Expansion of tumor-reactive t cells using Anti-CD28 antibodies

Abstract

Provided herein are methods for expansion of neoantigen expressing tumor-infiltrating lymphocytes for use in treating cancer, comprising culturing a tumor sample in a culture medium comprising an agonist of a costimulatory molecule, e.g., a CD28 agonist, to expand tumor-infiltrating lymphocytes with enriched tumor-reactivity and specificity.

Description

Docket No. 32757 / 59103EXPANSION OFTUMOR-REACTIVE T CELLS USING ANTI-CD28 ANTIBODIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 691 ,680, filed September 6, 2024, herein incorporated by reference in its entirety.FIELD

[0002] This disclosure relates to methods and compositions for activation and / or expansion of lymphocyte populations, e.g., tumor infiltrating lymphocytes (TILs), and compositions comprising such lymphocyte populations.BACKGROUND

[0003] The presence of lymphocytes in tumors is often associated with better clinical outcomes. Moreover, adoptive cell therapy (ACT) involving the transfer of ex vivo expanded autologous tumor infiltrating lymphocytes (TILs) has mediated durable, complete regressions of advanced stage cancers in a subset of patients. Although other forms of T-cell therapy involving the use of genetically engineered lymphocytes (e.g., TCR- and CAR- transduced T cells) have become available, TIL therapy has remained the superior form of therapy because it is able to target many tumor antigens recognized by a more heterogenous population of T cells rather than a single antigen that can be lost due to the high mutation rates in some cancers. In certain instances, TIL therapies in some patients have demonstrated a complete response while only targeting a single antigen.

[0004] TIL based therapy requires ex vivo production of billions of phenotypically effective TILs from fully or partially resected tumor tissues. Ex vivo expansion allows the T cells of interest to grow in an environment engineered to be free of immunosuppressive signals that are abundant within tumors. The standard TIL generation protocol consists of a primary expansion termed the pre-rapid expansion phase (pre-REP) also known as the TIL outgrowth phase, which is the initial growth stage of TILs from tumor material, followed by a large-scale expansion termed the rapid expansion phase (REP). The REP is used to generate sufficient TILs for patient infusion.

[0005] During pre-REP, tumor tissue is typically mechanically cut into small fragments which are cultured for 3-6 weeks in media containing a high dose (6000 lU / mL) of the T cell growth factor, interleukin-2 (IL-2), which is replenished as often as every 3-4 days. This allows TILs to gradually egress from the tumor tissue into the media and supports their initial expansion.

[0006] In the REP phase, TILs from the pre-REP phase are activated with anti-CD3 antibody in the presence of a large excess of irradiated autologous or allogeneic PBMCDocket No. 32757 / 59103 feeder cells. The feeder cells can activate TIL through HLA mismatch and release growth factors to promote the expansion of TILs. The cells are typically expanded for 2 weeks by feeding them with culture medium and IL-2. The final post-REP TIL population is then harvested, concentrated, cryopreserved or infused back into the same patient after a lymphodepleting preparative regimen (Wang, X., & Riviere, I. 2015. Cancer Gene Ther. 22(2):85-94).

[0007] Although widely used, this conventional TIL expansion protocol is not reliable for expanding all TIL populations, nor is it designed to enhance the generation of TILs with the optimum phenotype for infusion. For example, TIL outgrowth from many patient tumor samples is slow and insufficient due to suppressive effects of the tumor microenvironment on TIL proliferation and survival. Tumor inaccessibility can also limit the amount of tissue that can be surgically resected and the number of TILs that can be isolated.SUMMARY

[0008] Provided herein is a method that improves the current TIL expansion methods and provides ways to increase the expansion rate, and overall yield and frequency of neoantigen-reactive TILs.

[0009] The disclosure provides a method for producing tumor-reactive T cells, the method comprising:

[0010] (a) generating a tumor slurry from tumor fragments obtained from a tumor sample;

[0011] (b) culturing the tumor slurry in a cell culture medium comprising one or more cytokine and an anti-CD28 agonist for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs);

[0012] (c) harvesting the enhanced population of TILs obtained from step (b).

[0013] In various embodiments, generating a tumor slurry of step (a) comprises incubating the tumor sample in a digestion media comprising at least 3 enzymes. In various embodiments, the digestion media comprises enzymes selected from the group consisting of trypsin, chymotrypsin, trypsinogen, chymotrypsinogen, dispase, collagenase, accutase, thermolysin, pronase, hyaluronidase, elastase, papain, DNase, neuraminidase, pancreatin or a combination thereof. In various embodiments, the digestion media comprises dispase, collagenase, and hyaluronidase.

[0014] In various embodiments, generating a tumor slurry of step (a) further comprises disrupting the tumor sample mechanically so as to dissociate the tumor sample. In various embodiments, generating a tumor slurry of step (a) further comprises filtering the tumor slurry to remove any undigested tissue chunks.Docket No. 32757 / 59103

[0015] Also provided is a method for producing tumor-reactive T cells, the method comprising:

[0016] (a) generating tumor fragments from a tumor sample;

[0017] (b) culturing the tumor fragments in a cell culture medium comprising one or more cytokine and an anti-CD28 agonist for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs);

[0018] (c) harvesting the enhanced population of TILs obtained from step (b).

[0019] Also provided is a method for producing tumor-reactive T cells, the method comprising:

[0020] (a) generating a tumor slurry from tumor fragments obtained from a tumor sample;

[0021] (b) culturing the tumor slurry in a cell culture medium comprising one or more cytokine and an antibody to a costimulatory molecule for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs);

[0022] (c) harvesting the enhanced population of TILs obtained from step (b).

[0023] In certain embodiments, the disclosure provides a method for producing tumor- reactive T cells, the method comprising:

[0024] (a) generating tumor fragments from a tumor sample;

[0025] (b culturing the tumor slurry in a cell culture medium comprising one or more cytokine and an antibody to a costimulatory molecule for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs);

[0026] (c) harvesting the enhanced population of TILs obtained from step (b).

[0027] The disclosure further provides a method for producing tumor-reactive T cells comprising,

[0028] (a) isolating tumor reactive T cells from blood of a subject with cancer;

[0029] (b) culturing the tumor reactive T cells of (a) in a cell culture medium comprising one or more cytokine and an antibody to a costimulatory molecule for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs);

[0030] (c) harvesting the enhanced population of TILs obtained from step (b).

[0031] In various embodiments, the costimulatory molecule is CD28, 41 BB, 0X40, CD40L or ICOS.Docket No. 32757 / 59103

[0032] In various embodiments, the cytokine is interleukin (IL)-2, IL-7, IL-15, IL-21 or combinations thereof.

[0033] In various embodiments, the method comprises a rapid expansion phase in which TILs previously cultured in the presence of cytokine and a CD28 agonist or antibody to a costimulatory molecule are activated to proliferate in the presence of irradiated autologous or allogeneic feeder cells, optionally in culture with anti-CD3 antibody and additional growth factors or cytokines that stimulate T cell growth. In various embodiments, the culture is with a large excess of irradiated autologous or allogeneic feeder cells (such as PBMCs), e.g., 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20- or 25-fold more feeder cells compared to TIL. In various embodiments, the anti-CD3 antibody is OKT3.

[0034] In various embodiments, the rapid expansion phase can take place after harvesting the TIL from culture with cytokine and a CD28 agonist or antibody that binds a costimulatory molecule or before harvesting the TIL from culture with cytokine and a CD28 agonist or antibody that binds a costimulatory molecule.

[0035] In various embodiments, the TILs are cultured in the cell culture medium for 2-6 weeks prior to harvesting.

[0036] In various embodiments, the culturing of tumor reactive T cells is divided into a first expansion phase and a second (or rapid) expansion phase. In various embodiments the duration of the first expansion phase is 2, 3, 4, 5, or 6 weeks. In various embodiments, the duration of the second expansion phase is 2, 3, 4, 5 or 6 weeks. In various embodiments, the duration of the total culturing step(s) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks.

[0037] In various embodiments, the CD28 agonist is a CD28 agonist antibody. In various embodiments, the CD28 agonist antibody is present at a concentration in the range of 1 pg / ml to 10 pg / ml.

[0038] In various embodiments, the method further comprises adding an anti-PD-1 antibody to the second (or rapid expansion) culture medium.

[0039] In various embodiments, the IL-2 is present at a concentration of 100 lU / mL to 10,000 lU / mL.

[0040] In various embodiments, culturing the cells in a cell culture medium comprises spiking the cell culture medium with IL-2 between days 3-6 of culture. In various embodiments, the IL-2 spike comprises the addition of between 100 to 6,000 lU / mL of IL-2.

[0041] In various embodiments, culturing the cells in a cell culture medium comprises exchanging half of the spent cell culture medium with fresh replacement media (50:50 mediaDocket No. 32757 / 59103 change) containing twice the concentration of IL-2 and anti-CD28 agonist, including once a week or twice a week, or more as necessary.

[0042] In various embodiments, the IL-2 and or anti-CD28-agonist in culture is replenished every 3-4 days. In various embodiments, the media comprising IL-2 and or anti- CD28-agonist in culture is changed every 3-4 days.

[0043] In various embodiments, the TILs are analyzed for tumor neoantigen specificity by TCR vBeta and / or TCR vAlpha repertoire sequencing, and / or by functional screening using peptide-pulsed or tandem mini gene transfected APC, or autologous tumor suspension.

[0044] Also provided herein is a composition comprising enhanced TILs produced by the method described herein and a physiologically acceptable excipient.

[0045] Also provided is a population of enhanced TILs produced by the methods of producing enhanced TIL described herein.

[0046] The disclosure further provides tumor-reactive T cells comprising T cells with a phenotype selected from the group consisting of one or more of CD3+, CD4+, CD8+, ybTCR+, CD62L+, CD27+, CCR7+, CD45ro+, or CD45ra+, wherein the tumor reactive T cells are produced by a method comprising the steps of:

[0047] (a) generating a tumor slurry from tumor fragments obtained from a tumor sample or generating tumor fragments from a tumor sample;

[0048] (b) culturing the tumor slurry or tumor fragments in a cell culture medium comprising lnterleukin-2 (IL-2) and an anti-CD28 agonist for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs); and

[0049] (c) harvesting the enhanced population of TILs obtained from step (b).

[0050] Alternatively, the tumor reactive T cells comprising T cells with a phenotype selected from the group consisting of one or more of CD3+, CD4+, CD8+, ybTCR+, CD62L+, CD27+, CCR7+, CD45ro+, or CD45ra+, are produced by a method comprising the steps of:

[0051] (a) isolated tumor reactive T cells from blood of a subject with cancer;

[0052] (b) culturing the tumor slurry or tumor fragments in a cell culture medium comprising one or more cytokine, e.g., lnterleukin-2 (IL-2), and an anti-CD28 agonist or an antibody to a costimulatory molecule for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs); and

[0053] (c) harvesting the enhanced population of TILs obtained from step (b).Docket No. 32757 / 59103

[0054] The disclosure further provides tumor-reactive T cells comprising T cells with a phenotype selected from the group consisting of one or more of CD3+, CD4+, CD8+, y5TCR+, CD62L+, CD27+, CCR7+, CD45ro+, or CD45ra+, wherein the tumor reactive T cells are produced by a method comprising the steps of:

[0055] (a) generating a tumor slurry from tumor fragments obtained from a tumor sample or generating tumor fragments from a tumor sample;

[0056] (b) culturing the tumor slurry in a cell culture medium comprising one or more cytokine and an antibody to a costimulatory molecule for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs); and

[0057] (c) harvesting the enhanced population of TILs obtained from step (b).

[0058] In various embodiments, the cytokine is interleukin (IL)-2, IL-7, IL-15, IL-21 or combinations thereof.

[0059] In various embodiments, the method further comprises a rapid expansion phase in which TILs previously cultured in the presence of cytokine and a CD28 agonist or antibody to a costimulatory molecule are activated to proliferate in the presence of irradiated autologous or allogeneic feeder cells, optionally in culture with anti-CD3 antibody and additional growth factors or cytokines that stimulate T cell growth. In various embodiments, the culture is with a large excess of irradiated autologous or allogeneic feeder cells (such as PBMCs), e.g., 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20- or 25-fold more feeder cells compared to TIL. In various embodiments, the anti-CD3 antibody is OKT3.

[0060] In various embodiments, the rapid expansion phase can take place after harvesting the TIL from culture with cytokine and a CD28 agonist or antibody that binds a costimulatory molecule or before harvesting the TIL from culture with cytokine and a CD28 agonist or antibody that binds a costimulatory molecule.

[0061] Also provided is a method of treating cancer in a subject in need thereof, comprising administering to the subject the composition comprising TIL as described herein or the enhanced TIL cells described herein.

[0062] The disclosure also provides a method of treating cancer in a subject in need thereof, comprising the steps of:

[0063] (a) producing a population of enhanced TIL from a tumor fragment or from blood from the subject using a method described herein;

[0064] (b) treating the subject with nonmyleoablative lymphodepleting chemotherapy; and

[0065] (c) administering the enhanced population of TILs to the subject.Docket No. 32757 / 59103

[0066] In various embodiments, the tumor is a solid tumor.

[0067] In various embodiments, the cancer is selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancers, gastric cancer, glioblastoma, glioma, head and neck cancer, lung cancer, melanoma, mesothelioma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, testicular cancer, thyroid cancer, skin cancer, and uterine cancer. In some embodiments, the cancer is a sarcoma selected from the group consisting of undifferentiated pleomorphic sarcoma, epithelioid sarcoma, liposarcoma, and leiomyosarcoma, lymphoma and myeloma.

[0068] In various embodiments, the tumor is metastatic. In various embodiments, the tumor is recurrent.

[0069] In various embodiments, the administration reduces tumor volume in the subject. In various embodiments, the administration results in partial or full elimination of the tumor in the subject.

[0070] In various embodiments, between 1 x 106- 1 x 1013enhanced TILs cells / ml are administered to the subject to treat cancer.

[0071] It is understood that each feature or embodiment, or combination, described herein is a non-limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “various embodiments”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and / or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination. Such features or combinations of features apply to any of the aspects of the invention. Where examples of values falling within ranges are disclosed, any of these examples are contemplated as possible endpoints of a range, any and all numeric values between such endpoints are contemplated, and any and all combinations of upper and lower endpoints are envisioned.

[0072] The headings herein are for the convenience of the reader and not intended to be limiting. Additional aspects, embodiments, and variations of the invention will be apparent from the Detailed Description and / or Drawings and / or claims.BRIEF DESCRIPTION OF THE DRAWINGSDocket No. 32757 / 59103

[0073] Figure 1 is a graph showing that addition of anti-CD28 antibody to tumor slurries increases tumor outgrowth. The graph provides weekly total cell counts of 25,000 cells from TIL / tumor slurries cultured in the presence of the indicated antibodies or small molecules for 3 weeks.

[0074] Figures 2A-2C provides graph of cell counts after tumor slurry outgrowths following the addition of the indicated molecules. Figure 2A is a bar graph showing the total cell count, CD45+ cell count, CD3+ cell count, CD4+ cell count, and CD8+ cell count of TIL / tumor slurry after 3 weeks of outgrowth with the indicated antibody combinations.Figure 2B is a bar graph showing the percentage of CD45+ cells of the number of total TIL / tumor slurry cells, as well as the percentage of CD3+, CD4+, and CD8+ cells of the number of CD45+ cells. Figure 2C is a column graph of the cell counts of CD4+ cells in conditions that received anti-CD28 agonist antibody vs conditions that did not receive agonist anti-CD28 antibody.

[0075] Figures 3A-3D provide cell counts of different cell populations from tumor slurries from two different donors (Fig. 3A&3B) outgrown in the presence of the agonist antibodies against the indicated costimulatory molecules as well as a control culture. The total cell count (upper left panel), CD45+ cell count (upper middle panel), CD45+CD3+ cell count (upper right panel), CD45+CD3+CD4+ cell count (lower left panel), CD45+CD3+CD8+ cell count (lower middle panel), and CD45+CD56+ cell count (lower right panel) are shown after 1 , 2, and 3 weeks of outgrowth. Frequency of different cell populations from slurries from two different donors (Fig. 3C&3D) outgrown in the presence of the agonist antibodies against the indicated costimulatory molecules as well as a control culture. The frequency of CD45+ of total cells (upper middle panel), the frequency of CD3+ cells of the CD45+ cell population (upper right panel), the frequency of CD4+ cells of the CD45+ cell population (lower left panel), the frequency of CD8+ cells of the CD45+ cell population (lower middle panel), and the frequency of CD56+ cells of CD45+ cell population (lower right panel) are shown after 1 , 2, and 3 weeks of outgrowth.

