75 mg / m2, 15 mg / m2and 50 mg / m2, 20 mg / m2and 40 mg / m2” 24 mg / m2and 35 mg / m2, 20 mg / m2and 30 mg / m2, or 24 mg / m2and 26 mg / m2. In some instances, the subject is administered 15 mg / m2of Mesna (sodium 2-mercaptoethane sulfonate). In some instances, the subject is administered about 25 mg / m2of Mesna (sodium 2-mercaptoethane sulfonate). In some embodiments, the Mesna (sodium 2- mercaptoethane sulfonate)can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, Mesna (sodium 2- mercaptoethane sulfonate)is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 30 mg / m2of Mesna (sodium 2-mercaptoethane sulfonate), daily for 3 days, prior to initiation of the cell therapy.

[0197] In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and / or fludarabine and / or Mesna (sodium 2-mercaptoethane sulfonate). Thus, the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, fludarabine at any dose or administration schedule, such as those described above, and / or Mesna (sodium 2-mercaptoethane sulfonate) at any dose or administration schedule, such as those described above. For example, in some aspects, the subject is administered 60 mg / kg (~2 g / m2) of cyclophosphamide and 25 mg / m2fludarabine, and 15 mg / m2Mesna (sodium 2-mercaptoethane sulfonate) prior to the dose of cells (e.g., TIL composition). In some embodiments, the preconditioning administration schedule ends between 2 and 7, inclusive, such as at 2, 3, 4, 5, 6, or 7, days prior to the initiation of the administration of the dose of cells (e.g., TIL composition).

[0198] In one exemplary dosage regimen, prior to receiving the TIL composition, subjects receive a lymphodepleting preconditioning chemotherapy of cyclophosphamide and fludarabine (CY / FLU), which is administered at least two days before the TIL composition and generally no more than 7 days before administration of cells (e.g., TIL composition). After preconditioning treatment, subjects are administered the TIL composition as described above. In another exemplary dosage regimen, prior to receiving the TIL composition, subjects receive a lymphodepleting preconditioning chemotherapy of cyclophosphamide, fludarabine, and Mesna (sodium 2-mercaptoethane sulfonate), which is administered at least two days before the TIL composition and generally no more than 7 days before administration of cells (e.g., TIL composition). After preconditioning treatment, subjects are administered the TIL composition as described above.

[0199] In some embodiments, the administration of the preconditioning agent prior to administration of the dose of cells (e.g., TIL composition) improves an outcome of the treatment. For example, in some aspects, preconditioning improves the efficacy of treatment with the dose or increases the persistence of the receptor-expressing cells (e.g., TIL cells in a composition) in the subject. In some embodiments, preconditioning treatment increases disease-free survival, such as the percent of subjects that are alive and exhibit no minimal residual or molecularly detectable disease after a given period of time following the dose of cells. In some embodiments, the time to median disease-free survival is increased.

[0200] In some embodiments, the administration of the preconditioning agent prior to infusion of the dose of cells (e.g., TIL composition) improves an outcome of the treatment such as by improving the efficacy of treatment with the dose or increases the persistence of the cells (e.g., T cells in a TIL composition) in the subject. Therefore, in some embodiments, the dose of preconditioning agent given in the method which is a combination of a checkpoint inhibitor therapy (e.g., PD-1 inhibitors) and a T cell therapy (e.g., TIL composition) is higher or lower than the dose given in the method without the checkpoint inhibitor therapy (e.g., PD-1 inhibitors). In some embodiments, the dose of preconditioning agent given in the method which is a combination of a checkpoint inhibitor therapy (e.g., PD-1 inhibitors) and a cell therapy (e.g., TIL composition) is higher than the dose given in the method without the checkpoint inhibitor therapy. In some embodiments, the dose of preconditioning agent given in the method which is a combination of a checkpoint inhibitor therapy (e.g., PD-1 inhibitors) and a cell therapy (e.g., TIL composition) is lower than the dose given in the method without the checkpoint inhibitor therapy.F. Cytokine Therapy

[0201] In some embodiments, the provided methods include a cytokine therapy that is administered in combination with the TIL therapy and the one or more other therapies as described. In some embodiments, the cytokine therapy is an IL-2 regimen. In some embodiments, the TIL-2 is a recombinant human IL-2 have the amino acid sequence set forth in SEQ ID NO:1.

[0202] In some embodiments, the IL-2 is aldesleukin (Proleukin®). 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. Aldesleukin is available from commercial suppliers.IL-2 (SEQ ID NO:1)MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL EEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN RWITFCQSII STLTAldesleukin (SEQ ID NO:2) PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFSQSIIST LT

[0203] In some embodiments, the IL-2 is a pegylated forms of IL-2, such as the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and pegylated IL-2 include those as described in U.S. Patent Application Publication No. US 2014 / 0328791 Al and International Patent Application Publication No. WO 2012 / 065086 Al. The IL-2 also can be a conjugated IL-2 described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and4902,502. Examples of formulations of IL-2 suitable for use in the provided embodiments are described in U.S. Patent No. 6,706,289.

[0204] In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 lU / kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours.

[0205] In some embodiments, the methods include administering a cytokine therapy, such as IL-2 (e.g., Aldesleukin) , to a subject following the completion of the administration of the dose of cells (e.g., TIL composition). For example, the subject may be administered a cytokine therapy at least 2 hours after, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours following, completion of the first or subsequent dose of cells. In some embodiments, the subject is administered a cytokine therapy no more than 24 hours after, such as no more than 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 following the completion of the administration of the dose of cells (e.g., TIL composition). In some embodiments, the subject is administered a cytokine therapy that is IL-2 (e.g., Aldesleukin) between 2 and 8, inclusive, such as at 2, 3, 4, 5, 6, 7, or 8 hours following the completion of the administration of the dose of cells (e.g., TIL composition).

[0206] In some embodiments, the subject is administered a cytokine therapy that is IL-2 (e.g., Aldesleukin). In some embodiments, the subject is administered IL-2 at a dose between or between about 600,000 lU / kg and 1,000,000 lU / kg, such as between or between about 650,000 lU / kg and 700,000 lU / kg. In some embodiments, the IL-2 can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every 12 hours, every 8 hours, or every 6 hours. In some embodiments, IL-2 is administered daily, such as for 1-7 days, for example, for 1 to 6 days. In some embodiments, the IL-2 is administered every 8 hours for 48 hours. In some embodiments, IL-2 is administered every 6 hours, such as for 6-48 hours, for example, for 36 hours.

[0207] In some embodiments, where the cytokine therapy comprises IL-2, the subject is administered IL-2 at a dose between or between about 600,000 lU / kg and 1 ,000,000 lU / kg, such as between or between about 600,000 lU / kg to 650,000 lU / kg, inclusive. In some instances, the subject is administered about 600,000 lU / kg of IL-2 (e.g., Aldesleukin). In some instances, the subject is administered about 600,000 lU / kg of IL-2, every 6 hours for 36 hours, following completion of administration of the cell therapy.II. EX VIVO METHODS OF PRODUCING TIL COMPOSITIONS ENRICHED IN TUMOR- REACTIVE T CELLS

[0208] Various embodiments of the provided methods involve the ex vivo expansion and production of a T cell therapeutic composition, particularly for use in connection with treating cancer. In some embodiments, the method of manufacturing involves the growth and manipulation of patient cells outside of the body. In particular aspects, the provided methods can be used for the ex vivo production of a T celltherapy, including for the ex vivo expansion of autologous tumor-reactive T cells. In some embodiments, the methods produce or expand T cells for use in autologous cell therapy for treating cancer. In some embodiments, the tumor reactive T cells are obtained from a subject (e.g. a subject with the cancer to be treated) and are enriched ex vivo by processes of selection and expansion. In some embodiments, such methods results in a product containing tumor reactive T cells that can target many mutations and / or that are enriched in a multiclonal TCR TIL population that are reactive to different tumor antigens. Thus, such tumor reactive T cells offer advantages compared to existing methods in which cells are transduced to express a single neoepitope reactive TCR, or in which TILs are expanded in bulk or exhibit limited multiclonality.

[0209] In some embodiments, methods as described herein involve enrichment of tumor-reactive T cells, either by co-culture with peptide-pulsed antigen-presenting cells (APCs) followed by selection of activated T cells or by direct selection of tumor-reactive T cells from resected tumor fragments. This is in contrast to existing methods that involve passive expansion of bulk T cells in which all T cells from a tumor are subjected to expansion. In such other methods, while total viable cells (TVC) can be greatly expanded by these alternative processes, there is no step of actively ensuring that tumor reactive T cells are predominantly propagated.

[0210] In some embodiments, the stimulation or any culture or incubation of the cells can be further carried out with an apoptosis inhibitor, such as Fas decoys or caspase inhibitors or any combination thereof.

[0211] In some embodiments, the subject to be treated by the combination therapy methods is the same subject from with the biological sample is obtained for producing the TIL cell therapy. In some such embodiments, the provided method of treatment is an adoptive cell therapy with a therapeutic TIL composition containing T cells autologous to the subject.

[0212] In some embodiments, the TIL compositions provided herein are autologous to the subject to be treated. In such embodiments, the starting cells for expansion are isolated directly from a biological sample from the subject as described herein, in some cases including with enrichment for T cells positive for one or more selection marker as described, and cultured under conditions for expansion as provided herein. In some aspects, the biological sample from the subject is or includes a tumor or lymph node sample and such sample tumor and an amount of such tissue is obtained, such as by resection or biopsy (e.g. core needle biopsy or fine-needle aspiration). In some embodiments, following the culturing under conditions for expansion in accord with the provided methods the cells are formulated and optionally cryopreserved for subsequent administration to the same subject for treating the cancer.

[0213] In some embodiments, the TIL therapy is produced by a process that utilizes an unbiased identification and functional screening process to isolate and selectively expand the greatest breadth of tumor reactive TILs from the subject’s tumor. In some embodiments, greater than 60%, greater than 70% or greater than 80% of the cells of the cell therapy are functional and potent tumor reactive T cells. Insome embodiments, the TIL cell therapy is administered in an amount of least 109cells with greater than 70% functional and potent tumor reactive T cells.

[0214] The TIL therapy for use in the provided combination therapy methods and uses include any as described in PCT publication No. W02020 / 172202, W02020 / 205662, WO2021 / 108727, and WO2021 / 174208, each incorporated by reference in their entirety. Any of such methods can produce TILs with features as described herein that are enriched in tumor-reactive T cells, including any TIL compositions described in Section I.C. Exemplary methods are described in the following subsections.A. Tumor-Reactive T cells Enriched by Co-Culture

[0215] Provided methods include ex vivo co-culture methods in which a population of T cells that have been expanded from T cells present in or from a tumor sample is incubated in the presence of antigen-presenting cells that have been contacted with, or made to present, the neoantigenic peptides. In some embodiments of the provided methods a source of potential tumor peptides is used to identify TCRs that are reactive to neoantigens in a process that includes expansion of the T cells reactive to the tumor neoantigenic peptides. In particular aspects, the T cells and antigen-presenting cells are autologous to the tumor-bearing subject from which the peptides were identified. The provided methods further include steps to separate, enrich for and / or select for tumor-reactive T cells from the co-culture prior to or in connection with their further ex vivo expansion.

[0216] In some embodiments, the TILs are produced by a process in which a population containing T cells is obtained, selected or isolated from a tumor sample from a subject, such as a human subject. In some embodiments, the tumor sample is a tumor fragment or suspension of cell therefrom containing tumor infiltrating lymphocytes or TILs. In some embodiments, the provided methods include (1) Enriching a population containing T cells obtained from a tumor sample of a donor subject to produce a first population of T cells; (2) Stimulating the first population with one or more T-cell stimulating agents of lymphocytes to produce a second population of activated T cells; (3) Co-culturing cells from the second population of T cells in the presence of antigen presenting cells that present one or more peptide (e.g. peptide neoepitopes) on an MHC (MHC-associated non-native peptide), in which the co-culturing produces a third population of cells containing or enriched for T cells that are reactive to a peptides presented on the MHC of an APC (e.g. tumor reactive T cells); (4) from the third population, separating the APCs to produce a fourth population of T cells containing endogenous TCR that are reactive to peptides present on the APCs; and (5) expanding the fourth population of T cells containing tumor reactive T cells by incubation in the presence of T cell stimulating agents. In some embodiments, the separating of the T cells in (4) can involve depleting or removing the APCs and / or can include selection of T cells based on upregulation markers on reactive or activated T cells. In some embodiments, one or more of the steps can be carried out in a closed system using serum free medium.

[0217] In the provided methods, tumor reactive T cells are identified or enriched from the stimulated T cells expanded in the first step by one or more further steps that include ex vivo co-culture of the stimulated T cells (second population of T cells) with antigen presenting cells (APCs) and one or a plurality of peptides that include neoepitopes of a tumor antigen (APCs / peptide neoepitopes). In some embodiments, provided methods include ex vivo co-culture in which the second population of T cells are incubated with APCs, such as autologous APCs or artificial antigen presenting cells (aAPCs), that have been exposed to or contacted with one or more peptides, e.g. synthetic peptides, under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen. In some embodiments, the population of T cells are autologous T cells from a subject with a tumor and the source of synthetic peptides are tumor antigenic peptides from a tumor antigen of the same subject. In some embodiments, cells from the ex vivo co-culture are a population of cells (third population) that include tumor reactive T cells that recognize or are activated by a peptide presented on an MHC of an APC in the culture. In some embodiments, cells from the ex vivo co-culture represent a source of cells that are enriched for tumor reactive T cells.

[0218] In some cases, the tumor reactive T cells can be further enriched by separation or selection of cells that express one or more upregulation marker, such as an activation marker, associated with tumor- reactive T cells (the further separation or selection producing a fourth population of T cells of the enriched tumor reactive T cells). The T cell activation markers can include cell surface markers whose expression is upregulated or specific to T cells that have been exposed to antigen and activated. Exemplary of such markers are described below.

[0219] FIG. 1A depicts a schematic of an exemplary process for manufacturing a T cell therapeutic composition in accord with the provided methods. In an exemplary process, a tumor sample is obtained from a patient for identification and generation of peptides for use in co-culturing methods with antigen presenting cells (APCs) presenting the peptides and autologous antigen T cells obtained from the same subject. In some cases, a suspension of dissociated tumor cells containing T cells is obtained or provided from the patient, and subjected to an initial pre-expansion with recombinant IL-2 to expand T cells from the tumor, prior to co-culture with antigen presenting cells that have been loaded with a pool of 20mer- 40mer peptides identified from the neoantigens and manufactured for presentation on MHC class I and / or MHC class II molecules. Following co-culture, tumor-reactive T cells are selected for cells surface positive for CD134 and / or CD137 by sorting using fluorescence activated cell sorting (FACs), thereby removing potential bystander cells. The selected and sorted cells are then subjected to a rapid expansion protocol with irradiated peripheral blood mononuclear cells (iPBMCs), agonist anti-CD3 antibody (e.g. OKT3), and recombinant IL-2 until a threshold number of cells is obtained, typically 12 to 16 days, such as at or about 14 days. Upon reaching the threshold number of cells, the cells can be harvested and formulated, in some cases concentrated or cryopreserved, and used for administration to a subject such as by infusion.

[0220] The process can be carried out in the presence of serum-free media containing nutrients. One or more or all of the steps can be carried out in a closed system, such as without exposure of cells to the outside environment.

[0221] By virtue of the provided process, the initial small population of tumor reactive T cells expanded from the tumor are enriched for cells that are tumor reactive cells before a subsequent second expansion step, thereby promoting preservation and expansion of cells of interest and limiting expansion of bystander T cells that are not reactive to a tumor antigen and / or that may include cells that exhibit inhibitory activity. The provided methods maximize the number of tumor reactive cells that may be collected by co-culturing all of the cells propagated after the first expansion with peptide -presenting APCs, and then by selecting from among all of the bulk cells after the co-culturing for cells positive for CD134 and / or CD137 before the subsequent second expansion. As demonstrated herein, the provided methods can result in 1000-fold or more expansion of the selected tumor reactive cells with REP, and such cells exhibit robust neoantigen reactivity as demonstrated by cytokine (e.g. IFNy) production and secretion and production of factors necessary for cytotoxic killing (e.g. CD107a and Granzyme B).

[0222] In some embodiments, the provided methods for producing a TIL composition, such as any described herein, include: providing dissociated tumor cells from a tumor obtained from a donor subject, wherein the dissociated tumor cells are a first population of T cells that comprise CD4+ and CD8+ T cells; culturing the first population of T cells with recombinant IL-2 added at a concentration between 3000 lU / mL and 6000 lU / mL, inclusive, for 14 to 28 days to produce a second population of T cells; co- culturing the second population of T cells for 12 to 48 hours with autologous antigen presenting cells (APCs) with recombinant IL-2 added at a concentration of 100 lU / mL to 1000 lU / mL to produce a third population of T cells, wherein the APCs are loaded with a pool of peptide neoantigens from the tumor, wherein each peptide is 13-40 amino acids in length and is loaded at a concentration of 100 ng / mL per peptide, and wherein the ratio of the second population of T cells to APCs is 2: 1 to 10: 1 ; selecting cells from the third population of T cells that are surface positive for CD134 and / or CD137 to produce a fourth population of T cells; and expanding tumor infiltrating lymphocytes (TILs) by incubating the fourth population of T cells with irradiated human peripheral blood mononuclear cells (iPBMCs) at a ratio of 100 to 500 iPBMC to cells of the fourth population of T cells with recombinant IL-2 added at a concentration between 3000 lU / mL and 6000 lU / mL, inclusive, and 10 to 50 ng / mL anti-CD3 antibody (OKT3) for 12 to 16 days to produce a therapeutic composition of TILs enriched in tumor reactive cells.

