Expansion of tils utilizing AKT pathway inhibitors

Abstract

To achieve the expansion of TILs utilizing AKT pathway inhibitors.SOLUTION: In some embodiments, methods of expanding tumor infiltrating lymphocytes using AKT pathway inhibitors and methods of using the expanded tumor infiltrating lymphocytes in the treatment of human diseases, including cancers, are disclosed.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION

[0001] The invention described herein relates generally to the expansion of lymphocytes, and more particularly, but not exclusively, to the expansion of lymphocytes using inhibitors of kinases in the AKT signaling pathway. [Background technology]

[0002] Background of the Invention

[0002] Treating resistant cancers using adoptive transfer of tumor-infiltrating lymphocytes (TILs) may be a powerful approach to treating patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. Successful immunotherapy requires large quantities of TILs, thus necessitating a robust and reliable process for manufacturing and commercialization. This scaling for commercialization presents serious challenges due to numerous technical, logistical, and regulatory issues associated with cell expansion. IL-2-based TIL expansion followed by the "Rapid Expansion Process" (REP) is becoming the preferred method for TIL expansion due to its efficiency. Dudley, et al., Science 2002, 298, 850-54;Dudley, et al., J. Clin.Oncol.2005, 23, 2346-57;Dudley, et al., J. Clin.Oncol.2008, 26, 5233-39;Riddell, et al., Science 1992, 257, 238-41;Dudley, et al., J. Immunother.2003, 26, 332-42. REP can result in a 1,000-fold expansion of TILs in a 14-day period, but it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)) as feeder cells, often from multiple donors, as well as anti-CD3 antibodies (OKT3) and high doses of IL-2. (Dudley, et al., J. Immunother. 2003, 26, 332-42) TILs that have undergone the REP procedure have produced successful adoptive cell therapy in some melanoma patients after host immunosuppression.

[0003] Current TIL manufacturing processes are limited by time, expense, sterility concerns, and other factors described herein. Among the many limitations of current processes is the phenotypic variability of expanded lymphocytes. A method of expansion that enriches expanded lymphocyte populations for therapeutically useful phenotypes would greatly advance the art. Such a method is disclosed herein. Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention In one embodiment, the present invention provides a method of treating cancer in a patient suffering from cancer using a population of tumor infiltrating lymphocytes (TILs), comprising: (a) removing a tumor from a patient; (b) obtaining a first population of TILs from the tumor; (c) performing an initial expansion culture of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the second TIL population is at least 5 times greater in number than the first TIL population, the first cell culture medium comprising IL-2, a tumor necrosis factor receptor superfamily (TNFRSF) agonist, and at least one AKT pathway inhibitor (AKTi), and the initial expansion culture is performed for a period of 21 days or less; (d) performing a rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is at least 50-fold greater in number than the second TIL population 7 days after the initiation of the rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (anti-CD3 antibody), peripheral blood mononuclear cells (PBMCs), and optionally a TNFRSF agonist and at least one AKT pathway inhibitor (AKTi), and the rapid expansion is performed for a period of 14 days or less; (e) recovering a third population of TILs; and (f) administering a therapeutically effective portion of the third population of TILs to the patient. The present invention provides a method comprising:

[0005] A method of treating cancer in a patient suffering from cancer using a population of tumor infiltrating lymphocytes (TILs), comprising: (a) removing a tumor from a patient; (b) obtaining a first population of TILs from the tumor; (c) performing an initial expansion culture of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the second TIL population is at least 5 times more numerous than the first TIL population, the first cell culture medium comprising IL-2 and an AKT pathway inhibitor (AKTi), and the initial expansion culture is performed for a period of 21 days or less; (d) performing a rapid expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the third TIL population is at least 50-fold greater in number than the second TIL population 7 days after the initiation of the rapid expansion, the second cell culture medium comprising IL-2, OKT-3 (anti-CD3) antibody, peripheral blood mononuclear cells (PBMCs), and optionally an AKT pathway inhibitor and a second AKT pathway inhibitor (AKTi), and the rapid expansion is performed for a period of 14 days or less; (e) recovering a third population of TILs; and (f) administering a therapeutically effective portion of the third population of TILs to the patient. A method comprising:

[0006] In one embodiment, the present invention provides a method for expanding tumor infiltrating lymphocytes (TILs).

[0007] The present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a tumor sample from a patient, said tumor sample comprising a first population of TILs; (b) processing the tumor sample into a plurality of tumor fragments; (c) adding said tumor fragments to a closed container; (d) performing an initial expansion of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2 and at least one AKT pathway inhibitor (AKTi), and the initial expansion comprises at least 100 cm 2 wherein the initial expansion is performed within a first period of about 7 to 14 days to obtain a second TIL population, the second TIL population being at least 50-fold more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) expanding the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and at least one AKT pathway inhibitor (AKTi) and peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)), wherein the expansion is performed within a second period of about 7 to 14 days to obtain a third TIL population, wherein the third TIL population exhibits an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, and wherein the expansion is performed for a period of at least 500 cm. 2 and the transition from step (d) to step (e) occurs without opening the system; (f) recovering the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; and (g) transferring the recovered TIL population from step (f) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system. Includes.

[0008]

[0008] In some embodiments, the method is an in vitro or ex vivo method.

[0009] In some embodiments, the method further comprises recovering in step (f) via a cell processing system, such as the LOVO system manufactured by Fresenius Kabi. The term "LOVO cell processing system" refers to any device or apparatus manufactured by any vendor that allows for continuous flow and cell processing in a sterile and / or closed environment, where a solution containing cells can be pumped through a membrane or filter, such as a spinning membrane or spinning filter, and the supernatant or cell culture medium is removed without pelleting. In some cases, the cell processing system can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0010]

[0010] In some embodiments, the closed vessel is selected from the group consisting of a G container and a Xuri cell culture bag.

[0011]

[0011] In some embodiments, the infusion bag in step (g) is a HypoThermosol-containing infusion bag.

[0012] In some embodiments, the first period of step (d) and the second period of step (e) are each independently performed within a period of 10 days, 11 days, or 12 days.

[0013] In some embodiments, the first period of step (d) and the second period of step (e) are each carried out separately within a period of 11 days.

[0014] In some embodiments, steps (a) through (g) are carried out within a period of about 25 days to about 30 days.

[0015] In some embodiments, steps (a) through (g) are carried out within a period of about 20 days to about 25 days.

[0016] In some embodiments, steps (a) through (g) are carried out within a period of about 20 days to about 22 days.

[0017] In some embodiments, steps (a) through (g) are performed within 22 days.

[0018]

[0018] In some embodiments, steps (c) to (f) are performed in a single container, and performing steps (c) to (f) in a single container results in an increased number of TILs being generated per resected tumor compared to performing steps (c) to (f) in multiple containers.

[0019] In some embodiments, the PBMCs are added to the TILs during the second period of step (e) without releasing the system.

[0020] In some embodiments, effector T cells and / or central memory T cells obtained from the third TIL population have a higher CD27 expression level than effector T cells and / or central memory T cells obtained from the second TIL population. + Expression of CD28 + The cells exhibit one or more characteristics selected from the group consisting of expression of CD57, longer telomeres, increased CD57 expression, and decreased CD56 expression.

[0021]

[0021] In some embodiments, effector T cells and / or central memory T cells obtained from the third TIL population exhibit increased CD57 expression and decreased CD56 expression compared to effector T cells and / or central memory T cells obtained from the second cell population.

[0022]

[0022] In some embodiments, the risk of microbial contamination is reduced compared to open systems.

[0023] In some embodiments, the TILs from step (g) are infused into a patient. In some embodiments, the TILs from step (g) are infused into a patient in combination with an AKT pathway inhibitor. In some embodiments, the AKT pathway inhibitor is an AKT inhibitor. In some embodiments, the AKT inhibitor is afuresertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and combinations thereof. In some embodiments, the AKT pathway inhibitor (AKTi) is selected from the group consisting of afuresertib, uprosertib, ipatasertib, AT7867, AT13148, MK-2206, LY294002, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and combinations thereof.

[0024] The present invention also provides a method of treating cancer in a patient with a tumor-infiltrating lymphocyte (TIL) population, the method comprising: (a) obtaining a tumor sample from a patient, the tumor sample comprising a first population of TILs; (b) processing the tumor sample into a plurality of tumor fragments; (c) adding the tumor fragments to a first closed container within a closed system; (d) performing an initial expansion of the first TIL population in a first cell culture medium to obtain a second TIL population, wherein the first cell culture medium comprises IL-2 and at least one AKT pathway inhibitor (AKTi), and the initial expansion comprises at least 100 cm 2 wherein the initial expansion is carried out within a first period of about 7 to 14 days to obtain a second TIL population, the second TIL population being at least 50 times more numerous than the first TIL population, and the transition from step (c) to step (d) occurs without opening the system; (e) performing a second expansion of the second TIL population in a second cell culture medium comprising IL-2, OKT-3, and at least one AKT pathway inhibitor (AKTi) and peripheral blood mononuclear cells (PBMCs), the second expansion being performed within a second period of about 7 to 14 days to obtain a third TIL population, the third TIL population exhibiting an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, and the second expansion being performed for at least 500 cm 2 wherein the transition from step (d) to step (e) occurs without opening the system; (f) recovering the third population of TILs obtained from step (e), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the recovered TIL population from step (f) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; and (h) administering a therapeutically effective amount of TIL cells from the infusion bag of step (g) to the patient. Includes:

[0025] In some embodiments, the therapeutically effective amount of TIL cells from the infusion bag of step (h) are administered to the patient in combination with an AKT pathway inhibitor. In some embodiments, the at least one AKT pathway inhibitor is afuresertib or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof, and combinations thereof. In some embodiments, the at least one AKT pathway inhibitor is selected from the group consisting of afuresertib, uprosertib, ipatasertib, AT7867, AT13148, MK-2206, LY294002, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof, and combinations thereof.

[0026] In some embodiments, the method further comprises the culture medium of step (d) comprising one or more AKT pathway inhibitors. In some embodiments, the one or more AKT pathway inhibitors target different protein kinases in the AKT signaling pathway.

[0027] In some embodiments, the method further comprises the culture medium of step (e) comprising one or more AKT pathway inhibitors. In some embodiments, the one or more AKT pathway inhibitors target different kinases in the AKT signaling pathway.

[0028] In some embodiments, the one or more AKT pathway inhibitors are added only to the first culture medium. In some embodiments, the one or more AKT pathway inhibitors are added only to the second culture medium.

[0029] In some embodiments, the one or more AKT pathway inhibitors targeting different kinases in the AKT signaling pathway comprise an AKT inhibitor and an mTOR inhibitor. In some embodiments, the one or more AKT pathway inhibitors targeting different kinases in the AKT signaling pathway comprise an AKT inhibitor and a PI3K inhibitor. In some embodiments, the one or more AKT pathway inhibitors targeting different kinases in the AKT signaling pathway comprise an AKT inhibitor and a FOXO inhibitor. In some embodiments, the one or more AKT pathway inhibitors targeting different kinases in the AKT signaling pathway comprise an AKT inhibitor and a PDK1 inhibitor. In some embodiments, the one or more AKT pathway inhibitors targeting different kinases in the AKT signaling pathway comprise an AKT inhibitor and a CHK1 inhibitor. In some embodiments, the one or more AKT pathway inhibitors targeting different kinases in the AKT signaling pathway comprise an AKT inhibitor and a RAF1 inhibitor. In some embodiments, the one or more AKT pathway inhibitors targeting different kinases in the AKT signaling pathway comprise an AKT inhibitor and an AMPK inhibitor. In some embodiments, the one or more AKT pathway inhibitors that target different kinases in the AKT signaling pathway include an AKT inhibitor and a PIKfyve inhibitor.