[0076] Figures 4A-4C provide graphs of cell counts of different cell populations from three different donors (Fig. 4A, 4B & 4C) outgrown in the presence of anti-CD28 agonist antibody, or anti-CD28 agonist antibody & anti-PD1 antagonist antibody, or a no-antibody standard outgrowth control sample. The total cell counts (upper left panel), CD45+ cell count (upper middle panel), CD45+CD3+ cell count (upper right panel), CD45+CD3+CD4+ cell count (lower left panel), CD45+CD3+CD8+ cell count (lower middle panel), and CD45+CD56+ cell count (lower right panel) are shown after 1 , 2, and 3 weeks of outgrowth.Docket No. 32757 / 59103

[0077] Figures 5A-5C provide graphs of the frequency of different cell populations from large-scale slurries from three different donors (Fig. 5A, 5B & 5C) outgrown in the presence of anti-CD28 agonist antibody, or anti-CD28 agonist antibody & anti-PD1 antagonist antibody, or a no-antibody standard outgrowth control sample. Frequency of different cell populations from slurries from three different donors (Fig. 5A-C) outgrown in the presence of the agonist antibodies against the indicated costimulatory molecules as well as a control culture. The frequency of CD45+ of total cells (upper middle panel), the frequency of CD3+ cells of CD45+ cells (upper right panel), the frequency of CD4+ cells of CD45+ cells (lower left panel), the frequency of CD8+ cells of CD45+ cells (lower middle panel), and the frequency of CD56+ cells of CD45+ cells (lower right panel) are shown after 1 , 2, and 3 weeks of outgrowth.

[0078] Figures 6A-6B provide graphs of cell counts of TILs (A) outgrown in the presence or absence of anti-CD28 agonist antibodies as well as HLA-1 and HLA-2 antagonist antibodies. The total cell count (upper left panel), CD45+ cell count (upper middle panel), CD45+CD3+ cell count (upper right panel), CD45+CD3+CD4+ cell count (lower left panel), CD45+CD3+CD8+ cell count (lower middle panel), and CD45+CD56+ cell count (lower right panel) are shown after 1 , 2, and 3 weeks of outgrowth. Frequency of different cell populations from slurry (Fig. 6B) outgrown in the presence or absence of anti-CD28 agonist antibodies as well as HLA-1 and HLA-2 antagonist antibodies. The frequency of CD45+ of total cells (upper middle panel), the frequency of CD3+ cells of CD45+ cells (upper right panel), the frequency of CD4+ cells of CD45+ cells (lower left panel), the frequency of CD8+ cells of CD45+ cells (lower middle panel), and the frequency of CD56+ cells of CD45+ cells (lower right panel) are shown after 1 , 2, and 3 weeks of outgrowth.

[0079] Figure 7 provides scanned images of an enzyme-linked immunospot (ELISPOT) analysis of IFN-y and TNF secreted by outgrown TILs. Wells staining for IFN (red spots) and TNF (blue spots) production of TIL outgrown with anti-CD28 antibody or TIL outgrown with standard procedures cocultured with donor-matched tumor slurry as well as TIL stimulated with anti-CD3, anti-CD28 activation beads, and a no-stim TIL control in triplicate.

[0080] Figures 8A-8C are schematic diagrams illustrating the proposed mechanism by which the anti-CD28 antibody increases outgrowth of neoantigen reactive T cells from tumor slurries. Figure 8A shows standard T cell activation requiring signal 1 and signal 2 (Figure 8A, top left panel), non-activation of non-neoantigen reactive TIL (Figure 8A, middle left panel), non-activation of neoantigen reactive TIL (Figure 8A, lower left panel) as well as non-activation of non-neoantigen reactive TIL in the presence of anti-CD28 antibody (Figure 8B, upper right panel) and activation of neoantigen reactive TIL in the presence of anti-CD28Docket No. 32757 / 59103 antibody (Figure 8B, lower right panel). Figure 8C shows increased activation and outgrowth of neoantigen reactive TIL in the presence of anti-CD28 antibody.

[0081] Figure 9 shows TIL expansion from sarcoma tumor fragments. Fragments derived from a sarcoma were seeded into media containing 10% human serum and 6000 lU / mL IL-2 supplemented with, or without anti-CD28 agonist antibody. Outgrown TIL were counted over 21 days expansion.DETAILED DESCRIPTION

[0082] The present disclosure provides an improved method for isolating and expanding neoantigen-specific tumor infiltrating lymphocytes from a subject to improve cancer therapy.

[0083] Effectively isolating and expanding neoantigen-reactive T cells for patient treatment remains a challenge. Only a fraction of TILs (approximately 1 -10%) propagated by standard TIL isolation methods are tumor reactive. Neoantigens, which are antigens created by somatic mutation, are appealing TIL-targets as they are expressed specifically by tumor and not normal somatic tissues. Moreover, clinical responses have been associated with higher mutational load in the tumor, attributed to an increased frequency of mutation reactive T cells. The frequency of neoantigen-reactive TILs can decline dramatically when cells are expanded ex vivo possibly due to inter-clonal competition and overgrowth of nonreactive cells. The inadequate number of obtained neoantigen-reactive TILs is a limiting factor in ACT therapy.

[0084] Enrichment for tumor-reactive T cells has been achieved by assessing TILs isolated from each tumor fragment for known neoantigen reactivity and only expanding those that demonstrate neoantigen reactivity. However, this process is costly, labor-intensive, and time-consuming, which hampers its scalability and widespread application.I. Overview

[0085] Clinical success of adoptive TIL transfer has been demonstrated in multiple tumor indications; however, the efficacy of TILs varies and only subsets of cancer patients benefit from the treatment. The quality of the transferred cells, particularly with respect to their ability to recognize tumor-specific mutations (neoantigens) i.e., neoantigen-reactive TILs and their memory and effector properties are key determinants of their ability to exert antitumor effects. The expansion of TILs with optimal effector properties and long-term persistence in vivo for strong and durable antitumor responses remains elusive.

[0086] The activation of naive T cells, which leads to clonal expansion, effector and memory T-cell differentiation requires both the stimulation of the T-cell receptor (TCR) by a major histocompatibility complex (MHC)-peptide complex (signal 1) and co-stimulation by coDocket No. 32757 / 59103 stimulatory receptors (signal 2) with their corresponding ligands on antigen-presenting cells (APCs). T cell co-signaling receptors positively (co-stimulatory) or negatively (co-inhibitory) regulate TCR driven signals and therefore T-cell activation. Co-stimulatory signals are required for the activation, expansion and differentiation of naive T cells, notably as they prevent anergy and activation-induced cell death (AICD). The CD28 receptor on T cells is one of the best characterized co-stimulatory molecules. CD28 and its ligand B7-1 or B7-2 on activated APCs amplify TCR signaling interleukin-2 (IL2) production to promote T-cell proliferation and survival during the initial phases of T-cell activation. TNFR-like receptors such as 4-1 BB / CD137 and OX40 / CD134 play a prominent role as co-stimulatory molecules especially at the effector-memory stage.

[0087] In order to provide a mechanism to turn off T cell activation, as T cells are being activated and expanded, the expression of co-inhibitory receptors is upregulated (Schnell et al., 2020. Cell Res. 30(4):285-299). Multiple co-inhibitory receptors have been identified including CTLA-4, PD-1 , TIM-3, TIGIT, and LAG-3. Co-inhibitory receptors play an important role in several T-cell subsets including activated T cells, regulatory T cells, and exhausted T cells (Schnell et al., 2020. Cell Res. 30(4):285-299). In activated T cells, co-inhibitory receptors control and contract the expanded T-cell population and eventually attenuate and terminate T-cell responses (Schnell et al., 2020. Cell Res. 30(4):285-299). In regulatory T cells (Tregs), co-inhibitory receptors, such as CTLA-4 and PD-1 , promote the suppressive function of Tregs (Schnell et al., 2020. Cell Res. 30(4):285-299).

[0088] Accordingly, co-stimulatory pathway activation and coinhibitory pathway blockade ex vivo can increase TIL activation and expansion. For example, an agonistic anti-CD28 antibody can mimic B7 binding, thereby providing the CD28 costimulatory signal while a PD- 1 antagonistic antibody can block the PD-1 co-inhibitory signaling. Disclosed are agents that mimic these antigen-independent second signals to amplify T cell receptor (TCR)-mediated T cell activation ex vivo. Use of such agents can accelerate the rate of TIL expansion out of the tumor fragments and simultaneously enrich the overall yield and frequency of neoantigen-reactive TILs with increased therapeutic efficacy. The present disclosure also provides a method for treating a subject with cancer comprising administering tumor TILs expanded using the disclosed methods.II. Definitions

[0089] Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch,Docket No. 32757 / 59103 and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F. M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

[0090] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0091] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0092] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0093] As used herein, the terms “about” and “approximately” or "comprising essentially of” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "comprising essentially of” can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" or "comprising essentially of” can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of” should be assumed to be within an acceptable error range for that particular value or composition.Docket No. 32757 / 59103

[0094] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo, or cultured ex vivo or in vitro are also encompassed. The term "subject" may also refer to a human who has a tumor into which a population of lymphocytes that have left the human being's bloodstream have migrated and transformed into tumor infiltrating lymphocytes (TILs). In some embodiments, this human may be a patient in need of immunotherapy involving an expanded population of their own TILs (autologous TILs). In other embodiments, the human may be a patient in need of immunotherapy involving an expanded TILs from a person other than the patient (allogeneic TILs).

[0095] The term “in vivo” refers to an event that takes place in a subject's body.

[0096] The term “in vitro” refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0097] The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject's body. The cell, tissue and / or organ may be returned to the subject's body in a method of surgery or treatment.

[0098] As used herein, the phrase “tumor infiltrating lymphocytes” or “TILs” refers to a population of lymphocytes that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells, CD4+ T cells including Th1 and Th17 CD4+ T cells, yd T cells, natural killer T cells, and natural killer (NK) cells. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”). In some embodiments, primary TILs include tumor reactive T cells that are obtained from the peripheral blood of a patient. TIL cell populations can include genetically modified TILs. In some embodiments, the TILs are optionally genetically engineered to include additional functionalities, including, but not limited to, a high-affinity T cell receptor (TCR), e.g., a TCR targeted at a tumor-associated antigen such as MAGE-1 , HER2, or NY-ESO-1 , or a chimeric antigen receptor (CAR) which binds to a tumor-associated cell surface molecule (e.g., mesothelin) or lineage-restricted cell surface molecule (e.g., CD19).Docket No. 32757 / 59103

[0099] TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR op, CD27, CD28, CD56, CCR7, CD45Ra, CD95, 41 BB, CD39, PD-1 , and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs may further be characterized by potency — for example, TILs may be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. TILs may be considered potent if, for example, interferon (IFNy) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, greater than about 1000 pg / mL.

[0100] As used herein, the phrase “population of cells” or “population of TILs” refers to a number of cells or TILs that share common traits. In general, populations generally range from 1 x106to 1 x1010in number, with different TIL populations comprising different numbers. For example, initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of roughly 1 x108cells. REP expansion is generally done to provide populations of 2.0x101° to 1x 1011cells for infusion.

[0101] By “cryopreserved TILs” herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored in the range of about -150eC to -60eC. General methods for cryopreservation are also described elsewhere herein. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs.

[0102] As used herein, the phrase "tumor fragment," refers to small pieces of a tumor obtained by fragmentation of tumor tissue that has been surgically resected from a subject. The terms “fragmenting,” “fragment,” and “fragmented,” are used herein to describe processes for disrupting a tumor. In some embodiments, fragmentation includes mechanical fragmentation, including for example, dissecting, crushing, slicing, dividing, and morcellating of tumor tissue as well as any other method for disrupting the physical structure of tumor tissue. In some embodiments, TILs can be initially cultured from tumor fragments obtained from patients.

[0103] In some embodiments, the tumor is fragmented into 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, or 90 fragments or pieces. In some embodiments the firstDocket No. 32757 / 59103 expansion is seeded by incubating multiple tumor fragments. In one embodiment, the first expansion is seeded with 10, 20, 30, 40 or more tumor fragments or pieces. In another embodiment, the first expansion is seeded with 30 or 40 tumor fragments or pieces. In some embodiments, the multiple fragments comprise about 4 to about 50 fragments.

[0104] In some embodiments, the tumor fragment is between about 1 mm3and 20 mm3. In some embodiments, the tumor fragment is from about 1 mm3and 8 mm3. In some embodiments, the tumor fragment is from about 0.5 mm3to about 4 mm3. In some embodiments, the tumor fragment is about 1 mm3, about 2 mm3, about 3 mm3, about 4 mm3, about 5 mm3, about 6 mm3, about 7 mm3, about 8 mm3, about 9 mm3, about 10 mm3, about 1 1 mm3, about 12 mm3, about 13 mm3, about 14 mm3, about 15 mm3, about 16 mm3, about 17 mm3, about 18 mm3, about 19 mm3, or about 20 mm3.

[0105] As used herein, the phrase “expanding a population of TILs” is synonymous with “proliferating a population of TILs” and refers to increasing the number of cells in a TIL population. The phrase “expansion process” refers to the process whereby the number of cells in a TIL population is increased. Processes where TILs are merely isolated or enriched without substantial increase in the number of TILs are not expansion processes.

[0106] The term “rapid expansion” or “rapid expansion phase” means an increase in the number of tumor reactive TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of 1 -2 weeks, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of 1 -2 weeks, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are described herein. In various embodiments, culturing the cells in a cell culture medium comprises exchanging the media during culture to renew nutrients. In various embodiments, approximately one half or two thirds of the cell culture medium is exchanged with fresh culture medium comprising IL-2 and anti-CD28 agonist every 3-4 days.

[0107] As used herein, “T cell activation” refers to a process (e.g., during REP) in which T cells are activated by major histocompatibility complex (MHC) mismatch when their receptors (TCRs) interact with allogeneic MHC molecules on the irradiated allogeneic feeder cells used during REP. Alternatively, T cells can be activated by anti-CD3 mAbs.

[0108] The term "activation" and its grammatical equivalents as used herein can refer to a process whereby a cell transitions from a resting state to an active state. This process can comprise a response to an antigen, migration, and / or a phenotypic or genetic change to a functionally active state. For example, the term "activation" can refer to the stepwise process of T cell activation. For example, a T cell can require at least two signals to become fully activated. The first signal can occur after engagement of a TCR by the antigen-MHCDocket No. 32757 / 59103 complex, and the second signal can occur by engagement of co-stimulatory molecules. An anti-CD3 agonistic antibody can mimic the first signal and an anti-CD28 agonistic antibody can mimic the second signal in vitro. Activated T cells can express antigen-specific T cell receptors on their surface to recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating the cell-based functions of the immune system.

[0109] The term “antigen” or “Ag” as used herein refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.

[0110] The term “tumor-associated antigens” or “TAAs” as used herein refers to antigen molecules that are overexpressed in tumor cells compared to the corresponding normal tissue. The human tumor-associated antigens (TAAs) include overexpressed cellular antigens (such as HER2, renal antigen 1 (RAGE-1), carbonic anhydrase IX (CAIX)), which are mainly involved in the survival of the cancer cells, differentiation antigens (such as melanocyte differentiation antigens e.g., tyrosinase, Melan-A / MART-1 , gp100 / Pmel17), mutational antigens (such as p53), viral antigens (such as human papillomavirus proteins), and the cancer / testis (CT) antigens that are expressed in germ cells of the testis and ovary but are silent in normal somatic cells (such as melanoma antigen family A,1 (MAGEA1) and New York esophageal squamous cell carcinoma 1 (NY-ESO 1)).

[0111] The term “neoantigen” as used herein refers to an antigen that arises in a subject’s tumor, and is unique to the cancer cells (i.e., it is absent in the subject's corresponding normal cells or tissue). A neoantigen is a new protein that forms on cancer cells when certain mutations occur in tumor DNA, e.g., somatic mutations, transcriptomic variants, RNA splicing, post-translational modifications (PTMs), and integrated viral open reading frames for virally associated tumors. See e.g., Xie, et al. Sig Transduct Target Ther 8, 9 (2023). Neoantigens differ from TAAs in that TAAs are not unique to tumor tissue as they are also present in normal tissues albeit at a lower expression level. Emerging evidence has suggested that neoantigens play a critical role in tumor-specific T cell-mediated antitumor immune response and successful cancer immunotherapies. Neoantigens are an ideal immunotherapy target owing to their strong immunogenicity and lack of expression in normal tissues. The terms “neoantigen,” “tumor-specific mutation,” “tumor-specific neoantigen” and “tumor-specific antigen” or “TSA” are used interchangeably herein.

[0112] The term “agonist” refers to a chemical, a molecule, a macromolecule, a complex of molecules, or a complex of macromolecules that binds to a target, either on the surface of a cell or in soluble form. In certain embodiments, when an agonist binds to a target on theDocket No. 32757 / 59103 surface of a cell, the agonist triggers (e.g., initiates or promotes), partially or fully enhances, stimulates, or activates one or more biological activities. Agonists include, for example, hormones, neurotransmitters, antibodies, or antigen binding fragments thereof.

[0113] The term “antagonist” refers to a molecule which blocks (e.g., reduces or prevents) a biological activity.