[0223] In some embodiments methods for producing a TIL composition may include ex vivo coculture in which the second population of T cells are incubated with APCs, such as autologous APCs, that have been exposed to or contacted with one or more peptides, e.g. synthetic peptides, under conditions in which the APCs have been induced to present one or more peptides from a tumor- associated antigen. In some embodiments, the population of T cells are autologous T cells from a subject with a tumor and the source of synthetic peptides are tumor antigenic peptides from a tumorantigen of the same subject. In some embodiments, cells from the ex vivo co-culture are a population of cells (third population) that include tumor reactive T cells that recognize or are activated by a peptide presented on an MHC of an APC in the culture. In some embodiments, cells from the ex vivo co-culture represent a source of cells that are enriched for tumor reactive T cells.

[0224] In some cases, the tumor reactive T cells can be further enriched by separation or selection of cells that express one CD137, CD134 or CD137 and CD134 (also phrased “CD134 and / or CD137”).

[0225] In particular embodiments, a second expansion (also called “rapid expansion protocol” or REP) is performed on T cells enriched or isolated from the co-culture, such as after separation or selection of tumor reactive T cells for cells that express or are surface positive for CD134 and / or CD137. The second expansion involves incubation to further stimulate T cells for expansion with anti-CD3 antibody (e.g. OKT3), irradiated peripheral blood mononuclear cells (iPBMCs), and recombinant IL-2. The T cells are allowed to expand for a certain number of days as desired and / or until a therapeutic dose or harvest dose is met. The composition of expanded T cells can then be harvested and formulated for administration to a subject for treatment of a cancer in the subject.

[0226] In particular embodiments, the incubation or culture of T cells also is carried out with nutrient containing media so that the cells can survive outside of the body. In embodiments of the provided methods, one or more of the steps can be carried out in serum-free media. In one embodiment, the serum free medium is OpTmizer CTS (LifeTech), Immunocult XF (Stemcell technologies), CellGro (CellGenix), TexMacs (Miltenyi), Stemline (Sigma), Xvivol5 (Lonza), PrimeXV (Irvine Scientific), or Stem XVivo (RandD systems). The serum-free medium can be supplemented with a serum substitute such as ICSR (immune cell serum replacement) from LifeTech. The level of serum substitute (e.g., ICSR) can be, e.g., up to 5%, e.g., about 1%, 2%, 3%, 4%, or 5%. In some embodiments, the serum-free media contains 0.5 mM to 5 mM of a dipeptide form of L-glutamine, such L-alanyl-L-glutamine (Glutamax™). In some embodiments, the concentration of the dipeptide form of L-glutamine, such as L- alanyl-L-glutamine, is from or from about 0.5 mM to 5 mM, 0.5 mM to 4 mM, 0.5 mM to 3 mM, 0.5 mM to 2 mM, 0.5 mM to 1 mM, 1 mM to 5 mM, 1 mM to 4 mM, 1 mM to 3 mM, 1 mM to 2 mM, 2 mM to 5 mM, 2 mM to 4 mM, 2 mM to 3 mM, 3 mM to 5 mM, 3 mM to 4 mM or 4 mM to 5 mM, each inclusive. In some embodiments, the concentration of the dipeptide form of L-glutamine, such as L-alanyl-L- glutamine, is or is about 2 mM. a. Neoantigen identification and peptide generation

[0227] The provided methods include a step of generating or identifying in silico a plurality of peptides (also referred to as “P” or “n-mers”) that contain at least one cancer-specific cancer neoantigen. In some embodiments, at least one synthetic peptide is prepared using sequence information from all or a subset of neoantigen sequences, and the synthetic peptide is then employed in methods to enrich for tumor-reactive T cells in accord with the provided methods.

[0228] In some embodiments, the cancer-specific cancer neoepitope is determined by identifying or isolating a tumor-associated antigen or peptide sequence thereof from a cancer cell from a subject. The cancer cell may be obtained from any bodily sample derived from a patient which contains or is expected to contain tumor or cancer cells. The bodily sample may be any tissue sample such as blood, a tissue sample obtained from the primary tumor or from tumor metastases, a lymph node sample or any other sample containing tumor or cancer cells.

[0229] In some embodiments, the tumor is a hematological tumor. Non- limiting examples of hematological tumors include leukemias, including acute leukemias (such as 1 lq23- positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0230] In some embodiments, the tumor is a solid tumor. Non-limiting examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). In several examples, a tumor is melanoma, lung cancer, lymphoma breast cancer or colon cancer.

[0231] In some embodiments, the cancer is a gastrointestinal cancer involving a cancer of the gastrointestinal tract (GI tract), including cancers or the upper or lower digestive tract, or an accessory organ of digestion, such as esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum or anus. In some embodiments, the cancer is an esophageal cancer, stomach (gastric) cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, cancer of the mucosa- associated lymphoid tissue (MALT lymphoma), cancer of the biliary tree, colorectal cancer (including colon cancer, rectum cancer or both), anal cancer, or a gastrointestinal carcinoid tumor.

[0232] In particular embodiments, the cancer is a colorectal cancer.

[0233] In some embodiments, the tumor is from a breast cancer, such as a ductal carcinoma or a lobular carcinoma. In some embodiments, the tumor is from a prostate cancer. In some embodiments, tumor is from a skin cancer, such as a basal cell carcinoma, a squamous cell carcinoma, a Kaposi's sarcoma, or a melanoma. In some embodiments, the tumor is from a lung cancer, such as an adenocarcinoma, a bronchiolaveolar carcinoma, a large cell carcinoma, or a small cell carcinoma. In some embodiments, the tumor is from a brain cancer, such as a glioblastoma or a meningioma. In some embodiments, the tumor is from a gastrointestinal cancer, such as any described above. In some embodiments, the tumor is from a colon cancer. In some embodiments, the tumor is from a liver cancer, such as a hepatocellular carcinoma. In some embodiments, the tumor is from a pancreatic cancer. In some embodiments, the tumor is from a kidney cancer, such as a renal cell carcinoma. In some embodiments, the tumor is from a testicular cancer.

[0234] In some embodiments, the cancer is not a melanoma. Melanoma is a cancer that generally has a high mutational rate. High tumor mutation burden has been thought to be a particularly desired prognostic marker for success related to treatment with an immunotherapy targeting tumor neoantigens (Simpson et al., Journal of Clinical Oncology 2017, 35:15_suppl, 9567-9567; McGranahan et al. Science 2016, 351:1463-1469) In some embodiments, the provided methods can be used in cancers that have a lower tumor mutation burden, since the methods are carried out to actively (as opposed to passively) enrich for tumor reactive T cells.

[0235] In some embodiments, the subject is a subject with a tumor mutational burden (TMB) of less than 8 mutations. TMB includes the number of non-synonymous mutations per tumor. In some embodiments, TMB can be calculated by counting the number of synonymous and non-synonymous mutations across a 0.8- to 1.2-megabase (Mb) region, and reporting the result as mutations / Mb. In some embodiments, TMB can be determined by next generation sequencing (NGS) on tumor tissue samples. In some cases, whole exome sequencing can be used or computational germline status filtering can be used (Chalmers et al. Genome Med 2017 9:34). In some embodiments, the subject has a TMB of less than at or about 60 mutations / Mb, such as less than at or about 55 mutations / Mb, less than at or about 50 mutations / Mb, less than at or about 45 mutations / Mb, less than at or about 40 mutations / Mb, less then at or about 30 mutations / Mb, less than at or about 25 mutations per Mb, or less than at or about 20 mutations / Mb, or any value between any of the foregoing. In some embodiments, the subject has a TMB of less than at or about 41 mutations / Mb, less than at or about 40 mutations / Mb, less than at or about 39 mutations / Mb, less than at or about 38 mutations / Mb, less than at or about 37 mutations / Mb or less.

[0236] In some embodiments, the peptide (P) is a tumor-associated antigen derived from premalignant conditions, such as variants of carcinoma in situ, or vulvar intraepithelial neoplasia, cervical intraepithelial neoplasia, or vaginal intraepithelial neoplasia.

[0237] In some aspects, nucleic acid from such cells of the tumor or cancer is obtained and sequenced. In embodiments, the protein-coding region of genes in a genome is obtained, such as byomics analysis, such as by analysis of whole genomic sequencing data, exome sequencing data, and / or transcriptome data. To identify tumor-specific sequences, sequencing data can be compared to a reference sequencing data, such as data obtained from a normal cell or noncancerous cell from the same subject. In some embodiments, next-generation sequencing (NGS) methods are used.

[0238] In some embodiments, the methods include a step of using matched normal omics data of a tumor. In such methods, the in silica analysis involves an omics analysis to identify mutations in the tumor relative to normal tissue of the same patient, such as non-diseased tissue of the same patient. It is generally contemplated that matched normal omics data are whole genomic sequencing data, exome sequencing data, and / or transcriptome data, and that the matched normal omics data are matched against normal before treatment of the patient. In a particular embodiment, whole exome sequencing is performed on healthy and diseased tissue to identify somatic mutations associated with the tumor.

[0239] In some embodiments, omics data are obtained from one or more patient biopsy samples following standard tissue processing protocol and sequencing protocols. In particular embodiments, the data are patient matched tumor data (e.g., tumor versus same patient normal). In some cases, nonmatched or matched versus other reference (e.g., prior same patient normal or prior same patient tumor, or homo statisticus) are also deemed suitable for use herein. The omics data may be fresh omics data or omics data that were obtained from a prior procedure (or even different patient). For example, neoepitopes may be identified from a patient tumor in a first step by whole genome and / or exome analysis of a tumor biopsy (or lymph biopsy or biopsy of a metastatic site) and matched normal tissue (i.e., non-diseased tissue from the same patient such as peripheral blood). In some embodiments, genomic analysis can be processed via location-guided synchronous comparison of the so obtained omics information.

[0240] The genomic analysis can be performed by any number of analytic methods. In particular embodiments, the methods include WGS (whole genome sequencing) and exome sequencing of both tumor and matched normal sample using next generation sequencing such as massively parallel sequencing methods, ion torrent sequencing, pyrosequencing. Computational analysis of the sequence data may be performed in numerous manners. In some embodiments, the data format is in SAM, BAM, GAR, or VCF format. As an example, analysis can be performed in silica by location-guided synchronous alignment of tumor and normal samples as, for example, disclosed in US 2012 / 0059670 Al and US 2012 / 0066001 Al using BAM files and BAM servers. Alternative file formats for sequence analysis (e.g., SAM, GAR, FASTA, etc.) are also contemplated.

[0241] In some of any embodiments, peptides (P) comprising neoantigens arising from a missense mutation encompass the amino acid change encoded by 1 or more nucleotide polymorphisms. Peptides (P) comprising neoantigens that arise from frameshift mutations, splice site variants, insertions, inversions and deletions should encompass the novel peptide sequences and junctions of novel peptidesequences. Peptides (P) comprising neoantigens with novel post-translational modifications should encompass the amino acids bearing the post-translational modification(s), such as a phosphate or glycan.

[0242] Once these mutations are identified, neoepitopes are then identified. Neoepitopes are mutant peptides that are recognized by a patient’s T cells. These neoepitopes must be presented by a tumor or antigen presenting cell by the MHC complex and then be recognized by a TCR on the T cell. In some embodiments, the provided methods include a step of calculation of one or more neoepitopes to define neoepitopes that are specific to the tumor and patient. Consequently, it should be recognized that patient and cancer specific neoepitopes can be identified from omics information in an exclusively in silica environment that ultimately predicts potential epitopes that are unique to the patient and tumor type. In particular aspects, the so identified cancer neoepitopes are unique to the patient and the particular cancer in the patient (e.g., having a frequency of less than 0.1% of all neoepitopes, and more typically less than 0.01% in a population of cancer patients diagnosed with the same cancer), but that the so identified cancer neoepitopes have a high likelihood of being presented in a tumor.

[0243] In some of any embodiments, the length of the peptide (P) depends on the specific application and is typically between about 5 to about 50 amino acids. In particular embodiments, neoepitopes will be calculated to have a length of between 2-50 amino acids, such as 13-40 amino acids, for example 25 amino acids (25mer). In preferred embodiments, the peptide (P) is between about 13 to 40 amino acids, e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids.

[0244] In some embodiments, the methods are carried out with a pool of peptides. The pool of peptides can include tens to hundreds of individual peptides. The pool of peptides can include up to 200 different peptides containing predicted mutations. In some cases, the pool of peptides includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or more individual peptides, or any value between any of the foregoing. The pool of peptides can represent one neo-antigen or can represent several neo-antigens. In some cases, a pool of peptides can include multiple overlapping peptides of the same neo-antigen. Thus, for a tumor- associated antigen, the antigen may be divided into 13 to 40 amino acid, e.g., 25 amino acid, peptides (P) wherein each peptide (P) contains a unique composition of amino acids; or, the peptides (P) can be overlapping peptide pools wherein an antigen is divided into a set number of 13 to 40 amino acid, e.g., 25 amino acid, peptides (P) that have overlapping sequences. In some cases, each of the peptides of the overlapping pool of an antigen can be offset by a set number of amino acid residues, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 15 or 15 amino acids. In some embodiments, each of the peptides of the overlapping pool of an antigen is offset by 10 amino acids. In some embodiments, each of the peptides of the overlapping pool of an antigen is offset by 12 amino acids. For example, an overlapping peptide pool comprising a 100 amino acid antigen may be divided into eight 25 amino acid peptides (P) that are each offset by 12 amino acids (i.e., each subsequent 25 amino acid peptide comprising a 100 amino acid peptide sequencestarts at the 13thamino acid position from the prior peptide). Those skilled in the art understand that many permutations exist for generating a peptide pool from an antigen.

[0245] Various algorithms have been developed and can be used to map T cell epitopes (both MHC Class I and Class Il-restricted) within protein molecules of various origins. In some embodiments, many programs utilize availability of the large-scale peptide -MHC binding affinity matrix from experimental measurements to train machine learning (ML)-based classifiers to distinguish MHC-binders from nonbinders (see e.g., Zhao et al. (2018) PLoS Comput Biol 14(11): el006457). Exemplary predictor methods for MHC class I (e.g. 9-mer) include smm, smmpmbec, ann (NetMHC3.4), NetMHC4, PickPocket, consensus, NetMHCpan2.8, NetMHCpan3, NetMHCpan4, NetMHCcons, mhcflurry, mhcflurry_pan, or MixMHCpred. Exemplary predictor methods for MHC class II (e.g. 15-mer ) include NetMHCIIpan, NetMHCII2.3, nn_align, smm_align, consensus, comblib, tepitope, or mhcflurry. Any of such methods can be used.

[0246] After the in silico identification of suitable neoepitope sequences, corresponding synthetic peptides are then prepared in vitro (e.g. , using solid phase synthesis). In particular embodiments, a library of synthetic peptides is prepared representing a plurality of different neoepitopes from the subject.

[0247] Various methods can be used to prepare synthetic peptides. For example, peptides with cancer neoepitope sequences can be prepared on a solid phase (e.g., using Merrified synthesis), via liquid phase synthesis, or from smaller peptide fragments. Peptide epitopes can be obtained by chemical synthesis using a commercially available automated peptide synthesizer. In some embodiments, the peptides can be synthesized, for example, by using the Fmoc-polyamide mode of solid-phase peptide synthesis which is disclosed by Lu et al (1981).J. Org. Chem. 46,3433 and the references therein. In some aspects, peptides can be produced by expression of a recombinant nucleic acid in a suitable host and with a suitable expression system. In some aspects, recombinant methods can be used where multiple neoepitopes are on a single peptide chain, such as with spacers between neoepitopes or cleavage sites).

[0248] The peptides can be purified by any one, or a combination of techniques such as recrystallization, size exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, and reverse-phase high performance liquid chromatography using e.g. acetonitrile / water gradient separation. In some embodiments, peptides can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Analysis of peptides can be carried out using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF mass spectrometric analysis.b. Tumor Sample for First Expansion

[0249] The provided methods include obtaining and enriching or selecting a population of T cells from a biological sample for use as a first or input population of T cells. In some cases, the first population of T cells is one that is known or likely to contain T cells reactive to a tumor antigen or that are capable of being reactive to a tumor antigen, such as following an ex vivo co-culture with an autologous source of tumor antigen. For example, typically the first population of T cells is from a biological sample from a tumor or from a subject known or likely to have a tumor. In particular embodiments, the first population of T cells is further stimulated with one or more T cell stimulatory agent(s) (e.g. one or more recombinant cytokines, such as IL-2) to produce a second or stimulated population of T cells containing T cells that have expanded following the stimulation.