[0030] In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, melanoma, resistant melanoma, metastatic melanoma, and non-small cell lung cancer. The histology of solid tumors comprises interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells in which cancer cells may disperse and provide a supportive microenvironment.

[0031]

[0031] Other embodiments include combinations and variations of these compositions and methods.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. [Brief explanation of the drawings]

[0033] [Figure 1]

[0033] Figure 2 shows the main steps of an embodiment of process 2A, including, optionally, cryopreservation of fresh tumor tissue after fragmentation in step B. The total time from the start of the first expansion culture to the end of step E is approximately 22 days. [Figure 2]

[0034] Panels A-C show various steps of an embodiment of the TIL manufacturing process, including cryopreservation of fresh tumor fragments to allow for later initiation of pre-REP cultures. [Figure 3]

[0035] An embodiment of TIL production is shown, including cryopreservation of fresh tumor fragments that are later used to initiate pre-REP cultures, and indicates the point in the procedure where an AKT pathway inhibitor is added to the culture medium. [Figure 4]

[0036] 1 illustrates an embodiment of TIL production involving cryopreservation of fresh tumor fragments that are subsequently used to initiate pre-REP cultures. [Figure 5]

[0037] In general, compare the embodiment of Process 1C with an exemplary embodiment of Process 2A, which contemplates initiation of pre-REP cultures from either fresh tumor tissue fragments or thawed cryopreserved tumor tissue fragments. [Figure 6]

[0038] Additionally, the embodiment of Process 1C is compared to the embodiment of Process 2A. [Figure 7]

[0039] 1 illustrates an embodiment of a 2A process in which REP is started "early." [Figure 8A]

[0040] 1 shows a comparison of the 2A process (an approximately 22 day process) and an embodiment of the Gen3 process for TIL fabrication (an approximately 14-16 day process). [Figure 8B]

[0040] An exemplary Process Gen3 chart (approximately a 14-16 day process) is shown, providing an overview of steps A through F. [Figure 9]

[0041] 1 provides an experimental flow chart for comparing GEN2 (Process 2A) versus GEN3. [Figure 10A]

[0042] 1 shows the L4054-phenotypic characteristics of TIL products in the Gen2 and Gen3 processes. [Figure 10B]

[0042] L4055-phenotypic characteristics of TIL products in Gen2 and Gen3 processes. [Figure 10C]

[0042] M1085T-phenotypic characteristics of TIL products in Gen2 and Gen3 processes. [Figure 11A]

[0043] Memory marker analysis of TIL products from L4054-Gen2 and Gen3 processes. [Figure 11B]

[0043] Memory marker analysis of TIL products from L4055-Gen2 and Gen3 processes. [Figure 11C]

[0043] Memory marker analysis of TIL products from M1085T-Gen2 and Gen3 processes. [Figure 12]

[0044] L4054 activation and depletion markers. (A) Gated on CD4+, (B) Gated on CD8+. [Figure 13]

[0045] L4055 activation and depletion markers. (A) Gated on CD4+, (B) Gated on CD8+. [Figure 14]

[0046] IFNγ production (pg / mL): (A) L4054, (B) L4055, and (C) M1085T for Gen2 and Gen3 processes. Each bar represented here is the mean + SEM of IFNγ levels for stimulated, unstimulated, and medium controls. Optical density measured at 450 nm. [Figure 15]

[0047] ELISA analysis of IL-2 concentration in cell culture supernatants: (A) L4054 and (B) L4055. Each bar represented here is the mean + SEM of IL-2 levels in spent medium. Optical density measured at 450 nm. [Figure 16]

[0048] Quantification of glucose and lactate (g / L) in spent medium: (A) Glucose and (B) Lactate: A decrease in glucose was observed throughout the REP expansion culture for both tumor lines and both processes. Conversely, as expected, an increase in lactate was observed. Both the decrease in glucose and the increase in lactate were comparable between the Gen2 and Gen3 processes. [Figure 17A]

[0049] A) Quantification of L-glutamine in spent medium of L4054 and L4055. [Figure 17B] B) Quantification of Glutamax in spent medium of L4054 and L4055. [Figure 17C] C) Determination of ammonia in spent medium for L4054 and L4055. [Figure 18]

[0050] Telomere length analysis: Relative telomere length (RTL) values ​​indicate the average telomere fluorescence per chromosome / genome in Gen2 and Gen3 processes of the control cell line (1301 leukemia cell line) using the DAKO kit. [Figure 19]

[0051] Unique CDR3 sequence analysis of TIL end products L4054 and L4055 in the Gen2 and Gen3 processes. The columns show the number of unique TCR B clonotypes identified from 1 x 10 cells collected on the harvest day for the Gen2 (e.g., day 22) and Gen3 processes (e.g., days 14-16). Gen3 demonstrates higher clonal diversity compared to Gen2 based on the number of unique peptide CDRs within the sample. [Figure 20]

[0052] Frequency of unique CDR3 sequences in the final cell product (Gen2 (e.g., day 22) and Gen3 process (e.g., days 14-16)) harvested in L4054 IL. [Figure 21]

[0053] Frequency of unique CDR3 sequences in the final cell product (Gen2 (e.g., day 22) and Gen3 process (e.g., days 14-16)) collected from L4055 TILs. [Figure 22]

[0054] Diversity index of the TIL final products of L4054 and L4055 in the Gen2 and Gen3 processes. The Shannon entropy diversity index is a more reliable and common indicator for comparison. Gen3 L4054 and L4055 showed slightly higher diversity than Gen2. [Figure 23]

[0055] Raw cell count data for Day 7 - Gen3REP initiation shown in Table 22 (see Example 6 below). [Figure 24]

[0056] Day 11 - Gen2REP initiation and Gen3 scale-up cell count raw data shown in Table 38 (see Example 6 below). [Figure 25]

[0057] Day 16 - Gen2 scale-up and Gen3 harvest (e.g., day 16) cell count raw data shown in Table 39 (see Example 6 below). [Figure 26]

[0058] The raw cell count data for Day 22 - Gen2 harvest (e.g., Day 22) are shown in Table 39 (see Example 5 below). For L4054 Gen2, the post-LOVO count was extrapolated to 4 flasks since that was the total number for the study. One flask was contaminated and extrapolated to a total of 6.67E+10. [Figure 27]

[0059] Raw data of flow cytometry results shown in Figures 10A, 11A and 11B. [Figure 28]

[0060] Raw data of flow cytometry results shown in Figures 10C and 11C. [Figure 29]

[0061] Raw data of flow cytometry results shown in Figures 12 and 13. [Figure 30]

[0062] Raw data of IFNγ production assay results for L4054 samples shown in FIG. [Figure 31]

[0063] Raw data of IFNγ production assay results for L4055 sample shown in Figure 7 . [Figure 32]

[0064] Raw data of IFNγ production assay results for M1085T samples shown in FIG. [Figure 33]

[0065] Raw data of IL-2 ELISA assay results shown in FIG. [Figure 34]

[0066] The raw data of metabolic substrates and metabolic analysis results shown in Figures 16 and 17. [Figure 35]

[0067] Raw data from the relative telomere length analysis shown in Figure 18. [Figure 36]

[0068] Raw data of unique CD3 sequences and results of clonal diversity analysis shown in Figures 19 and 22. [Figure 37]

[0069] 1 shows a comparison of various Gen2 (2A process) and Gen3.1 process embodiments. [Figure 38]

[0070] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen 3.0 processes. [Figure 39]

[0071] Summary of media conditions for an embodiment of the Gen3 process, referred to as Gen3.1. [Figure 40]

[0072] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen 3.0 processes. [Figure 41]

[0073] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 42]

[0074] 1 is a table providing media uses in various embodiments of the described expansion process. [Figure 43]

[0075] Phenotypic Comparison: The Gen3.0 and Gen3.1 embodiments of the process showed comparable CD28, CD27 and CD57 expression. [Figure 44]

[0076] Higher IFNγ production in the Gen3 final product. To compare both processes, IFNγ analysis (by ELISA) was evaluated on frozen culture supernatants. Fresh TIL products from each Gen2 (e.g., day 22) and Gen3 process (e.g., day 16) were used, and for each tumor, overnight stimulation was performed on anti-CD3 coated plates. Each bar represents the IFNγ levels for stimulated, unstimulated, and medium control. [Figure 45]

[0077] Top: Unique CDR3 sequence analysis of TIL end products: The columns show the number of unique TCR B clonotypes identified from 1 x 10 cells collected in the Gen2 (e.g., day 22) and Gen3 processes (e.g., days 14-16). Gen3 shows higher clonal diversity compared to Gen2, based on the number of unique peptide CDRs within the sample. Bottom: Diversity index of TIL end products: The Shannon entropy diversity index is a more reliable general indicator for comparison. Gen3 showed slightly higher diversity than Gen2. [Figure 46]

[0078] 199 sequences were shared between the Gen3 and Gen2 final products, representing 97.07% of the top 80% of unique CDR3 sequences from Gen2 shared with the Gen3 final product. [Figure 47]

[0079] 1833 sequences were shared between the Gen3 and Gen2 final products and correspond to 99.45% of the top 80% of unique CDR3 sequences from Gen2 shared with the Gen3 final product. [Figure 48]

[0080] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 49]

[0081] Schematic of an exemplary embodiment of expanding TILS from hematopoietic malignancies using the Gen3 process. On day 0, a T cell fraction (CD3+, CD45+) is isolated from lymphocyte-, whole blood-, or tumor digest-enriched apheresis products (fresh or thawed) using positive or negative selection methods, i.e., T cell depletion using T cell markers (CD2, CD3, etc., or other cell depletion leaving T cells), or gradient centrifugation. [Figure 50]

[0082] The schematic diagrams show structures IA and IB, with cylinders indicating individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains, e.g., derived from an antibody that binds to 4-1BBL or 4-1BB, which fold to form a trivalent protein and then link to a second trivalent protein via IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link the two trivalent proteins via disulfide bonds (small, elongated ovals), stabilizing the structure and providing an agonist that can combine six receptor intracellular signaling domains and signaling proteins to form a signaling complex. The TNFRSF-binding domain, shown as a cylinder, can be, for example, an scFv domain comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility and Glu and Lys for solubility. DETAILED DESCRIPTION OF THE INVENTION

[0034] Brief description of the sequence listing

[0083] SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0084] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0085] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0086] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0087] SEQ ID NO: 5 is the amino acid sequence of recombinant human IL-4 protein.

[0088] SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

[0089] SEQ ID NO: 7 is the amino acid sequence of recombinant human IL-15 protein.

[0090] SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

[0091] SEQ ID NO: 9 is the amino acid sequence of human 4-1BB.

[0092] SEQ ID NO: 10 is the amino acid sequence of mouse 4-1BB.