[0114] As used herein, the term “ligand” refers to a molecule that forms a complex with a biomolecule (e.g., a receptor) to serve a biological purpose. In a narrower sense, it is a signal triggering molecule, binding to a site on a target protein. The binding occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and van der Waals forces. The docking (association) is usually reversible (dissociation). Actual irreversible covalent binding between a ligand and its target molecule is rare in biological systems. Ligand binding to a receptor alters the receptor’s conformation. The conformational state of a receptor protein determines its functional state.

[0115] The term “antibody,” as used herein, refers to an immunoglobulin molecule, or a fragment thereof, with the ability to bind an antigen, and includes monoclonal antibodies, polyclonal antibodies, multivalent antibodies, chimeric antibodies, multispecific antibodies, diabodies and fragments or parts of an immunoglobulin molecule or combinations thereof that have the ability to specifically bind to the antigenic region of another molecule with the desired affinity.

[0116] The phrase "specifically binds," as used herein with respect to an antibody, is meant an antibody which recognizes and binds to a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.

[0117] In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally.

[0118] The methods of the present disclosure may involve use of forms of antibody including, for example, polyclonal antibodies, monoclonal antibodies, humanized antibodies,Docket No. 32757 / 59103 a Fab, Fab', F(ab')2, Fv, a single-chain antibody (scFv) or a polypeptide that contains at least a portion of an immunoglobulin (or a variant of an immunoglobulin) that is sufficient to confer specific antigen binding, such as a molecule including one or more Complementarity Determining Regions (CDRs) (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0119] Agonistic antibodies directed towards co-stimulatory pathways and antagonistic antibodies directed towards co-inhibitory pathways can directly enhance T cell activation, expansion, and survival as well as increase effector numbers, functions or persistence.

[0120] In various embodiments, the co-stimulatory signal is provided by agonist antibodies that bind CD28, ICOS, HVEM, CD27, DR3, CD30, CD2, 2B4, CD226, CD40L, Glucocorticoid-Induced TNFR-Related (GITR), CD40, CD137 (also known as 4-1 BB), CD134 (also known as 0X40) or a combination thereof.

[0121] In certain embodiments, the antibody (or an antigen binding fragment or part thereof) is reactive to one or more of PD1 , CD3, CD28, CD5, CD2, CD44, CD137, CD95, CD278, an alpha integrin or a beta integrin and isoforms thereof. In certain embodiments, the antibody is an anti-CD3 agonistic antibody, an anti-CD28 agonistic antibody, an anti-PD1 antagonistic antibody or fragments thereof. In other embodiments, the antibody can bind NKG2D. Antibodies to the aforementioned molecules may be produced by any method known in the art or are commercially available, such as anti- human CD3 (Affymetrix eBioscence; Cat. No: 16-0039-81 ) and anti-human CD28 (BD Pharmingen Cat. No: 555725).

[0122] The term “CD28,” as used herein, refers to a protein which is expressed on T cells that provides costimulatory signals required for T cell activation and survival. In addition to the T Cell Receptor (TCR), the ligation of CD28 and its ligands CD80 / CD86 on the APC provides a critical costimulatory signal for T cell activation and the production of various interleukins, particularly IL-6. The CD28 costimulatory signal can also be delivered by the interaction between CD28 and its agonist antibody. Human CD28 comprises the amino acid sequence as set forth in NCBI accession No. NP 006130.1. Soluble CD28 includes natural CD28 proteins as well as recombinant CD28 protein variants such as, e.g., monomeric and dimeric CD28 constructs, that lack a transmembrane domain or are otherwise unassociated with a cell membrane.

[0123] As used herein, the expression “cell surface-expressed CD28” means one or more CD28 protein(s) that is / are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CD28 protein is exposed to the extracellular side of the cell membraneDocket No. 32757 / 59103 and is accessible to an antigen-binding portion of an antibody. "Cell surface-expressed CD28" includes CD28 proteins contained within the context of a functional T cell costimulatory receptor in the membrane of a cell. The expression "cell surface-expressed CD28" includes CD28 protein expressed as part of a homodimer on the surface of a cell. A "cell surface-expressed CD28" can comprise or consist of a CD28 protein expressed on the surface of a cell which normally expresses CD28 protein. Alternatively, "cell surface- expressed CD28" can comprise or consist of CD28 protein expressed on the surface of a cell that normally does not express human CD28 on its surface but has been artificially engineered to express CD28 on its surface.

[0124] As used herein, “an antibody that binds CD28” or an “anti-CD28 antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize a monomeric CD28, as well as antibodies and antigen-binding fragments thereof e.g., Fab and F(ab')2 fragments that specifically recognize a dimeric CD28. The antibodies and antigen-binding fragments may bind soluble CD28 and / or cell surface expressed CD28.

[0125] The term “agonistic anti-CD28 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD28 receptor, thus mimicking the costimulatory signal. A number of antibodies which bind human CD28 have been described in the art or they may be produced by methods known in the art. These can be broadly classified as either conventional (agonistic) anti-CD28 antibodies, or non-conventional (super-agonistic) anti- CD28 antibodies (Tacke et al., 1997. Eur J Immunol. 27(1):239-247; Luhder et al., 2003. J Exp Med. 197:955-966).

[0126] Combination of agonistic antibodies against both the TCR / CD3 complex and CD28 are sufficient to fully activate T cells through cross-linking and therefore can replace MHC and CD80 / 86 signaling, respectively. Conventional anti-CD28 mAbs bind close to the natural binding site of CD80 / CD86 and provide co-stimulation only in the presence of TCR / CD3 signaling (Luhder et al., 2003. J Exp Med. 197:955-966).

[0127] Conversely, super-agonistic anti-CD28 mAbs bind to the laterally exposed C”D loop of CD28 and can fully activate T cells without the need for TCR / CD3 complex engagement (Tacke et al., 1997. Eur J Immunol. 27(1):239-247; Luhder et al., 2003. J Exp Med. 197:955-966). In rat models, this unusual class of antibodies induces potent proliferation of T cells without clear toxicity (Tacke et al., 1997. Eur J Immunol. 27(1):239- 247; Rodnguez-Palmero et al., 1999. Eur J Immunol. 29:3914-3392). At low doses, only regulatory T cells (Tregs) were activated, while at high doses both Tregs and conventional T cells were expanded and therefore preferential activation of T cells at different doses wasDocket No. 32757 / 59103 hypothesized (Lin CH, Hunig T. 2003. Eur J Immunol. 33:626-638.; Beyersdorf et al., 2005. J Exp Med. 202:445—455).

[0128] Accordingly, anti-CD28 agonistic antibodies or fragments thereof useful in stimulating proliferation of TILs in the disclosed methods include but are not limited to the anti-human CD28 super agonistic monoclonal antibody TGN1412 a humanized lgG4k antibody, anti-CD28 monoclonal antibody clone 10F3, anti-human CD28 antibody clone 15E8 an lgG1 antibody, CD28.2 monoclonal antibody clone CD28.2, monoclonal antibody clone 9.3, an lgG2a antibody (Bristol Myers Squibb Corporation, Seattle, Wash.), monoclonal antibody clone KOLT-2, an lgG1 antibody, 248.23.2, an IgM antibody and EX5.3D10, an lgG2a antibody, antiCD28 clone L293, an lgG1 antibody, as well as polyclonal antibodies.

[0129] CD134, also known as 0X40, is a member of the TNFR receptor superfamily and unlike CD28, it is not constitutively expressed on resting naive T cells. 0X40 is a secondary costimulatory molecule, expressed after 24 to 72 hours after activation; its ligand OX40L is also not expressed on resting antigen-presenting cells, but rather after its activation. Expression of 0X40 is dependent on complete activation of T cells; without CD28, expression of 0X40 was delayed and levels decreased four-fold.

[0130] CD137, also known as 4-1 BB, is a member of the Tumor Necrosis Factor (TNF) receptor family. CD137 may be expressed by activated T cells, but to a greater extent on CD8 than on CD4T cells. In addition, CD137 expression was found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. The most characteristic activity of CD137 is its co-stimulatory activity on activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion survival and cytolytic activity.

[0131] As used herein, the phrase “T cell-stimulating cytokine” refers to a cytokine that stimulates and / or activates T cell lymphocytes. In some embodiments, the T-cell stimulating cytokine is IL-2, IL-7, IL-15 or IL-21 . In certain embodiments, T cell-stimulating cytokines are produced in a cell from a viral vector.

[0132] As used herein, the term “IL-2” (also referred to herein as “IL2”) refers to the cytokine known as interleukin-2. IL-2 was first described in the 1980s as a T cell growth factor based on its ability to promote the growth of lymphocytes in vitro. IL-2 is a pleiotropic cytokine that plays essential roles in key functions of the immune system, tolerance and immunity, primarily via its direct effects on T cells. The cytokine mediates its effects by binding to IL-2 receptors, which are expressed by lymphocytes. IL-2 enhances activation- induced cell death (AICD). IL-2 also promotes the differentiation of T cells into effector TDocket No. 32757 / 59103 cells and into memory T cells when the initial T cell is also stimulated by an antigen, thus playing a role in immune activation. Together with other polarizing cytokines, IL-2 stimulates naive CD4+ T cell differentiation into Th1 and Th2 lymphocytes while it impedes differentiation into Th17 and follicular Th lymphocytes. Its expression and secretion are tightly regulated and functions as part of both transient positive and negative feedback loops in mounting and dampening immune responses. Through its role in the development of T cell immunologic memory, which depends upon the expansion of the number and function of antigen-selected T cell clones, IL-2 plays a role in enduring cell-mediated immunity. Human IL-2 is a 15.5-16 kDa glycoprotein whose gene is identified by NCBI Gene ID 3558. An exemplary nucleotide sequence for a human IL2 gene is the NCBI Reference Sequence: NG_016779.1.

[0133] IL-2 includes all forms of IL-2, including human and mammalian forms, forms with conservative amino acid substitutions, glycoforms, biosimilars, mimetics, and variants thereof. IL-2 is described, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference in their entireties. The term IL-2 encompasses human, recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1 , serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The term IL-2 also encompasses pegylated forms of IL-2, including the pegylated IL-2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, Calif., USA. NKTR-214 and pegylated IL-2 suitable for use in the disclosure is described in U.S. Patent Application Publication No. US 2014 / 0328791 A1 and International Patent Application Publication No. WO 2012 / 065086 A1 , the disclosures of which are incorporated herein by reference in their entireties. Alternative forms of conjugated IL-2 suitable for use in the disclosure are described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated herein by reference in their entireties.Formulations of IL-2 suitable for use in the methods of the disclosure are described in U.S. Pat. No. 6,706,289, incorporated herein by reference in its entirety.

[0134] A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent, such as the TILs of the disclosure, is any amount of TILs that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity ofDocket No. 32757 / 59103 disease symptoms, an increase in frequency and duration of disease symptom- free periods, amelioration of one or more symptoms of disease, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0135] By way of example, an anti-cancer agent promotes cancer regression in a subject. In preferred embodiments, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administering an effective amount of the drug, alone or in combination with an anti- neoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a decrease in severity of at least one disease symptom, an increase in frequency and duration of disease symptom- free periods, or a prevention of impairment or disability due to the disease affliction. In addition, the terms "effective" and "effectiveness" with regard to a treatment includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the drug to promote cancer regression in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and / or organism level (adverse effects) resulting from administration of the drug.

[0136] In some embodiments, therapeutically effective amount of the treatment prevents the development of a cancer, reduces the symptoms of cancer, inhibits the growth of an established cancer, prevents metastasis and / or invasion of an existing cancer, promotes or induces regression of the cancer, inhibits or suppresses the proliferation of cancerous cells, and / or reduces angiogenesis or increases the amount of apoptotic cancer cells. Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, lessening in severity or amelioration of one or more symptoms of disease. In various embodiments, a therapeutically effective amount of the treatment inhibits cell growth, tumor growth or tumor volume by at least about 20%, at least about 40%, at least about 50%, at least about 60%, or at least about 80% relative to untreated subjects. In various embodiments, the treatment leads to a durable, complete response (CR). In various embodiments of the disclosure, tumor regression may be observed and continue for a period of at least about 3 months, 6 months, 9 months, 1 year, 18 months, 2 years, 36 months, 4 years or more. Notwithstanding these ultimate measurements of therapeutic effectiveness, evaluation of immunotherapeutic drugs must also make allowance for "immune-related" response patterns.Docket No. 32757 / 59103

[0137] An "immune-related" response pattern refers to a clinical response pattern often observed in cancer patients treated with immunotherapeutic agents that produce antitumor effects by inducing cancer-specific immune responses or by modifying native immune processes. This response pattern is characterized by a beneficial therapeutic effect that follows an initial increase in tumor burden or the appearance of new lesions, which in the evaluation of traditional chemotherapeutic agents would be classified as disease progression and would be synonymous with drug failure. Accordingly, proper evaluation of immunotherapeutic agents may require long-term monitoring of the effects of these agents on the target disease.

[0138] A therapeutically effective amount of a drug includes a "prophylactically effective amount," which is any amount of the drug that, when administered alone or in combination with an anti-neoplastic agent to a subject at risk of developing a cancer (e.g., a subject having a pre-malignant condition) or of suffering a recurrence of cancer, inhibits the development of or recurrence of the cancer. "Inhibiting" the development or recurrence of a cancer means either lessening the likelihood of the cancer's development or recurrence, or preventing the development or recurrence of the cancer entirely.III. Methods and Assays

[0139] The present disclosure provides a method for producing tumor-reactive T cells. In one embodiment, the method comprises one or more or all of the following steps, (a) generating a tumor slurry from tumor fragments from a tumor sample, or generating tumor fragment from a tumor sample; (b) culturing the tumor slurry or tumor fragments in a cell culture medium comprising lnterleukin-2 (IL-2) and / or a costimulatory agonist, e.g., an anti- CD28 agonist, for a time sufficient to obtain a population of tumor infiltrating lymphocytes (TILs); (c) harvesting the enhanced population of TILs obtained from step (c).A. Tumor Processing

[0140] As outlined herein, TILs are generally taken from a patient tumor sample ("primary TILs") and manipulated to expand their number prior to transplant into a patient. In some embodiments, the TILs may be optionally genetically manipulated as discussed below.

[0141] A patient tumor sample may be obtained using methods known in the art for obtaining a sample that contains a mixture of tumor and TIL cells. In some embodiments, the sample may be obtained by non-invasive methods including but not limited to scraping of the skin or cervix, or swabbing of the cheek. In other embodiments, the sample is obtained by an invasive procedure including but not limited to surgical resection, biopsy, alveolar or pulmonary lavage, needle aspiration, or phlebotomy. The method of biopsy may further include incisional biopsy, excisional biopsy, punch biopsy, shave biopsy, or skin biopsy. TheDocket No. 32757 / 59103 method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some embodiments, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material. Generic methods for obtaining biological samples are also known in the art and further described in for example Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001 which is herein incorporated by reference in its entirety.

[0142] For the “infiltrating” tumor cells, the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. For tumor reactive T cells, the tumor sample is a liquid tumor, such as a tumor obtained from a hematological malignancy.

[0143] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign or malignant. Malignant solid tumors or cancers include, but are not limited to bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancers, gastric cancer, glioblastoma, glioma, head and neck cancer, lung cancer, melanoma, mesothelioma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, testicular cancer, thyroid cancer, skin cancer, and uterine cancer. In some embodiments, the cancer is a sarcoma selected from the group consisting of undifferentiated pleomorphic sarcoma, epithelioid sarcoma, liposarcoma, and leiomyosarcoma. In some embodiments, the cancer is a hematopoietic cancer such as, lymphoma and myeloma. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed and which may provide a supporting microenvironment.

[0144] The term "hematological malignancy" refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and nonHodgkin's lymphomas. The term "B cell hematological malignancy" refers to hematological malignancies that affect B cells.

[0145] In some embodiments, a TIL can be isolated from an organ afflicted with a cancer. One or more cells can be isolated from an organ with a cancer that can be a brain, heart, lungs, eye, stomach, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails,Docket No. 32757 / 59103 ears, glands, nose, mouth, lips, spleen, gums, teeth, tongue, salivary glands, tonsils, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid gland, thymus gland, bones, cartilage, tendons, ligaments, suprarenal capsule, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureter, urethra, hypothalamus, pituitary, pylorus, adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands, testes, seminal vesicles, penis, prostate, cervix, lymph, lymph nodes or lymph vessels.

[0146] Once obtained, the tumor sample is generally fragmented using sharp dissection into small pieces referred herein as “tumor fragments.” In one embodiment, the tumor fragments are less than 20 mm3. In some embodiments, the tumor fragment is from about 1 mm3and 8 mm3. In some embodiments, the tumor fragment is from about 0.5 mm3to about 4 mm3. In some embodiments, the tumor fragment is about 1 mm3, about 2 mm3, about 3 mm3, about 4 mm3, about 5 mm3, about 6 mm3, about 7 mm3, about 8 mm3, about 9 mm3, about 10 mm3, about 11 mm3, about 12 mm3, about 13 mm3, about 14 mm3, about 15 mm3, about 16 mm3, about 17 mm3, about 18 mm3, about 19 mm3, or about 20 mm3. Optionally, the tissue sample is dissected with a scalpel.