[0250] In some embodiments, the provided methods involve obtaining cells from the tumor fragments, such as by enzymatic digestion of tumor fragments to obtain TILs. Enzymatic digestion can be carried out using a collagenase, such as a type IV collagenase or a type I / II collagenase. The enzyme, such as a collagenase, can be present in media for the enzymatic digestion at a concentration of from at or about 1 mg / mL to at or about 5 mg / mL, such as at or about 1 mg / mL, at or about 2 mg / mL, at or about 3 mg / mL, at or about 4 mg / mL or at or about 5 mg / mL, or any value between any of the foregoing. In some embodiments, the enzymatic digestion is with a media that includes type IV collagenase, such as from at or about 1 mg / mL to at or about 5 mg / mL. In some embodiments, the enzymatic digestion is with a media that includes type I / II collagenase, such as from at or about 1 mg / mL to at or about 5 mg / mL. In other embodiments, enzymes from the Miltenyi human tumor dissociation kit can be used (e.g. Cat. O. 130-095-929; Miltenyi Biotec). The enzymatic media containing the enzyme can be a serum-free media, such as any as described. In particular embodiments, enzymatic media includes collagenase, e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate (e.g. GlutaMAX), 10 mg / mL gentamicin, 30 units / mL of DNase and 1.0 mg / mL of collagenase). In some embodiments, enzymatic media includes a serum free media (e.g. OpTmizer) containing 2 mM glutamate (e.g. GlutaMAX), 10 pg / mL gentamicin, an immune cell serum replacement (e.g. CTS Immune Cell Serum Replacement) and 1.0 mg / mL to 5.0 mg / mL of collagenase). In some embodiments, the collagenase is a type IV collagenase. In some embodiments, the collagenase is a type I / II collagenase.

[0251] The tumor fragment is then mechanically dissected to dissociate the TILs, e.g., using a tissue dissociator. An example of a tissue dissociator is GentleMACs™ (Miltenyi Biotec) to homogenize the tissue. Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37 °C in 5% CO2, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL canbe performed to remove these cells. In some cases, separation can be achieved by centrifugation, in which case the cell pellet can be resuspended and strained through a e.g. 70 pm strainer to remove debris. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 Al, 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 obtaining TILs for use in the provided methods.

[0252] In particular embodiments, a T cell population is one that includes both CD4+ and CD8+ T cells. Many cancers, including solid tumors, such as many common epithelial indications (e.g. GI), express class I and class II restricted mutations. In order for a T cell product to target such indications, e.g. common epithelial indications, it is contemplated that both CD8+ T cells to recognize class I MHC- restricted molecules and CD4+ T cells to recognize Class II MHC -restricted molecules are necessary.

[0253] In some aspects, the subject is a human. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. In some embodiments, the sample is autologous to a subject to be treated, such as a subject who is a patient in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or expanded in accord with the provided methods. In some embodiments, the sample is allogenic to a subject to be treated.

[0254] In some embodiments, digested cells from the tumor fragments are placed into culture media under conditions and with appropriate nutrients to mediate T cell activation and / or sustain T cell expansion, such as any of the conditions described below for stimulation and pre-expansion of T cells. The cells are seeded at a particular density suitable for the particular culture vessel. The culture vessel can be a microwell, flask, tube, bag or other closed system device. In some embodiments the culture vessel is a closed container that provides a gas-permeable surface area, such as a gas permeable flask. An exemplary culture vessel that provides a gas-permeable surface area include G-Rex plates or flasks. For a G-Rex 6M (single well) or 10M vessel, it is ideal to inoculate with between IxlO7and 4xl07total cells. The particular culture vessel can be chosen based on the number of cells available and / or the desired yield of cells. The choice of culture vessel (e.g. G-Rex) can be chosen by linearly scaling the number of cells seeded to the surface area of the culture vessel. In some embodiments, the surface area of a culture vessel is about 100 cm2(e.g. G-Rex 100 M / 100M-CS)!. In some embodiments, the surface area of a culture vessel is about 500 cm2(e.g. G-Rex 500 M / 500M-CS).

[0255] In some of any of the provided embodiments, the biological sample is a tumor sourced sample, and wherein: the number of cells at the initiation of the culturing is between at or about 10 x 106and 100 x 106total viable cells, 20 x 106and 100 x 106total viable cells, or 12 x 106and 43 x 106total viable cells; or is at or about 10 x 106total viable cells, at or about 12 x 106total viable cells, 20 x 106total viable cells, 40 x 106total viable cells, 60 x 106total viable cells, or 100 x 106total viable cells, , or any value between any of the foregoing.

[0256] In some embodiments, IL-2 is added to the culture media for expansion. In some embodiments, the culture media is a complete media. In some embodiments, the culture media is a serum free media. In some embodiments, the culture media is a serum-free media containing recombinant IL-2.

[0257] In some embodiments, recombinant IL-2 is present in the cell culture medium. IL-2 is a cytokine that supports T cell recovery and proliferation. IL-2 also supports the homeostasis of T cells, thereby supporting their phenotype, differentiation status, and immune memory. In some cases, induction of regulatory T cells in the tumor microenvironment may lead to low bioavailability of IL-2. Recombinant IL-2 has been regularly used in broad expansion of T cells in various contexts. Recombinant IL-2 is commercially available. In particular embodiments, recombinant IL-2 is GMP grade (e.g. MACS GMP Recombinant Human IL-2, Miltenyi Biotec).

[0258] In some embodiments, the recombinant IL-2 is added to the culture medium at a concentration between at or about 1000 lU / mL at or about 8000 lU / mL, such as between at or about 1000 lU / mL and at or about 7000 lU / mL, between at or about 1000 lU / mL and at or about 6000 lU / mL, between at or about 1000 lU / mL and at or about 5000 lU / mL, between at or about 1000 lU / mL and at or about 4000 lU / mL, between at or about 1000 lU / mL and at or about 2000 lU / mL, 2000 lU / mL at or about 8000 IIJ / mL, between at or about 2000 lU / mL and at or about 7000 lU / mL, between at or about 2000 lU / mL and at or about 6000 lU / mL, between at or about 2000 lU / mL and at or about 5000 IIJ / mL, between at or about 2000 lU / mL and at or about 4000 lU / mL, 4000 lU / mL at or about 8000 IIJ / mL, between at or about 4000 lU / mL and at or about 7000 lU / mL, between at or about 4000 lU / mL and at or about 6000 IIJ / mL, between at or about 4000 lU / mL and at or about 5000 lU / mL, between at or about 5000 lU / mL at or about 8000 lU / mL, between at or about 5000 lU / mL and at or about 7000 IIJ / mL, between at or about 5000 lU / mL and at or about 6000 lU / mL, between at or about 6000 lU / mL at or about 8000 IIJ / mL, between at or about 6000 lU / mL and at or about 7000 lU / mL or between at or about 7000 lU / mL and at or about 8000 lU / mL. In some embodiments, recombinant IL-2 is present in an amount that is or is about 6000 lU / mL. In some embodiments, recombinant IL-2 is present in an amount that is or is about 3000 lU / mL.

[0259] In some embodiments, the incubation with the T cell stimulatory agent(s) is carried out under conditions for initial expansion of T cells from the biological sample. In some embodiments, the cells are cultured at about 37 °C with about 5% CO2.

[0260] In some embodiments, the incubation with the T cell stimulatory agent(s) is carried out for 7 to 28 days, such as 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or any value between any of the foregoing. In some embodiments, the incubation is carried out for 7-28 days. In some embodiments, the incubation is carried out for 7-14 days.

[0261] In some embodiments, the first population of T cells are cultured with recombinant IL-2 added at a concentration between 3000 lU / mL and 6000 lU / mL, inclusive, for 14 to 28 days to produce asecond population of T cells. In some embodiments, the first population of T cells are cultured with recombinant IL-2 added at a concentration of about 3000 lU / mL for 14 to 28 days to produce a second population of T cells. In some embodiments, the first population of T cells are cultured with recombinant IL-2 added at a concentration of about 6000 lU / mL for 14 to 28 days to produce a second population of T cells. In some embodiments, the first population of T cells are cultured for about 14 days. In some embodiments, the first population of T cells are cultured for about 15 days. In some embodiments, the first population of T cells are cultured for about 16 days. In some embodiments, the first population of T cells are cultured for about 17 days. In some embodiments, the first population of T cells are cultured for about 18 days. In some embodiments, the first population of T cells are cultured for about 19 days. In some embodiments, the first population of T cells are cultured for about 20 days. In some embodiments, the first population of T cells are cultured for about 21 days. In some embodiments, the first population of T cells are cultured for about 22 days. In some embodiments, the first population of T cells are cultured for about 23 days. In some embodiments, the first population of T cells are cultured for about 24 days. In some embodiments, the first population of T cells are cultured for about 25 days. In some embodiments, the first population of T cells are cultured for about 26 days. In some embodiments, the first population of T cells are cultured for about 27 days. In some embodiments, the first population of T cells are cultured for about 28 days.

[0262] In some cases, media can be exchanged daily, every other day, every third day, every 5thday or once a week during the time of the culture or incubation. In some embodiments, the recombinant IL-2 replenished (added) at each media exchange. In some embodiments, about 3000 lU / mL recombinant IL- 2 is replenished (added) at each media exchange. In some embodiments, about 6000 lU / mL recombinant IL-2 is replenished (added) at each media exchange.

[0263] The incubation, such as for initial expansion of T cells in the biological sample, can be carried out under GMP conditions. In some embodiments, the incubation is in a closed system, which in some aspects may be a closed automated system. In some embodiments, the culture media containing the T cell stimulatory agent(s) can be a serum-free media. In some embodiments, the incubation is carried out in a closed automated system and with serum-free media.

[0264] In some embodiments, the initial expansion of cells under the one or more stimulatory conditions is in a culture vessel suitable for cell expansion. In some embodiments, the culture vessel is a gas permeable culture vessel, such as a G-Rex system (e.g. G-Rex 10, G-Rex 10M, G-Rex 100 M / 100M- CS or G-Rex 500 M / 500M-CS). In some embodiments the culture vessel is a microplate, flask, bar or other culture vessel suitable for expansion of cells in a closed system. In some embodiments, expansion can be carried out in a bioreactor. In some embodiments, the initial expansion can be carried out using a cell expansion system by transfer of the cells to gas permeable bags, such as in connection with a bioreactor (e.g. Xuri Cell Expansion System W25 (GE Healthcare)). In an embodiment, the cell expansion system includes a culture vessel, such as a bag, e.g. gas permeable cell bag, with a volume thatis about 50 mL, 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, or any value between any of the foregoing.In some embodiments, the process is automated or semi-automated. Examples of suitable bioreactors for the automated perfusion expansion include, but are not limited to, GE Xuri W25, GE Xuri W5, Sartorius BioSTAT RM 20 | 50, Finesse SmartRocker Bioreactor Systems, and Pall XRS Bioreactor Systems, or Miltenyi Prodigy. In some aspects, the expansion culture is carried out under static conditions. In some embodiments, the expansion culture is carried out under rocking conditions. The medium can be added in bolus or can be added on a perfusion schedule. In some embodiments, the bioreactor maintains the temperature at or near 37°C and CO2 levels at or near 5% with a steady air flow at, at about, or at least 0.01 L / min, 0.05 L / min, 0.1 L / min, 0.2 L / min, 0.3 L / min, 0.4 L / min, 0.5 L / min, 1.0 L / min, 1.5 L / min, or 2.0 L / min or greater than 2.0 L / min. In certain embodiments, at least a portion of the culturing is performed with perfusion, such as with a rate of 290 ml / day, 580 ml / day, and / or 1160 ml / day.

[0265] In some embodiments, the cells are seeded in an appropriate culture vessel (e.g. gas permeable bag) at a density of from 0.5 x 106cells / mL to 1.5 x 106cells / mL. In some embodiments, the density is at or about 0.5 x 106cells / mL, 0.75 x 106cells / mL, 1 x 106cells / mL, 1.25 x 106cells / mL or 1.5 x 106cells / mL, or any value between any of the foregoing.

[0266] In some aspects, cells are expanded in an automated closed expansion system that is perfusion enabled. Perfusions can continuously add media to the cells to ensure an optimal growth rate is achieved.

[0267] In some embodiments, the stimulated cells are collected and are cryofrozen. The provision of an intermediate hold step by cryopreservation after the initial expansion phase can be used to coordinate timing with the neoepitope identification and peptide generation, such as described in Section LA and / or the generation of APCs as described in Section I.C. In some embodiments, for cryopreservation, the stimulated cells are formulated as a composition with a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or s glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, such as ultra low temperatures, for example, storage with temperature ranges from -40 °C to -150 °C, such as or about 80 °C ± 6.0 ° C.

[0268] In some embodiments, the cryopreserved cells are prepared for subsequent steps by thawing. In some cases, the cells can be ready for subsequent culturing with APCs and peptides immediately after thawing following one or more wash steps.

[0269] The expansion methods can be carried out under GMP conditions, including in a closed automated system and using serum free medium. In some embodiments, any one or more of the steps of the method can be carried out in a closed system or under GMP conditions. In certain embodiments, all process operations are performed in a GMP suite. In some embodiments, a closed system is used forcarrying out one or more of the other processing steps of a method for manufacturing, generating or producing a cell therapy. In some embodiments, one or more or all of the processing steps, e.g., isolation, selection and / or enrichment, processing, culturing steps including incubation in connection with expansion of the cells, and formulation steps is carried out using a system, device, or apparatus in an integrated or self-contained system, and / or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and / or adjust various aspects of the processing, isolation, engineering, and formulation steps. c. Co- Culture with APCs

[0270] In embodiments of the provided methods, once the neopitopes that encode for proteins are synthesized to a plurality of the synthetic peptides, a pool of synthetic peptides are contacted with antigen presenting cells under conditions to present peptides in the context of an MHC molecule. Antigen presenting cells are used to present these peptides. T cells that recognize these peptides on the surface of the APC can then be isolated, such as by methods described below. The loaded APCs (presenting peptides) are incubated with T cells from the first population of pre-expansion T cells above for recognition of the peptides presented on the APCs.

[0271] In some embodiments, of the provided methods the method can include co-culturing the T cells with the APCs over the course of several hours to days and then separating antigen presenting cells from the population of T cells for the expansion of the T cells under conditions to enrich or expand tumor-reactive T cells. In some embodiments, the separating can include isolating or selecting reactive T cells from culture based on one or more T cell activation markers on T cells, such as CD134 and / or CD137.

[0272] Various methods can be used for culturing cells for antigen-specificity, see e.g. US published application No. US2017 / 0224800.

[0273] In some embodiments, the tumor reactive T cells are co-cultured with APCs that have been contacted or exposed to present a peptide, e.g. containing a mutated amino acid sequence, such as neoepitope peptides as described above. The method may comprise inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequence. The APCs may include any cells which present peptide fragments of proteins in association with major histocompatibility complex (MHC) molecules on their cell surface. The MHC molecule can be any MHC molecule expressed by the patient including, but not limited to, MHC Class I, MHC Class II, HLA-A, HLA-B, HLA-C, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR molecules. The APCs may include, for example, any one or more of macrophages, DCs, Langerhans cells, B -lymphocytes, and T- cells. In particular embodiments, the APCs are DCs. In some particular embodiments, the APCs are B cells. In some embodiments, the APCs are artificial APCs.

[0274] In particular embodiments, the APCs include cells that are able to present Class I and Class II restricted molecules. For example, B cells and DCs both have the ability to present MHC class I and MHC class II restricted molecules. In some embodiments, the APC cell sample includes B cells and DCs. In some embodiments, the APC cell sample is enriched for B cells, such as by selection or isolation from a primary cell sample. In some embodiments, the APC cell sample is enriched for DCs, such as by selection or isolation from a primary cell sample.

[0275] In some embodiments, the APCs express MHC class I and / or MHC class II molecules with a matched HL A from which the source of T cells has been obtained. In particular embodiments, both the APCs and T cells have been isolated from the same subject, i.e. are autologous to the cancer patient. In some embodiments, the method may comprise inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequence. By using autologous APCs from the patient, the methods may identify T cells that have antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation that is presented in the context of an MHC molecule expressed by the patient.

[0276] In some embodiments, the APCs are cells from a blood or apheresis sample from a subject, such as the patient. In some embodiments, the APCs include cells present in a peripheral blood mononuclear cell (PBMC) sample. Typically, APCs function in a PBMC culture primarily involves monocytes and B cells. In some embodiments, a population of isolated PBMCs can be used as APCs in the provided methods. PBMCs can be obtained using standard methods such as Ficoll-Paque gradient separation. In some cases, the APCs are or include B cells that are isolated from the blood or apheresis sample or from a PBMC sample. In other cases, the APCs are or include monocytes isolated from the blood or apheresis sample or from a PBMC sample. In some aspects, the monocytes can be used as a source for preparing monocyte-derived DCs for use as APCs. In some embodiments, a source of monocyte-derived DCs (e.g. CDllchlghMHCIIhlghCD14lowcells) can be generated ex vivo from isolated monocytes, by culture with GM-CSF and IL-4 for 4 to 6 days to produce monocyte-derived dendritic cells. In particular embodiments, the monocytes are isolated from PBMCs such as by CD14 selection, and then are cultured with GM-CSF and IL-4 for 4 to 6 days.

[0277] In some embodiments, the APCs are primary cells (e.g. B cells or monocyte-derived DCs) that are replication competent, for example, the cells are not subjected to irradiation, heat treatment or other method that would result in their inactivation. In particular embodiments, the provided methods do not use irradiated APCs. In some embodiments, the APCs are freshly isolated primary cells obtained from the subject, or are derived from primary cells obtained from the subject. In some embodiments, the APCs have been cryopreserved and subsequently thawed prior to the co-culture with the stimulated T cells in accord with provided methods.