[0093] SEQ ID NO: 11 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0094] SEQ ID NO: 12 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0095] SEQ ID NO: 13 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0096] SEQ ID NO: 14 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0097] SEQ ID NO: 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0098] SEQ ID NO: 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0099] SEQ ID NO: 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0100] SEQ ID NO: 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0101] SEQ ID NO: 19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0102] SEQ ID NO: 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0103] SEQ ID NO: 21 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0104] SEQ ID NO: 22 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0105] SEQ ID NO: 23 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0106] SEQ ID NO: 24 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0107] SEQ ID NO: 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0108] SEQ ID NO: 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0109] SEQ ID NO: 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0110] SEQ ID NO: 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0111] SEQ ID NO: 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0112] SEQ ID NO: 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0113] SEQ ID NO: 31 is the Fc domain of the TNFRSF agonist fusion protein.

[0114] SEQ ID NO: 32 is the linker of the TNFRSF agonist fusion protein.

[0115] SEQ ID NO: 33 is the linker of the TNFRSF agonist fusion protein.

[0116] SEQ ID NO: 34 is the linker of the TNFRSF agonist fusion protein.

[0117] SEQ ID NO: 35 is the linker of the TNFRSF agonist fusion protein.

[0118] SEQ ID NO: 36 is the linker of the TNFRSF agonist fusion protein.

[0119] SEQ ID NO: 37 is the linker of the TNFRSF agonist fusion protein.

[0120] SEQ ID NO: 38 is the linker of the TNFRSF agonist fusion protein.

[0121] SEQ ID NO: 39 is the linker of the TNFRSF agonist fusion protein.

[0122] SEQ ID NO: 40 is the linker of the TNFRSF agonist fusion protein.

[0123] SEQ ID NO: 41 is the linker of the TNFRSF agonist fusion protein.

[0124] SEQ ID NO: 42 is the Fc domain of the TNFRSF agonist fusion protein.

[0125] SEQ ID NO: 43 is the linker of the TNFRSF agonist fusion protein.

[0126] SEQ ID NO: 44 is the linker of the TNFRSF agonist fusion protein.

[0127] SEQ ID NO: 45 is the linker of the TNFRSF agonist fusion protein.

[0128] SEQ ID NO: 46 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0129] SEQ ID NO:47 is the soluble portion of the 4-1BBL polypeptide.

[0130] SEQ ID NO: 48 is the heavy chain variable region (VH) of 4-1BB agonist antibody 4B4-1-1 version 1.

[0131] SEQ ID NO: 49 is the light chain variable region (VL) of 4-1BB agonist antibody 4B4-1-1 version 1.

[0132] SEQ ID NO: 50 is the heavy chain variable region (VH) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0133] SEQ ID NO: 51 is the light chain variable region (VL) of the 4-1BB agonist antibody 4B4-1-1 version 2.

[0134] SEQ ID NO: 52 is the heavy chain variable region (VH) of the 4-1BB agonist antibody H39E3-2.

[0135] SEQ ID NO: 53 is the light chain variable region (VL) of the 4-1BB agonist antibody H39E3-2.

[0136] SEQ ID NO: 54 is the amino acid sequence of human OX40.

[0137] SEQ ID NO: 55 is the amino acid sequence of mouse OX40.

[0138] SEQ ID NO: 56 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0139] SEQ ID NO: 57 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0140] SEQ ID NO: 58 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0141] SEQ ID NO: 59 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0142] SEQ ID NO: 60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0143] SEQ ID NO: 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0144] SEQ ID NO: 62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0145] SEQ ID NO: 63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0146] SEQ ID NO: 64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0147] SEQ ID NO: 65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0148] SEQ ID NO: 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0149] SEQ ID NO: 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0150] SEQ ID NO: 68 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 11D4.

[0151] SEQ ID NO: 69 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 11D4.

[0152] SEQ ID NO: 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0153] SEQ ID NO: 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0154] SEQ ID NO: 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0155] SEQ ID NO: 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0156] SEQ ID NO: 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0157] SEQ ID NO: 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0158] SEQ ID NO: 76 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0159] SEQ ID NO: 77 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0160] SEQ ID NO: 78 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody 18D8.

[0161] SEQ ID NO: 79 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody 18D8.

[0162] SEQ ID NO: 80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0163] SEQ ID NO: 81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0164] SEQ ID NO: 82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0165] SEQ ID NO: 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0166] SEQ ID NO: 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0167] SEQ ID NO: 85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0168] SEQ ID NO: 86 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu119-122.

[0169] SEQ ID NO: 87 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu119-122.

[0170] SEQ ID NO: 88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0171] SEQ ID NO: 89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0172] SEQ ID NO: 90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0173] SEQ ID NO: 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0174] SEQ ID NO: 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0175] SEQ ID NO: 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0176] SEQ ID NO: 94 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody Hu106-222.

[0177] SEQ ID NO: 95 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody Hu106-222.

[0178] SEQ ID NO: 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0179] SEQ ID NO: 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0180] SEQ ID NO: 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0181] SEQ ID NO: 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0182] SEQ ID NO: 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0183] SEQ ID NO: 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0184] SEQ ID NO: 102 is the OX40 ligand (OX40L) amino acid sequence.

[0185] SEQ ID NO: 103 is the soluble portion of the OX40L polypeptide.

[0186] SEQ ID NO: 104 is an alternative soluble portion of the OX40L polypeptide.

[0187] SEQ ID NO: 105 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 008.

[0188] SEQ ID NO: 106 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 008.

[0189] SEQ ID NO: 107 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 011.

[0190] SEQ ID NO: 108 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 011.

[0191] SEQ ID NO: 109 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 021.

[0192] SEQ ID NO: 110 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 021.

[0193] SEQ ID NO: 111 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 023.

[0194] SEQ ID NO: 112 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 023.

[0195] SEQ ID NO: 113 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0196] SEQ ID NO: 114 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0197] SEQ ID NO: 115 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0198] SEQ ID NO: 116 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody.

[0199] SEQ ID NO: 117 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0200] SEQ ID NO: 118 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0201] SEQ ID NO: 119 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0202] SEQ ID NO: 120 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0203] SEQ ID NO: 121 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0204] SEQ ID NO: 122 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

[0205] SEQ ID NO: 123 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0206] SEQ ID NO: 124 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

[0207] SEQ ID NO: 125 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

[0208] SEQ ID NO: 126 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

[0209] SEQ ID NO: 127 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0210] SEQ ID NO: 128 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0211] SEQ ID NO: 129 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0212] SEQ ID NO: 130 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0213] SEQ ID NO: 131 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0214] SEQ ID NO: 132 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0215] SEQ ID NO: 133 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0216] SEQ ID NO: 134 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0217] SEQ ID NO: 135 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0218] SEQ ID NO: 136 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0219] SEQ ID NO: 137 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0220] SEQ ID NO: 138 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0221] SEQ ID NO: 139 is the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0222] SEQ ID NO: 140 represents the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0223] SEQ ID NO: 141 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0224] SEQ ID NO: 142 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0225] SEQ ID NO: 143 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0226] SEQ ID NO: 144 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0227] SEQ ID NO: 145 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0228] SEQ ID NO: 146 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0229] SEQ ID NO: 147 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0230] SEQ ID NO: 148 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0231] SEQ ID NO: 149 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0232] SEQ ID NO: 150 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0233] SEQ ID NO: 151 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0234] SEQ ID NO: 152 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0235] SEQ ID NO: 153 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0236] SEQ ID NO: 154 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0237] SEQ ID NO: 155 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0238] SEQ ID NO: 156 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0239] SEQ ID NO: 157 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0240] SEQ ID NO: 158 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0241] SEQ ID NO: 159 is the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0242] SEQ ID NO: 160 represents the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0243] SEQ ID NO: 161 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0244] SEQ ID NO: 162 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0245] SEQ ID NO: 163 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0246] SEQ ID NO: 164 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0247] SEQ ID NO: 165 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0248] SEQ ID NO: 166 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0249] SEQ ID NO: 167 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0250] SEQ ID NO: 168 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0251] SEQ ID NO: 169 is the heavy chain variable region (V H ) amino acid sequence.

[0252] SEQ ID NO: 170 represents the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0253] SEQ ID NO: 171 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0254] SEQ ID NO: 172 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0255] SEQ ID NO: 173 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0256] SEQ ID NO: 174 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0257] SEQ ID NO: 175 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0258] SEQ ID NO: 176 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0035] Detailed Description of the Invention

[0259] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

[0036] definition

[0260] The term "AKT pathway inhibitor" refers to an inhibitor of the kinases and phosphatases that make up the AKT signaling network, which includes PI3K, AKT, PDK1, mTORC2, GSK3, FOXO (including FOXO1, FOXO3A, and FOXO4), mTORC1, PTEN, BAD, ASK1, MERIT40, MDM2, CHK1, IKKα, p21CIP1, p27KIP1, RAF1, PFKFB2, PDE3B, AMPK, ACLY, PIKfyve, eNOS, and WNK1.

[0037]

[0261] The terms "LOVO cell processing system" and "LOVO" refer to a cell processing system manufactured by Fresenius Kabi USA, LLC. These two terms also refer to any device or apparatus manufactured by any vendor that allows for continuous flow and cell processing in a sterile and / or closed environment, where a solution containing cells can be pumped through a membrane or filter, such as a spinning membrane or spinning filter, and the supernatant or cell culture medium is removed without pelleting. In some embodiments, such cell harvesters and / or cell processing systems can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0038]

[0262] The term "fungin" refers to the antifungal reagent Fungin™ sold by InvitroGen, San Diego, CA, USA (catalog numbers ant-fn-1 and ant-fn-2). Fungin is a soluble formulation of pimaricin, CAS 7681-93-8. As used herein, "fungin" encompasses any commercially available formulation of pimaricin or natamycin.

[0039]

[0263] The term "Fungizone" is a trademark of ERSquibb and Sons, LLC, and refers to the antifungal amphotericin B, CAS 1397-89-3. Amphotericin B is commercially available, for example, from SIGMA-Aldrich, St. Louis, MO, USA (catalog number A2942, as a 250 μg / mL solution in deionized water). As used herein, "Fungizone" encompasses any commercially available formulation of amphotericin B.

[0040]

[0264] The term "physiologically buffered isotonic saline" refers to any one of many salt solutions known to those skilled in the art in which the solution is made at physiological pH and isotonic salt concentration. In the art, these are commonly referred to as balanced salt solutions. Without limitation, such physiologically buffered isotonic saline can include Hank's balanced salt solution ("HBSS"), Tris-buffered saline ("TBS"), phosphate-buffered saline ("PBS"), or Dulbecco's phosphate-buffered saline ("DPBS" or "dPBS").

[0041]

[0265] The term "in vivo" refers to an event that takes place inside a subject's body.

[0042]

[0266] The term "in vitro" refers to events that take place outside a subject's body. In vitro assays include cell-based assays, utilizing live or dead cells, and can also include cell-free assays, which do not utilize intact cells.

[0043]

[0267] The term "ex vivo" refers to events involving the treatment or implementation of treatment on cells, tissues and / or organs that have been removed from a subject's body. Suitably, the cells, tissues and / or organs may be returned to the subject's body by way of surgery or treatment.