[0147] In some embodiments, tumor fragmentation may initially be done mechanically (e.g., by dissection) in the presence of a tumor collection media to generate a “tumor slurry” and optionally followed by enzymatic digestion of the tumor fragments into a single cell suspension. As used herein, the term “medium” or “media” refers to a liquid or gel designed to support the survival, growth, and / or proliferation of cells in an artificial environment. A medium generally comprises a defined set of components. Such components may include an energy source, growth factors, hormones, stimulants, activators, sugars, salts, vitamins, and / or amino acids, and / or a combination of these. In many embodiments, the medium is cell culture medium.

[0148] Mechanical means of separating cells which are attached to one another (e.g., in a tumor fragment) include but are not limited to trituration through a narrow bore pipette (Reynolds, B. A., & Weiss, S. 1992. Science. 255(5052):1707-1710; Sen et al., 2001. J Cell Biochem. 81 (2):312-319), fine needle aspiration (Ottesen et al., 1996. Cytometry. 26(1 ):65- 68), vortex disaggregation (Vos et al., 2003. Cytometry B Clin Cytom. 52(1 ):20-31 ) and forced filtration through a fine nylon or stainless steel mesh. Whereas all these methods are effective in creating single cell suspensions, the excessive physical forces involved often result in a significant amount of cell death and cell damage. In situations where the generation of a suspension of viable single cells is the ultimate goal, cell death and cell damage are extremely undesirable.Docket No. 32757 / 59103

[0149] To avoid the negative consequences of mechanical dissociation, enzymes (either alone or in combination), can be used to dissociate or digest primary tissues and aggregates. The dissociation or digestion enzymes include but are not limited to collagenases (e.g., collagenases type I, II, III, IV, and others), elastase, dispase, trypsin, trypsinogen, chymotrypsinogen, accutase, papain, thermolysin, TrypLE™, hyaluronidase, chymotrypsin, neuraminidase, pancreatin, neutral protease, pronase, liberase, clostripain, caseinase, neutral protease (Dispase), DNAse, RNase, and protease XIV.

[0150] In some embodiments, the tumor fragments are digested using ready-to-use cell dissociation solutions. Examples of ready-to-use cell dissociation solutions include but are not limited to ACCUMAX™ (Innovative Cell Technologies, Inc., San Diego, Calif.), ACCUTASE® (Innovative Cell Technologies, Inc., San Diego, Calif.), and LIBERASE™ (Roche).

[0151] In one embodiment, TILs are cultured directly from the tumor fragments. In another embodiment, the tumor fragments may be subjected to an initial mild pretreatment with dissociation enzymes before the immersion of these fragments in culture medium.Such pretreatment does not aim at an extensive dissociation of the tissue, but only at a small relaxation of tissue coherence to facilitate the movement of TILs when immersed in the culture medium.

[0152] In an alternative embodiment TILs are cultured from the single-cell suspensions obtained through mechanical fragmentation followed by the complete dissociation of the tumor fragments by enzymatic digestion. After dissociation, the resulting cell suspension may be subject to additional separation techniques to remove contaminating cells such as red blood cells.

[0153] The term “digestion media,” as used herein refers to cell culture medium that contains tissue dissociation enzymes. The term “tumor digests” refers to tumor fragments that have been subjected to partial or complete enzymatic dissociation.

[0154] In one embodiment, the digestion media comprises one or more dissociation enzymes in Roswell Park Memorial Institute (RPMI) 1640 (Complete Media (CM)), 10% Human AB serum, 25 mM HEPES, 10 pg / mL Gentamicin, 1x Penicillin / Streptomycin (Pen / Strep), 150 pL Antibiotic-Antimycotic (100X), and digestion enzymes.

[0155] In some embodiments, the digestion media comprises a combination of at least 2, at least 3, at least 4, or at least 5 digestion enzymes.

[0156] In one embodiment, the tumor fragments are incubated in a digestion media containing collagenase, dispase and hyaluronidase.Docket No. 32757 / 59103

[0157] In one embodiment, the tissue sample is enzymatically disaggregated for about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes or about 90 minutes.

[0158] In various embodiments, the tumor digests are subjected to one or more rounds of mechanical dissociation or disaggregation to produce a tumor slurry.

[0159] In one embodiment, the mechanical disaggregation of the tumor digests is performed via pipette trituration, wherein the tumor digests are repeatedly passed through a pipette tip, e.g., a plastic pipette tip. Here, the inner diameter of a pipette tip is utilized for separation of the tumor digests and progressively smaller pipette tips are used to dissociate the tumor digests into single cells, e.g., 25 ml pipette tips, 10 ml pipette tips, 5 ml pipette tips, and 1 ml pipette tips are used to dissociate the tumor sample. In some cases, the pipette gauge diameter is progressively decreased for better disaggregation of tissue. In this manner, the tissue size is reduced to a single cell suspension. Optionally, the tumor sample stays within the same tube, i.e., only the pipette tip itself is exchanged for one with a smaller diameter. In this manner, cell loss is reduced. Alternatively (or in addition), shredding spikes (i.e., teeth) are placed within a pipette tip for better disaggregation of tissue. Subsequently, red blood cells are removed, the sample is filtered, and single cells are isolated.

[0160] Optionally, the method further comprises filtering the tissue sample and discarding residual cell clumps and undigested tissue chunks. Suitable filter sizes include 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 105 pm, 110 pm, 115 pm, 120 pm, 125 pm, 130 pm, 135 pm, 140 pm, 145 pm, and 150 pm. For example, the tissue sample is filtered with a 70 pm cell strainer or a 100 pm cell strainer.B. Initial Expansion Phase

[0161] After the sample processing described above, the resulting tumor fragments, tumor slurry, or the single cell suspensions are cultured under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, the culture is done in two expansion phases. In some embodiments, this TIL expansion phase is also referred to as a first expansion, or a first TIL expansion and this can include an expansion step referred to as pre-REP as described below and herein.

[0162] In some embodiments, during pre-REP, tumor fragments or cells from tumor fragments are grown in a culture medium comprising standard lab media (including without limitation RPMI 1640) supplemented with reagents such as T cell costimulatory molecules, agonists to T cell costimulatory molecules, antagonists to coinhibitory molecules, and / or TDocket No. 32757 / 59103 cell-stimulating cytokines to achieve a desired effect, such as an increase in the number of TILs and / or an enrichment of the population for cells containing desired cell surface markers or other structural, biochemical or functional features. Pre-REP may utilize lab grade reagents (under the assumption that the lab grade reagents get diluted out during a later REP stage), making it easier to incorporate alternative strategies for improving TIL production.

[0163] In some embodiments, the first expansion during pre-REP is performed in a closed system bioreactor, such as G-REX-10 or a G-REX-100. In some embodiments, the first expansion is performed in GRex 24-well cell culture plates.

[0164] In embodiments where TIL cultures are initiated in 24-well plates, for example, using GRex 24-well cell culture plates, (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA) each well can be seeded with about 6x106cells from the tumor slurry / single cell suspension or one small tumor fragment (~2 mm3) in 2 mL culture medium comprising complete medium (CM) and inactivated human AB serum containing T cell costimulatory molecules , antagonists to coinhibitory molecules, T cell-stimulating cytokines. In some embodiments, the tumor fragment is between about 0.5 mm3and 10 mm3. In some embodiments, multiple tumor fragments are included in a single well. The cultures are maintained at cell concentrations from 5x105to 2x106cells per mL until several million TIL cells are available, usually in 2-6 weeks.

[0165] In some embodiments, the culture medium comprises one or more T cellstimulating cytokines selected from IL-2, IL- 7, IL-15, IL-21 , and combinations thereof under conditions that favor the growth of TILs over tumor and other cells.

[0166] In some embodiments, the culture medium comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rh IL-2). In some embodiments, the first culture medium comprises IL-2 at a concentration of about 100 lU / mL, about 500 lU / mL, about 1 ,000 lU / mL, about 2,000 lU / mL, about 3,000 lU / mL, about 4,000 lU / mL, about 5000 lU / mL or about 10,000 lU / ml.

[0167] In some embodiments, the culture medium further comprises T cell costimulatory molecules, agonists to T cell costimulatory molecules, antagonists to coinhibitory molecules or a combination thereof. In one embodiment, T cell co-stimulatory molecules and antagonists to coinhibitory molecules are agonist and antagonist antibodies respectively, or fragments thereof.

[0168] In various embodiments, the culture medium comprises an anti-CD28 agonistic antibody. In some embodiments, the anti-CD28 agonistic antibody in the cell culture medium is utilized at a final concentration of about 5 pg / mL or less, such as about 4.9 pg / mL or less,Docket No. 32757 / 59103 about 4.85 pg / mL or less, about 4.8 pg / mL or less, about 4.75 pg / mL or less, about 4.7 pg / mL or less, about 4.65 pg / mL or less, about 4.6 pg / mL or less, about 4.55 pg / mL or less, about 4.5 pg / mL or less, about 4.45 pg / mL or less, about 4.4 pg / mL or less, about 4 pg / mL or less, about 3.9 pg / mL or less, about 3.85 pg / mL or less, about 3.8 pg / mL or less, about 3.75 pg / mL or less, about 3.7 pg / mL or less, about 3.65 pg / mL or less, about 3.6 pg / mL or less, about 3.55 pg / mL or less, about 3.5 pg / mL or less, about 3.0 pg / mL or less, about 2.9 pg / mL or less , about 2.85 pg / mL or less, about 2.80 pg / mL or less, about 2.75 pg / mL or less, about 2.7 pg / mL or less, about 2.65 pg / mL or less, about 2.6 pg / mL or less, about 2.55 pg / mL or less, about 2.50 pg / mL or less, about 2.45 pg / mL or less, about 2.4 pg / mL or less, about 2.35 pg / mL or less, about 2.3 pg / mL or less, about 2.25 pg / mL or less, about 2.2 pg / mL or less, about 2.15 pg / mL or less, about 2.1 pg / mL or less, about 2.0 pg / mL or less, about 1 .9 pg / mL or less, about 1 .85 pg / mL or less, about 1 .8 pg / mL or less, about 1 .75 pg / mL or less, about 1 .70 pg / mL or less, about 1 .65 pg / mL or less, about 1 .60 pg / mL or less, about 1 .55 pg / mL or less, about 1 .50 pg / mL or less, about 1 .45 pg / mL or less, about 1 .40 pg / mL or less, about 1 .35 pg / mL or less, about 1 .30 pg / mL or less, about 1 .25 pg / mL or less, about 1 .2 pg / mL or less, about 1.15 pg / mL or less , about 1 .0 pg / mL or less, about 0.9 pg / mL or less, about 0.85 pg / mL or less, about 0.8 pg / mL or less, about 0.75 pg / mL or less, about 0.7 pg / mL or less, about 0.65 pg / mL or less, about 0.6 pg / mL or less, about 0.55 pg / mL or less, about 0.5 pg / mL or less, about 0.45 pg / mL or less, about 0.4 pg / mL or less, about 0.35 pg / mL or less, about 0.3 pg / mL or less, about 0.25 pg / mL or less, about 0.2 pg / mL or less, about 0.15 pg / mL or less, about 0.1 pg / mL or less, or about 0.05 pg / mL or less. In various embodiments of the disclosure, the final concentration of the anti-CD28 agonistic antibody is from about 1 .5 pg / mL to 2.5 pg / mL.

[0169] In some embodiments, the culture medium utilized in the described methods is changed at a time interval selected from 3 days, 4 days, 5 days, and 6 days. In one embodiment, 30% to 99% of the culture medium is changed at a time interval selected from 3 days, 4 days, 5 days, and 6 days.

[0170] In some embodiments, feeding the cells comprises a ratio change of the old media with fresh media to allow the cells to acclimate to the difference in nutrient content. In one embodiment, the media change is performed at a ration selected from, 75:25, 50:50, or 25:75. In one embodiment 30% to 99% of the first culture medium is changed. In one embodiment 50% of the media is changed every 3-4 days. In various embodiments, culturing the cells in a cell culture medium comprises exchanging half of the spent cell culture medium with fresh replacement media (50:50 media change) containing twice the concentration of IL2 and anti-CD28 agonist, e.g. once or twice a week. Those of skill in the art appreciate that the frequency of the media change depends on cell culture type cellDocket No. 32757 / 59103 density and volume of medium used per unit of surface. In one embodiment, the old media is changed with complete media comprising IL-2 and / or a costimulatory agonist, such as an CD28 agonist. In one embodiment, the CM for the media change is supplemented with IL-2 at a concentration of about 100 lU / mL, about 250 lU / mL, about 500 lU / mL, about 1 ,000 lU / mL, about 2,000 lU / mL, about 3,000 lU / mL, about 4,000 lU / mL, about 5000 lU / mL. or about 10,000 lU / ml.

[0171] In some embodiments, the first TIL expansion can proceed for between 7 and 42 days, e.g., 7 days, 14 days, 21 days, 28 days, or 42 days. In some embodiments, the first TIL expansion can proceed for 14 days to 21 days. In some embodiments, the first TIL expansion can proceed for 14 days to 28 days. In some embodiments, the first TIL expansion can proceed for 28 days to 42 days. In some embodiments, the first TIL expansion can proceed for 28 days. In some embodiments, the first TIL expansion can proceed for 42 days.

[0172] In various embodiments, the first TIL expansion results in 1 x106to 1 x107TILs, such as 2x106to 5x106TILs. In various embodiments, the expansion results in 5x106to 1 x107cells. In various embodiments, the expansion results in 1 x106to 5x107TILs. In various embodiments, the expansion results in 1.0x108to 1 x101° TILs.

[0173] In some embodiments, the bulk TIL population obtained from the first expansion, can be cryopreserved immediately, using the protocols discussed herein. In other embodiments, the TIL population obtained from the first expansion can be subjected to a second expansion (which can include expansions sometimes referred to as REP). In some embodiments, the TILs obtained from the first expansion are not stored and proceed directly to the second expansion or REP.

[0174] The expression “bulk TIL population,” as used herein refers to all TILs that are not otherwise enriched or purified.C. The Second TIL Expansion

[0175] In a multi-step TIL production, the TIL cell population obtained in the first expansion (i. e., pre-REP) is further expanded in number in a second expansion, or rapid expansion phase. This second expansion can include expansion processes generally referred to in the art as a rapid expansion protocol (REP). The second expansion or REP is generally accomplished using a cell culture medium comprising a number of components; a cytokine source, T cell costimulatory molecules, agonists to T cell costimulatory molecules, antagonists to coinhibitory molecules, and T cell-stimulating cytokines. In various embodiments, the second or rapid expansion comprises culture of TILs previously cultured in the presence in of cytokine and a CD28 agonist in the presence of a large excess ofDocket No. 32757 / 59103 irradiated autologous or allogeneic feeder cells (e.g., PBMC), optionally in culture with anti- CD3 antibody and additional growth factors or cytokines that stimulate T cell growth. In various embodiments, the second or rapid expansion phase can take place after harvesting the TIL from culture with cytokine and a CD28 agonist or antibody that binds a costimulatory molecule or before harvesting the TIL from culture with cytokine and a CD28 agonist or antibody that binds a costimulatory molecule.

[0176] In some cases, the second expansion or REP can be performed using any TIL containers known by those of skill in the art incubated at 37°C in 5% CC^and can proceed for 28-42 days or longer. Containers currently relied upon extensively in the field of Adoptive Cell Therapy are static cell culture devices, namely cell culture plates, flasks, and gas permeable bags.

[0177] The second expansion is generally accomplished by passaging the TILs from the first expansion once they reach 100% confluency. In some embodiments, the cells are passaged at a split ratio of 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7 1 :8, 1 :9, 1 :10, 1 :12, 1 :14, 1 :16, 1 :18 or 1 :20. In some embodiments, the cells are passaged at a split ratio of 1 :3, 1 :9, or 1 :20. After splitting, the cells may be replated in the cell culture medium.

[0178] In an embodiment, the culture medium comprises a T-cell receptor co-stimulator in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). The T-cell receptor costimulator can include, for example, an anti-CD28 agonistic antibody as described above. TILs can be expanded to induce further TILs activation in vitro by blocking a coinhibitory pathway for example, adding a PD-1 antagonistic antibody can block the PD-1 co-inhibitory pathway. In some embodiments, the second expansion can include the addition of anti-PD1 antagonistic antibody, at a concentration of about 1 pg / mL, about 2 pg / mL, about 3 pg / mL, about 4 pg / mL, about 5 pg / mL, about 6 pg / mL, about 7 pg / mL, about 8 pg / mL, about 9 pg / mL, about 10 pg / mL, about 12 pg / mL, about 15 pg / mL, about 18 pg / mL, or about 20 pg / mL.