[0278] In some particular embodiments, B cells are used as a source of APCs and are generated from a patient apheresis, such as an apheresis autologous to the subject from which the tumor fragmentsand / or T cells were obtained. In other particular embodiments, monocyte-derived dendritic cells are used as a source of APCs and are generated from monocytes from a patient apheresis, such as an apheresis autologous to the subject from which the tumor fragment and / or T cells are obtained.

[0279] In some embodiments, the isolated or generated APCs are collected and are cryofrozen. The provision of an intermediate hold step by cryopreservation after the isolation or generation of APCs can be used to coordinate timing with the neoepitope identification and peptide generation such as described in Section LA and / or initial expansion of T cells, such as described in Section I.B. In some embodiments, for cryopreservation, the isolated or generated APCs are formulated as a composition with a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or s glycerol. In some embodiments, compositions formulated for cry opreservation can be stored at low temperatures, such as ultra low temperatures, for example, storage with temperature ranges from -40 °C to -150 °C, such as or about 80 °C ± 6.0 ° C.

[0280] In some embodiments, the cryopreserved cells are prepared for subsequent steps by thawing. In some cases, the cells can be ready for subsequent culturing with T cells and peptides immediately after thawing following one or more wash steps.

[0281] In some embodiments, the APCs (e.g. PBMCs, B cells or monocyte-derived DCs) are contacted with a pool of peptides. The pool of peptide can represent many different mutated amino acid sequences, such as 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 100 peptides, or any value between any of the foregoing.

[0282] The peptides or pool of peptides are loaded onto antigen presenting cells (e.g. dendritic cells or B cells), such as by peptide pulsing, at a concentrations suitable for their presentation on the surface of a major histocompatibility complex (MHC).

[0283] in some embodiments, the peptide concentration representing an individual or single peptide in the pool of peptide is in a range between at or about 0.1 pg / mL and at or about 1 pg / mL, or at or about 1 pg / mL and at or about 10 pg / mL. In some embodiments, the concentration representing an individual or single peptide can be at or about 0. 1 pg / mL, at or about 0. 25 pg / mL, at or about 0.5 pg / mL, at or about 1 pg / mL, at or about 2.5 pg / mL, at or about 5 pg / mL, at or about 10 pg / mL, or any value between any of the foregoing.

[0284] n an embodiment, inducing APCs (e.g. B cells or monocyte-derived DCs) to present the mutated amino acid sequence comprises introducing a nucleotide sequence encoding the mutated amino acid sequence into the APCs. The nucleotide sequence is introduced into the APCs so that the APCs express and display the mutated amino acid sequence, bound to an MHC molecule, on the cell membrane. The nucleotide sequence encoding the mutated amino acid may be RNA or DNA. Introducing a nucleotide sequence into APCs may be carried out in any of a variety of different ways. Non-limiting examples of techniques that are useful for introducing a nucleotide sequence into APCs include transformation, transduction, transfection, and electroporation. In some cases, peptides for binding MHCclass II restricted molecules are presented as a gene encoding DNA of the mutation and electroporated into the antigen presenting cell. This DNA will then be in-vitro transcribed into RNA encoding peptides on the surface for recognition by CD4+ cells. In some cases, Tandem Mini Gene methods can be employed to do this for MHC class II restricted molecules, see e.g. published PCT Patent Application Number WO2016 / 053338 and Parkhurst et al. (2016) Clin Cancer Res., 23:2491-505. In an embodiment in which more than one gene is identified, the method may comprise preparing more than one nucleotide sequence, each encoding a mutated amino acid sequence encoded by a different gene, and introducing each nucleotide sequence into a different population of APCs. In this regard, multiple populations of APCs, each population expressing and displaying a different mutated amino acid sequence, may be obtained. For example, in the case where tandem minigenes are used, APCs (e.g. B cells or monocyte- derived DCs) are electroporated with a mixture of DNA (plurality of DNA) encoding a different mutated amino acid sequences, which will then be in-vitro transcribed into RNA encoding peptides for surface recognition by CD4+ T cells. In some embodiments, APCs (e.g. B cells or monocyte-derived DCs) are electroporated using the Lonza 4D Nucleofector continuous electroporation system.

[0285] The methods include adding T cells (e.g. from patient having a tumor) with the culture of APCs presenting the peptides and co-culturing the APCs and T cells for a period of time to allow presentation and recognition of the peptide on the surface of APCs by one or more T cells in the population. In provided embodiments, the T cells include a population of the stimulated T cells.

[0286] In some embodiments, for APCs are first contacted or incubated with peptides to prepare peptide-presenting APCs (also referred to as “loading” or “pulsing” with peptides). In some embodiments, APCs (e.g. B cells or monocyte-derived DCs) are incubated with peptides for between at or about 2 hours and at or about 48 hours, such as between at or about 2 hours and at or about 36 hours, between at or about 2 hours and at or about 24 hours, between at or about 2 hours and at or about 24 hours, between at or about 2 hours and at or about 18 hours, between at or about 2 hours and at or about 12 hours, between at or about 2 hours and at or about 6 hours, between at or about 6 hours and at or about 48 hours, between at or about 6 hours and at or about 36 hours, between at or about 6 hours and at or about 24 hours, between at or about 6 hours and at or about 24 hours, between at or about 6 hours and at or about 18 hours, between at or about 6 hours and at or about 12 hours, between at or about 12 hours and at or about 48 hours, between at or about 12 hours and at or about 36 hours, between at or about 12 hours and at or about 24 hours, between at or about 12 hours and at or about 18 hours, between at or about 18 hours and at or about 48 hours, between at or about 18 hours and at or about 36 hours, between at or about 18 hours and at or about 24 hours, between at or about 24 hours and at or about 48 hours, between at or about 24 hours and at or about 36 hours, or between at or about 36 hours and at or about 48 hours. In some embodiments, the APCs (e.g. B cells or monocyte-derived DCs) are incubated with peptides for at or about 4 hours, at or about 6 hours, at or about 7 hours, at or about 8 hours, at or about 9 hours, at or about 10 hours, at or about 12 hours, at or about 14 hours, at or about 16 hours, at or about 18hours, at or about 20 hours, at or about 22 hours, at or about 24 hours, or any value between any of the foregoing. In some embodiments, the APCs (e.g. PBMCs, B cells or monocyte -derived DCs) are incubated with peptides overnight, such as for between at or about 8 to 12 hours. In some embodiments, the co-culture incubation is for at or about 6 hours.

[0287] In some embodiments, the T cells (e.g. stimulated T cells) and APCs (e.g. B cells or monocyte-derived DCs) can be present in a culture at a ratio of T cells to APC of 1:100 to 100:1, such as 1:50 to 50:1, 1:25 to 25:1, 1:10 to 10:1, or 1:5 to 5:1. In some embodiments, the ratio of T cells (e.g. stimulated T cells) to APC is at or about 1:100, at or about 1:50, at or about 1:25, at or about 1:10, at or about 1:5, at or about 1:2.5, at or about 1:1, at or about 2:5:1, at or about 5:1, at or about 10:1, at or about 25:1, at or about 50:1 or at or about 100:1, or any value between any of the foregoing. In some embodiments, the ratio of T cells (e.g. stimulated T cells) to APC is between 20:1 and 1:1, between 15:1 and 1:1, between 10:1 and 1:1, between 5:1 and 1:1, or between 2.5:1 and 1:1. In some embodiments, the ratio of T cells (e.g. stimulated T cells) to APC is between 1 :20 and 1:1, between 1:15 and 1:1, between 1:10 and 1:1, between 1:5 and 1:1, or between 1:2.5 and 1:1. In particular embodiments, coculture will be performed by mixing the T cells, e.g. population of stimulated T cells, and APC (e.g. B cells or monocyte-derived DC) at approximately a 3:1 ratio. In some embodiments, coculture will be performed by mixing the T cells, e.g. population of stimulated T cells, and APC (e.g. B cells or monocyte-derived DC) at approximately a 1 : 1 ratio.

[0288] In some embodiments, recombinant IL-2 is present in the cell culture medium. In some embodiments, recombinant IL-2 is added at a concentration of 100 lU / mL to 1000 lU / mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration between at or about 10 lU / mL and at or about 600 lU / mL, such as between at or about 10 lU / mL and at or about 400 lU / mL, between at or about 10 lU / mL and at or about 200 lU / mL, between at or about 10 lU / mL and at or about 100 lU / mL, between at or about 10 lU / mL and at or about 50 lU / mL, between at or about 50 lU / mL and at or about 400 lU / mL, between at or about 50 lU / mL and at or about 200 lU / mL, between at or about 50 lU / mL and at or about 100 lU / mL, between at or about 100 lU / mL and at or about 400 lU / mL, between at or about 100 lU / mL and at or about 200 lU / mL, between at or about 200 lU / mL and at or about 400 HJ / mL, between at or about 400 HJ / mL and at or about 600 lU / m. In some embodiments, recombinant IL-2 is present in an amount that is at or about 300 lU / mL.

[0289] In some embodiments, the co-culture of APCs and T cells can be incubated at a temperature suitable for the presentation of peptides on MHC and the activation of T cells in the culture, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius.

[0290] In some embodiments, the co-culture incubation of peptide -presenting APCs and T cells is carried out for up to 96 hours. The co-culture incubation of peptide -presenting APC can be carried out for 12 hours to 96 hours, such as at or about 12 hours, at or about 16 hours, at or about 20 hours, at or about24 hours, at or about 36 hours, at or about 48 hours, at or about 60 hours, at or about 72 hours, at or about 84 hours or at or about 96 hours, or for a time between any of the foregoing. In some embodiments, the co-culture is incubated for 12 to 48 hours. In particular embodiments, the co-culture is incubated for 20 to 24 hours. In some embodiments, the co-culture is incubated for about 20 hours. In some embodiments, the co-culture is incubated for about 24 hours.

[0291] In some embodiments, the methods include co-culturing the second population of T cells for about 12 to 48 hours with autologous antigen presenting cells (APCs) with recombinant IL-2 added at a concentration of 100 lU / rnL to 1000 lU / mL to produce a third population of T cells, wherein the APCs are loaded with a pool of peptide neoantigens from the tumor, wherein each peptide is 13-40 amino acids in length and is loaded at a concentration of 100 ng / mL per peptide, and wherein the ratio of the second population of T cells to APCs is about 2: 1 to 10: 1. In some embodiments, the recombinant IL-2 is added at about 300 lU / mL. In some embodiments, the co-culture of T cells and APCs is at a ratio of about 5:1 T cells to APCs and is carried out for about 20 hours with IL-2 added at about 300 lU / mL. In some embodiments, the co-culture of T cells and APCs is at a ratio of about 5:1 T cells to APCs and is carried out for about 24 hours with recombinant IL-2 added at about 300 lU / mL. In some embodiments, the pool of peptide neoantigens includes up to 200 peptides, such as at least 100 peptides, 110 peptides, 120 peptides, 130 peptides, 140 peptides, 150 peptides, 160 peptides, 170 peptides, 180 peptides, 190 peptides or 200 peptides, or any value between any of the foregoing. In some embodiments, the pool of peptides includes about 190 peptides.

[0292] In some embodiments, at the end of the co-culturing tumor reactive T cells are separated from APCs present in the co-culture. In some embodiments, the separation can include methods that select away or remove the APCs. In some embodiments, the separation can include methods that positively select or retain the T cells present in the co-culture. In some embodiments, total T cells in the co-culture can be selected. In particular embodiments, tumor reactive T cells or T cells that express one or more upregulation marker, e.g. activation markers, associated with tumor-reactive T cells can be selected. d. Selection for Tumor Reactive T Cells

[0293] In embodiments of the provided methods, prior to the further expansion of T cells from the co-culture, the methods involve enrichment or selection of tumor reactive T cells that are surface positive for CD134 (0X40) and / or CD137 (41BB). The selected cells surface positive for CD134 and / or CD137 are a fourth population of cells in accord with provided methods. The selection of the cells may be through antibody binding for CD134 and / or CD137 markers and subsequent enrichment by flow cytometry, including by methods involving magnetic separation or fluorescence-activated cell sorting (FACS). In some embodiments, T cells selected from the co-culture results in a population of T cells enriched for CD3+ T cells, or CD4+ cells and CD8+ cells, that are positive for one of more of such T cellactivation marker. In some embodiments, such cells include or are enriched for tumor-reactive T cells. For example, such CD3+ T cells, or CD4+and / or CD8+populations, can be further sorted into subpopulations by positive selection for markers expressed or expressed to a relatively higher degree on tumor-reactive T cells. In particular embodiments, the enriched population of cells is cultured under conditions for expansion.

[0294] Methods of isolating, selecting and / or enriching for cells can be by any of a variety of methods, such as by positive or negative selection based, such as by using any methods as described in Section I.B above. In some embodiments, methods can include immunoaffinity-based selections. In some embodiments, the T cells can be enriched or sorted a variety of ways including, but not limited to, magnetic bead separation, fluorescent cell sorting, and disposable closed cartridge based cell sorters. In particular aspects, one or more T cell activation markers can be used to select reactive cells using, but not limited to, florescent antibodies, nanoparticles or beads on cell selection equipment, but not limited to, the CliniMACS, Sony FX500 or the Tyto cell sorting systems (Miltenyi).

[0295] In some embodiments, the T cells can be enriched or sorted a variety of ways including, but not limited to, magnetic bead separation, fluorescent cell sorting, and disposable closed cartridge based cell sorters. In particular aspects, one or more reagents specific to CD134 and / or CD137 can be used including, but not limited to, florescent antibodies, nanoparticles or beads on cell selection equipment, but not limited to, the CliniMACS, Sony FX500 or the Tyto cell sorting systems (Miltenyi).

[0296] In certain embodiments, the sample is contacted with a binding agent, e.g., a detectably labeled binding agent, that specifically binds to the cell surface marker CD134 and / or CD137. In certain embodiments, the detectably labeled binding agent(s) are fluorescently labeled. In certain embodiments, T cells labeled with binding agents specific to the cell surface marker are identified by flow cytometry. In certain embodiments, the method further includes separating any resultant T cells labeled with the binding agent(s) from other components of the sample to produce a composition enriched for T cells surface positive for the one or more cell surface marker. Cell selection sorting equipment can be used that has a sufficiently high-throughput to handle large volumes and cell numbers. Non-limiting cell sorting equipment includes, for example, Sony FX500 or the Tyto cell sorting systems (Miltenyi).

[0297] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead and / or are detectably labeled, specifically bind to cell surface molecules if present on cells within the sample. In some aspects, cells bound to the antibodies can be recovered or separated from non-bound cells in the sample.

[0298] The separation need not result in 100 % enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.

[0299] In some embodiments, the selections produces an enriched population of cells, such as a population of cells enriched for CD3+ T cells or CD4+ cells and CD8+ cells, that are further positive for CD134 and / or CD137. In some embodiments, such cells include or are enriched for tumor-reactive T cells or T cells associated with tumor-reactive T cells. In some embodiments, the enriched population of cells is used in subsequent processing steps, such as subsequent processing steps involving incubation, stimulation or activation, and / or expansion in accord with one or more steps of any of the provided methods.

[0300] In some embodiments, the enriched population of cells are enriched cells from a starting sample as describe above, in which the percentage of cells of a particular phenotype, e.g. tumor-reactive CD3+ T cells or CD3+ T cells surface positive for CD134 and / or CD 137, in the enriched population of cells in increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000% or more greater than the percentage of such cells in the starting sample. In some embodiments, the purity of tumor-reactive CD3+ T cells or CD3+ T cells surface positive for CD134 and / or CD 137 in the enriched composition, i.e. the percentage of cells positive for the selected cell surface marker versus total cells in the population of enriched cells, is at least 90%, 91%, 92%, 93%, 94%, and is generally at least 95%, 96%, 97%, 98%, 99% or greater. e. Further Expansion and Harvesting

[0301] In some embodiments, the selected T cells from the co-culture (e.g. fourth population of cells) are further incubated under conditions to expand the cells ex vivo following the co-culture. The incubation is carried out in the presence of one or more T cell stimulatory agent(s) under conditions for stimulating the T cells, such as to expand the T cells. In some embodiments, the incubation is a rapid expansion protocol (REP).

[0302] In some of any of the provided embodiments, the T cell stimulatory agent(s) is selected from an agent that initiates TCR / CD3 intracellular signaling and / or an agent that initiates signaling via a costimulatory receptor. An anti-CD3 antibody can include any antibody directed against or that can specifically bind the CD3 receptor on the surface of T cells, typically human CD3 on human T cells. Anti-CD3 antibodies include OKT3, also known as muromonab. Anti-CD3 antibodies also include the UHCTI clone, also known as T3 and CD3E. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab. The anti-CD3 antibody can be added as a soluble reagent or bound to a bead. In particular embodiments, the anti-CD3 antibody is soluble. In some of any of the provided embodiments, the agent that initiates TCR / CD3 intracellular signaling is an anti-CD3 antibody, such as OKT3.