[0044]

[0268] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold) over a one-week period, more preferably at least about 10-fold (or 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold) over a one-week period, or most preferably at least about 100-fold over a one-week period. Several rapid expansion protocols are outlined below.

[0045]

[0269] As used herein, "tumor-infiltrating lymphocytes" or "TILs" refer to a population of cells originally obtained as white blood cells that have left a subject's bloodstream and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell populations expanded or grown as discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0046]

[0270] As used herein, a "cell population" (including TILs) refers to a large number of cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The range is 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion cultures typically yield a bulk TIL population of 1.5 x 10 cells. 9 ~1.5×10 10 This is done to provide a population of cells for injection.

[0047]

[0271] As used herein, "cryopreserved TILs" refers to TILs, either primary, bulk, or expanded culture (REP TILs), that are processed and stored at temperatures ranging from about -150°C to -60°C. General cryopreservation methods are described elsewhere herein, including in the Examples. For clarity and to prevent misinterpretation, "cryopreserved TILs" are distinguishable from frozen tissue samples that may be used as a source of primary TILs.

[0048]

[0272] As used herein, "thawed cryopreserved TILs" refers to a population of TILs that have been previously cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperatures or temperatures at which the TILs can be administered to a patient.

[0049]

[0273] TILs can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and affect treatment. TILs can generally be classified by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0050]

[0274] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 5% v / v DMSO to 10% v / v DMSO, and can also include media containing 7% v / v DMSO to 10% v / v DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium is sometimes referred to by the trade name "CryoStor® CS10." CS10 medium is a serum-free, animal-component-free medium containing DMSO.

[0051]

[0275] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 hi ) and CD62L (CD62 hi) is a subset of T cells that constitutively express the CD4 receptor. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0052]

[0276] The term "effector memory T cells" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+ but lack constitutive expression of CCR7 (CCR7lo) and have heterogeneous or low CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BLIMP1. After antigen stimulation, effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestine in humans. CD8+ effector memory T cells possess high amounts of perforin.

[0053]

[0277] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Examples of closed systems include, but are not limited to, closed G-containers. After tumor segments are added to the closed system, the system is not open to the external environment until immediately before the TILs are administered to a patient.

[0054]

[0278] The terms "fragmentation," "fragments," and "fragmented" as used herein to describe the process of disrupting tumors include mechanical fragmentation methods such as crushing, slicing, splitting, and mincing tumor tissue, as well as any other method that disrupts the physical structure of tumor tissue.

[0055]

[0279] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are a type of antigen-presenting cell.

[0056]

[0280] The term "anti-CD3 antibody" refers to an antibody or variant thereof, such as a monoclonal antibody, including human, humanized, chimeric, or murine antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0057]

[0281] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody or biosimilar or variant thereof, including a human, humanized, chimeric, or murine antibody directed against the CD3 receptor in the T cell antigen receptor of mature T cells, including OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or commercially available forms thereof, such as variants, conservative amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0058] [Table 1]

[0059] [Table 2]

[0060]

[0282] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, 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. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple sources at 22 million IU per single-use vial) and commercially available forms of recombinant IL-2 from CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Catalog No. CYT-209-b), as well as other commercially available equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2, as described herein, also encompasses pegylated forms of IL-2, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. 2014 / 0328791 A1 and WO 2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4902,502, the disclosures of which are incorporated herein by reference.Formulations of IL-2 suitable for use in the present invention are described in US Pat. No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0061]

[0283] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell expansion and class II MHC expression and induces class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 5).

[0062]

[0284] The term "IL-7" (also referred to herein as "IL7") refers to the glycosylated tissue-derived cytokine known as interleukin-7, which is available from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, in a series of signals important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 6).

[0063]

[0285] The term "IL-15" (also referred to herein as "IL15") refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 7).

[0064]

[0286] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc., 13:379-95 (2014), the disclosure of which is incorporated herein by reference. IL-21 is produced primarily by natural killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several sources, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, catalog number 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 8).

[0065]

[0287] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve the intended use, including but not limited to disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro, in vivo, and ex vivo) or the subject and disease state being treated (e.g., the subject's weight, age, and sex), the severity of the disease state, or the method of administration, among other factors, which can be readily determined by one of ordinary skill in the art. The term also applies to a dose that elicits a specific response in target cells (e.g., decreased platelet adhesion and / or cell migration). The specific dose will vary depending on the particular compound selected, the administration regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system by which the compound is delivered.

[0066]

[0288] A "therapeutic effect," as that term is used herein, encompasses a therapeutic benefit and / or a prophylactic benefit. A prophylactic benefit includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0067]

[0289] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersants, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0068]

[0290] When ranges are used herein to describe physical or chemical properties, such as, for example, molecular weight or chemical formula, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The use of the term "about" when referring to a numerical value or numerical range means that the referenced numerical value or numerical range is an approximation within experimental variation (or within statistical experimental error), and thus the numerical value or numerical range may vary. Variation is typically 0% to 15%, preferably 0% to 10%, and more preferably 0% to 5% of the stated numerical value or numerical range. The term "comprise" (and related terms such as "comprise" or "comprises," or "having," or "including") includes embodiments, such as, for example, embodiments of any composition of matter, method, or process that "consists of" or "consists essentially of" the described property.

[0069]

[0291] The compounds of the present invention also include antibodies. The term "antibody" or "antibodies" refers to whole immunoglobulins and any antigen-binding fragments ("antigen-binding portions") or single chains thereof. "Antibody" also refers to a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which can be interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigen epitopes. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0070]

[0292] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody (e.g., IL-33, ST2, CD20, PD-1, PD-L1, or PD-L2) that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L(ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or V L and (vi) isolated complementarity-determining regions (CDRs). In addition, two domains of an Fv fragment, V, and VB, may be included. L and V H are encoded by separate genes, which can be synthesized by recombinant methods. L and V H Pairs of domains can be linked by a synthetic linker, allowing them to be produced as a single protein chain forming a monovalent molecule known as a single-chain Fv (scFv); see, e.g., Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883. Such scFv antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0071]

[0293] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0072]

[0294] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas obtained from transgenic non-human animals, e.g., transgenic mice, whose genomes include human heavy chain and light chain transgenes fused to an immortalized cell.

[0073]

[0295] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express human antibodies; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used) to thereby modify the V and V sequences of the recombinant antibodies. H and V L The amino acid sequence of the region is similar to that of the human germline V H and V L These are sequences that, while derived from and related to sequences, may not naturally occur within the human antibody germline repertoire in vivo.

[0074]

[0296] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. Five antibody isotypes exist in mammals: IgA, IgD, IgG, IgM, and IgE. In humans, the IgG isotype has four subclasses: IgG1, IgG2, IgG3, and IgG4, and the IgA isotype has two subclasses: IgA1 and IgA2.

[0075]

[0297] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0076]

[0298] The term "human antibody derivative" refers to any mutated form of a human antibody, for example, a conjugate of an antibody with another active pharmaceutical ingredient or antibody. The terms "conjugate," "antibody-drug conjugate," "ADC," or "immunoconjugate" refer to an antibody or fragment thereof conjugated to a therapeutic moiety, such as a bacterial toxin, a cytotoxic drug, or a radionuclide-containing toxin. Toxic moieties can be conjugated to the antibodies of the invention using methods available in the art.

[0077]

[0299] The terms "humanized antibody" and "humanization" are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies containing minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable regions have been replaced by residues from 15 hypervariable regions of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol 1992, 2, 593-596.

[0078]

[0300] The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0079]

[0301] A "diabody" is a small antibody fragment with two antigen-binding sites. The fragments are bound to the same polypeptide chain (V H -V L or V L -V H ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain and create two antigen-binding sites. Diabodies are further described in, for example, EP 404,097; WO 93 / 11161; and Bolliger et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.

[0080]

[0302] The term "glycosylation" refers to an engineered derivative of an antibody. An aglycosylated antibody lacks glycosylation. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. As described in U.S. Pat. Nos. 5,714,350 and 6,350,861, aglycosylation can increase the affinity of an antibody for an antigen. Additionally or alternatively, antibodies can be generated with altered glycosylation types, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase antibody potency. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha(1,6) fucosyltransferase), and therefore antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. Ms704, Ms705, and Ms709 FUT8 - / -The cell line was generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see, e.g., U.S. Patent Application Publication No. 2004 / 0110704 or Yamane-Ohnuki et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, European Patent No. 1,176,195 describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating alpha-1,6 bond-related enzymes. The document also describes cell lines, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662), with reduced or no enzymatic activity that adds fucose to N-acetylglucosamine linked to the Fc region of antibodies. WO 03 / 035835 describes a mutant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates and results in hypofucosylation of antibodies expressed in the host cells (see also Shields, et al., J. Biol Chem. 2002, 277, 26733-26740). WO 99 / 54342 describes cell lines that have been engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibody (Umana, et al., Nat. Biotech. 1999, 17, (See also, e.g., 176-180.) Alternatively, the fucose residues of antibodies can be cleaved using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem. 1975, 14, 5516-5523.

[0081]

[0303] "PEGylation" refers to a modified antibody or fragment thereof that is reacted with polyethylene glycol (PEG), typically a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. PEGylation can, for example, increase the biological (e.g., serum) half-life of the antibody. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to a mono (C1-C 10 The term "PEG" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody to be pegylated may be an aglycosylated antibody. Methods of pegylation are known in the art and can be applied to the antibodies of the invention, for example, as described in European Patent Nos. 0154316 and 0401384.

[0082]