[0179] In some embodiment, the cells in the second expansion are grown in a culture media with high doses of IL-2, as is known in the art. Alternatively, using combinations of cytokines for the second expansion of TILs is additionally possible, with combinations of two or more of IL-2, IL-7, IL-15 and IL-21 , as described in International Publication No. WO 2015 / 189356 and International Publication No. WO 2015 / 189357, hereby expressly incorporated by reference in their entirety. Thus, possible combinations include IL-2 and IL- 15, IL-2 and IL-21 , IL-15 and IL-21 , IL-2, IL-15 and IL-21 , IL-2 and IL-7, IL-7 and IL-21 , IL-15 and IL-7, IL-2, IL-7 and IL-21 , IL-15, IL-7 and IL-21 , IL-2, IL-7 and IL-15, IL-2, IL-7, IL-15 and IL-21 , and other combinations.Docket No. 32757 / 59103

[0180] In one embodiment, the cell culture medium lacks Pen / Strep and is further supplemented with IL-2 at a concentration of about 100 ILI / mL, about 250 ILI / mL about 500 ILI / mL, about 1 ,000 ILI / mL, about 2,000 ILI / mL, about 3,000 ILI / mL, about 4,000 ILI / mL, or about 5000 ILI / mL.

[0181] In some embodiments, the first and second expansion processes described herein result in an expanded population of TILs exhibiting an increased subpopulation of effector T cells, central memory T cells and / or CD45RA re-expressing memory T cells relative to the initial TILs at the start of the expansion process.

[0182] In some embodiments, the second expansion is performed one or more times, i.e., the second expansion is repeated.

[0183] In some embodiments, the TIL population obtained after the second expansion is removed from the supplemented second cell culture media and optionally cryopreserved in a storage media (for example, media containing 5% DMSO or CRYOSTOR® CS-10).

[0184] After the second expansion step, cells can be harvested. TILs can be harvested in any appropriate and sterile manner, including for example by centrifugation. Methods for TIL harvesting are well known in the art and any such known methods can be employed with the present process.D. Isolation of Neoantigen-Specific T Cells from Peripheral Blood Lymphocytes (PBLS)

[0185] Besides tumor resident lymphocytes, neoantigen-specific T cells have also been detected in peripheral blood, which represents an alternative source for tumor specific T cells and their TCRs (see e.g., Chatani et al. 2023. J Immunother Cancer 11 (5):e006264). This circumvents the need for invasive surgery and overcomes the problem of limited specimen availability in certain tumors. Moreover, isolation of neoantigen-specific T cells from blood is a useful alternative in situations where tumor-resident lymphocytes might be undetectable in the TIL compartment, possibly owing to; presentation of neoantigens in a non-inflammatory context, impaired T cell infiltration because of the sparse distribution of adhesion molecules on these cells, and presence of immunosuppressive cytokines and cells (e.g., regulatory T cells) in the tumor microenvironment.

[0186] In some embodiments, neoantigen-specific T cells in blood are identified based on an immunogenomic approach or an immunopeptidomic strategy. In some embodiments, neoantigen reactivity is identified by sorting PD1+, 41 BB+, CD39+ CD56+, CCR7+, and / or CD45Ra+ circulating T cells from blood to enrich for neoantigen reactive T cells.Docket No. 32757 / 59103

[0187] One approach involves whole genome sequencing (WGS) or whole exome sequencing (WES) of paired tumor and normal samples to identify tumor-specific mutations. RNA-sequencing (RNA-seq) is subsequently performed to enable prioritization of highly expressed mutations over nonexpressed variants.

[0188] In silico tools for mutation identification include but are not limited to INTEGRATE- neo, neoFusion, pVACtools, Epidisco, GATK and Antigen. garnish, Spliceman, MutPred, REVEL, rMATS, pVACseq, Neopepsee, MuPeXI, RepeatMasker, CloudNeoA, Tlminer, MuTect / MuTect2, Strelka / Strelka2, SMIIFIN, VarScan2, SomaticSniper, CaVEMan, MuSE, cgpPindel, SvABA, RADIA, NeuSomatic, NeoantigenR, MutPred, JuncBase, Splice, SpliceGrapher, rMATS, SplAdder, ASGAL, REVEL, TSNAD, HERVd, HESAS and EnHERV, and hervQuant (See, Xie et al. 2023. Signal Transduct Target Ther. 8(1 ):9).

[0189] For HLA typing, computational tools include but are not limited to Polysolver, OptiType, HLAreporter, PHLAT, HLAScan, and HLAProfiler (Xie et al. supra).

[0190] For HLA binding affinity prediction, tools include but are not limited to NetMHCpan, NetMHCIIpan4.0, MixMHC2pred, MARIA, neomhc2, pVAC-Seq, Tlminer, HLAthena, DeepHLApan, TEPITOPEpan, NetMHClIpan, SYFPEITHI, RNAKPEP, MULTIPRED2, ProPred, MHCPred, MARIA, Neonmhc2, and EDGE (Xie et al. supra).

[0191] In an immunopeptidomic strategy, peptide / MHC complexes are purified from biological samples e.g., blood, by immunoprecipitation using anti-MHC antibodies. The bound peptides are eluted and separated by size by high-pressure liquid chromatography then, mass spectrometry is performed to determine the molecular weight and identify the corresponding mutated peptides and their abundance. Since the patient sample contains multiple HLA alleles, identified peptides should be validated and matched to their corresponding HLA allele by predication algorithms.

[0192] For all approaches, TILs determined to react to the immunodominant epitopes can then be isolated and expanded from the patient's peripheral blood before adoptive TIL transfer. This method is termed the "selected TIL" approach.E. Administration

[0193] The compositions to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0194] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of agent or agents, the type of cells or recombinant receptors, the severity and course of the disease, whether the agent or cells are administered for preventive or therapeutic purposes, previous therapy, the subject'sDocket No. 32757 / 59103 clinical history and response to the agent or the cells, and the discretion of the attending physician. The compositions are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0195] In some cases, the cell therapy is administered as a single pharmaceutical composition comprising the TILs. In some embodiments, a given dose is administered by a single bolus administration of the TILs. In some embodiments, it is administered by multiple bolus administrations of the TILs, for example, over a period of no more than 3 days, or by continuous infusion administration of the TILs.

[0196] In some embodiments, a dose of TILs is administered to subjects in accord with the provided combination therapy methods. In some embodiments, the size or timing of the doses is determined as a function of the particular disease or condition in the subject. It is within the level of a skilled artisan to empirically determine the size or timing of the doses for a particular disease in view of the provided description.

[0197] In some embodiments, the TILs are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0198] Any suitable dose of TILs can be administered. In some embodiments, a therapeutically sufficient number of TILs are needed for a suitable dosage. In some embodiments, the therapeutically effective dosage is about 1 x 106, 2x 106, 3x 106, 4x 106, 5x106, 6x106, 7x106, 8x106, 9x106, 1 x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 1 x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1 x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1 x1 O10, 2x1 O10, 3x1 O10, 4x1 O10, 5x1 O10, 6x1 O10, 7x1 O10, 8x1 O10, 9x1 O10, 1 x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1 x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1 x1013, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x1013, 8x1013and 9x1013TILs. In various embodiments, between 1 x 109- 1 x 1011enhanced TIL cells / ml are administered to the subject.

[0199] In some embodiments, the number of the TILs provided in the pharmaceutical compositions of the disclosure is about 1 x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1 x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 1 x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1 x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1 x1 O10, 2x1 O10, 3x1 O10, 4x1 O10, 5x1 O10, 6x1 O10, 7x1 O10, 8x1 O10, 9x1 O10, 1 x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1 x1012, 2x1012,Docket No. 32757 / 591033x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1 x1013, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x1013, 8x1013, and 9x1013.

[0200] In an embodiment, the number of the TILs provided in the pharmaceutical compositions of the invention is in the range of 1 x 106to 5x 106, 5x 106to 1 x 107, 1 x 107to 5x107, 5x107to 1 x108, 1 x108to 5x108, 5x108to 1 x109, 1 x109to 5x109, 5x109to 1 x1 O10, 1 x101° to 5x1 O10, 5x101° to 1 x1011, 5x1011to 1 x1012, 1 x1012to 5x1012, and 5x1012to 1 x1013. In various embodiments, between 1 x 109- 1 x 1011enhanced TIL cells / ml are in the pharmaceutical composition.

[0201] The TILs provided in the pharmaceutical compositions of the disclosure are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of the TILs may also be used if appropriate. The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of TILs, will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.

[0202] In some embodiments, TILs may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, TILs 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. Administration of TILs may continue as long as necessary.F. Adoptive Cell Transfer

[0203] In some embodiments, once a therapeutically sufficient number of TILs are obtained using the expansion methods described above, they are usually frozen in cryopreservation media and administered to a patient immediately after thawing, which is also known as adoptive cell transfer (ACT).

[0204] ACT is an effective form of immunotherapy and involves the transfer of immune cells with antitumor activity into cancer patients. ACT is a treatment approach that involves the identification, in vitro, of lymphocytes with antitumor activity, the in vitro expansion of these cells to large numbers and their infusion into the cancer-bearing host. Lymphocytes used for adoptive transfer can be derived from the stroma of resected tumors (tumor infiltrating lymphocytes or TILs). They can also be derived from a solid tumor or from bloodDocket No. 32757 / 59103 if they are genetically engineered to express antitumor T cell receptors (TCRs) or chimeric antigen receptors (CARs), enriched with mixed lymphocyte tumor cell cultures (MLTCs), or cloned using autologous antigen presenting cells and tumor derived peptides. ACT in which the lymphocytes originate from the cancer-bearing host to be infused is termed autologous ACT. US 2011 / 0052530 relates to a method for performing adoptive cell therapy to promote cancer regression, primarily for treatment of patients suffering from metastatic melanoma, which is incorporated by reference in its entirety for these methods.

[0205] In an embodiment, the disclosure provides a method of treating a cancer in a subject in need thereof, comprising producing tumor-reactive T cells, e.g., an enhanced population of TILs from a tumor sample from the subject as disclosed herein, preconditioning subjects with immunodepleting (e.g., lymphodepleting) therapies, and administering the cells to the subject.

[0206] In some embodiments, preconditioning subjects with immunodepleting (e.g., lymphodepleting) therapies can improve the effects of the ACT. Preconditioning with lymphodepleting agents, including combinations of cyclosporine and fludarabine, have been effective in improving the efficacy of the transferred TILs, including to improve response and / or persistence of the transferred cells. See, e.g., Dudley et al., Science, 298, 850-54 (2002); Rosenberg et al., Clin Cancer Res, 17(13):4550-4557 (2011).

[0207] Such preconditioning 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 for homeostatic 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. See, Muranski et al., Nat Clin Pract Oncol. 3(12): 668-681 (2006).

[0208] 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 administration of the dose of cells. In some embodiments, the lymphodepleting therapy contains a chemotherapeutic agent such as fludarabine and / or cyclophosphamide. In some embodiments, the administration of the cells and / or the lymphodepleting therapy is carried out via outpatient delivery.

[0209] In some embodiments, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the administration of the dose ofDocket No. 32757 / 59103 cells. 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 administration of the dose of cells.

[0210] In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg / kg and 100 mg / kg of body surface area of the subject, such as between or between about 40 mg / kg and 80 mg / kg. In some embodiments, the subject is preconditioned with or with about 60 mg / kg of cyclophosphamide. 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, the cyclophosphamide is administered once daily for one or two days. In some embodiments, where the lymphodepleting agent comprises cyclophosphamide, the subject is administered cyclophosphamide at a dose between or between about 100 mg / m2and 500 mg / m2body surface area of the subject, such as between or between about 200 mg / m2 and 400 mg / m2, or 250 mg / m2and 350 mg / m2, inclusive. In some embodiments, the subject is administered about 300 mg / m2of cyclophosphamide. 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 -days, for example, for 2 to 4 days. In some instances, the subject is administered about 300 mg / m2body surface area of the subject, of cyclophosphamide, daily for 3 days, prior to initiation of the cell therapy.

[0211] 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 / m2of body surface area of the subject, such as between or between about 10 mg / m2and 75 mg / m2, 15 mg / m2and 50 mg / m2, 20 mg / m2and 30 mg / m2, 20 mg / m2and 40 mg / m2, 24 mg / m2and 35 mg / m2, or 24 mg / m2and 26 mg / m2, each inclusive. In some instances, the subject is administered about 30 mg / m2of fludarabine. In some instances, the subject is administered 25 mg / m2of fludarabine. 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, e.g., for 1 -5 days, for 3-5 days or for 3-4 days. In some embodiments, the subject is administered about 30 mg / m2body surface area of the subject, of fludarabine, daily for 3 days, prior to initiation of the cell therapy.

[0212] In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combinationDocket No. 32757 / 59103 of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some embodiments, the subject is administered 60 mg / kg (-2 g / m2) of cyclophosphamide and 3 to 5 doses of 25 mg / m2fludarabine prior to the dose of cells. In some embodiments, the subject is administered fludarabine at or about 30 mg / m2body surface area of the subject, daily, and cyclophosphamide at or about 300 mg / m2body surface area of the subject, daily, for 3 days.

[0213] In various embodiments, cyclophosphamide and fludarabine are administered intravenously.

[0214] In some embodiments, the administration of the preconditioning agent prior to infusion of the dose of cells improves an outcome of the treatment. For example, in some aspects, preconditioning improves the efficacy of treatment or increases the persistence of the infused TILs 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.

[0215] An embodiment of the method comprises, after administering the nonmyeloablative lymphodepleting chemotherapy, administering to the subject the expanded T cells. The cell solution to be used for in vivo administration should be sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0216] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of agent or agents, the type of cells or recombinant receptors, the severity and course of the disease, whether the agent or cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the agent or the cells, and the discretion of the attending physician. The cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0217] In some embodiments, the TILs are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

[0218] Any suitable dose of TILs can be administered. In some embodiments, a therapeutically sufficient number of TILs are needed for a suitable dosage. In some embodiments, the therapeutically effective dosage is about 1 x 106, 2x 106, 3x 106, 4x 106, 5x106, 6x106, 7x106, 8x106, 9x106, 1 x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107,Docket No. 32757 / 591039x107, 1 x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1 x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1 x1 O10, 2x1 O10, 3x1 O10, 4x1 O10, 5x1 O10, 6x1 O10, 7x1 O10, 8x1 O10, 9x1 O10, 1 x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1 x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1 x1013, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x1013, 8x1013and 9x1013TILs.

[0219] The TILs produced by methods of the disclosure are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of the TILs may also be used if appropriate. The dosages of TILs to be administered will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active ingredients and the discretion of the prescribing physician.

[0220] In some embodiments, TILs may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, TILs 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. Administration of TILs may continue as long as necessary.

[0221] The expanded TILs that are administered to the subject in accordance with an embodiment of the disclosure advantageously have features associated with in vivo persistence, proliferation, and antitumor activity. For example, the TILs have a higher expression of CD27 and / or CD28 than T cells produced by other methods. Without being bound to a particular theory, it is believed that CD27 and CD28 are associated with proliferation, in vivo persistence, and a less differentiated state of T cells (the increased differentiation of T cells is believed to negatively affect the capacity of T cells to function in vivo). T cells expressing higher levels of CD27 are believed to have better antitumor activity than CD27-low cells. In various embodiments, the TILs produced by the present methods have a higher frequency of CD4+ cells than T cells produced by other methods.

[0222] The T-cells can be administered by any suitable route as known in the art. Preferably, the T-cells are administered as an intra-arterial or intravenous infusion, which preferably lasts about 30 to about 60 minutes. Other examples of routes of administration include intratumoral, intraperitoneal, intrathecal and intralymphatic. Likewise, any suitable dose of T-cells can be administered as indicated above.Docket No. 32757 / 59103

[0223] Prior to, during or following administration of the TILs, the biological activity of the T cell therapy, e.g. the biological activity of the cells, in some embodiments is measured by any number of known methods. Parameters to assess include specific tumor reactivity, persistence and other measures of T cell activity, measured using any suitable method known in the art, such as assays described further below. In some embodiments, the biological activity of the cells, e.g., T cells administered for the T cell-based therapy, is measured by assaying cytotoxic cell killing, expression and / or secretion of one or more cytokines, proliferation or expansion, such as upon restimulation with antigen. In some embodiments, the biological activity is measured by assessing the disease burden and / or clinical outcome, such as reduction in tumor burden or load.

[0224] Once the cells are administered to the subject (e.g., human), the biological activity of the TILs in some embodiments is measured by any of a number of known methods, e.g., by ELISA or flow cytometry. In certain embodiments, the biological activity of the cells also can be measured by assaying expression and / or secretion of certain cytokines, such as IFNy, IL-2, and TNF, or by CD107a, a marker of degranulation. In some embodiments, the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load. In some embodiments, toxic outcomes, persistence and / or expansion of the cells, presence or absence of a host immune response, alteration of the patient's immune repertoire, emergence of new TCRs, including those not in the infusion product, increased immune infiltration of the tumor, alteration of tumor neoantigen profile, alteration of MHC expression and / or the MHC processing pathway are assessed.