[0303] In particular embodiments, the T cell stimulatory agent(s) include an anti-CD3 antibody, which is added to the cell culture medium during the incubation. In some embodiments, the anti-CD3 antibody is added at a concentration ranging between at or about 0.1 ng / mL and 50 ng / mL, such betweenat or about 0.5 ng / mL and at or about 50 ng / mL, between at or about 0.5 ng / mL and at or about 30 ng / mL, between at or about 0.5 ng / mL and at or about 15 ng / mL, between at or about 0.5 ng / mL and at or about 5 ng / mL, between at or about 0.5 ng / mL and at or about 1 ng / mL, between at or about 1 ng / mL and at or about 50 ng / mL, between at or about 1 ng / mL and at or about 30 ng / mL, between at or about 1 ng / mL and at or about 15 ng / mL, between at or about 1 ng / mL and at or about 5 ng / mL, between at or about 5 ng / mL and at or about 50 ng / mL, between at or about 5 ng / mL and at or about 30 ng / mL, between at or about 5 ng / mL and at or about 15 ng / mL, between at or about 15 ng / mL and at or 50 ng / mL, between at or about 15 ng / mL and at or about 30 ng / mL or between at or about 30 ng / mL and at or about 50 ng / mL, each inclusive. In an embodiment, the anti-CD3 is added to the cell culture medium at 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. In some embodiments, the anti-CD3 antibody is added at a concentration of about 30 ng / mL. In particular embodiments, the anti-CD3 antibody is OKT3.

[0304] In some embodiments, the T cell stimulatory agent can include adding the anti-CD3 antibody and additionally include adding to the population of T cells feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC). In some aspects, the PBMCs provide a CD28-mediated signal to provide a costimulatory signal to the T cells. In some aspects, the non-dividing feeder cells can be irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some embodiments, the non-dividing PBMCs, such as irradiated PBMCs, are added at a ratio of PBMCs to T cells that is between 100:1 to 500:1. In some embodiments, the non-dividing PBMCs, such as irradiated PBMCs, are added at a ratio of PBMCs to T cells that is 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1 or 500:1, or any value between any of the foregoing. In some embodiments, the non-dividing PBMCs, such as irradiated PBMCs, are added at a ratio of PBMCs to T cells that is about 200:1.

[0305] In general, the culturing and incubations can occur in the presence of recombinant IL-2. In some embodiments, recombinant IL-2 s added or is exogenous to the culture media. In some embodiments, the recombinant IL-2 is added to the culture medium at a concentration between at or about 1000 lU / mL at or about 8000 HJ / mL, such as between at or about 1000 lU / mL and at or about 7000 HJ / mL, between at or about 1000 IIJ / mL and at or about 6000 lU / mL, between at or about 1000 HJ / mL and at or about 5000 lU / mL, between at or about 1000 IIJ / mL and at or about 4000 lU / mL, between at or about 1000 lU / mL and at or about 2000 lU / mL, 2000 lU / mL at or about 8000 IIJ / mL, between at or about 2000 lU / mL and at or about 7000 lU / mL, between at or about 2000 lU / mL and at or about 6000 lU / mL, between at or about 2000 lU / mL and at or about 5000 IIJ / mL, between at or about 2000 lU / mL and at or about 4000 lU / mL, 4000 lU / mL at or about 8000 lU / mL, between at or about 4000 lU / mL and at or about 7000 lU / mL, between at or about 4000 lU / mL and at or about 6000 lU / mL, between at or about 4000 lU / mL and at or about 5000 lU / mL, between at or about 5000 lU / mL at orabout 8000 lU / mL, between at or about 5000 lU / mL and at or about 7000 lU / mL, between at or about 5000 lU / mL and at or about 6000 lU / mL, between at or about 6000 lU / mL at or about 8000 lU / mL, between at or about 6000 lU / mL and at or about 7000 lU / mL or between at or about 7000 lU / mL and at or about 8000 lU / mL. In some embodiments, recombinant IL-2 is present in an amount that is or is about 6000 lU / mL.

[0306] In some embodiments, the selected cells from the co-culture (e.g. fourth population of cells) are expanded by culture of the T cells with irradiated PBMCs added at a ratio of 200: 1 iPBMC to TIL, with 6000 lU / mL human recombinant IL-2 and 30 ng / mL anti-CD3 antibody (OKT3).

[0307] The sorted or selected T cells can be expanded under the one or more stimulatory conditions in a culture vessel suitable for cell expansion. In some embodiments, the culture vessel is a gas permeable culture vessel, such as a G-Rex system (e.g. G-Rex 10, G-Rex 10M, G-Rex 100 M / 100M-CS or G-Rex 500 M / 500M-CS). In some embodiments the culture vessel is a microplate, flask, bar or other culture vessel suitable for expansion of cells in a closed system. In some embodiments, expansion can be carried out in a bioreactor. In some embodiments the composition of expanded T cells is removed from a closed system and placed in and / or connected to a bioreactor for expansion. The sorted or selected T cells can be expanded using a cell expansion system by transfer to the cell to gas permeable bags, such as in connection with a bioreactor (e.g. Xuri Cell Expansion System W25 (GE Healthcare)). In an embodiment, the cell expansion system includes a culture vessel, such as a bag, e.g. gas permeable cell bag, with a volume that is about 50 mb, 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, or any value between any of the foregoing. In some embodiments, the process is automated or semi-automated. Examples of suitable bioreactors for the automated perfusion expansion include, but are not limited to, GE Xuri W25, GE Xuri W5, Sartorius BioSTAT RM 20 | 50, Finesse SmartRocker Bioreactor Systems, and Pall XRS Bioreactor Systems, or Miltenyi Prodigy. In some aspects, the expansion culture is carried out under static conditions. In some embodiments, the expansion culture is carried out under rocking conditions. The medium can be added in bolus or can be added on a perfusion schedule. In some embodiments, the bioreactor maintains the temperature at or near 37°C and CO2 levels at or near 5% with a steady air flow at, at about, or at least 0.01 L / min, 0.05 L / min, 0.1 L / min, 0.2 L / min, 0.3 L / min, 0.4 L / min, 0.5 L / min, 1.0 L / min, 1.5 L / min, or 2.0 L / min or greater than 2.0 L / min. In certain embodiments, at least a portion of the culturing is performed with perfusion, such as with a rate of 290 ml / day, 580 ml / day, and / or 1160 ml / day.

[0308] In some embodiments, the cells are seeded in an appropriate culture vessel (e.g. gas permeable bag) at a density of from 0.5 x 106cells / mL to 1.5 x 106cells / mL. In some embodiments, the density is at or about 0.5 x 106cells / mL, 0.75 x 106cells / mL, 1 x 106cells / mL, 1.25 x 106cells / mL or 1.5 x 106cells / mL, or any value between any of the foregoing.

[0309] In some aspects, cells are expanded in an automated closed expansion system that is perfusion enabled. Perfusions can continuously add media to the cells to ensure an optimal growth rate is achieved.

[0310] The expansion methods can be carried out under GMP conditions, including in a closed automated system and using serum free medium. In some embodiments, any one or more of the steps of the method can be carried out in a closed system or under GMP conditions.

[0311] some embodiments, the incubation with the T cell stimulatory agent(s) for expansion of tumor-reactive cells is carried out until a threshold number of cells is achieved. In some embodiments, the threshold number of cells is about 100-fold or more greater in number than the number of selected TILs in the fourth population of T cells prior to the expansion. In some embodiments, the threshold number of cell is a number of TILs that is greater than the number of selected TILs in the fourth population prior to the expansion by about 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1250-fold, 1500-fold or 2000-fold, or any value between the foregoing.

[0312] In some embodiments, the incubation with the T cell stimulatory agent(s) for expansion is for 7 to 21 days, such as 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14, days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days, or any value between any of the foregoing. In some embodiments, the incubation is carried out for 7-14 days. In some embodiments, the incubation is for about 10 days. In some embodiments, the incubation is for about 11 days. In some embodiments, the incubation is for about 12 days. In some embodiments, the incubation is for about 13 days. In some embodiments, the incubation is for about 14 days. In some cases, media can be exchanged daily, every other day, every third day, every 5thday or once a week during the time of the culture or incubation. In some embodiments, the media exchange is at Day 5 and Day 10. In some embodiments, the recombinant IL-2 is replenished (added) at each media exchange. In some embodiments, about 6000 lU / mL recombinant IL-2 is replenished (added) at each media exchange.

[0313] In some of any of the provided embodiments, the culturing is carried out until a threshold amount of cells is achieved that is between at or about 0.5 x 108and at or about 50 x 109total cells or total viable cells, between at or about 0.5 x 108and at or about 30 x 109total cells or total viable cells, between 0.5 x 108and at or about 12 x 109total cells or total viable cells, between at or about 0.5 x 108and at or about 60 x 108total cells or total viable cells, between at or about 0.5 x 108and at or about 15 x 108total cells or total viable cells, between at or about 0.5 x 108and at or about 8 x 10stotal cells or total viable cells, between at or about 0.5 x 108and at or about 3.5x 108total cells or total viable cells, between at or about 0.5 x 108and at or about 1 x 108total cells or total viable cells, between 1 x 108and at or about 50 x 109total cells or total viable cells, between at or about 1 x 108and at or about 30 x 109total cells or total viable cells, between 1 x 108and at or about 12 x 109total cells or total viable cells, between at or about 1 x 108and at or about 60 x 108total cells or total viable cells, between at or about 1 x 108and at or about 15 x 108total cells or total viable cells, between at or about 1 x 10sand at or about 8 x 108total cells or total viable cells, between at or about 1 x 108and at or about 3.5x 108total cells or total viable cells, between at or about 3.5 x 108and at or about 50 x 109total cells or total viable cells, between at or about 3.5 x 10sand at or about 30 x 109total cells or total viable cells, between at or about 3.5 x 10sand at or about 12 x 109total cells or total viable cells, between at or about 3.5 x 108and at or about 60 x 108total cells or total viable cells, between at or about 3.5 x 108and at or about 15 x 108total cells or total viable cells, between at or about 3.5 x 108and at or about 8 x 108total cells or total viable cells, between at or about 8 x 108and at or about 50 x 109total cells or total viable cells, between at or about 8 x 108and at or about 30 x 109total cells or total viable cells, between at or about 8 x 108and at or about 12 x 109total cells or total viable cells, between at or about 8 x 108and at or about 60 x 108total cells or total viable cells, between at or about 8 x 108and at or about 15 x 108total cells or total viable cells, between at or about 15 x 108and at or about 50 x 109total cells or total viable cells, between at or about 15 x 108and at or about 30 x 109total cells or total viable cells, between at or about 15 x 108and at or about 12 x 109total cells or total viable cells, between at or about 15 x 108and at or about 60 x 108total cells or total viable cells, between at or about 60 x 108and at or about 50 x 109total cells or total viable cells, between at or about 60 x 108and at or about 30 x 109total cells or total viable cells, between at or about 60 x 108and at or about 12 x 109total cells or total viable cells, between at or about 12 x 109and at or about 50 x 109total cells or total viable cells, between at or about 12 x 109and at or about 30 x 109total cells or total viable cells, or between at or about 30 x 109and at or about 60 x 109total cells or total viable cells, each inclusive.

[0314] In some of any of the provided embodiments, the method results in a fold-expansion of T cells that is at least at or about 100-fold, at least at or about 250-fold, at least at or about 500-fold, at least at or about 1000-fold, or more compared to the number of cells in the fourth population of cells before the expansion.

[0315] Upon reaching a therapeutic dose after expansion the product can be concentrated and frozen in cryopreservation medium. In some embodiments, the cryoprotectant is or comprises DMSO and / or s glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, such as ultra low temperatures, for example, storage with temperature ranges from -40 °C to -150 °C, such as or about 80 °C ± 6.0 ° C.

[0316] Also provided herein are populations of T cells produced by methods described herein and pharmaceutical compositions thereof. The composition of cells are enriched in tumor-reactive T cells. In some embodiments, the composition of cells is characterized by one or more features as described in Section I.C.B. Direct Selected TILs

[0317] In some embodiments, the methods for preparing a therapeutic TIL composition enriched for tumor reactive T cells involves a direct selection of cells to yield a tumor reactive cell population that isfurther expanded. In the provided methods, a TIL tumor sample is obtained that contains or is expected to contain tumor reactive T cells (e.g. first population). In one embodiment, this population can be processed by digestion to create a single cell suspension (e.g. second population). In some embodiments, this second population is sorted to select for cells that are enriched for tumor reactive T cells and to minimize the presence of bystander cells, such as regulatory T cells. In some embodiments, the selection is for cells positive for T cell activation markers, such as two or more of PD-1, CD39 and / or TIGIT. In some embodiments, the selection is for cells positive for PD-1 and CD39. In provided methods, the T cells are sorted directly after tumor digest or after a short period of cell culture for cells surface positive for the two or more markers to create a population of selected T cells. This process removes the nonreactive and inhibitory ‘bystander’ cells, resulting in T cell product enriched in neoantigen reactive T cells. The selection yields a selected or sorted population of cells (e.g. in some cases also called a third population), which then can be expanded to create a therapeutic composition containing an expanded population of tumor specific reactive cells (e.g. in some cases also called a fourth population). In some embodiments, cells are then expanded into clinically relevant numbers of tumor specific T cells. In aspects of the provided methods, the T cells from the selected population (third population), such as present from the sorted T selected cells from a resected tumor fragment or a single cell suspension therefrom, are expanded by culture in the presence of one or more T cell stimulatory agent(s) under conditions for stimulating the T cells. In some embodiments, the final expanded therapeutic composition is formulated with a cryoprotectant for cryopreservation.

[0318] In particular embodiments, the provided methods include enriching from a biological sample (directly sourced from a sample in vivo or from an ex vivo coculture with antigen presenting cells (APCs)) T cells that have an endogenous TCR that recognize tumor-associated antigens, e.g. neoantigens, such as by selecting for T cells that are surface positive for one or more T cell activation marker (e.g. PD-1, TIGIT, CD107, CD107a, CD39, CD134, CD137, CD59, CD69, CD90, CD38, or CD103). In some embodiments, the selection is for cells positive for two or more of such T cell activation markers.

[0319] In some embodiments, the TILs are produced by a method that includes the steps of (1) selecting, from a population of cells containing T lymphocytes obtained from a donor subject, cells positive for an exhaustion marker from among PD-1, CD39 and / or TIGIT; and (2) stimulating the population by incubation or culture of selected cells with one or more T-cell stimulating agents of lymphocytes to produce a population of expanded T cells. In some embodiments, the methods for selection and / or stimulation are performed in a closed system. In some embodiments, only a single expansion step is carried out in the method. In some embodiments, an initial expansion (e.g. first expansion) is carried out prior to selecting the cells. In some embodiments, the provided methods further can include a secondary stimulation to further expand cells in which the further stimulation is by incubation or culture with one or more T-cell stimulating agents. In some embodiments, one or more ofthe steps is carried out in a closed system. In some embodiments, all of the steps is carried out in a closed system. In some embodiments, the T cell stimulating agent can include any one or more recombinant cytokines IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27 or IL-25, such as generally at least IL-2 or IL-15. In some embodiments, the T cell stimulating agent can further include an anti-CD3 antibody (e.g. 0KT3). In some embodiments, the T cell stimulating agents include an anti-CD3 antibody (0KT3) and / or a recombinant cytokine such as IL-2, IL-7, IL- 15, IL-21, IL-25, IL-23. / . Sample Containing C ceils

[0320] The provided methods include selecting or obtaining an input sample of T cells from a biological sample, which can be used as the source or input of T cells for stimulation with one or more T cell stimulatory agents(s) (e.g. recombinant IL-2 or other T cell stimulating cytokines and / or anti-CD3). In some embodiments, the T cells are from a biological sample from a subject that is known or likely to contain tumor reactive T cells. In some embodiments, the biological sample provides an input population of cells that is a single cell suspension (SCS), which can be used for subsequent selection of TILs for expansion as described in Section II.B.2 and II.B.3. In some embodiments, the biological sample is processed to provide an input population of cells that is a single cell suspension, which can be used for subsequent selection of TILs for expansion as described in Section II.B.2 and II.B.3. For instance, particular methods include processing tumor fragments by dissociation using homogenization and / or enzymatic methods and filtering the dissociated cells to prepare a single cell suspension as an input population of cells.

[0321] In aspects of any of the provided embodiments, a suitable biological sample from a subject, such as from a patient of interest, i.e., a patient suspected of having or known to have cancer, is obtained. In some embodiments, the sample is one that is known or suspected of containing T cells, such as T cells that may be or may likely express an endogenous T cell receptor (TCR). The biological sample may be derived from any initial source that would contain or is suspected of containing such T cells. In some aspects, biological sample sources of interest include, but are not limited to, many different physiological sources, e.g. tissue derived samples, e.g. homogenates, and blood or derivatives thereof.

[0322] Any of a variety of biological samples can be used as a source of potentially reactive T cells. Although the tumor and downstream lymph nodes may have the highest frequency of reactive T cells (Powell et al., Clin. Cancer. Res., 2014), other sample sources also can be used. In some cases the sample is a tumor sample, a tertiary lymphoid site, a draining lymph node, peripheral blood or bone marrow. In some embodiments, the biological sample is a tumor sample. In some embodiments, the biological sample is a lymph sample. In some embodiments, the biological sample is a peripheral blood sample.