[0304] The term "biosimilar" refers to a biological product that is highly similar to a reference biological product approved in the United States, despite minor differences in clinically inactive components, and there are no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. Furthermore, a similar biological or "biosimilar" drug is a biological product similar to another biological product already approved for use by the European Medicines Agency. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions. Biological products or biological products are medicines made by or derived from biological sources, such as bacteria or yeast. They can consist of relatively small molecules, such as human insulin or erythropoietin, or complex molecules, such as monoclonal antibodies. For example, if the reference anti-CD20 monoclonal antibody is rituximab, an anti-CD20 biosimilar monoclonal antibody approved by a drug regulatory agency for rituximab is a "biosimilar to" or "biosimilar of" rituximab. In Europe, a similar biological or "biosimilar" medicinal product is a biological product similar to another biological product already authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological uses in Europe is Article 6 of Regulation (EC) No. 726 / 2004, as amended, and Article 10(4) of Directive 2001 / 83 / EC. Therefore, in Europe, a biosimilar may be authorized, authorized, or the subject of an authorization application under Article 6 of Regulation (EC) No. 726 / 2004 and Article 10(4) of Directive 2001 / 83 / EC. In Europe, the original biological product already authorized is sometimes referred to as the "reference medicinal product." Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP guidelines on similar biological medicinal products. Additionally, product-specific guidelines, including those for monoclonal antibody biosimilars, are provided by the EMA on a product-by-product basis and are published on its website. The biosimilars described herein may be similar to the reference drug in terms of quality characteristics, biological activity, mechanism of action, safety profile, and / or efficacy.Furthermore, biosimilars may be used or intended for use to treat the same condition as the reference medicinal product. Thus, biosimilars described herein may be considered to have similar or very similar quality characteristics to the reference medicinal product. Alternatively or additionally, biosimilars described herein may be considered to have similar or very similar biological activity to the reference medicinal product. Alternatively or additionally, biosimilars described herein may be considered to have a similar or very similar safety profile to the reference medicinal product. Alternatively or additionally, biosimilars described herein may be considered to have similar or very similar efficacy to the reference medicinal product. As described herein, biosimilars in Europe are compared to a reference medicinal product authorized by the EMA. However, in some cases, biosimilars may be compared to biological medicinal products authorized outside the European Economic Area (non-EEA "comparators") in specific studies. Such studies include, for example, specific clinical trials and in vivo non-clinical studies. As used herein, the term "biosimilar" also refers to biological medicinal products that have been or can be compared to a non-EEA-authorized comparator. Particular biosimilars are proteins, such as antibodies, antibody fragments (e.g., antigen-binding portions), and fusion proteins. Protein biosimilars may have amino acid sequences with minor modifications to the amino acid structure (e.g., including amino acid deletions, additions, and / or substitutions) that do not significantly affect the function of the polypeptide. A biosimilar may include an amino acid sequence with 97% or greater sequence identity, e.g., 97%, 98%, 99%, or 100%, to the amino acid sequence of its reference drug. A biosimilar may include one or more post-translational modifications, such as, but not limited to, glycosylation, oxidation, deamidation, and / or cleavage, that differ from those of the reference drug, provided that the differences do not result in changes in the drug's safety and / or efficacy. A biosimilar may have the same or different glycosylation pattern as the reference drug. Although not exclusively, a biosimilar may have a different glycosylation pattern, particularly if the differences address or are intended to address safety concerns related to the reference drug.Furthermore, a biosimilar may deviate from a reference drug, for example, in its strength, pharmaceutical form, formulation, excipients, and / or presentation, provided that the safety and efficacy of the drug are not compromised. A biosimilar may contain, for example, differences in its pharmacokinetic (PK) and / or pharmacodynamic (PD) profile compared to the reference drug, but still be deemed sufficiently similar to the reference drug to be approved or considered suitable for approval. In certain circumstances, a biosimilar may exhibit different binding properties compared to the reference drug, where the different binding properties are not considered by regulatory authorities, such as the EMA, to be a barrier to approval as a similar biological product. The term "biosimilar" is also used interchangeably by regulatory agencies in other countries and regions.

[0083]

[0305] The term "hematologic malignancies" refers to mammalian cancers and tumors of hematopoietic and lymphoid tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematologic malignancies are also referred to as "liquid tumors." Hematologic malignancies include, but are not limited to, ALL, CLL, SLL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematologic malignancies" refers to hematologic malignancies affecting B cells.

[0084]

[0306] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. The histology of a solid tumor comprises interdependent tissue compartments, including parenchyma (cancer cells), and supporting stromal cells in which cancer cells may be dispersed and provide a supportive microenvironment.

[0085]

[0307] The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors are also referred to herein as bone marrow-infiltrating lymphocytes (MILs).

[0086]

[0308] The term "microenvironment" as used herein may refer to the solid or hematologic tumor microenvironment as a whole or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to the complex mixture of cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, cultivate therapeutic resistance, and provide a niche for successful and dominant metastasis, as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that are recognized by T cells, elimination of tumors by the immune system is rare due to immunosuppression by the microenvironment.

[0087]

[0309] In certain embodiments, the present invention includes a method of treating cancer with a TIL population, wherein the patient is conditioned with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In some embodiments, a TIL population can be provided, wherein the patient is conditioned with non-myeloablative chemotherapy prior to infusion of TILs according to the present invention. In one embodiment, the non-myeloablative chemotherapy comprises cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m for 5 days (27-23 days before TIL infusion). 2 In one embodiment, after non-myeloablative chemotherapy and TIL infusion according to the present invention (day 0), the patient receives an intravenous infusion of 720,000 IU / kg of IL-2 intravenously every 8 hours to a physiologically tolerated dose.

[0088]

[0310] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (also referred to as "immunosuppressive conditioning") on patients prior to introducing the rTILs of the present invention.

[0089]

[0311] The terms "sequence identity," "percent identity," and "percent sequence identity" in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparisons between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign available from DNASTAR are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine appropriate parameters for maximal alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0090]

[0312] Certain embodiments of the present invention include variants of antibodies, such as anti-IL-33 or anti-ST2 antibodies and / or anti-CD20 antibodies and / or anti-PD-1 antibodies, anti-PD-L1 and / or anti-PD-L2 antibodies. As used herein, the term "variant" encompasses, but is not limited to, antibodies comprising an amino acid sequence that differs from that of a reference antibody by one or more substitutions, deletions, and / or additions at specific positions within or adjacent to the amino acid sequence of the reference antibody. A variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example, substitutions of similarly charged or uncharged amino acids. A variant retains the ability of the reference antibody to specifically bind to the antigen.

[0091]

[0313] For the avoidance of doubt, it is intended that a particular feature (e.g., an integer, property, value, use, disease, formula, compound, or group) described herein in connection with a particular aspect, embodiment, or example of the invention is to be understood as also being applicable to any other aspect, embodiment, or example described herein, unless incompatible. Accordingly, such features may be used, where appropriate, with any of the definitions, claims, or embodiments defined herein. All features disclosed herein (including the accompanying claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except for combinations in which at least some features and / or steps are mutually exclusive. The invention is not limited to any of the details of the disclosed embodiments. The invention extends to any novel or novel combination of features disclosed herein (including the accompanying claims, abstract, and drawings) or any novel or novel combination of steps of any method or process so disclosed.

[0092] AKT pathway inhibitors

[0314] The Akt family of serine / threonine kinases includes several members, including Akt1 (also known as PKB or RacPK), Akt2 (also known as PKBβ or RacPK-β), and Akt3 (also known as PKBγ or thymic viral proto-oncogene 3). Akt1 and Akt2 are activated by PDGF stimulation. AKT kinases are tethered to the membrane by a pleckstrin homology domain through interaction with the PIP3 lipid. Manning and Toker summarize the past 25 years of research into this complex signaling pathway in “AKT / PKB Signaling: Navigating the Network,” Cell 169:381–405 (2017).

[0093] Afresertib

[0315] In one embodiment, the AKT pathway inhibitor is afuresertib. Afuresertib is also known by names such as CAS1047644-62-1, GSK2110183, ZINC43197674, and CHEMBL2219422. Afuresertib is an orally bioavailable inhibitor of the serine / threonine protein kinase Akt (protein kinase B). In one embodiment, the AKT pathway inhibitor is N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methylpyrazol-3-yl)thiophene-2-carboxamide.

[0094]

[0316] In one embodiment, the AKT pathway inhibitor has the formula: [ka] is a compound of

[0095]

[0317] Synthetic methods are disclosed, for example, in Lin et al., "Discovery of 2-pyrimidyl-5-amidothiophenes as potent inhibitors for AKT: Synthesis and SAR studies", Bioorg.Med.Chem.Let. 16:4163-4168 (2006) and U.S. Patent Application Publication No. 20100041726A1 (Seefeld et al.), both of which are incorporated by reference in their entireties, particularly the synthetic methods and methods for identifying and analyzing such compounds detailed, for example, in Schemes 1-3.

[0096] Uprosertib

[0318] In one embodiment, the AKT pathway inhibitor is uprosertib. In one embodiment, the AKT pathway inhibitor is N-[(2S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methylpyrazol-3-yl)furan-2-carboxamide. In one embodiment, the AKT pathway inhibitor is represented by the formula: [ka] is a compound of

[0097]

[0319] Synthetic methods are disclosed, for example, in US Patent Application Publication No. 20100041726A1 (Seefeld et al.).

[0098] Ipatasertib

[0320] In one embodiment, the AKT pathway inhibitor is ipatasertib. In one embodiment, the AKT pathway inhibitor is 2-(4-chlorophenyl)-1-(4-(7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta(d)pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one. In one embodiment, the AKT pathway inhibitor is a compound represented by the formula: [ka] is a compound of

[0099]

[0321] Synthetic methods are disclosed, for example, in US Pat. No. 8,063,050, which is incorporated by reference in its entirety, with particular attention to Formula I and Schemes AJ.

[0100] AT7867

[0322] In one embodiment, the AKT pathway inhibitor is AT7867. In one embodiment, the AKT pathway inhibitor is 4-(4-chlorophenyl)-4-(4-(1h-pyrazol-4-yl)phenyl)piperidine. In one embodiment, the AKT pathway inhibitor has the formula: [ka] is a compound of

[0101]

[0323] Synthetic methods are disclosed, for example, in US Pat. No. 8,247,576, which is incorporated by reference in its entirety, with particular attention to Examples 57 and 63-68.

[0102] AT13148

[0324] In one embodiment, the AKT pathway inhibitor is AT13148, also known as CS-3136 and ZINC95920747. In one embodiment, the AKT pathway inhibitor is (S)-1-(4-(1H-pyrazol-4-yl)phenyl)-2-amino-1-(4-chlorophenyl)ethanol. In one embodiment, the AKT pathway inhibitor has the formula: [ka] is a compound of

[0103]

[0325] Synthetic methods are disclosed, for example, in US Pat. No. 8,247,576, which is incorporated by reference in its entirety.

[0104] MK-2206

[0326] In one embodiment, the AKT pathway inhibitor is MK-2206, also known as 1032349-93-1. In one embodiment, the AKT pathway inhibitor is 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one. In one embodiment, the AKT pathway inhibitor is a compound represented by the formula: [ka] is a compound of

[0105]

[0327] Synthetic methods are disclosed, for example, in US Pat. No. 8,609,688, which is incorporated by reference in its entirety, with particular attention to General Procedure A.

[0106]

[0328] Therapeutic uses are disclosed by Yap et al., "First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 in patients with advanced solid tumors," J Clin Oncol. 29:4688-95 (2011).

[0107] LY294002

[0329] In one embodiment, the AKT pathway inhibitor is LY294002, also known as CAS154447-36-6. In one embodiment, the AKT pathway inhibitor is 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one. In one embodiment, the AKT pathway inhibitor is a compound represented by the formula: [ka] is a compound of

[0108]

[0330] Synthetic methods are disclosed, for example, in U.S. Patent Nos. 6,800,620 and 7,511,041, which are incorporated by reference in their entireties. Particular attention is directed to the synthetic methods taught in the General Procedures disclosed in U.S. Patent No. 6,800,620. LY294002 is a PI3K kinase inhibitor, which is a component kinase of the AKT pathway.

[0109] AZD8055

[0331] In one embodiment, the AKT pathway inhibitor is AZD8055, also known as CAS1009298-09-2. In one embodiment, the AKT pathway inhibitor is [5-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-2-methoxyphenyl]methanol. In one embodiment, the AKT pathway inhibitor has the formula: [ka] is a compound of

[0110]

[0332] AZD8055 is an mTOR kinase inhibitor, which is a component kinase of the AKT pathway. Synthetic methods are disclosed, for example, in U.S. Patent No. 7,902,189, which is incorporated by reference in its entirety, with particular attention to Examples 1-3. Other mTOR kinase inhibitors are taught, for example, by Barlaam et al. in U.S. Patent No. 9,657,008, which is incorporated by reference in its entirety.