[0225] With respect to the disclosed methods, the cancer can be any cancer, including a carcinoma, sarcoma, or hematopoietic cancer. In some embodiments, the cancer is a carcinoma including any of bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancers, gastric cancer, glioblastoma, glioma, head and neck cancer, lung cancer, melanoma, mesothelioma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, testicular cancer, thyroid cancer, skin cancer, and uterine cancer. In some embodiments, the cancer is a sarcoma selected from the group consisting of undifferentiated pleomorphic sarcoma, epithelioid sarcoma, liposarcoma, and leiomyosarcoma. In some embodiments, the cancer is a hematopoietic cancer such as, lymphoma and myeloma.

[0226] Further, the cancer may be primary, metastatic, or recurrent, and may be of any type (as described above), any stage (e.g., Stage I, II, III, or IV or an equivalent of other staging system), and / or histology. The subject may be of any age, gender, treatment history and / or extent and duration of remission.Docket No. 32757 / 59103

[0227] In certain embodiments, the treatments disclosed herein produce one or more therapeutic effects selected from the group consisting of tumor regression, reduction in metastatic lesions over time, reduction in one or more cancer symptoms, reduced use of chemotherapeutic or cytotoxic agents, reduction in tumor burden, increase in progression- free survival, increase in overall response rate, increase in overall survival, increase in progression-free survival, complete response, partial response, and stable disease.

[0228] The term "regression" does not necessarily imply 100% or complete regression. Rather, there are varying degrees of regression of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. The term also encompasses delaying the onset of the disease, or a symptom or condition thereof.IV. Additional Methods and Optional Cell Analyses

[0229] Success of immunotherapy regimens, including ACT, is influenced by T cell- intrinsic factors, including cell differentiation states, self-renewing capability, and resistance to tumor microenvironment inhibition, as well as tumor-specific factors, such as mutational burden, human leukocyte antigen (HLA) expression, and tumor clonality. Notwithstanding, antitumor T-cell reactivity remains a common denominator to successful immunotherapies. However, tumor-infiltrating lymphocytes (TILs) are a heterogeneous population of T cells comprising tumor-specific reactive T lymphocytes and bystander T cells. Therefore, fast and reliable identification of tumor-reactive T cells and the repertoire of their cognate T-cell receptor (TCRs) remains a top priority for immune monitoring and therapeutic targeting.

[0230] Conventional means of identifying tumor-reactive T cells and their cognate TCRs have generally relied on i) detection of the upregulation of cell surface protein markers of T cell activation following recognition of autologous tumor-antigens; the most common markers of tumor-specific reactive T cells (TILs) are CD137, PD-1 , CD103, and CD39. Other markers include CXCL13, CD69, CD25, and FOXP3+, ii) in vitro T cell functional assays wherein tumor reactivity is detected e.g., by lymphoproliferation assays, cytotoxic T cell assays using chromium release, CD107a Degranulation Assay, cytokine production assays such as the enzyme-linked immunospot (ELISpot) assays, cytokine capture assays and intracellular cytokine staining — or by antigen-binding methods, for example when the minimal epitope is known, single-cell sorting of T cells from bulk populations using antigen-specific HLA multimers and then reconstructing the TCRs expressed by the sorted T cells, iii) transcriptomic profiling of intratumoral neoantigen-specific and tumor-reactive T cells using single-cell RNA and TCR sequencing (scRNA-seq and scTCR-seq).Docket No. 32757 / 59103

[0231] In various embodiments, tumor-reactive T cells produced by the methods herein comprise T cells expressing one or more the biomarkers selected from the group consisting of CD3, CD4, CD8, ybTCR, CD62L, CD27, CCR7, CD45ro, or CD45ra,

[0232] Exomic and RNA-seq approaches have been developed to identify the relatively rare T cells that recognize neoantigens expressed in tumors. One approach that has been used to identify neoantigen-reactive T cells involves the screening of TIL cultures from different resected tumors from the same patient or different parts of the same metastatic tumor. Whole exome sequences using those tumor specimens and normal tissue are performed to identify the somatic mutations present in the cancer. In this approach, all the mutant gene products are expressed on the patient’s own antigen presenting cells using either tandem mini genes (TMGs) or peptides and cocultured with TIL specimens (grown in 24-well plate) to identify tumor reactive lymphocytes. The reactivities to mutations are assessed by immunological assays combining functional marker such as Interferon-y and Tumor Nercrosis Factor alpha (IFNy and / or TNFa secretion measured by ELISPOT) and T cell activation markers (e.g. 4-1 BB, CD69 and / or 0x40 upregulation measured by flow cytometry).

[0233] Additional details for the various assays are provided below.

[0234] Cell Counts and Viability: In some embodiments, cell counts and / or viability are measured. In one embodiment, a cell viability assay can be performed after the expansion step using standard assays known in the art. The cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL) and viability can be assessed using any method known in the art. For example, a trypan blue exclusion assay can be performed on a sample of the bulk TILs, which selectively labels dead cells and allows a viability assessment. Other assays for use in testing viability can include but are not limited to the Alamar blue assay, the ethidium bromide acridine orange staining assay, and the MTT assay. In some cases, the bulk TIL population can be cryopreserved immediately, using the protocols discussed below. Alternatively, the bulk TIL population can be subjected to REP and then cryopreserved as discussed below.

[0235] Cell Cultures: In an embodiment, a method for expanding TILs may include using about 5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cell medium. In an embodiment, expanding the number of TILs uses no more than one type of cell culture medium. Any suitable cell culture medium may be used, e.g., RPMI 1640 medium with or without 1x Penicillin / Streptomycin (Pen / Strep) supplemented with 10% Human AB serum, 25 mM HEPES, 10 pg / mL Gentamicin, and 150 pL Antibiotic-Antimycotic (100X), or AIM-V cellDocket No. 32757 / 59103 medium (GIBCO™) supplemented with L-glutamine, 50 pM streptomycin sulfate, and 10 pM gentamicin sulfate cell culture medium. In an embodiment, expanding the number of TIL may comprise adding fresh cell culture media to the cells (also referred to as feeding the cells) no more frequently than every third or fourth day.

[0236] Optional Cryopreservation of TILs: As discussed above, cryopreservation can occur at numerous points throughout the TIL expansion process. In some embodiments, the bulk TIL population after the first expansion or the expanded population of TILs after the one or more second expansions can be cryopreserved. Cryopreservation can be generally accomplished by placing the TIL population into a freezing solution, e.g., 85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO). Alternatively, cryopreservation can be done using CRYOSTOR® CS10 cryopreservation freeze media (BioLife Solutions, Bothell, WA, USA). For example, in some cases, a TIL can be harvested, washed, and resuspended in a buffer, such as Cryostor buffer. This preparation can be mixed with an equal volume of CRYOSTOR® CS10. The cells in solution are placed into cryogenic vials and stored for 24 hours at -80°C., with optional transfer to gaseous nitrogen freezers for cryopreservation. See, Sadeghi, et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, the TILs are cryopreserved in 5% DMSO. In some embodiments, the TILs are cryopreserved in cell culture media plus 5% DMSO.

[0237] A cryopreservation can be at a freeze density from about 7.5x107cells / mL to about 1 .5x108cells / mL. A freezing density can be from about 1 x107cells / mL, 1 .5x107cells / mL, 2x107cells / mL, 2.5x107cells / mL, 3x107cells / mL, 3.5x107cells / mL, 4x107cells / mL, 4.5x107cells / mL, 5x107cells / mL, 5.5x107cells / mL, 6x107cells / mL, 6.5x107cells / mL, 7x107cells / mL, 7.5x107cells / mL, 8x107cells / mL, 8.5x107cells / mL, 9x107cells / mL, 9.5x107cells / mL, 1x108cells / mL, 1 .5x108cells / mL, 2x108cells / mL, 2.5x108cells / mL, 3x108cells / mL, 3.5x108cells / mL, 4x108cells / mL, 4.5x108cells / mL, 5x108cells / mL, 5.5x108cells / mL, 6x108cells / mL, 6.5x108cells / mL, 7x108cells / mL, 7.5x108cells / mL, or up to about 8x108cells / mL.

[0238] In some cases, a cellular composition is thawed prior to an introducing into a subject in need thereof. When appropriate, the cells are removed from the freezer and thawed in a 37° C. water bath until approximately4 / s of the solution is thawed. The cells are generally resuspended in complete media and optionally washed one or more times with 1X PBS. In some embodiments, the thawed TILs can be counted and assessed for viability as is known in the art.

[0239] For example, cellular viability can be determined by flow cytometry and trypan blue exclusion. In some embodiments, a forward scatter and side scatter on a flow cytometer can identify percent viable cells. In other embodiments, cells can be stained with Annexin V toDocket No. 32757 / 59103 determine a percent of dead / live cells. Trypan blue exclusion may also be utilized to determine cellular viability with a hemocytometer. In some embodiments, at least about 50% cells can be viable for administration. In some embodiments, from about 50%, 60%, 70%, 80%, 90%, 95%, or up to about 100% cells can be viable.

[0240] Phenotypic Characteristics of Expanded TILs: In some embodiments, a method can further comprise performing a pre-infusion testing on TILs. Pre- infusion testing can comprise at least one of: phenotypic testing, potency testing, microbiological testing, endotoxin testing, viability testing, and tumor cell testing. In some embodiments, phenotypic testing comprises detecting the presence of CD28 on TILs. In some embodiments, potency testing comprises detecting a level of IFNy upon anti-CD28 antibody stimulation of TILs. In some embodiments, microbiological testing comprises detecting growth of an aerobic culture, anaerobic culture, gram status, fungal status, or mycoplasma status. Endotoxin testing can comprise performing a limulus assay. In some embodiments, viability testing comprises performing a trypan blue exclusion assay. In some embodiments, tumor cell testing comprises a cytopathology assay. In some embodiments, TILs can be administered when pre-infusion testing is negative for a microbiological testing. In some embodiments, TILs are administered when pre-infusion testing is over at least about 70% viable cells for said viability testing. In some embodiments, TILs are administered when pre-infusion testing is at least about 80% CD28 positive for a phenotypic testing. In some embodiments, TILs are administered when pre-infusion testing is at least about 200 pg / mL per 105cells of IFNy upon anti-CD28 stimulation of TILs in a potency testing. In some embodiments, TILs are administered when pre-infusion testing is negative for tumor cells per at least about 200 TILs examined in a cytopathology testing.

[0241] Various assays for determining qualitative and / or quantitative characteristics of cells or components thereof are known in the art and may be utilized in assessing the TILs of the disclosure.

[0242] Flow cytometry. Cell surface immunophenotyping using fluorescent flow cytometry, for example, is used in both diagnostics and research laboratories to differentiate, count, or analyze cells of interest in a cell sample containing many different cell types.

[0243] A general description of standard flow cytometry apparatus and methods is provided in U.S. Pat. No. 4,284,412, which is incorporated by reference herein. Further, commonly used flow cytometers, such as the Becton-Dickinson Immunocytometry Systems “FACSCAN” (San Jose, Calif.), for example, can measure forward light scatter (generally correlated with the refractive index and size of the particle being illuminated), side light scatter (generally correlated with the particle's size), and fluorescence at one or moreDocket No. 32757 / 59103 wavelengths. Flow cytometers and various techniques for their use are described, generally in “Practical Flow Cytometry” by Howard M. Shapiro (Alan R. Liss, Inc., 1985) and “Flow Cytometry and Sorting, Second Edition” edited by Melamed et al. (Wiley-Liss, 1990).

[0244] In some embodiments, the expression of one or more phenotypic markers is examined. In some embodiments, the markers CD3, CD4, CD8, CD25, ydTCR, CD27, CCR7, CD45ra, CD45ro, CD62L, CD69, LAG3, TIM3, PD1 , 0X40, 4-1 BB, as well as any other disclosed or described herein, can be measured by flow cytometry with antibodies, for example but not limited to those commercially available from BD Bio-sciences (BD Biosciences, San Jose, Calif.) using a CytoFlex S™ flow cytometer (Beckman Coulter, Brea, Calif., USA). Additional markers include but are not limited to TCR alpha / beta (i.e., TCRa / p), TIGIT, KLRG1 , HLA-DR, IL-2R, CCR7, NKp46, CD11b, CD27, CD28, CD56, CD57, CD69, CD122, CD137, and CD154. In some embodiments, expression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen markers is examined.

[0245] In some embodiments, the phenotypic characteristics of the TILs are analyzed after the expansion phase.

[0246] In certain embodiments, TILs are evaluated for cytokine release, especially IFN-y, IL-2 or TNF-a, using cytokine release assays that are known in the art including but not limited to ELISA, ELISPOT, intracellular cytokine staining and flow cytometry, and the like. In some embodiments, IFN-y is measured using a Quantikine ELISA kit. In some embodiments, TILs can be evaluated for interferon-y (IFN-y) secretion. Such secretion can be detected using the ELISPOT assay described in WO 98 / 23960 incorporated by reference in its entirety.

[0247] In some embodiments, IFN-y secretion is indicative of active TILs. IFN-y production is also another measure of cytotoxic potential. Moreover, the measure of cytokine release (e.g., interferon-y) following co-culture of TILs with tumor cells is an indication of specific tumor reactivity. To determine the fold change in IFN-y production, the levels of the IFN-y cytokine in the media of TILs stimulated with antibodies to CD28 and / or PD1 compared to the cytokine level in the absence of any antibody. The term “fold change” refers to the extent, as compared to the control, that the IFN-y produced by the TILs being assayed increased or decreased in response to the antibody stimulation. A two- fold change thus means 200% cytokine production (e.g. by anti-CD28 agonist antibody stimulation) when compared to no antibody (set at 100%). In some embodiments, IFN-y is measured in TILs ex vivo, including TILs produced by the methods of the present disclosure, as well as freshly harvested TILs or those TILs produced by other methods. In some embodiments, anDocket No. 32757 / 59103 increase in IFN-y production in the expansion as compared to initially harvested TILs is indicative of an increase in cytotoxic potential of the expanded TILs.

[0248] In some embodiments, I FN-y secretion increases 2-fold, 5-fold, 10-fold, 15-fold, 20- fold, 25-fold, 50-fold or 100-fold or more between the unstimulated TILs and stimulated TILs. In some embodiments, I FN-y secretion increases 5-fold. In some embodiments, IFN-y secretion increases 10-fold. In some embodiments, IFN-y secretion increases 20-fold. In some embodiments, IFN-y secretion increases 50-fold. In some embodiments, IFN-y secretion increases 100-fold.

[0249] Co-culture assays for assessment of the cytotoxic potential of TILs: In some embodiments, the cytotoxic potential of TIL to lyse target cells is assessed using a co-culture assay of TIL with the bioluminescent cell line, P815 (Clone G6), according to a bioluminescent redirected lysis assay (potency assay) for TIL assay which measures TIL cytotoxicity in a highly sensitive dose dependent manner.

[0250] In various embodiments, use of autologous tumor cell or organoid assays are used to measure potency or functionality of TILs.

[0251] In some embodiments, the phenotypic characterization and viability of the TILs is examined after cryopreservation.

[0252] TCR vBeta repertoire sequencing: The present disclosure provides a method for generating TILs with increased T-cell repertoire diversity (sometimes referred to as polyclonality). T-cell receptors (TCRs) are highly diverse heterodimeric plasma membrane proteins located the surface of T cells, and they are responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an inflammatory response. TCR heterodimers consist of a combination of a and p chains (TCR-ap) expressed by the majority of T cells, or y and 8 chains (TCR-y8) expressed by T cells in peripheral blood (1-5%) and T cells found at mucosal sites. The loci of TCR a and chains are organized as gene segments comprising a variable (V), joining (J), and constant (C) segments. Further, the TCR a and p chains possess three hypervariable regions termed complementarity determining regions (CDR1 , 2 and 3). CDR3 is responsible for recognizing processed antigen peptides. The TCR y and 8 chains are also made up of V, J, and C regions, with an additional diversity (D) gene segment.