[0323] The biological samples include tissue, fluid, and other samples taken directly from the subject to obtain an input sample, or can undergo one or more processing steps, such as separation, e.g.selection or enrichment, centrifugation, washing, and / or incubation, to obtain or produce an input sample. The input sample containing T cells can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, and tumor samples, including processed samples derived therefrom.

[0324] In some aspects, the sample is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0325] In many embodiments, the sample may be derived from fluids in which the T cells of interest are at least suspected of being present. In many embodiments, a suitable initial source for the sample is blood. In some embodiments, the biological sample is a blood-derived sample. The blood-derived sample may be derived from whole blood or a fraction thereof, e.g. serum, plasma, etc., where in many embodiments the sample is derived from blood cells harvested from whole blood. In some aspects, the sample source contains mononuclear cells. For example, a biological sample is or contains peripheral blood mononuclear cells (PBMCs) or is derived from PBMCs.

[0326] In some embodiments in which the sample is a PBMC derived sample, the sample is generally a fluid PBMC derived sample. Any convenient methodology for producing a fluid PBMC sample may be employed. In many embodiments, the fluid PBMC derived sample is prepared by separating PBMCs from whole blood, i.e., collecting PBMCs, e.g., by centrifugation (such as by Ficoll- Hypaque density gradient centrifugation, where representative protocols for such separation procedures are disclosed in WO 98 / 15646 and U.S. Pat. No. 5,985,565).

[0327] In some embodiments, the sample is a tumor sample and thereby provides a source of tumorinfiltrating lymphocytes (TILs). In some aspects, TILs are T cells that have left the bloodstream of a subject and migrated into or infiltrated a tumor. In particular aspects, TILs are reactive to a tumor antigen.

[0328] A patient tumor sample may be obtained by any of a variety of methods in which the method obtains a sample that contains a mixture of tumor and TIL cells. In some embodiments, the tumor sample is obtained by surgical resection. In some embodiments, the tumor sample is obtained by needle biopsy. In general, 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.

[0329] In some embodiments, the solid tumor may be of any cancer type, including, but not limited to, ovarian, vulva, endometrial, urothelial, breast, pancreatic, prostate, colorectal, lung, brain, renal, stomach (gastrointestinal), and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the tumor is from a patient with a cancer, including, but not limited to, ovarian, vulva, endometrial, urothelial, breast, colorectal, lung, renal, and skin (including but not limited to melanoma). In some embodiments, the tumor is from a patient with an ovarian cancer. In some embodiments, the tumor is from a patient with cancer of the vulvar. In some embodiments, the tumor is from a patient with an endometrial cancer. In some embodiments, the tumor is from a patient with a urothelial cancer. In some embodiments, the tumor is from a patient with a breast cancer. In some embodiments, the tumor is from a patient with a colorectal cancer. In some embodiments, the tumor is from a patient with a lung cancer. In some embodiments, the tumor is from a patient with a renal cancer. In some embodiments, the tumor is from a patient with melanoma. In particular embodiments, the tumor is from a patient to be treated.

[0330] In particular embodiments, a T cell population is one that includes both CD4+ and CD8+ T cells. Many cancers, including solid tumors, such as many common epithelial indications (e.g. GI), express class I and class II restricted mutations. In order for a T cell product to target such indications, e.g. common epithelial indications, it is contemplated that both CD8+ T cells to recognize class I MHC- restricted molecules and CD4+ T cells to recognize Class II MHC -restricted molecules are necessary.

[0331] The sample may be obtained from a variety of different subjects / patients / hosts. Generally such hosts are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class Mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans.

[0332] In some aspects, the subject is a human. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. In some embodiments, the sample is autologous to a subject to be treated, such as a subject who is a patient in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or expanded in accord with the provided methods. In some embodiments, the sample is allogenic to a subject to be treated.

[0333] In provided embodiments, the obtained tumor sample is fragmented into small pieces of between at or about 1 mm3and at or about 8 mm3in size, such as between at or about 1 mm3and at or about 3 mm3, between at or about 1 mm3and at or about 4 mm3, between at or about 1 mm3and at or about 2 mm3. In some embodiments, the tumor fragment is from about -3 mm3. In some embodiments, the tumor fragment is from about 1-3 mm3. In some embodiments, the tumor fragment is obtained by physical fragmentation, such as by dissection. In some embodiments, the tumor fragment is obtained by sharp dissection.

[0334] In some of any of the provided embodiments, the obtained tumor sample is fragmented into small pieces of between at or about 1 mm and at or about 8 mm in diameter, such as between at or about 1 mm and at or about 6 mm in diameter, between at or about 1 mm and at or about 4 mm in diameter, between at or about 1 mm and at or about 2 mm in diameter. In some embodiments, the tumor fragment is from about 3 mm in diameter. In some embodiments, the tumor fragment is from about 1-2 mm in diameter. In some embodiments, the tumor fragment is obtained by physical fragmentation, such as by dissection. In some embodiments, the tumor fragment is obtained by sharp dissection.

[0335] In some embodiments, the tumor sample is cryopreserved prior to fragmentation. In some embodiments, the tumor fragments are cryopreserved.

[0336] In some embodiments, tumor fragments are used as a source to prepare a single cell suspension for use as an input sample of T cells in the provided methods. In some embodiments, the provided methods involve obtaining cells from the tumor fragments, such as by enzymatic digestion of tumor fragments to obtain TILs. Enzymatic digestion can be carried out, in part, using a collagenase, such as a type IV collagenase or a type I / II collagenase. Collagenase is an enzyme that degrades the collagen network embedded in the extracellular matrix of the cell (Eikenes et al. Anticancer Research, 2010). The enzyme, such as a collagenase, can be present in media for the enzymatic digestion at a concentration of from at or about 1 mg / mL to at or about 5 mg / mL, such as at or about 1 mg / mL, at or about 2 mg / mL, at or about 3 mg / mL, at or about 4 mg / mL at or about 5 mg / mL, at or about 6 mg / mL, at or about 7 mg / mL, at or about 8 mg / mL or at or about 9 mg / mL, at or about 10 mg / mL or any value between any of the foregoing. In some embodiments, collagenase is present in the media at a concentration of from about 5 mg / mL to about 10 mg / mL. In some embodiments, the concentration is about 5 mg / mL. In some embodiments, the concentration is 10 mg / mL. In some embodiments, the collagenase is a type IV collagenase. In some embodiments, the collagenase is a type I / II collagenase. In some embodiments, the enzymatic digestion is with a media that includes type IV collagenase, such as from at or about 5 mg / mL to at or about 10 mg / mL. In some embodiments, the enzymatic digestion is with a media that includes type I / II collagenase, such as from at or about 5 mg / mL to at or about 10 mg / mL. In some embodiments, if a more gentle digestion is desired at or about 5 mg / mL collagenase is used. In some embodiments, if a more complete digestion is desired a higher concentration of collagenase is used, such as at or abut 10 mg / mL collagenase. In some embodiments, the collagenase is a type IV collagenase. In some embodiments, the collagenase is a type I / II collagenase.

[0337] In some embodiments, enzymatic digestion can be carried out, in part, using a hyaluronidase. Hyaluronidase is a hyaluronic acid-metabolizing enzyme, subsequently enhancing cell membrane permeability (Eikenes et al. Anticancer Research, 2010). The enzyme, such as a hyaluronidase, can be present in media for the enzymatic digestion at a concentration of from at or about 5 mg / mL to at or about 10 mg / mL, such as at or about 5 mg / mL, at or about 6 mg / mL, at or about 7 mg / mL, at or about 8 mg / mL or at or about 9 mg / mL, at or about 10 mg / mL or any value between any of the foregoing. Insome embodiments, the enzymatic digestion is with a media that includes type II hyaluronidase, such as from at or about 5 mg / mL to at or about 10 mg / mL. In some embodiments, if a more gentle digestion is desired at or about 5 mg / mL hyaluronidase is used. In some embodiments, if a more complete digestion is desired a higher concentration of hyaluronidase is used, such as at or abut 10 mg / mL hyaluronidase.

[0338] In some embodiments, DNase is also present in the media for during the enzymatic digestion. DNase is an enzyme that degrades any free DNA released into the media as a result of the tumor fragment digestion process. The enzyme, such as a DNase I, can be present in media for the enzymatic digestion at a concentration of from at or about 5,000 units / mL to at or about 10,000 units / mL, such as at or about 5,000 units / mL, at or about 6,000 units / mL, at or about 7,000 units / mL, at or about 8,000 units / mL or at or about 9,000 units / mL, at or about 10,000 units / mL or any value between any of the foregoing. In some embodiments, the enzymatic digestion is with a media that includes DNase I, such as from at or about 5,000 units / mL to at or about 10,000 units / mL.

[0339] In some embodiments, enzymes from the Miltenyi human tumor dissociation kit can be used (e.g. Cat. O. 130-095-929; Miltenyi Biotec). The enzymatic media containing the enzyme can be a serum-free media, such as any as described. In particular embodiments, enzymatic media includes hyaluronidase and / or collagenase, e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate (e.g. GlutaMAX), 10 mg / mL gentamicin, 10,000 units / mL of DNase I, 10 mg / mL of collagenase and 10 mg / mL of hyaluronidase).

[0340] The tumor fragment is then mechanically dissected to dissociate the TILs, e.g., using a tissue dissociator. An example of a tissue dissociator is GentleMACs™ (Miltenyi Biotec) to homogenize the tissue. Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37 °C in 5% CO2, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. In some embodiments, tumor digests are subjected to homogenization and enzymatic digestion by incubation in the enzyme cocktail for 15 minutes to 2 hours, such as for at or about 30 minutes to 60 minutes. In some embodiments, tumor digests are subjected to homogenization and enzymatic digestion by incubation in the enzyme cocktail for about 60 minutes. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL can be performed to remove these cells. In some embodiments, a single cell suspension is prepared following processing of the tumor fragments by straining the cells through a filter to remove debris, such as a 70 pm strainer. In some cases, separation can be achieved by centrifugation, in which case the cell pellet can be resuspended and strained through a e.g. 70 pm strainer to remove debris. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 Al, 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 obtaining TILs for use in the provided methods.

[0341] In some embodiments, a single cell suspension for use as an input sample comprises from at or about 1 x 106dissociated tumor cells to at or about 1000 x 106dissociated tumor cells, such as 1 x 106to 500 x 106dissociated tumor cells, 1 x 106to 100 x 106dissociated tumor cells, 1 x 106to 50 x 106dissociated tumor cells, 1 x 106to 10 x 106dissociated tumor cells, 10 x 106to 1000 x 106dissociated tumor cells, 10 x 106to 100 x 106dissociated tumor cells, 10 x 106to 500 x 106dissociated tumor cells, 10 x 106to 50 x 106dissociated tumor cells, 50 x 106to 1000 x 106dissociated tumor cells, 50 x 106to 500 x 106dissociated tumor cells, 50 x 106to 100 x 106dissociated tumor cells, 100 x 106to 1000 x 106dissociated tumor cells, 100 x 106to 500 x 106dissociated tumor cells, or 500 x 106to 1000 x 106dissociated tumor cells. In some embodiments, a single cell suspension for use as an input sample of T cells comprises from at or about or at least at or about 10 x 106dissociated tumor cells , 20 x 106dissociated tumor cells, 30 x 106dissociated tumor cells, 40 x 106dissociated tumor cells, 50 x 106dissociated tumor cells, 60 x 106dissociated tumor cells, 70 x 106dissociated tumor cells, 80 x 106dissociated tumor cells, 90 x 106dissociated tumor cells, or 100 x 106dissociated tumor cells In some embodiments, a single cell suspension for use as an input sample of T cells comprises from at or about 10 x 106dissociated tumor cells to at or about 100 x 106dissociated tumor cells.2. Selection of Celis

[0342] In embodiments of the provided methods, the methods involve selecting or enriching from the first population of T cells (e.g. dissociated tumor cells), cells that are likely or suspected of being tumor reactive T cells by selecting or isolating T cells that are surface positive for the T cell activation markers, e.g. PD-1 and CD39, among a lymphocyte subset of cells. In some embodiments, T cells that are positive for the T cell activation markers, e.g., PD-1 and CD39, are selected or enriched from a first population of T cells that have been obtained from a biological sample, such as described in Section II. B.2. In particular embodiments, the selection is from cells present in the single cell suspension dissociated tumor cell sample. In some embodiments, the enriched or selected population of cells is used in subsequent processing steps, such as subsequent processing steps involving expansion in accord with the provided methods.

[0343] Methods for selection of surface receptors on cells can be by any of a number of techniques, such as generally involving antibody binding with an antibody specific reagent and subsequent enrichment by magnetic separation or fluorescence-activated cell sorting (FACS).

[0344] In some embodiments, cells are selected directly from a single cell suspension input sample prepared by enzymatic or mechanical digestion of tumor fragments, in which the selection is carried out using a CD39 / PD1 positive selection. In some embodiments, the selected cells are then stimulated for expansion using methods as described, such as by incubation or culture in the presence of one or more of IL-2, IL-7, IL-15 or IL-21. In some embodiments, the stimulation would not include culture with an anti-CD3 antibody (0KT3) or other costimulatory molecules. In some embodiments, the stimulation may include culture with an anti-CD3 antibody (0KT3) or other costimulatory molecules.

[0345] In some embodiments, selection is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker4") at a relatively higher level (markerhlgh) on the positively or negatively selected cells, respectively.

[0346] In particular embodiments, a T cell population is one that includes both CD4+ and CD8+ T cells. Many cancers, including solid tumors, such as many common epithelial indications (e.g. GI), express class I and class II restricted mutations. In order for a T cell product to target such indications, e.g. common epithelial indications, it is contemplated that both CD8+ T cells to recognize class I MHC- restricted molecules and CD4+ T cells to recognize Class II MHC -restricted molecules are necessary.

[0347] In some embodiments, the methods include selection of PD-1+ and CD39+ cells from among a lymphocyte subject of cells that includes CD4+ and CD8+ T cells. In one method, lymphocytes can be selected by positive selection for CD3+ cells. In another method, lymphocytes can be selected by positive selection for CD4+ and / or CD8+ cells. In some aspects, a CD4+or CD8+selection step, such as positive selection for CD4 and positive selection for CD8, is used to separate CD4+helper and CD8+cytotoxic T cells. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some embodiments, the methods include enriching for CD4+ and CD8+ T cells by selecting for T cells surface positive for CD3 or by sequential or simultaneous selection for T cells surface positive for CD4 and T cells surface positive for CD8. Such CD3+ T cells, or CD4+and / or CD8+populations, can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on tumor -reactive T cells or on T cells having expression of T cell markers associated with tumor-reactive T cells, e.g. as described above.

[0348] In some embodiments, the selections produces an enriched population of cells, such as a population of cells enriched for CD3+ T cells or CD4+ cells and CD8+ cells, that are further positive for PD-1 and CD39. In some embodiments, such cells include or are enriched for tumor -reactive T cells or T cells associated with tumor-reactive T cells. I

[0349] In some embodiments, enriching for a T cell that is surface positive for one or more cell surface marker includes any method for separation based on such markers. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation ofcells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Methods of selection of cells include, but are not limited to, bead selection (e.g. serial bead passage for positive / negative selection of cells), immunoaffinity chromatography (e.g. serial elution for positive / negative selection, and flow cytometry sorting. For use in accord with the provided methods, the selection method meets GMP standards.

[0350] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead and / or are detectably labeled, specifically bind to cell surface molecules if present on cells within the sample. In some aspects, cells bound to the antibodies can be recovered or separated from non-bound cells in the sample.

[0351] In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0352] In some embodiments, the T cells for use in connection with the provided methods can be enriched or sorted a variety of ways including, but not limited to, magnetic bead separation, fluorescent cell sorting, and disposable closed cartridge based cell sorters. In particular aspects, one or more reagents specific to T cells or a subset thereof, such as reagents specific to T cell activation markers for selecting reactive cells, can be used including, but not limited to, florescent antibodies, nanoparticles or beads on cell selection equipment, but not limited to, the CliniMACS, Sony FX500 or the Tyto cell sorting systems (Miltenyi).

[0353] In particular embodiments, selection of cells is carried out by flow cytometry-based cell sorting. Compared to other methods, flow cytometry-based cell sorting has the advantage that cells can be isolated in a single step on the basis of multiple parameters for each cell, thereby achieving a higher yield of cells and a higher purity that may not be possible with bead-based (e.g. magnetic bead-based) separations. Further, multiparameter cell staining and separation allows simultaneous labeling and identification and sorting of a plurality of antigens and characteristic fluorescent signals. Using flow cytometry sorting, a single process can remove and isolate specific populations based on a complex cell surface phenotype. Cell selection sorting equipment can be used that has a sufficiently high-throughput to handle large volumes and cell numbers. Non-limiting cell sorting equipment includes, for example, Sony FX500 or the Tyto cell sorting systems (Miltenyi). For use in provided methods, the flow cytometer instrument is GMP compliant. Method of cell sorting to achieve multiparameter sorting for two or morecell surface markers (e.g. CD39 and PD-1) can be carried out using multicolor fluorophore reagents that are compatible. It is within the level of a skilled artisan to choose appropriate fluorophores and reagents, such as by choosing a bright fluorophore and choosing fluorophores that have minimal to no spectral overlap.