[0111] Identification of functionally useful AKT pathway inhibitors

[0333] Small molecules that inhibit kinases are commonly known to those skilled in the art. For example, Bhullar et al. summarized approaches to identifying useful inhibitors of 538 known protein kinases in "Kinase-targeted cancer therapies: progress, challenges, and future directions," Mol. Cancer 17:48 (2018). Bhullar emphasized that kinase inhibitors may target the ATP-binding site and be ATP-competitive compounds (type I inhibitors), target the inactive conformation of the kinase (type II inhibitors), act via allosteric inhibition (type III inhibitors), target the substrate-binding site (type IV inhibitors), or react covalently with the kinase (type V inhibitors). Due to the several means by which kinases interact, predictability from structure to binding mode and inhibitory mechanism is limited.

[0112]

[0334] As noted above, the AKT signaling pathway is composed of many distinct protein kinases that interact through a complex network to result in changes in cellular metabolism, transcription, and translation as a result of extracellular signals transmitted through a receptor-linked network. See, e.g., Manning and Toker, "AKT / PKB Signaling: Navigating the Network," Cell 169:381-405 (2017).

[0113]

[0335] Useful inhibitors of AKT pathway protein kinases are identified by the functional screens described herein. Example 6 describes such a functional screen for AKT pathway protein kinase inhibitors, which, when used in lymphocyte culture, enhances the population of lymphocytes with memory T cell phenotypes and increases the population of lymphocytes with immature TIL phenotype characteristics. Among other characteristics, the target phenotype includes increased expression within the expanded or cultured lymphocyte population that expresses memory T cell surface markers (e.g., CD27, CD28, IL7R, CCR9, SELL (L-selectin or CD62L), FCER1G, SATB1, LEF1, and KLF2) and decreased expression within the expanded or cultured lymphocyte population that expresses T cell effector surface markers (e.g., IFNG, XCL1, ACL2, CD300A, and KLRG1). CD62L (L-selectin) is known to be an important marker for memory T cells, which preferentially respond to antigens. Hengel et al.,“Cutting Edge:L-Selectin (CD62L) Expression Distinguishes Small Resting Memory CD4 + “T Cells That Preferentially Respond to Recall Antigen,” J. Immunol. 170:28-32 (2003). A less useful population is one that does not express CD62L but does express CD45RO, e.g., CD8 + CD62L - CD45RO + Includes lymphocytes. See, e.g., Booth et al., "Different Proliferative Potential and Migratory Characteristics of Human CD4+ Regulatory T Cells That Express either CD45RA or CD45RO," J. Immunol. 184:4317-4326 (2010).

[0114]

[0336] Without being limited to a particular theory, it is believed that the use of Akt inhibitors according to the methods of the present invention enables the preparation of TILs that retain a "more immature" phenotype, and therefore, it is expected that the TILs prepared according to the present invention will exhibit greater cytotoxicity against cancer cells than TILs prepared by other methods. In particular, it is believed that inhibition of the Akt pathway during the initial expansion of TILs obtained from tumors inhibits or attenuates the maturation of TILs in the initial expansion, and that exposure of TILs to OKT-3, IL-2, and antigen-presenting cells (APCs) in the second expansion activates the TILs while inhibiting or attenuating their maturation, resulting in a TIL cell population with a less mature phenotype that is less depleted by expansion in culture and exhibits greater cytotoxicity against cancer cells.

[0115]

[0337] In some embodiments, the present invention provides methods of using Akt inhibitors during the initial expansion of TILs and during the second expansion of TILs.

[0116]

[0338] In another embodiment, the invention provides a method using a prime initial expansion culture comprising exposing TILs in culture to additional OKT-3, IL-2 and APC to boost the initial activation of TILs, and exposing TILs in culture to an Akt inhibitor, OKT-3 and IL-2 to achieve initial activation and a second rapid expansion culture.

[0117]

[0339] In another embodiment, the invention provides a method using a primed initial expansion culture comprising exposing TILs in culture to additional OKT-3, IL-2 and APC to further stimulate the initial activation of TILs, and exposing TILs in culture to an Akt inhibitor, OKT-3, IL-2 and APC to achieve the initial activation of TILs and a second rapid expansion culture.

[0118]

[0340] In another embodiment, the invention provides a method using a primed initial expansion culture comprising exposing TILs in culture to an additional Akt inhibitor, OKT-3, IL-2 and APC to further stimulate the initial activation of the TILs, and exposing TILs in culture to an Akt inhibitor, OKT-3 and IL-2 to achieve the initial activation of the TILs and a second rapid expansion culture.

[0119]

[0341] In another embodiment, the invention provides a method using a primed initial expansion culture comprising exposing TILs in culture to an additional Akt inhibitor, OKT-3, IL-2 and APC to further stimulate the initial activation of the TILs, and exposing TILs in culture to an Akt inhibitor, OKT-3, IL-2 and APC to achieve the initial activation of the TILs and a second rapid expansion culture.

[0120]

[0342] In another embodiment, the invention provides a method using a primed initial expansion culture comprising exposing TILs in culture to an additional Akt inhibitor, OKT-3, IL-2 and APC to further stimulate the initial activation of the TILs, and exposing TILs in culture to OKT-3 and IL-2 to achieve the initial activation and second rapid expansion culture.

[0121]

[0343] In another embodiment, the invention provides a method using a primed initial expansion culture comprising exposing TILs in culture to an additional Akt inhibitor, OKT-3, IL-2 and APC to further stimulate the initial activation of the TILs, and exposing TILs in culture to OKT-3, IL-2 and APC to achieve the initial activation of the TILs and a second rapid expansion culture.

[0122] TIL manufacturing method - Process 2A

[0344] There are various methods for expanding TILs known to those skilled in the art. For example, Jin et al., J. Immunother. 35(3):283-292 (2012), "Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes in Gas-permeable Flasks to Numbers Needed for Patient Treatment," the disclosure of which is incorporated herein by reference, teaches a simplified method for producing TILs for clinical use. Jin et al. teach a first TIL culture followed by a rapid expansion (REP) protocol, which, in combination, allows one skilled in the art to produce clinically useful quantities of TILs. In some embodiments, the present invention provides a method for producing TILs, comprising the steps of cryopreserving a tumor, thawing the tumor, and carrying out the process described in Jin et al. Briefly, this process includes the following steps: TILs are first prepared by cryopreserving tumor fragments (approximately 1-8 mm) obtained by enzymatic tumor digestion and sharp dissection. 3Tumors can be cultured from 1000 cells / well. Tumor digests are produced by incubation in enzyme medium (RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). Immediately after placing the tumor in the enzyme medium, it is mechanically dissociated for approximately 1 minute. The material is then incubated for 30 minutes at 37°C in 5% CO2, then mechanically disrupted again for approximately 1 minute, and incubated again for 30 minutes at 37°C in 5% CO2. The tumor is then mechanically disrupted a third time for approximately 1 minute. After the third mechanical disruption, if large tissue fragments are present, the sample may be subjected to one or two additional mechanical dissociations, with or without an additional 30-minute incubation at 37°C in 5% CO2. At the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells. If TIL cultures are initiated in 24-well plates (Costar 24-well cell culture cluster, flat bottom; Corning Incorporated, Corning, NY), each well contains 1 × 10 cells in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 1 tumor digestive cell or approximately 1-8 mm 3 The CM contains RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and approximately 10 μg / mL gentamicin. The volume is 40 mL and the CM is 10 cm. 2 When cultures were initiated in gas-permeable flasks (G-Rex 10; Wilson Wolf Manufacturing, New Brighton, MN) with gas-permeable silicone bottoms, each flask contained 10–40 × 10 cells in 10–40 mL of CM containing IL-2. 6Viable tumor digested cells or 5-30 tumor fragments are loaded into G-Rex 10-well and 24-well plates. Both plates are incubated at 371°C in a humidified incubator with 5% CO2. Five days after the start of culture, half of the medium is removed and replenished with fresh CM and IL-2. From day 5 onward, half of the medium is replaced every 2-3 days. TIL REP is performed using T-175 flasks with gas-permeable bags or gas-permeable G-Rex flasks. For TIL REP in T-175 flasks, 1 x 10 cells suspended in 150 mL of medium are used. 6 TILs were added to each T-175 flask. The TILs were cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells, and the cells were cultured in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium) supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks were incubated at 37°C under 5% CO2. Half of the medium was replaced on day 5 with 50 / 50 medium containing 3000 IU / mL IL-2. On day 7, cells from two T-175 flasks were combined in a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 were added to the 300 mL TIL suspension. The number of cells in each bag was counted daily or every other day, and fresh medium was added to keep the cell count at 0.5–2.0 × 10 6 cells / mL. 100cm 2 For TIL REP in a 500 mL volumetric flask with gas-permeable silicone bottom (G-Rex 100, Wilson Wolf), 5 × 10 cells were cultured in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. 6 ~10×10 6TILs were cultured at a 1:100 ratio with irradiated allogeneic PBMCs. G-Rex 100 flasks were incubated at 37°C under 5% CO2. On day 5, 250 mL of supernatant was removed and placed in a centrifuge bottle. The flask was centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet was resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL IL-2 and added back to the original G-Rex 100 flask. If TILs were to be continuously expanded in the G-Rex 100 flasks, on day 7, the TILs in each G-Rex 100 flask were suspended in the 300 mL of medium present in each flask. The cell suspension was divided into three 100 mL aliquots, which were then used to seed three G-Rex 100 flasks. 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to each flask. The G-Rex 100 flasks were incubated at 37°C under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 was added to each G-Rex 100 flask. Cells were harvested on day 14 of culture.

[0123]

[0345] In some embodiments, the present invention provides a method for producing TILs, comprising the steps of cryopreserving tumors, thawing the tumors, and carrying out the following process: TILs can be produced by rapid expansion using in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with a cancer antigen (including one or more antigenic portions thereof, such as epitopes or cells), optionally expressed from a vector such as an HLA-A2-binding peptide, e.g., 0.3 μM MART-1:26-35(27L) or gp100:209-217(210M), in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15 (preferably IL-2). In vitro-induced TILs can also be rapidly expanded by restimulation with the same cancer antigen or antigens pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be rapidly expanded by, for example, irradiated autologous lymphocytes or by irradiating HLA-A2-binding peptides. +TILs can be restimulated with allogeneic lymphocytes and IL-2. TILs can be selected for highly active recognition of any of the unique antigens generated as a result of the estimated 10,000 genetic mutations encoded by each tumor cell genome. However, the antigen need not be unique. T cells can be selected for highly active recognition of one or more antigens of the cancer, including antigenic portions of one or more antigens, such as epitopes, or cells of the cancer. "Cancer antigen" and "cancer antigen" are intended to encompass all of the aforementioned antigens. When the cancer is melanoma, such as metastatic melanoma, TILs are preferably selected for highly active recognition of "unique" or patient-specific antigens derived from MART-1 (e.g., MART-1:26-35(27L)), gp100 (e.g., gp100:209-217(210M)), or tumor-encoded mutations. Other suitable melanoma antigens that can be selected for highly active recognition by TILs include, but are not limited to, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, and MAGE. For the treatment of lung cancer, breast cancer, colon cancer, prostate cancer, and the like, antigens such as NY-ESO-1, telomerase, p53, HER2 / neu, carcinoembryonic antigen, or prostate-specific antigen can be used to select for highly active recognition by TILs. TILs that can be selected include, but are not limited to, tyrosinase and tyrosinase-related proteins.