[0253] Effective T-cell responses rely on highly diverse TCR repertoires which ensure the capability to identify a wide range of antigens. TCR chains belong structurally to the immunoglobulin gene superfamily. Thus, similar to B lymphocytes, the high diversity of antigen receptors of T lymphocytes (i.e., TCR chains) is generated by somatic recombinationDocket No. 32757 / 59103 of a limited number of variable, diversity, and joining segments, called V(D)J recombination. Additionally, random nucleotides are added and / or deleted at the junction sites between the gene segments. This process leads to strong combinatorial (depending on which gene segments will recombine) and junctional diversity (which and how many nucleotides will be added / deleted), resulting in a large and highly variable TCR repertoire, ensuring the recognition of a plethora of antigens. Additional diversity is achieved by the pairing of a and P or y and 5 chains to form a functional TCR. The sum of all TCRs by the T cells of one individual is termed the TCR repertoire or TCR profile. The lower limit of distinct TCRs in the peripheral blood of a healthy individual is around 1.1 million, and the theoretical diversity for op Tcells, the most abundant T cell type in humans, is up to1016types. This large repertoire of T cells with structurally divergent TCRs is required to recognize cells expressing foreign or mutated proteins, including neoantigens in cancer cells. Therefore, characterizing the repertoire of tumor-infiltrating T cells can help identify the tumor-reactive T cell clones and facilitate the clinical practice of cancer immunotherapies.

[0254] Within the TCRp chain, the complementarity-determining region (CDR)1 and CDR2 loops of the TCR contact the MHC alpha-helices while the hypervariable CDR3 regions interact mainly with the peptide. In both TCRa and TCRp chains, CDR3 loops have the highest sequence diversity and are the principal determinants of receptor binding specificity. The CDR1 and CDR2 are determined by the V gene used in the TCR, while CDR3 is determined both by V, (D in the TCRp) and J, and by the addition and removal of nucleotides at the VD and DJ junctions (VJ in the alpha chain). Thus, since the vast majority of TCR variation is within CDR3, which encompasses the VDJ recombination junctions, the sequence of CDR3 and the identity of the flanking V and J gene segments are widely used to classify TCR variants.

[0255] Accordingly, in one embodiment, the TCR repertoire analysis of the TILs produced by the disclosed methods can be performed by amplifying the cDNA of the CDR3 locus of the p chain (i.e., P-CDR3) using predesigned PCR primers followed by deep sequencing to determine the TCR p clonal frequency measurement. In some embodiments, the TCR repertoire analysis comprises performing a single multiplex amplification reaction with primer sets which target the hypervariable CDR3 of the TCR beta chain followed by sequencing and automated analysis. In some embodiments, the multiplex amplification reaction is carried out with a ready-to-use panel of primers. In some embodiments, the ready-to-use panel of primers is the AmpliSeq for Illumina TCR beta-SR (Short Read) Panel (Illumina, Inc). Methods of the disclosure further comprise preparing a TCR repertoire library using the amplified target immune receptor sequences through introducing adapter sequences to theDocket No. 32757 / 59103 termini of the amplified target sequences. In some embodiments, the adapter-modified immune receptor repertoire library is clonally amplified.

[0256] TCR vAlpha repertoire sequencing: TCR alpha chain analysis is also carried out. Methods for analyzing alpha chain sequence repertoire include Chromium 10X (1 OX Genomics), iRepertoire or similar single cell technologies that allow for paired alpha beta TCR profiling

[0257] The methods further comprise detecting sequences of the immune repertoire of each of the immune receptors in the sample and / or expression of each of the plurality of target immune receptor sequences, wherein a change in the level of repertoire sequences and / or expression of one or more target immune receptor markers as compared with a second sample or a control sample determines a change in immune repertoire activity in the sample. In certain embodiments sequencing of the immune receptor amplicon molecules is carried out using next generation sequence analysis to determine sequence of the immune receptor amplicons. In one embodiment, bioinformatics analysis is carried out to determine the sequence of the immune receptor amplicon molecules, which includes obtaining initial sequence reads, aligning and identifying productive reads and correcting errors to generate rescued productive reads and determining the sequences of the resulting total productive reads, thereby providing sequence of the immune repertoire in the sample.

[0258] In various embodiments, the bioinformatics analysis is carried out using MiXCR software (Bolotin et al., 2015 MiXCR: Software for comprehensive adaptive immunity profiling. Nat. Methods. 12:380-381). MiXCR is a universal software for fast and accurate extraction of T- and B- cell receptor repertoires from any type of sequencing data. It handles paired- and single-end reads, considers sequence quality, corrects PCR errors and identifies germline hypermutations. The software supports both partial- and full-length profiling and employs all available RNA or DNA information, including sequences upstream of V and downstream of J gene segments. Accordingly, MiXCR identifies and counts the V, D, and J genes, filters nonproductive sequences and reports and tracks T cell clonality. In some embodiments, subsequent analysis can be carried out with custom R scipts using the Immunarch package (ImmunoMind Team., 2019. Immunarch: An R Package for Painless Bioinformatics Analysis of T-Cell and B-Cell Immune Repertoires. Zenodo. 10). Immunarch offers data loading, analysis and visualization for all popular TCR and BCR analysis and post-analysis formats, including from MiXCR. Immunarch implements most of the commonly used analysis methods, such as clonality analysis; estimation of repertoire similarities in the distribution of clonotypes; gene usage and kmer distribution measures; repertoire diversity analysis; clonotype tracking between samples and across time points; and annotation of clonotypes, using external immune receptor databases.Docket No. 32757 / 59103

[0259] TIL therapy relies on the infiltration of reactive T cells, capable of recognizing tumor-associated antigen (TAAs) or unknown antigens. Accordingly, the present disclosure provides a method for generating TILs which exhibit and increase the T-cell repertoire diversity. In some embodiments, the TILs, e.g., a population of TILs, obtained in the expansion exhibit an increase in the T-cell repertoire diversity. In some embodiments, the increase in T-cell repertoire diversity in the TILs produced in the expansion is as compared to freshly harvested TILs and / or TILs prepared using other methods than those provided herein.

[0260] In some embodiments, the increase in diversity is an increase in the immunoglobulin diversity and / or the T-cell receptor diversity. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin heavy chain. In some embodiments, the diversity is in the immunoglobulin is in the immunoglobulin light chain. In some embodiments, the diversity is in the T-cell receptor. In some embodiments, the diversity is in one of the T-cell receptors selected from the group; alpha, beta, gamma, and delta receptors. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha and / or beta. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) alpha. In some embodiments, there is an increase in the expression of T-cell receptor (TCR) beta. In some embodiments, there is an increase in the expression of TCRab (i.e., TCRa / p).

[0261] Peptide-MHC (pMHC) multimer staining: Major histocompatibility complex class I(MHC-I) proteins display the cellular proteome, as small peptide fragments, at the cell surface of antigen presenting cells (APC) to mediate T-cell immune surveillance. This surveillance is essential for countering cellular abnormalities, such as viral infections and cancer. TCRs mediate the recognition of such pMHC epitopes by T cells. As such, an important parameter to describe T cell functionality is the affinity of their TCRs for a given antigen peptides bound to MHC molecules. Screening of T cell specificity using large libraries of pMHC molecules is suitable for analyses of T cell recognition. Such analysis provides insights into the immune specificities involved in response to immunotherapy and extends knowledge related to T cell recognition patterns and cross-recognition by TCRs. In general, a high affinity TCR is a TCR with above-average affinity for antigen peptides bound to major histocompatibility complex (MHC) molecules. Affinity is defined as the probability of a receptor (TCR)-ligand (pMHC) interaction. T cells with high affinity TCRs for example could be shown to be superior in inducing tumor regression, in this sense, high affinity TCR positively correlates with high tumor protection. In this context, by tumor protection it is meant the ability of T cells of the invention to cause tumor regression. Tumor protection can be measured with many techniques known to the skilled artisan both in preclinical mouseDocket No. 32757 / 59103 models wherein survival of animals is measured and in clinical studies in humans. In one embodiment, the TILs generated by the present methods have high affinity TCRs.

[0262] Cytopathology testing: In some cases, a population of TILs is tested for the presence of tumor cells in a composition. TILs that can be administered can be negative for tumor cells per at least about 200 TILs examined in a cytopathology testing. In other cases, less than 1% of tumor cells exist per at least about 200 TILs examined in a cytopathology testing. In other cases, less than 2% of tumor cells exist per at least about 200 TILs examined in a cytopathology testing. In other cases, less than 3% of tumor cells exist per at least about 200 TILs examined in a cytopathology testing. In other cases, less than 4% of tumor cells exist per at least about 200 TILs examined in a cytopathology testing. In other cases, less than 5% of tumor cells exist per at least about 200 TILs examined in a cytopathology testing.EXAMPLESExample 1 : Materials and Methods

[0263] Tumor and blood source

[0264] Ovarian tumors were obtained from de-identified patients following surgical resection of primary ovarian tumors. Signed informed consent was obtained from all donors and the study was approved by the University of Minnesota Institutional Review Board (IRB study number 1602E84302). All methods were performed in accordance with the relevant guidelines and regulations.

[0265] Isolation of PBMCs

[0266] Whole blood from matched de-identified, normal, healthy human donors were obtained by venipuncture. PBMCs were isolated by diluting whole blood samples with sterile PBS at a 1 :1 v / v ratio and layering on a Ficoll gradient before centrifugation at 1800xg for 25 mins. Buffy coats were then collected and washed 3 times with 1x PBS.

[0267] Tumor processing

[0268] Upon receiving the tumor, it was transferred to a petri dish and washed 2 times with 1x PBS and then transferred to a new petri dish. Two 2 mm3fragments were cut from the tumor and made into slurries using scissors. These slurries were frozen at -80 °C and used for downstream DNA or RNA isolation. The remaining tumor fragment was finely minced with 2 scalpels. For every 250 mm3of tumor, 5 mL of Digestion Media (Complete Media (CM) RPMI 1640, Human AB serum (10%), HEPES (25 mM), Gentamicin (10 pg / mL), with Pen / Strep (1x) w / digestion enzymes, 0.094 U / mL Dispase, 725 U / mL Collagenase, 50 U / mL Hyalronidase, 150 pL Anti-fungal / Anti-biotic (100x)) was added, and then placed in aDocket No. 32757 / 59103 tissue culture incubator at 37 °C with 5% CO2. The digesting tumor was removed every 15- 30 minutes and a 10 mL or 5 mL pipette was used to pipette the slurry up and down. This was done until the slurry easily passed through a sterile 5 mL pipette. The slurry was then filtered through a 100 pm cell strainer and washed twice in 50 mL of 1x PBS. Cells were then counted and resuspended in Cryostor CS10 media to a concentration of 1 -20 x 106cells per mL and frozen in 1 mL aliquots.

[0269] Sorting of B cells and MDDCs

[0270] Peripheral blood mononuclear cells (PBMCs) were resuspended in sorting buffer and then B cells were positively sorted with an EASYSEP™ Release Human CD19 Positive Selection kit (StemCell Cat#17754) in accordance with the manufacturer’s instructions. The positive fraction, containing B cells, was then used for lymphoblastoid cell line (LCL) generation. Monocytes were then sorted from the negative fraction using an EASYSEP™ Human Monocyte Isolation Kit (StemCell Cat#19359) and used to generate Monocyte Derived Dendritic Cells (MDDCs).

[0271] Generation of LCLs

[0272] Irradiated MCR-5 feeder cells (ATCC, fibroblast) were plated in a T25 flask for 1 -5 days. Then EBV (ATCC VR-194) and 1 -5e6 sorted B cells are added to the flask. Media is changed every 2-5 days until transformation of the cells is observed, usually 2-6 weeks.

[0273] Generation of MDDCs

[0274] Monocytes were cultured in GM-CSF and IL-4 for 7 days to generate MDDCs.

[0275] Small scale TIL outgrowth from tumor

[0276] T umor dissociations were thawed, counted, then plated at 25,000-100,000 cells in100 pL of CM (RPMI 1640, 10% Human AB serum, 25 mM HEPES, 10 pg / mL Gentamicin, with 1x Pen / Strep) and 6,000 lU / mL IL-2 in triplicates, and in triplicate 96 well plates for harvest at weeks 1 , 2, and 3. Small molecules or antibodies were added to the wells as listed in Table 1.

[0277] Table 1Docket No. 32757 / 59103

[0278] On day 4, an IL-2 spike was performed by adding 50 pL CM, 1x Pen / Strep, and 18,000 ILI / mL of IL-2. After 1 week, one of the triplicate plates was counted, harvested, and phenotyped by flow cytometry. The remaining two plates were spun at 400 g for 5 min to pellet the cells and media was carefully aspirated without disrupting the pellet. 100 pL fresh CM and 6,000 lU / mL IL-2 were added to each well as well as additional small molecules as indicated in Table 1 . Four days later, an IL-2 spike was performed by adding 50 pL CM, 1x Pen / Strep, and 18,000 lU / mL IL-2. At the end of week 2, the second replicate plate 2 was counted, harvested, and phenotyped by flow cytometry. The final replicate plate was spun at 400 g for 5 min to pellet the cells and media carefully aspirated without disrupting the pellet. 100 pL fresh CM and 6,000 of lU / mL IL-2 were added to each well, as well as additional small molecules as indicated in Table 1 . Four days later, an IL-2 spike was performed by adding 50 pL CM ,1x Pen / Strep, and 18,000 lU / mL IL-2. At the end of week 3, the final replicate plate was counted, harvested, and phenotyped by flow cytometry.

[0279] Expansion

[0280] Tumor dissociations were thawed, counted, then plated in a 24 well GRex plate (Wilson Wolf) at a cell density of between 7.5 x 106- 8.0 x 106cells in 6 mL CM containing 6,000 lU / ml IL-2. Antibodies were then added to wells as indicated in Table 2.

[0281] Table 2Docket No. 32757 / 59103

[0282] Every 3-4 days cultures were supplemented with 100 pL of CM containing 18,000 ILI / mL IL-2. The tumor dissociations were then cultured for 5 weeks. During culture, once the media began to appear orange / yellow the confluency of the cells in the well was checked by observing the bottom of the G-Rex flasks. If the well was 100% confluent, the well was split 1 :2 after a 50:50 media exchange. The 50:50 media exchanges were performed by gently removing the top 3 mL of media from each well, so as not to disturb the cells. 3 mL CM (without Pen Strep) supplemented with 6000 ILI / mL IL-2 and 2x anti-CD28 antibody or anti-CD28 antibody and anti-PD1 antibody was then added back to each well. The cells were counted in weekly intervals for 5 weeks and 100,000 cells were removed for flow cytometric analysis. At week 0, and once there were more than 3 x 106cells in a well (generally weeks 3, 4, and 5), 5.0 x 105cells were removed weekly for gDNA extraction and vBeta sequence analysis. After 5 weeks of outgrowth TILs were harvested, counted, and frozen viably in Cryostor at between 5 x 106- 2 x 107cells per vial.

[0283] Flow cytometry

[0284] T cell samples were washed in 1x PBS and incubated with Fixable Viability Dye eFluor780 (eBioscience) for 10 mins at room temperature. Cells were then washed in 1x PBS containing 0.5% BSA and stained with an amine reactive viability dye and a combination of fluorescently labeled antibodies against CD3, CD4, CD8, CD45ro, CD62L, LAG3, TIM3, PD1 , CD25, CD69, 0x40, and 41 BB for 15 mins at room temperature (Supplementary Table 2). Samples were then analyzed on a CytoFlex S flow cytometer (Beckman Coulter). Data analysis was performed using FlowJo version 10.6.1 (FlowJo LLC).

[0285] TCR vBeta repertoire sequencing

[0286] T cell receptor repertoire analysis was performed using AmpliSeq for Illumina TCR beta-SR Panel (Illumina, Inc), which is a highly multiplexed targeted resequencing panel that measures T cell diversity and clonal expansion by sequencing T cell receptor (TCR) beta chain rearrangements. Following confirmation of amplification and a successful final library preparation from Bioanalyzer, sequencing was performed on the Illumina MiSeq. The data were then analyzed using MiXCR software (MiLaboratories, Inc), which identifies and counts the V, D and J genes, filters nonproductive sequences, and reports and tracks T cell clonality. Subsequent analysis was done with custom R scripts using the Immunarch package (ImmunoMind Team. (2019). Immunarch: An R Package for Painless Bioinformatics Analysis of T-Cell and B-Cell Immune Repertoires).

[0287] Cocultures and ELISPOTDocket No. 32757 / 59103

[0288] 25,000 TILs outgrown in the presence or absence of anti-CD28 antibody were cultured in triplicate with either 25,000 donor-matched tumor slurry cells, alone as a negative control or in the presence of anti-CD3 / anti-CD28 stimulation beads as a positive control, in a 96 well dual color IFNy / TNF ELISPOT plate for 18 hours at 37C, 5%CC>2 before processing according to the manufacturer’s instructions.Example 2 - Addition of anti-CD28 antibody to tumor slurries increases tumor outgrowth.

[0289] To test the hypothesis that the addition of a small molecule, agonist antibodies, or antagonist antibodies can increase the outgrowth of cells from tumors, a tumor slurry was created from primary ovarian tumors and 25,000 slurry cells were cultured in the presence of an anti-PD1 antagonist antibody, an anti-CTLA agonist antibody, a DcR3 Fas decoy receptor, a TGFp inhibitor named SB431542, zoledronate, and an anti-CD28 agonist antibody, as well as a no-treatment control (Figure 1). While the results yielded no statistically significant differences in cell outgrowth, some of the individual samples treated with anti-CD28 antibody showed strongly increased outgrowth.Example 3 - Addition of anti-CD28 antibody to tumor slurries specifically increases CD4+ cell outgrowth.