[0354] In certain embodiments, the sample is contacted with a binding agent, e.g., a detectably labeled binding agent, that specifically binds to a cell surface marker. In certain embodiments, the detectably labeled binding agent(s) are fluorescently labeled. In certain embodiments, T cells labeled with binding agents specific to a cell surface marker are identified by flow cytometry. In certain embodiments, the method further includes separating any resultant T cells labeled with the binding agent(s) from other components of the sample to produce a composition enriched for T cells surface positive for the one or more cell surface marker. Cell selection sorting equipment can be used that has a sufficiently high-throughput to handle large volumes and cell numbers. Non-limiting cell sorting equipment includes, for example, Sony FX500 or the Tyto cell sorting systems (Miltenyi).

[0355] In some embodiments, any antibody reagent used to select cells in accord with the provided methods is a GMP antibody reagent. In some embodiments, the reagent is an analyte specific reagent (ASR).

[0356] In some embodiments, cells are selected that are surface positive for CD39 and PD-1. In some cases, staining methods also can include selecting CD45, CD4 and / or CD8 T cells from a sample. In some embodiments, multiparameter flow cytometry is carried out. In some embodiments, a multiparameter flow cytometry involves a sequential gating strategy. In some cases, CD45 expression, which optionally can be coupled with side scatter, can be used to exclude CD45 negative cells such as red blood cells. In some embodiments, only lymphocytes are gated based on CD45 expression and their scattering (e.g. FSClow, SSClow). In some embodiments, T cells can be gated based on positive expression of CD4+ and CD8+ cells. Within the T cell population, cells positive for CD39 and PD-1 can be identified for sorting.

[0357] Methods and antibody reagents to select for cells positive for these markers are known and commercially available. Any of a variety of fluorophores can be coupled to the antibodies and used in the multiparameter flow cytometry. In some embodiments, multi-color staining or labeling is carried out using multiple fluorophores in which multiple staining reagents against different cell surface markers are incubated with cells. In some embodiments, the fluorescent marker, e.g., conjugated to the one or more staining reagents, such as antibodies, are selected to minimize energy transfer between them, such as to avoid or minimize overlapping emission and absorption spectra. In some embodiments, each fluorescent marker has a different emission spectra. In some embodiments, the multiple fluorescent marker may be excited with a single wavelength or multiple wavelengths, but detection occurs in regions where the peak emission spectra do not overlap. In some embodiments, excitation of one or more of the fluorescentmarkers may be by light at a single or the same wavelength, but whereby different wavelengths of light are emitted therefrom.

[0358] In some embodiments, any fluorescent marker or fluorophore suitable for use with flow cytometry analysis can be used. Some non-limiting examples of fluorescent markers include fluorescent proteins (e.g., GFP, YFP, RFP), fluorescent moieties (e.g., fluorescein isothiocyanate) (FITC), Phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor (AF)), nucleic acid colorants (e.g., 4 ', 6- diamidino-2-phenylindole (DAPI), SYT016, propidium iodide (PI), cell membrane stain (e.g., FMI-43), cell functional dyes (e.g., Fluo-4, Indo-1), and synthetic dyes (e.g., Brilliant Violet (BV)). Exemplary fluorophores include, but are not limited to, hydroxycoumarin, Cascade Blue, Dylight 405 Pacific Orange, Alexa Fluor 430, Fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493, 2,7- Diochlorofluorescien, ATTO 488, Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 532, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, Rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700 (AF700), Cy5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, PE-Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, BV421, BV510, BV570, BV605, BV650, BV711, BV750, BV785, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705, or Qdot® 800. In some embodiments, the one or more fluorescent markers each individually comprise a fluorophore selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.

[0359] Table 1 lists exemplary antibodies for use in staining and selection or sorting of cells as described herein.

[0360] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and / or microelectromechanical systems (MEMS) chips, e.g., in combination with a flowcytometric detection system. In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers (e.g., with a antibody-coupled fluorescent peptide) are carried in a fluidic stream.

[0361] In some embodiments, the cell staining involves incubation with an antibody or binding partner that specifically binds to such markers as described, which in some embodiments is followed by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. In some aspects of such processes, a volume of cells is mixed with an amount of a desired staining reagent and incubated under conditions for staining of the cells. In some embodiments, the staining or labelling is carried out at a temperature between 0°C and 25°C, such as at or about 4°C. In some embodiments, the staining or labelling is carried out for greater than 5 minutes, typically greater than 15 minutes. In some embodiments, the staining or labelling is carried out for between 15 minutes and 6 hours, such as between 30 minutes and 2 hours. In some embodiments, the staining or labelling is carried out for example, at or about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or any value between any of the foregoing. In some embodiments, the labeling with the one or more staining reagents is carried out simultaneously. In some embodiments, one or more wash steps are carried out prior to introducing the sample into the flow cytometer for analysis.

[0362] In some embodiments, the cell sample is prepared by suspending single cells at a density of 1 x 106to 5 x 107cells / ml in order to allow the cells to pass through the flow cytometer for reading. In some embodiments, the density of cells for sorting is 5 x 106cells / mL to 50 x 106cells / mL, such as at or about 20 x 106cells / mL. In some embodiments, this concentration of cells is called the fluid sheath. In some embodiments, the fluid sheath influences the rate of flow sorting, which typically progresses at around 2,000-20,000 cells (events) per second. The cell sample's) fluid sheath is typically made of a phosphate buffered saline solution, but other solutions are available as will be known and understood by those skilled in the art.

[0363] In some embodiments, the flow cytometry sorting rate is from 2000 events / second to 10,000 event / second. In some embodiments, the flow cytometry sorting rate is about 2000 events / second, about 3000 events / second, about 4,000 events / second, about 5000 events / second, about 6000 events / second, about 7000 events / second, about 8000 events / second, about 9000 events / second, about 10000 events / second, about 15000 events / second, or about 20000 events per second, or any value between any of the foregoing. In some embodiments, the flow cytometry sorting rate is about 6,000 events / second.

[0364] In some embodiments, the sample is introduced into a flow cytometer. The cell sample is typically narrowed down to a single stream through a fluidics system with the application of hydro pressure. This stream is then passed through the one or more beams of light scattering or fluorescence emission. Lasers typically serves as the light source in flow cytometers. The laser produces a single wavelength of light that once contacted with the cell sample produces scattered light in the forward direction as a measure of cell size, scattered light in the side direction as a measure of cell complexity, and fluorescent light, also emitted in the side direction which is proportional to the relative amount of a particular cell marker. Fluorescent channels are usually indicated by the designations FL1, FL2, FL3,etc., depending on the number of channels in the instrument. Each fluorescent channel is set with barrier filters to detect a selected specific dye while filtering out all others. The channel in which a specific dye is predominantly detectable may be referred to as its primary fluorescent channel while other fluorescent channels may be designated as secondary channels. Scattered and fluorescent emitted light signals are converted to electronic pulses that are processed by the flow cytometry engine and displayed on a graphical user interface “GUI.”

[0365] Methods of analyzing flowcytometric or FACS data can involve a “gating” for data to separate specific groups of cells. Different cell types can be identified by the scatter parameters and the fluorescence emissions resulting from labeling various cell proteins with dye-labeled antibodies as described above. The identification of clusters and, thereby, populations can be carried out by “gating” of the cells. In some embodiments, gates corresponding to subsets of particles of interest, such as TIL expressing markers of neoantigen reactivity, are defined by users with the aid of software operationally associated with the flow system as described above.

[0366] In some embodiments, a gate may be a “threshold” gate, which is a gate for only one optical parameter that defines an open region within the multidimensional space. In some embodiments, “threshold” gating can be used for forward light scatter to remove high frequency low level signals caused by interference, such as debris in the sample. In some embodiments, “window” gating is employed, e.g., by defining upper and lower bounds for signal values. In some embodiments, gating is carried out on a 2D-plot of two parameters, such as side scatter (e.g., on vertical axis) and a fluorescence signal (e.g., on horizontal axis).

[0367] In some embodiments, flow cytometry for a cell surface marker includes gating for an “F- minus one” (FMO) control. FMO gating includes separate portions of the same sample stained with a panel of detectably labeled binding agents that contains all the agents but one. The distribution of the signal of the removed fluorophore can be used to define the positive threshold for the missing label as it is known that all cells are negative in the control. The position of all gates can be determined using FMO controls in which the antibody against the investigated marker is substituted with an appropriate isotype control. In an exemplary method, a gate can be drawn using cells stained with the FMO cocktail around cells positive for CD45, CD4 and / or CD8, PD1 and CD39. In some embodiments, sequential gates can be used to arrive at the selected subpopulation. For example, methods can be carried out in which cells are gated on CD45 positive cells, then CD4 and / or CD8 positive cells, then on cells positive for PD-1 and CD39. In some embodiments, a viability dye also can be added. An exemplary viability dye is 7-ADD. In some embodiments, a gate can be drawn around cells negative for 7- A AD (7-AADneg)-

[0368] In some embodiments, the cells are sorted into a single population of cells and collected. The selected population of cells are used as input for expansion, such as described in Section II. B.3.

[0369] The selection need not result in 100 % enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of aparticular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.

[0370] In some embodiments, the enriched population of cells are enriched cells from a starting sample as describe above, in which the percentage of cells of a particular phenotype, e.g. tumor-reactive CD3+ T cells or CD3+ T cells surface positive for one or more T cell marker, e.g. PD-1 andCD39, in the enriched population of cells in increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000% or more greater than the percentage of such cells in the starting sample. In some embodiments, the purity of tumor-reactive CD3+ T cells or CD3+ T cells surface positive for PD-1 andCD39) versus total cells in the population of enriched cells, is at least 90%, 91%, 92%, 93%, 94%, and is generally at least 95%, 96%, 97%, 98%, 99% or greater..7. Stimulation of T cells for Expansion

[0371] The provided methods further include expanding the population of selected TILs by culture with one or more T-cell stimulating agent of lymphocytes under conditions to produce a population of expanded T cells. The provided TIL composition is thus produced following the ex vivo expansion of the selected cells.

[0372] In some embodiments, the incubation or culturing with one or more T cell stimulatory agent(s) results in expansion or outgrowth of selected T cells, or a desired subset or subtype thereof or for viable cells thereof, for use in subsequent steps of the provided methods. Non-limiting examples of T cell stimulatory agent(s) and conditions for incubation or culture are described herein.

[0373] Thus, among the provided methods are methods of culturing T cells for manufacture of tumor reactive T cells in which T cells are cultured or incubated in the presence of a T cell stimulatory agent under conditions to expand T cells.

[0374] In some embodiments, the T cell stimulatory agent(s) include a recombinant T cell stimulating c...

Claims

CLAIMSWHAT IS CLAIMED:

1. A method for treating a solid tumor malignancy in a subject, the method comprising: a. administering a radiation therapy to a subject that has a solid tumor malignancy and b. administering a T lymphocyte infiltrating (TIL) composition enriched in tumor reactive T cells to the subject.

2. The method of claim 1, wherein the radiation therapy is a low dose radiation therapy.

3. The method of claim 1 or claim 2, wherein the radiation therapy is administered as a total dose of no more than about 30 Gy.

4. The method of any of claims 1-3, wherein the radiation therapy is administered as a total dose of from 0.5 Gy to 20 Gy.

5. The method of any of claims 1-4, wherein the radiation therapy is administered as a total dose of from 0.5 Gy to 10 Gy.

6. The method of any of claims 1-5, wherein the radiation therapy is administered as a total dose of about 4 Gy.

7. The method of any of claims 1-6, wherein the radiation therapy is administered as a total dose to induce infiltration of T cells into the tumor of the subject.

8. The method of any of claims 1-7, wherein the radiation therapy is administered as a total dose that does not damage DNA to reduce or prevent cell division of cancer cells.

9. The method of any of claims 1-8, wherein the radiation therapy is administered as a total dose that does not kill cancer cells.

10. The method of any of claims 1-9, wherein the total dose of the radiation therapy is administered prior to and / or after administering the TIL composition to the subject.

11. The method of any of claims 1-10, wherein the total dose of the radiation therapy is administered prior to administering the TIL composition to the subject.

12. The method of any of claims 1-11, wherein the total dose of the radiation therapy is administered after administering the TIL composition to the subject.

13. The method of any of claims 1-12, wherein the total dose of the radiation therapy is administered as a divided dose given as a plurality of fraction doses.

14. The method of claim 13, wherein the plurality of fraction doses are 2, 3, 4, 5 or 6 doses.

15. The method of claim 13 or claim 14, wherein the plurality of fraction doses are 4 doses.

16. The method of any of claims 13-15, wherein the plurality of fraction doses are administered prior to and / or after administering the TIL composition to the subject.

17. The method of any of claims 13-16, wherein each of the plurality of fraction doses are administered prior to administering the TIL composition to the subject.

18. The method of any of claims 13-16, wherein each of the plurality of fraction doses are administered after administering the TIL composition to the subject.

19. The method of any of claims 13-16, wherein at least one of the plurality of fraction doses is administered prior to administration of the TIL composition and at least one of the plurality of fraction doses is administered after administration of the TIL composition.

20. The method of any of claims 13-16, and 19, wherein 2 fraction doses are administered prior to administration of the TIL composition and 2 fraction disease are administered after administration of the TIL composition.

21. The method of any of claims 13-20, where each of the plurality of fraction doses is administered at a dose of no more than 6 Gy per lesion per day.

22. The method of any of claims 13-21, wherein each of the plurality of fraction doses is administered at a dose of from 0.5 Gy to 4 Gy.

23. The method of any of claims 13-22, wherein each of the plurality of fraction doses is administered at a dose of from 0.5 Gy to 2 Gy.

24. The method of any of claims 13-23, wherein each of the plurality of fraction doses is administered at a dose of about 1 Gy.

25. The method of any of claims 13-24, wherein administration of the radiation therapy prior to administration of the TIL composition is initiated no more than 30 days prior to the harvesting of tumor to derive the TIL composition.

26. The method of any of claims 13-24, wherein the administration of the radiation therapy prior to administration of the TIL composition is initiated no more than 2 days prior to the harvesting of tumor to derive the TIL composition.

27. The method of any of claims 10-24, wherein the administration of the radiation therapy prior to administration of the TIL composition is initiated no more than 10 days prior to the administration of the TIL composition.

28. The method of any of claims 10-27, wherein the administration of the radiation therapy prior to administration of the TIL composition is initiated no more than 2 days prior to the administration of the TIL composition.

29. The method of any of claims 1-28, wherein the radiation therapy comprises administering a first total dose of radiation and a second total dose of radiation.

30. The method of claim 29, wherein the administration of the first total dose of radiation is initiated no more than 30 days prior to the harvesting of tumor to derive the TIL composition and the second total dose of radiation is initiated no more than 10 days prior to the administration of the TIL composition.

31. The method of any one of claims 1-30, wherein the radiation therapy is an external beam radiation therapy, a brachytherapy or a systemic radioisotope therapy.

32. The method of any of claims 1-31, wherein the TIL composition comprises a population of T cells that is enriched in at least 10 different T cell receptor (TCR) clonotypes each with a frequency in the population of at least 2.0%.

33. The method of claim 32, wherein at least 11 different TCR clonotypes have a frequency in the population of at least 2.0%.

34. The method of claim 32 or 33, wherein at least 12 different TCR clonotypes have a frequency in the population of at least 2.0%.

35. The method of any of claims 32-34, wherein 8 to 15 different T cell receptor (TCR) clonotypes make up at least 50 % of the TCR frequency in the population.

36. The method of any of claims 32-35, wherein 9 to 12 different TCR clonotypes make up at least 50% of the TCR frequency in the population.

37. The method of any of claims 32-36, wherein the TCR clonotypes exhibit reactivity to at least one CD4 antigen and at least one CD8 antigen.

38. The method of any of claims 32-37, wherein the TCR clonotypes exhibit reactivity for 2 to 6 different peptide antigens.

39. The method of any of claims 32-38, wherein the TCR clonotypes exhibit reactivity for 2 to 4 peptide antigens40. The method of any of claims 32-39, wherein the TCR clonotypes exhibit reactivity for 2 peptide antigens.

41. The method of any of claims 32-40, wherein the TCR clonotypes exhibit reactivity for one CD8 antigen and one CD4 antigen.

42. The method of any of claims 32-41, wherein at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity.

43. The method of any of claims 32-42, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is at or about 10:1 to 25:1, optionally at or about 20:1.

44. The method of any of claims 32-42, wherein greater than 75% of the CD3+ T cells, optionally greater than 75% of the CD4 and CD8+ T cells, express markers of an effector memory phenotype.

45. The method of any of claims 32-42, wherein greater than 80% of the CD3+ T cells, optionally greater than 80% of the CD4 and CD8+ T cells, express markers of an effector memory phenotype.

46. The method of any of claims 32-42, wherein greater than 85% of the CD3+ T cells, optionally greater than 85% of the CD4 and CD8+ T cells, express markers of an effector memory phenotype.

47. The method of any of claims 32-42, wherein greater than 90% of the CD3+ T cells, optionally greater than 90% of the CD4 and CD8+ T cells, express markers of an effector memory phenotype.

48. The method of any of claims 44-47, wherein the effector memory phenotype is characterized by surface marker expression of one or more of CD45RO+, CD40RO-, CD62L , CCR7-, CD28- and CD27-.