[0124]

[0346] IL-2-based TIL expansion followed by the "rapid expansion process" (REP) is becoming the preferred method for TIL expansion due to its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-fold expansion of TILs in a 14-day period, but it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs) as feeder cells, often from multiple donors, as well as anti-CD3 antibodies (OKT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. Cryopreserved tumor fragments are a suitable starting point for initial or primary cultures to produce TILs for therapeutic or other purposes.

[0125]

[0347] An example of a TIL process known as Process 2A that includes some of these features is shown in Figure 1. Another exemplary TIL process known as Process 1C is described and compared to Process 2A in Figures 5 and 6. An embodiment of Process 2A is shown in Figure 1.

[0126]

[0348] As discussed herein, the invention may include steps relating to restimulating cryopreserved TILs prior to transplantation into a patient to increase their metabolic activity, and therefore relative health, and methods for testing said metabolic health. As generally outlined herein, TILs are typically obtained from a patient sample and manipulated to expand their numbers prior to transplantation into a patient. In some embodiments, TILs may optionally be genetically engineered, as discussed below.

[0127]

[0349] In some embodiments, TILs can be cryopreserved and thawed for administration to a patient. After thawing, the TILs can be restimulated to enhance their metabolism before infusion into a patient.

[0128]

[0350] In some embodiments, as discussed in detail below and in the Examples and Figures, the first expansion culture (including a process referred to as Pre-REP and shown as step A in FIG. 1 ) is shortened to 3-14 days, and the second expansion culture (including a process referred to as REP and shown as step B in FIG. 1 ) is shortened to 7-14 days. In some embodiments, the first expansion culture (e.g., the expansion culture described as step B in FIG. 1 ) is shortened to 11 days, and the second expansion culture (e.g., the expansion culture described as step D in FIG. 1 ) is shortened to 11 days. In some embodiments, as discussed in detail below and in the Examples and Figures, the combined first and second expansion cultures (e.g., the expansion cultures described as steps B and D in FIG. 1 ) are shortened to 22 days.

[0129]

[0351] The "step" designations A, B, C, etc. below refer to Figure 1 and to specific embodiments described herein. The order of steps below and in Figure 1 is exemplary, and any combination or order of steps, as well as additional steps, repeated steps, and / or omission of steps, are contemplated by the present application and methods disclosed herein.

[0130] Step A. Obtain patient tumor samples

[0352] Generally, TILs are initially obtained from patient tumor samples ("primary TILs") and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein, and optionally have phenotypic and metabolic parameters determined as indicators of TIL health.

[0131]

[0353] Patient tumor samples can be obtained using methods known in the art, generally by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be from liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be of any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, renal, gastric, and skin cancers (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from melanoma tumors, which have been reported to have particularly high levels of TILs.

[0132]

[0354] The term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The histology of a solid tumor comprises interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, in which cancer cells may disperse and provide a supportive microenvironment.

[0133]

[0355] The term "hematologic malignancies" refers to mammalian cancers and tumors of hematopoietic and lymphoid tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematologic malignancies are also referred to as "liquid tumors." Hematologic malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematologic malignancies" refers to hematologic malignancies affecting B cells.

[0134]

[0356] Once obtained, tumor samples are typically cut into 1 to approximately 8 mm sections using sharp dissection. 3 fragmented into small pieces of about 2-3 mm 3 are particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociation agent). Tumor digests can be produced by placing the tumor in the enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C under 5% CO2 for 30 minutes, and then repeating the cycle of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension contains a large number of red blood cells or dead cells at the end of this process, density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the embodiments described herein for the methods of expanding TILs or treating cancer.

[0135]

[0357] The harvested cell suspension is generally referred to as a "primary cell population" or a "freshly harvested" cell population.

[0136]

[0358] In some embodiments, fragmentation comprises physical fragmentation, including, for example, dissection and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient. In one embodiment, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient.

[0137]

[0359] In some embodiments, if the tumor is a solid tumor, after obtaining the tumor sample, for example, in step A (provided in FIG. 1), the tumor is subjected to physical fragmentation. In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor without any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for first expansion culture. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment being about 27 mm. 3 In some embodiments, the plurality of pieces has a volume of about 1300 mm 3 ~about 1500mm 3 In some embodiments, the plurality of pieces comprises about 30 to about 60 pieces having a total volume of about 1350 mm 3 In some embodiments, the plurality of fragments comprises about 50 fragments having a total mass of about 1 g to about 1.5 g. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0138]

[0360] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are about 1 mm 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm 3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 In some embodiments, the tumor is 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumor is 1 mm x 1 mm x 1 mm. In some embodiments, the tumor is 2 mm x 2 mm x 2 mm. In some embodiments, the tumor is 3 mm x 3 mm x 3 mm. In some embodiments, the tumor is 4 mm x 4 mm x 4 mm. In embodiments where the tumor tissue is first frozen before initiation of culture, the tumor is approximately 6 mm x 6 mm x 6 mm.

[0139]

[0361] In some embodiments, the tumor is resected to minimize the amount of bleeding, necrotic, and / or fatty tissue on each segment. In some embodiments, the tumor is resected to minimize the amount of bleeding tissue on each segment. In some embodiments, the tumor is resected to minimize the amount of necrotic tissue on each segment. In some embodiments, the tumor is resected to minimize the amount of fatty tissue on each segment.

[0140]

[0362] In some embodiments, tumor fragmentation is performed to maintain the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without sawing with a scalpel. In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are created by incubation in an enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 μg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C under 5% CO2 for 30 minutes, after which it may be mechanically disrupted again for approximately 1 minute. After another 30-minute incubation at 37°C under 5% CO2, the tumor may be mechanically disrupted a third time for approximately 1 minute. In some embodiments, if large tissue debris was present after the third mechanical disruption, the sample was subjected to one or two additional rounds of mechanical separation, with or without an additional 30-minute incubation at 37° C. under 5% CO. In some embodiments, if the cell suspension at the end of the final incubation contained a large number of red blood cells or dead cells, density gradient separation using Ficoll may be performed to remove such cells.

[0141]

[0363] In some embodiments, the harvested cell suspension before the first expansion step is referred to as a "primary cell population" or a "freshly harvested" cell population.

[0142]

[0364] In some embodiments, cells may optionally be frozen after sample collection and stored frozen before proceeding to expansion as described in step B, described in more detail below and illustrated in FIG. 1.

[0143] Step B: First expansion culture

[0365] In some embodiments, the methods provide for obtaining young TILs that are capable of increasing replication cycles upon administration to a subject / patient, and thus may provide additional therapeutic advantages over mature TILs (e.g., "mature TILs" that have undergone more rounds of ex vivo replication before administration to a subject / patient). Characteristics of young TILs have been described in the literature. For example, Donia, at al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010), Huang et al., J Immunother, 28(3):258-267 (2005), Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013), Besser et al., J Immunother 32:415-423 (2009), Robbins, et al., J Immunol 2004; 173:7125-7130, Shen et al., J Immunother, 30:123-129 (2007), Zhou, et al., J Immunother, 28:53-62 (2005) and Tran, et al., J Immunother, 31:742-751 (2008), all of which are incorporated herein by reference in their entireties.

[0144]

[0366] The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but numerous gene segments. The V (variable), D (diversity), J (joining), and C (constant) gene segments determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly collected TILs and / or TILs prepared using methods other than those provided herein, including, for example, methods other than those embodied in FIG. 1. In some embodiments, as illustrated in FIG. 5 and / or FIG. 6, TILs obtained by the present methods exhibit increased T cell repertoire diversity compared to freshly collected TILs and / or TILs prepared using a method referred to as Process 1C. In some embodiments, TILs obtained in the first expansion culture exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is an increase in immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is in immunoglobulin heavy chains. In some embodiments, the immunoglobulin diversity is in immunoglobulin light chains. In some embodiments, the diversity is in T cell receptors. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCR α / β) is increased.

[0145]

[0367] After dissection or digestion of tumor fragments, for example, as described in step A of Figure 1, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for several days, generally 3-14 days, thereby generating a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 7-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of 10-14 days, resulting in a bulk TIL population, generally about 1 x 10 8 In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about 1 x 10 8 Bulk TIL cells are obtained.

[0146]

[0368] In a preferred embodiment, as described below and herein, expansion of TILs can be performed using an initial bulk TIL expansion step (e.g., such as that described in step B of FIG. 1, which may include a process referred to as pre-REP), followed by a second expansion (step D, which includes a process referred to as a rapid expansion protocol (REP) step), as described below under step D and herein, followed by optional cryopreservation and then a second step D (which includes a process referred to as a restimulation REP step). TILs obtained by this process can optionally be characterized for phenotypic characteristics and metabolic parameters as described herein.

[0147]

[0369] In an embodiment, when TIL cultures are initiated in 24-well plates, e.g., using Costar 24-well cell culture clusters, flat bottom (Corning Incorporated, Corning, NY), each well contains 1 x 10 cells in 2 mL of complete medium (CM) containing IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA). 6 Tumor digest cells or 1 tumor fragment can be seeded. In some embodiments, tumor fragments are about 1 mm 3 ~10mm 3 is.

[0148]

[0370] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 μg / mL gentamicin. The culture is grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicone bottoms (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10 x 10 cells in 10-40 mL of IL-2-containing CM. 6 ~40×10 6 Live tumor digest cells or 5–30 tumor fragments were loaded into G-Rex10 and 24-well plates. Both were incubated in a humidified incubator at 37°C under 5% CO2. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From day 5 onward, half of the medium was replaced every 2–3 days.

[0149]

[0371] After preparation of tumor fragments, the resulting cells (i.e., fragments) are cultured in serum-containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum with 6000 IU / mL of IL-2 (or in some cases in the presence of an aAPC cell population as outlined herein). This primary cell population is cultured for several days, generally 10-14 days, thereby generating a bulk TIL population, generally about 1 x 10 8 In some embodiments, the growth medium during the first expansion culture contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution contains 20-30 x 10 cells per 1 mg vial. 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 20×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 25×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 30×10 6 IU / mg specific activity. In some embodiments, the IL-2 stock solution contains 4-8 x 10 6 In some embodiments, the IL-2 stock solution has a final concentration of 5-7 x 10 IU / mg of IL-2. 6 In some embodiments, the IL-2 stock solution has a final concentration of 6×10 IU / mg of IL-2. 6IU / mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example E. In some embodiments, the first expansion culture medium contains about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6,000 IU / mL IL-2, or about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 9,000 IU / mL to about 5,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 8,000 IU / mL to about 6,000 IU / mL IL-2. In some embodiments, the first expansion culture medium contains about 7,000 IU / mL to about 6,000 IU / mL IL-2. In some embodiments, the first expansion culture medium comprises about 6,000 IU / mL of IL-2. In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3,000 IU / mL of IL-2. In one embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3,000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium contains 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or about 8000 IU / mL of IL-2.