[0290] These observations in Example 2 led to the examination of the outgrowth of TILs specifically cultured with anti-CD28 antibody in a more strongly statistically powered experiment.

[0291] The tumor slurries were noted to contain only a small fraction of TILs, with the majority of cells coming from the tumor itself, non-lymphoid immune cells, or other non-tumor cells. Thus, it was likely that any increase in cell count due to TIL outgrowth from these samples could have been offset by die-off from other cells in the culture. To rule out this possibility, the tumor slurries were cultured for 3 weeks with various combinations of anti- CD28 agonist antibody, anti-4-1 BB agonist antibody, anti-PD1 antagonist antibody, and anti- CD2 agonist antibody, as well as a no-antibody control, in media also containing IL-2 and flow cytometry was used to measure the cell counts and frequencies of immune cell subsets (Figure 2A+B). In general, the samples in the anti-CD28 treatment group had higher CD3+ and CD4+ populations as measured both by cell count and cell frequency. Notably, taken together, all conditions that included the anti-CD28 antibody significantly outgrew CD3+ and CD4+ cells compared to samples that did not include the anti-CD28 antibody.

[0292] In contrast, the anti-CD28 antibody treatment had no effect on CD8+ cell count and a reduced cell frequency. The reduced CD8+ cell frequency is likely due to the outgrowth of CD4+ cells. However, it is important to note that the total count of CD8+ cells was extremelyDocket No. 32757 / 59103 low, with fewer than 20 cells in most cultures. Thus, it was difficult to discern the impact of CD28 stimulation on CD8+ TIL from this data set, as the frequency of CD8+ cells of the TIL from these donors was too low for analysis, even in the control samples.Example 4 - CD28 costimulation strongly promotes TIL outgrowth from tumor slurries.

[0293] To determine if targeting other costimulatory molecules has a similar impact as the anti-CD28 antibody, additional agonist antibodies against 4-1 BB, ICOS, and NKG2D were tested. 100,000 tumor slurry cells from two donors were cultured with antibodies targeting CD28, 41 BB, ICOS, and NKG2D, as well as a no-antibody control, and the total number of cells in each subset and their overall count were measured. TIL outgrowth was observed in all treatment conditions, as well as the no-antibody control, by the three-week time point. However, in the anti-CD28 antibody-treated samples, a strong increase in the frequency of CD3+ T cells was observed in both the CD4 and CD8 compartments and, surprisingly, an increase in the number of NK cells were observed from one donor (Figure 3A). However, when examining the total cell counts of each compartment, it was found that samples treated with anti-ICOS or NKG2D antibody also showed an increased outgrowth in the CD3+ compartment (Figure 3B).

[0294] In the second donor, samples treated with anti-CD28 antibody were observed to have an increased CD3+ cell frequency, this was however limited to the CD4+ subset, with no difference in CD8+ cell frequency (Figure 3C). Similar to the first donor, samples treated with anti-ICOS or NKG2D antibody showed an increase in the total cell number in the CD3+ and CD4+ compartments (Figure 3D).

[0295] These data demonstrate that anti-CD28 agonist antibodies can increase TIL outgrowth in both the CD4 and CD8 T cell subsets, depending on the donor. This variability is likely due to innate characteristics of each individual tumor, including the number and health of neoantigen reactive CD8+ TIL located in the tumor. Furthermore, since the ICOS and NKG2D antibody-treated samples also increased in cell number, it is likely that different costimulatory molecules can be used to increase the rate of TIL outgrowth, though some may be more efficacious than others. CD28 antibody's relatively large effect might be due to innate characteristics of CD28 costimulation, antibody titer optimization differences, or differences in the binding affinity of the antibodies.Example 5 - Large-scale outgrowth of TIL from tumor slurries and TIL cell freguency is enhanced in samples treated with anti-CD28 antibodies.

[0296] To determine if CD28 costimulation could be used to outgrow TIL at clinically relevant levels, tumor slurry cells from three donors (Figures 4A, B, & C) were seeded in a 24 well gRex at 6.3 x 106, 7.5 x 106, or 7.9 x 106cells per well. For all donors, CD3+ T cellsDocket No. 32757 / 59103 treated with anti-CD28 antibody expanded more rapidly than the control wells. Notably, there was significant variability in both the total number of CD3+ cells outgrown and the CD4 / CD8 ratio. However, this is consistent with normal TIL outgrowths where there can be substantial variability between patients and even between TIL isolated from different tumor segments from the same patient.

[0297] The frequency of TIL expansion as a fraction of CD45+ cells was also examined, and it was found that the fraction of CD3+ cells from samples treated with anti-CD28 antibody exceeded the fraction from the control samples (Figures 5A, B, & C). Thus, the anti-CD28 antibody is specifically outgrowing TIL.

[0298] The growth kinetics of different populations from different donors varied significantly. Notably, it appears that the CD8 TIL generally takes longer to outgrow than the CD4 population. Additionally, it appeared that the CD3+, CD4+, and CD8+ TIL populations crash at the 4-week timepoint (Figure 5C). However, this was not the case, as the reduction in frequency was due to a massive outgrowth of CD56+ NK cells in all treatment conditions, with the highest outgrowth being in the control wells. Thus, the proportion of CD3+, CD4+, and CD8+ TIL is lower, where the absolute number of TIL remains consistent or increases (Figure 4C).Example 6 - HLA blocking can reduce the increase in cell counts in cultures treated with anti-CD28 antibodies.

[0299] To confirm if the cultures treated with anti-CD28 antibody had increased growth due to T cell activation, cells were cultured in the presence or absence of anti-CD28 antibody with anti-HLA1 or anti-HLA2 antagonist antibody.

[0300] T cell activation requires a “signal 1” provided by the antigen presented on the TCR, in this case tumor neoantigen, and a “signal 2”, in this case provided by the anti-CD28 antibody. Thus, if the increased outgrowth is due to traditional T cell activation by tumor neoantigen presented on tumor cells or professional antigen presenting cells (pAPCs) and costimulation provided by the anti-CD28 antibody, blocking the MHC complex will block the antigen presentation and inhibit the outgrowth seen in cultures treated with anti-CD28 antibody.

[0301] The untreated samples and the samples treated with anti-MHC1 or anti-MHC2 antibody alone had no increased TIL growth (Figures 6A & B). The anti-CD28 antibody treated samples had the highest level of TIL growth, with the samples treated with anti-CD28 antibody as well as anti-MHC1 or MHC2 antibody having severely reduced CD3+ T cell counts and reduced cell outgrowth. This indicates that the increased cell outgrowth is due toDocket No. 32757 / 59103T cell activation, which could be specifically increasing the outgrowth of neoantigen reactive TILExample 7 - TIL outgrown with anti-CD28 antibodies have increased reactivity to tumor slurry.

[0302] To further investigate the potential outgrowth of neoantigen-specific TIL caused by the addition of anti-CD28 antibody to tumor slurry cultures, TILs grown in the presence or absence of anti-CD28 antibody were cocultured with a matched tumor slurry on ELISPOT plates. Wells with TILs alone were used as a negative control and TILs cultured with anti- CD3 / anti-CD28 stimulation beads were used as a positive control. Both the control TILs and the TILs outgrown in the presence of anti-CD28 antibody were negative for interferon gamma production in the negative control, TILs-only wells were positive in well culture with stimulation beads (Figure 7). However, in the samples cocultured with tumor slurry, the amount of TIL producing interferon gamma was much higher in wells containing TIL outgrown with anti-CD28 antibody than in wells containing TIL outgrown without anti-CD28 antibody. This indicated a greater frequency of TIL capable of activating and producing cytokine in response to tumor cells, lending support to the idea that CD28 costimulation during TIL outgrown specifically increases the expansion of neoantigen reactive TIL. Thus, neoantigen reactive TIL may be preferentially outgrown when outgrowth with aCD28.Example 8 - Anti-CD28 antibodies have a potential mechanistic role in the increased outgrowth of neoantigen reactive T cells from the tumor slurries.

[0303] Taken together, the above data indicate that anti-CD28 increases overall TIL numbers and specifically enriches the frequency of tumor neoantigen reactive TIL. Thus, a model is proposed where anti-CD28 antibody, combined with the neoantigen peptides present on the tumor cells in the tumor slurry specifically activate neoantigen TIL causing an overall increase in TIL numbers as well as enriching for neoantigen reactive TIL. Typical T cell activation requires both TCR activation (“signal 1 ”) and costimulatory activation (“signal 2”) (Figure 8A, upper panel). For standard TIL / tumor outgrowth, non-neoantigen reactive TIL do not activate and expand due to missing both signal 1 and signal 2 (Figure 8A, middle panel). Similarly, neoantigen reactive TIL will also not expand even though they will receive signal 1 , because there is no signal 2 (Figure 8A, lower panel). In the present tumor slurries cultured with CD28, non-neoantigen reactive TIL do not become activated due to an absence of signal 1 , whereas tumor neo-antigen reactive TIL are activated as they receive both signal 1 and signal 2 (Figure 8B). This allows for increased expansion of neoantigen reactive TIL in cultures treated with anti-CD28 antibody compared with standard TIL outgrowth methods (Figure 8C).Example 9- CD28 costimulation promotes TIL outgrowth from sarcoma cellsDocket No. 32757 / 59103

[0304] The ability of CD28 agonism to enhance TIL expansion in other tumor types was tested. Sarcoma tumor fragments were obtained from patient samples and seeded as tumor fragments into media containing 10% human serum and 6000 ILI / mL IL-2 supplemented with, or without anti-CD28 agonist antibody. Outgrown TIL were counted over 21 days expansion. Results show that the number of TIL from sarcoma cells is increased in the present of CD28 antibody after 21 days (Figure 9).

[0305] It is understood that every embodiment of the disclosure described herein may optionally be combined with any one or more of the other embodiments described herein. Every patent literature and every non-patent literature cited herein are incorporated herein by reference in their entirety.

[0306] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description, and / or in the attached drawings. Consequently, only such limitations as appear in the appended claims should be placed on the disclosure.

Claims

Docket No. 32757 / 59103What is claimed is:1 . A method for producing tumor-reactive T cells, the method comprising(a) generating a tumor slurry from tumor fragments obtained from a tumor sample;(b) culturing the tumor slurry in a cell culture medium comprising one or more cytokine and an anti-CD28 agonist for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs);(c) harvesting the enhanced population of TILs obtained from step (b).

2. The method of claim 1 wherein the cytokine is interleukin (IL)-2, IL-7, IL-15, IL-21 or combinations thereof.

3. The method of claim 1 or 2, wherein generating a tumor slurry of step (a) comprises incubating the tumor sample in a digestion media comprising at least 3 enzymes.

4. The method of claim 3, wherein the digestion media comprises enzymes selected from the group consisting of trypsin, chymotrypsin, trypsinogen, chymotrypsinogen, dispase, collagenase, accutase, thermolysin, pronase, hyaluronidase, elastase, papain, DNase, neuraminidase, pancreatin or a combination thereof.

5. The method of claim 3 or 4, wherein the digestion media comprises dispase, collagenase, and hyaluronidase.

6. The method of any one of claims 1 to 5, wherein generating a tumor slurry of step (a) further comprises disrupting the tumor sample mechanically so as to dissociate the tumor sample.

7. The method of any one of claims 1 to 6, wherein generating a tumor slurry of step (a) further comprises filtering the tumor slurry to remove any undigested tissue chunks.

8. The method of any one of claims 1 to 7, wherein the method comprises a rapid expansion phase in which TILs previously cultured in the presence of cytokine and a CD28 agonist are activated to proliferate in the presence of irradiated autologous or allogeneic feeder cells.

9. The method of claim 8 further comprising culture of the TIL with anti-CD3 antibody and / or additional growth factors or cytokines that stimulate T cell growth.Docket No. 32757 / 5910310. The method of claim 8 or 9, wherein the rapid expansion phase can take place after harvesting the TIL from culture with cytokine and a CD28 agonist or before harvesting the TIL from culture with cytokine and a CD28 agonist.

11. A method for producing tumor-reactive T cells, the method comprising(a) generating tumor fragments from a tumor sample;(b) culturing the tumor fragments in a cell culture medium comprising one or more cytokines and an anti-CD28 agonist for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs);(c) harvesting the enhanced population of TILs obtained from step (b).

12. The method of claim 11 , wherein the cytokine is interleukin ( I L)-2, IL-7, IL-15, IL-21 or combinations thereof.

13. The method of claim 11 or 12, wherein the method comprises a rapid expansion phase in which TILs previously cultured in the presence in of cytokine and a CD28 agonist are activated to proliferate in the presence of irradiated autologous or allogeneic feeder cells.

14. The method of claim 13 further comprising culture of the TIL with anti-CD3 antibody and / or additional growth factors or cytokines that stimulate T cell growth.

15. The method of claim 13 or 14, wherein the rapid expansion phase can take place after harvesting the TIL from culture with cytokine and a CD28 agonist or before harvesting the TIL from culture with cytokine and a CD28 agonist.

16. The method of any one of claims 1 to 15, wherein the TILs are cultured in the cell culture medium for 2-6 weeks prior to step (c).

17. The method of any one of claims 1 to 16, wherein CD28 agonist is a CD28 agonist antibody.

18. The method of claim 17, wherein the CD28 agonist antibody is present at a concentration in the range of 1 pg / ml to 10 pg / ml.

19. The method of any one of claims 1 to 18, further comprising adding an anti-PD-1 antibody to the culture medium.

20. The method of any one of claims 1 to 19, wherein the IL-2 is present at a concentration of about 100 lU / mL to about 10,000 lU / mL.Docket No. 32757 / 5910321 . The method of any one of claims 1 to 20, wherein culturing in a cell culture medium comprises spiking the cell culture medium with IL-2 between days 3-6 of culture.

22. The method of claim 21 , wherein the IL-2 spike comprises the addition of between 100 to 6,000 lU / mL of IL-2.

23. The method of any one of claims 1 to 22, wherein culturing in a cell culture medium comprises exchanging one half of the spent cell culture medium with fresh replacement media (50:50 media change) containing twice the concentration of IL2 and anti-CD28 agonist, weekly or twice weekly.

24. The method of any one of claims 1 to 23, wherein the cell culture medium comprising IL-2 and or anti-CD28-agonist in culture is replenished every 3-4 days.

25. The method of any one of claims 1 to 24, wherein the TIL are analyzed for tumor neoantigen specificity TCR vBeta repertoire sequencing.

26. A composition comprising enhanced TILs produced by the method of any one of claims 1-25 and a physiologically acceptable excipient.

27. A population of enhanced TILs produced by the method of any one of claims 1 -25.

28. Tumor-reactive T cells comprising T cells with a phenotype selected from the group consisting of one or more of CD3+, CD4+, CD8+, y5TCR+, CD62L+, CD27+, CCR7+, CD45ro+, or CD45ra+, wherein the tumor reactive T cells are produced by a method comprising the steps of:(a) generating a tumor slurry from tumor fragments obtained from a tumor sample or generating tumor fragments from a tumor sample;(b) culturing the tumor slurry or tumor fragments in a cell culture medium one or more cytokine and an anti-CD28 agonist for a time sufficient to obtain an enhanced population of tumor infiltrating lymphocytes (TILs);(c) harvesting the enhanced population of TILs obtained from step (b).

29. A method of treating cancer in a subject in need thereof, comprising administering to the subject the composition of claim 26 or the cells of claim 27 or 28.

30. A method of treating cancer in a subject in need thereof, comprising the steps of:(a) producing a population of enhanced TIL from a tumor fragment from the subject using the method of any one of claims 1 to 25;Docket No. 32757 / 59103(b) treating the subject with nonmyleoablative lymphodepleting chemotherapy; and(c) administering the enhanced population of TILs to the subject.31 . The method of claim 30, wherein the tumor is a solid tumor.

32. The method of claim 30 or 31 , wherein the cancer is selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancers, gastric cancer, glioblastoma, glioma, head and neck cancer, lung cancer, melanoma, mesothelioma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, testicular cancer, thyroid cancer, skin cancer, and uterine cancer.

33. The method of claim 30 or 31 , wherein the cancer is a sarcoma selected from the group consisting of undifferentiated pleomorphic sarcoma, epithelioid sarcoma, liposarcoma, and leiomyosarcoma, lymphoma and myeloma.

34. The method of any one of claims 31 to 33, wherein the tumor is metastatic.

35. The method of any one of claims 31 to 34, wherein the tumor is recurrent.

36. The method of any one of claims 29 to 35, wherein the administration reduces tumor volume in the subject.

37. The method of any one of claims 29 to 36, wherein the administration results in partial or full elimination of the tumor in the subject.

38. The method of any one of claims 29 to 37, wherein between 1 x 106- 9 x 1013enhanced TIL cells / ml are administered to the subject.

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