49. The method of any of claims 44-48, wherein the effector memory phenotype is characterized by surface marker expression CD45RO+, CD40RO , CD62L , and CCR7 .

50. The method of any of claims 44-47, wherein the effector memory phenotype is characterized by surface marker expression CD45RO+, CD40RO , CD62L , CCR7 , CD28 and CD27 .

51. The method of any of claims 1-50, wherein greater than 95% of the CD4+ and CD8+ T cells in the composition are PD-1 .

52. The method of any of claims 1-51, wherein greater than 80% of the CD4+ and CD8+ T cells in the composition LAG3 .

53. The method of any of claims 1-52, wherein the population of T cells of the TIL composition are able to produce IFNgamma at a concentration of greater than at or about 30 pg / mL, optionally greater than at or about 60 pg / mL, following antigen-specific stimulation.

54. The method of claim 1 or claim 2, wherein the TIL composition is produced by an ex vivo method comprising ex vivo expansion of tumor-reactive T cells that have been obtained from the subject and co-cultured with autologous antigen presenting cells and peptide neoantigens.

55. The method of claim 54, wherein the ex vivo method for producing the TIL composition comprises: i. processing a biological sample containing a population of T lymphocyte cells obtained from the subject that has a tumor to produce a first population of T lymphocyte cells; ii. stimulating the first population with one or more first T-cell stimulating agents of lymphocytes under conditions for expansion of T cells in the population to produce a second population of activated T cells; iii. co-culturing the second population of T cells in the presence of antigen presenting cells that present one or more peptides on a major histocompatibility complex (MHC), said one or more peptides corresponding to peptide neoantigens associated in the tumor of the subject, to produce a third population of cells containing T cells comprising endogenous T cell receptors reactive to mutation encoding peptides of the tumor; iv. selecting, from the third population of cells, the population of T cells containing endogenous TCR that are reactive to peptides present on the APCs based on one or more upregulation markers expressed on reactive or activated T cells to produce a fourth population containing the selected T cells; v. expanding the fourth population of selected cells in the presence of one or more second T-cell stimulating agents of lymphocytes under conditions to expand T cells in the population to produce the TIL composition.

56. The method of claim 55, wherein the antigen presenting cells are dendritic cells.

57. The method of claim 55 or claim 56, wherein the antigen presenting cells.

58. The method of any of claims 55-57, wherein the MHC molecule is a class I molecule.

59. The method of any of claims 55-57, wherein the MHC molecule is a Class II molecule.

60. The method of any of claims 55-59, wherein the MHC molecule is MHC class I and II.

61. The method of any of claims 55-60, wherein the T cells are CD4+ cells.

62. The method of any of claims 55-60, wherein the T cells are CD8+ cells.

63. The method of any of claims 55-60, wherein the T cells are CD4+ cells and CD8+ cells.

64. The method of any of claims 55-63, wherein the one or more first T cell stimulating agents are selected from one or more of an anti-CD3 antibody; an anti-CD28 antibody; or a recombinant cytokine selected from among IL-2, IL-7, IL- 15 and IL-21.

65. The method of any of claims 55-64, wherein the one or more first T-cell stimulating agents is or comprises recombinant IL-2.

66. The method of claim 65, wherein the concentration of recombinant IL-2 is from 100 lU / mL to 6000 lU / mL, optionally at from 1000 lU / mL to 6000 lU / mL.

67. The method of claim 65 or claim 66, wherein the concentration of recombinant IL-2 is from 3000 lU / mL to 6000 lU / mL, optionally about 3000 lU / mL or 6000 lU / mL.

68. The method of any of claims 55-67, wherein the one or more second T cell stimulating agents are selected from one or more of an anti-CD3 antibody; an anti-CD28 antibody; or a recombinant cytokine selected from among IL-2, IL-7, IL- 15 and IL-21.

69. The method of any of claims 55-68, wherein the one or more second T-cell stimulating agents is or comprises recombinant IL-2.

70. The method of claim 69, wherein the concentration of recombinant IL-2 is from 100 lU / mL to 6000 lU / mL, optionally at from 1000 lU / mL to 6000 lU / mL.

71. The method of claim 69 or claim 70, wherein the concentration of recombinant IL-2 is from 3000 lU / mL to 6000 lU / mL, optionally about 3000 lU / mL or 6000 lU / mL.

72. The method of any of claims 69-71, wherein the one or more second T cell stimulating agent further comprises an anti-CD3 antibody, optionally OKT3, optionally wherein the concentration of the anti-CD3 antibody is at or about 50 ng / mL.

73. The method of any of claims 55-72, wherein the one or more second T cell stimulating agent further comprises feeder cells that are non-dividing peripheral blood mononuclear cells (PBMC), optionally wherein the feeder cells are gamma irradiated.

74. The method of any of claims 55-73, where the one or more non-native peptides are loaded on antigen presenting cells by peptide pulse, optionally by electroporation.

75. The method of claim 74, wherein the one or more non-native peptide is 5-30 amino acids, optionally 12-25 amino acids, optionally at or about 25 amino acids in length.

76. The method of claim 74 or claim 75, wherein: the one or more non-native peptides are a pool of peptides and the concentration of peptides in the pool of peptides for the peptide pulse is between at or about 0.00001 pg / mL and about 0.01 pg / mL, 0.0001 pg / mL and at or about 0.001 pg / mL, 0.001 pg / mL and at or about 40 pg / mL, 0.01 pg / mL and at or about 40 pg / mL, at or about 0.1 pg / mL and at or about 40 pg / mL, at or about 1 pg / mL and at or about 40 pg / mL, at or about 0.01 pg / mL and at or about 10 pg / mL or at or about 1 pg / mL and at or about 10 pg / mL.

77. The method of any of claims 55-76, wherein the co-culture ratio of antigen presenting cells to T Cells is between 20:1 and 1:1, between 15:1 and 1:1, between 10:1 and 1:1, between 5:1 and 1:1, between 2.5:1 and 1:1, between 1:20 and 1:1, between 1:15 and 1:1, between 1:10 and 1:1, between 1:5 and 1:1, or between 1:2.5 and 1:1.

78. The method of any of claims 55-77, wherein the co-culture ratio of dendritic cells to T cells is between 5:1 and 1:5 or is between 3:1 and 1:3, optionally is or is about 1:1.

79. The method of any of claims 55-78, wherein the nonsynonymous somatic mutations associated in the tumor of the subject are identified using Whole exome sequencing (WES) and RNAseq.

80. The method of any of claims 55-79, wherein the co-culturing is for 2 hours to 24 hours, optionally wherein the co-culturing is for at or about 6 hours.

81. The method of any of claims 55-80, where the selection is performed using a florescence based cell sorter.

82. The method of any of claims 55-81, wherein the one or more upregulation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD137 (4-1BB), CD59, CD69, CD90, CD38, CD30, CD154, CD252, CD134 (0X40), CD258, CD256, PD-1, TIM-3 and LAG-3.

83. The method of any of claims 55-82, wherein the one or more upregulation maker is CD134 and / or CD137, optionally wherein the one or more upregulation maker is CD134 and CD137.

84. The method of any of claims 55-83, wherein the stimulating the first population is for 7 to 21 days, optionally 7 to 14 days.

85. The method of any of claims 55-84, wherein the stimulating the first population in a closed system is using a gas permeable culture vessel.

86. The method of any of claims 55-85, wherein the stimulating the first population in a closed system is performed using a bioreactor.

87. The method of any of claims 55-86, wherein the expanding the fourth population is for 7 to 21 days, optionally 7 to 14 days.

88. The method of any of claims 55-87, wherein the expanding the fourth population is performed in a gas permeable culture vessel.

89. The method of any of claims 55-88, wherein the expanding the fourth population is performed using a bioreactor.

90. The method of any of claims 1-52, wherein the TIL composition is prepared by an ex vivo method comprising selecting cells surface positive for at least two activation markers from the group consisting of PD-1, CD39 and TIGIT from cells obtained from a tumor of a donor subject and expanding the cells ex vivo.

91. The method of claim 90, wherein the ex vivo method comprises: contacting cells from an input sample comprising T cells with antibodies that bind to at least two activation markers from the group consisting of PD-1, CD39 and TIGIT, wherein the input sample comprises cells from a tumor of a subject; selecting cells surface positive for the at least two activation markers to obtain selected cells from the sample; andperforming an expansion by culture of the selected cells with one or more T-cell stimulating agents of lymphocytes under conditions to produce a population of expanded T cells.

92. The method of claim 90 or claim 91, wherein the at least two activation markers are PD- 1 and CD39.

93. The method of claim 91 or claim 92, wherein the input sample comprising T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from a resected tumor.

94. The method of any of claims 91-93, wherein the one or more T-cell stimulating agents of lymphocytes comprises one or more recombinant cytokines selected from the group consisting of IL-2, IL-7, IL-15 and IL-21, optionally wherein at least one of the one or more T-cell stimulating agents is recombinant IL-2.

95. The method of any of claims 92-94, wherein at least one recombinant cytokine is IL-2 and wherein the concentration of recombinant IL-2 is from 100 lU / mL to 6000 IIJ / mL.

96. The method of any of claims 92-95, wherein the expansion is carried out in a closed system.

97. The method of claim 96, wherein the closed system comprises at least one of a gas permeable culture vessel or a bioreactor.

98. The method of any of claims 92-97, wherein the expansion to produce the expanded population of T cells is performed for about 7 to 35 days.

99. The method of any of claims 1-98, wherein the method further comprises administration of a lymphodepleting therapy.

100. The method of claim 99, wherein the lymphodepleting therapy is or comprises cyclophosphamide, ifosfamide, altretamine, busulfan, carboplatin, carmustine, cisplatin, dacarbazine, lomustine, melphalan, temozolomide, trabectedin, sodium 2-mercaptoethane sulfonate (Mensa), fludarabine, floxuridine, or any combination thereof.

101. The method of any of claims 99-100, wherein the lymphodepleting therapy is or comprises cyclophosphamide, sodium 2-mercaptoethane sulfonate (Mensa), fludarabine, or a combination thereof.

102. The method of any of claims 99-101, wherein the lymphodepleting therapy comprises administration of cyclophosphamide from about 30 mg / kg to about 200 mg / kg, optionally wherein the cyclophosphamide is administered at or about 60 mg / kg, optionally wherein the cyclophosphamide is administered within about 5 to 9 days prior to administering the TIL composition, more optionally wherein the cyclophosphamide is administered about 7 days prior to administering the TIL composition (Day -7).

103. The method of any of claims 99-102, wherein the lymphodepleting therapy comprises administration of Mensa from about 5 mg / kg / day to 25 mg / kg / day, optionally wherein the Mensa is administered at or about 15 mg / kg / day, optionally wherein the Mensa is administered within about 4 to 8 days prior to administering the TIL composition, more optionally wherein the Mensa is administered about 6 days prior to administering the TIL composition (Day -6).

104. The method of any of claims 99-103, wherein the lymphodepleting therapy comprises administration of fludarabine from about 20-40 mg / m2, optionally at or about 25 mg / m2daily, optionally wherein the fludarabine is administered for three consecutive days, optionally wherein the fludarabine is administered 3 to 5 days prior to administering the TIL composition (Day -5 to Day -3).

105. The method of any of claims 99-104, wherein the administration of the lymphodepleting therapy is completed within about 2 to 7 days prior to initiation of the administration of the TIL composition, optionally 2 to 5 days prior to initiation of the administration of the TIL composition.

106. The method of any of claims 1-105, further comprising administering recombinant IL-2 to the subject.

107. The method of claim 106, wherein the initiation of administration of the recombinant IL- 2 is on the same day as the TIL composition.

108. The method of claim 106 or claim 107, wherein the initiation of administration of the recombinant IL-2 is 2 hour to 24 hours after administration of the TIL composition.

109. The method of any of claims 106-108, wherein initiation of administration of the recombinant IL-2 is 2-3 hours after administration of the TIL composition.

110. The method of any of claims 106-109, wherein the recombinant IL-2 is administered as a plurality of doses.

111. The method of claim 110, wherein the plurality of doses are 2 to 12 doses, optionally 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 doses.

112. The method of any of claims 110 or claim 111, wherein the plurality of doses is 6 doses.

113. The method of any of claims 106-112, wherein the IL-2 is administered twice a day, three times a day or four times a day.

114. The method of any of claims 106-113, wherein the recombinant IL-2 is administered three times a day, optionally every 8 hours.

115. The method of any of claims 106-114, wherein each dose of the recombinant IL-2 is from 200,000 lU / kg to 800,000 lU / kg.

116. The method of any of claims 106-115, wherein each dose of the recombinant IL-2 is about 600,000 lU / kg.

117. The method of any of claims 106-116, wherein the recombinant IL-2 is administered intravenously.

118. The method of any of claims 106-117, wherein the recombinant IL-2 is Aldesleukin.

119. The method of any of claims 1-118, wherein the method further comprises administration of an anti-PDl antibody.

120. The method of claim 119, wherein the anti-PD-1 antibody is administered at a dose between 100 mg and 600 mg.

121. The method of claim 119 or claim 120, wherein the anti-PD-1 antibody is administered in a dosing regimen comprising administration of the dose every other week dosing (Q2W), once monthly (Q4W) or once every six weeks (Q6W) or combinations thereof.

122. The method of claim 119, wherein a first dose of the anti-PDl antibody is administered every two weeks (Q2W) for 3 months.

123. The method of claim 120, wherein the first dose of the anti-PD-1 antibody is administered at Day 14, Day 35 and Day 56.

124. The method of claim 119 or claim 120, wherein the first dose of the anti-PDl antibody is about 100 mg to about 300 mg.

125. The method of any of claims 119-124, wherein the first dose of the anti-PDl antibody is about 200 mg.

126. The method of any of claims 119-125, further comprising administering a second dose of the anti-PDl antibody every six weeks (Q6W).

127. The method of claim 126, wherein the second dose is about 300 mg to about 600 mg.

128. The method of claim 126 or claim 127, wherein the second dose is about 400 mg.

129. The method of claims 119-126, wherein the anti-PDl antibody is administered for up to six months, 12 months, 18 months, 24 months, 30 months, or 36 months post administration of the TIL composition.

130. The method of any of claims 119-129, wherein the anti-PDl antibody is administered for up to about 24 months.

131. The method of any of claims 119-130, wherein the anti-PDl antibody is administered intravenously.

132. The method of any of claims 119-131, wherein the anti-PDl antibody is pembrolizumab.

133. The method of any of claim 1-132, wherein the composition of expanded cells are used to treat a cancer patient.

134. The method of any of claims 1-133, wherein the tumor is a tumor of an epithelial cancer.

135. The method of any of claims 1-134, wherein the tumor is a tumor of a melanoma, lung squamous, lung adenocarcinoma, bladder cancer, lung small cell cancer, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, stomach cancer or uterine cancer.

136. The method of any of claims 1-135, wherein the tumor is a tumor of a non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma cancer, endometrial cancer, optionally wherein the breast cancer is HR+ / Her2- breast cancer, triple negative breast cancer (TNBC) or HER2+ breast cancer.

137. The method of claims 1-136, wherein the solid tumor malignancy is selected from the group comprising: breast cancer, colorectal carcinoma (CRC), uveal melanoma, cutaneous carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, or any advanced solid tumor.

138. The method of any of claims 1-137, wherein the subject to be treated is a subject selected as having a solid tumor malignancy that is a breast cancer.

139. The method of any of claims 1-137, wherein the subject to be treated is a subject selected as having a solid tumor malignancy that is a colorectal carcinoma.

140. The method of any of claims 1-137, wherein the subject to be treated is a subject selected as having a solid tumor malignancy that is a uveal melanoma.

141. The method of any of claims 1-137, wherein the subject to be treated is a subject selected as having a solid tumor malignancy that is a cutaneous melanoma.

142. The method of any of claims 1-137, wherein the subject to be treated is a subject selected as having a solid tumor malignancy that is a non-small cell lung cancer (NSCLC).

143. The method of any of claims 1-137, wherein the subject to be treated is a subject selected as having a solid tumor malignancy that is a head and neck squamous cell carcinoma (HNSCC).

144. The method of any of claims 1-143, wherein the solid tumor malignancy is an advanced solid tumor malignancy.

145. The method of any of claims 1-144, wherein the solid tumor malignancy is a metastatic disease.

146. The method of any of claims 1-145, wherein the subject has failed, is refractory to, or is unable to tolerate a standard of care therapy for treating the solid tumor malignancy.

147. The method of any of claims 1-146, wherein the subject has relapsed or is refractory to treatment of one or more prior treatments for the solid tumor malignancy.

148. The method of any of claims 1-147, wherein the subject has pathologically confirmed, unresectable, and / or metastatic solid tumor or tumors.

149. The method of any of claims 1-148, wherein the subject is a human subject.

150. The method of any of claims 1-149, wherein the subject is an adult human of at least 18 years of age.

151. The method of any of claims 1-150, wherein the therapeutically effective dose of the TIL composition is between 1 x 108and 1 x 1012T cells.

152. The method of any of claims 1-151, wherein the therapeutically effective dose of the TIL composition is between 1 x 109and 1 x 1012T cells.

153. The method of any of claims 1-152, wherein the therapeutically effective dose of the TIL composition is between 1 x 10sand 1 x IO10T cells, optionally between 1 x 10sand 1 x 109T cells.

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