[0150]

[0372] In some embodiments, the first expansion culture medium contains about 500 IU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium contains about 500 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium contains about 400 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium contains about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the first expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In one embodiment, the cell culture medium further comprises IL-15. In one preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0151]

[0373] In some embodiments, the first expansion culture medium contains about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 20 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 15 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the first expansion culture medium contains about 2 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 1 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium further contains IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU / mL of IL-21.

[0152]

[0374] In one embodiment, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In one embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT-3 antibody. In one embodiment, the cell culture medium contains 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0153]

[0375] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg / mL to 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg / mL to 40 μg / mL in the cell culture medium.

[0154]

[0376] In some embodiments, in addition to one or more TNFRSFs, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and an OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0155]

[0377] In some embodiments, the first expansion culture medium is referred to as "CM," an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, the CM consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. The culture is grown in a 40 mL volume and 10 cm 2 In embodiments initiated in gas-permeable flasks with gas-permeable silicone bottoms (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10 x 10 cells in 10-40 mL of IL-2-containing CM. 6 ~40×10 6 Live tumor digest cells or 5-30 tumor fragments were loaded. Both G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C under 5% CO2. Five days after the start of culture, half of the medium was removed and replenished with fresh CM and IL-2. From day 5 onwards, half of the medium was replaced every 2-3 days. In some embodiments, the CM is CM1 as described in the Examples (see Example 1). In some embodiments, the first expansion culture is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

[0156]

[0378] In some embodiments, as discussed in the Examples and Figures, the first expansion culture (e.g., including the process described in step B of Figure 1, which may include what is sometimes referred to as pre-REP) process is shortened to 3-14 days. In some embodiments, as discussed in the Examples, including, for example, Figures 4 and 5 and the Examples described in step B of Figure 1, the first expansion culture (e.g., including the process described in step B of Figure 1, which may include what is sometimes referred to as pre-REP) is shortened to 7-14 days. In some embodiments, the first expansion culture in step B is shortened to 10-14 days. In some embodiments, as discussed in the Examples, for example, the expansion culture described in step B of Figure 1, the first expansion culture is shortened to 11 days.

[0157]

[0379] In some embodiments, the first TIL expansion culture may be continued for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first TIL expansion culture may be continued for 1 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 2 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 3 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 4 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 5 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 6 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 7 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 8 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 9 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 10 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 11 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 12 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 13 to 14 days. In some embodiments, the first TIL expansion culture may be continued for 14 days. In some embodiments, the first TIL expansion culture may be continued for 1 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 2 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 3 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 4 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 5 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 6 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 7 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 8 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 9 to 11 days. In some embodiments, the first TIL expansion culture may be continued for 10 to 11 days.In some embodiments, the first TIL expansion culture may continue for 11 days.

[0158]

[0380] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination in the first expansion culture. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and optional combinations thereof, may be included in the first expansion culture, including in step B process, for example, according to Figure 1 and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination in the first expansion culture. In some embodiments, IL-2, IL-15, and IL-21, and optional combinations thereof, may be included in step B process, for example, according to Figure 1 and as described herein.

[0159]

[0381] In some embodiments, as discussed in the Examples and Figures, the first expansion culture (including, for example, the process described in step B of FIG. 1, referred to as Pre-REP) process is shortened to 3-14 days. In some embodiments, the first expansion culture in step B is shortened to 7-14 days. In some embodiments, the first expansion culture in step B is shortened to 10-14 days. In some embodiments, the first expansion culture is shortened to 11 days.

[0160]

[0382] In some embodiments, the first expansion, e.g., step B according to FIG. 1, is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion, as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed bioreactor is a single bioreactor.

[0161] Step C: Transition from the first expansion culture to the second expansion culture

[0383] In some cases, the bulk TIL population obtained from the first expansion, including, for example, the TIL population obtained from step B, as shown in Figure 1, may be immediately cryopreserved using the protocols discussed herein below. Alternatively, the TIL population obtained from the first expansion, referred to as the second TIL population, may be subjected to a second expansion (which may include an expansion sometimes referred to as REP) and then cryopreserved as discussed below. Similarly, when genetically modified TILs are used for therapy, the first TIL population (which may also be referred to as the bulk TIL population) or the second TIL population (which in some embodiments may include a population referred to as the REP TIL population) may be subjected to genetic modification for an appropriate treatment before expansion or after the first expansion but before the second expansion.

[0162]

[0384] In some embodiments, TILs obtained from the first expansion culture (e.g., from step B as shown in FIG. 1 ) are stored until phenotyping for selection. In some embodiments, TILs obtained from the first expansion culture (e.g., from step B as shown in FIG. 1 ) are not stored but proceed directly to the second expansion culture. In some embodiments, TILs obtained from the first expansion culture are not cryopreserved after the first expansion culture and before the second expansion culture. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 3 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 4 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 4 to 10 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 7 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 14 days after fragmentation.

[0163]

[0385] In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 1 day to 14 days after fragmentation. In some embodiments, the first TIL expansion culture may continue for 2 days to 14 days. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 3 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 4 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 5 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 6 days to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 7 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 8 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 9 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 10 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 11 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 12 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 13 to 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 14 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 1 to 11 days after fragmentation.In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 2 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 3 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 4 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 5 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 6 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 7 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 8 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture is carried out 9 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 10 to 11 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs 11 days after fragmentation.

[0164]

[0386] In some embodiments, the TILs are not stored after the first expansion and before the second expansion, and the TILs proceed directly to the second expansion (e.g., in some embodiments, no storage occurs during the transition from step B to step D, as shown in FIG. 1). In some embodiments, the transfer occurs in a closed system, as described herein. In some embodiments, the TILs from the first expansion are TILs from a second TIL population and proceed directly to the second expansion without a transfer period.

[0165]

[0387] In some embodiments, the transition from the first expansion culture to the second expansion culture, e.g., step C according to FIG. 1, is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed bioreactor is a single bioreactor.

[0166] cytokines

[0388] The expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as is known in the art.

[0167]

[0389] Alternatively, it is further possible to use combinations of cytokines in the rapid and / or secondary expansion of TILs, such as combinations of two or more of IL-2, IL-15, and IL-21, generally as outlined in WO 2015 / 189356 and WO 2015 / 189357, which are expressly incorporated herein by reference in their entireties. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15, IL-21 and IL-2, and IL-15 and IL-21, the latter finding particular use in many embodiments. As described therein, the use of combinations of cytokines is particularly advantageous for the generation of lymphocytes, particularly T cells.

[0168] Step D: Second expansion culture

[0390] In some embodiments, the TIL cell population is expanded in number after harvesting and initial bulk processing, following a transition referred to as steps A and B and C, e.g., as described in FIG. 1. This further expansion is referred to herein as a second expansion, which may include an expansion process commonly referred to in the art as rapid expansion (REP, and the process depicted in step D of FIG. 1). The second expansion can generally be accomplished using culture medium in a gas-permeable vessel containing several components, including feeder cells, a cytokine source, and an anti-CD3 antibody.

[0169]

[0391] In some embodiments, the second expansion of TILs or the second TIL expansion culture (which may also be referred to as REP; and may include the process shown in step D of FIG. 1 ) can be performed using any TIL flask or vessel known to those of skill in the art. In some embodiments, the second TIL expansion culture may continue for 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL expansion culture may continue for about 7 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 8 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 9 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 10 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 11 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 12 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 13 to about 14 days. In some embodiments, the second TIL expansion culture may continue for about 14 days.

[0170]

[0392] In one embodiment, the second expansion can be performed in a gas-permeable container using the methods of the present disclosure (e.g., including the expansion referred to as REP and the process shown in step D of FIG. 1). For example, TILs can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulation can include, for example, about 30 ng / ml of an anti-CD3 antibody such as OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA), or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded to induce further stimulation in vitro by introducing one or more antigens of the cancer, including one or more antigenic portions thereof, such as human leukocyte antigen A2 (HLA-A2)-binding peptides, e.g., 0.3 μM MART-1:26-35(27L) or gpl 00:209-217(210M), optionally expressed from a vector, during the second expansion culture, optionally in the presence of a T cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same one or more antigens of the cancer that were pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be further restimulated, for example, with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of a second expansion culture. In some embodiments, the second expansion culture occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0171]

[0393] In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In one embodiment, the cell culture medium contains 1000 to 2000 IU / mL, 2000 to 3000 IU / mL, 3000 to 4000 IU / mL, 4000 to 5000 IU / mL, 5000 to 6000 IU / mL, 6000 to 7000 IU / mL, 7000 to 8000 IU / mL, or 8000 IU / mL of IL-2.

[0172]

[0394] In one embodiment, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of the OKT-3 antibody. In one embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 μg / mL of the OKT-3 antibody. In one embodiment, the cell culture medium contains 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, or 50 ng / mL to 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0173]

[0395] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 0.1 μg / mL to 100 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration of 20 μg / mL to 40 μg / mL in the cell culture medium.

[0174]

[0396] In some embodiments, in addition to one or more TNFRSFs, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and an OKT-3 antibody at an initial concentration of about 30 ng / mL, and the one or more TNFRSF agonists comprise a 4-1BB agonist.

[0175]

[0397] In some embodiments, a combination of IL-2, IL-7, IL-15, and / or IL-21 is used as a combination in the second expansion culture. In some embodiments, IL-2, IL-7, IL-15, and / or IL-21, and optional combinations thereof, may be included in the second expansion culture, including in step D process, for example, according to Figure 1 and as described herein. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination in the second expansion culture. In some embodiments, IL-2, IL-15, and IL-21, and optional combinations thereof, may be included in step D process, for example, according to Figure 1 and as described herein.

[0176]

[0398] In some embodiments, the second expansion culture may be performed in a supplemented cell culture medium comprising IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion culture is performed in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen-presenting cells (APCs; also referred to as antigen-presenting feeder cells). In some embodiments, the second expansion culture occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

[0177]

[0399] In some embodiments, the second expansion culture medium contains about 500 IU / mL of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium contains about 500 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium contains about 400 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium contains about 300 IU / mL to about 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium comprises about 200 IU / mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / mL of IL-15. In one embodiment, the cell culture medium further comprises IL-15. In one preferred embodiment, the cell culture medium comprises about 180 IU / mL of IL-15.

[0178]

[0400] In some embodiments, the second expansion culture medium contains about 20 IU / mL of IL-21, about 15 IU / mL of IL-21, about 12 IU / mL of IL-21, about 10 IU / mL of IL-21, about 5 IU / mL of IL-21, about 4 IU / mL of IL-21, about 3 IU / mL of IL-21, about 2 IU / mL of IL-21, about 1 IU / mL of IL-21, or about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 20 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 15 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 12 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 10 IU / mL to about 0.5 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 5 IU / mL to about 1 IU / mL of IL-21. In some embodiments, the second expansion culture medium contains about 2 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 1 IU / mL of IL-21. In some embodiments, the cell culture medium contains about 0.5 IU / mL of IL-21. In one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU / mL of IL-21.

[0179]

[0401] In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of TILs to PBMCs and / or antigen-presenting cells in the rapid expansion culture and / or second expansion culture is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, the ratio of TILs to PBMCs in the rapid expansion culture and / or second expansion culture is 1:50 to 1:300. In one embodiment, the ratio of TILs to PBMCs in the rapid expansion culture and / or second expansion culture is 1:100 to 1:200.

[0180]

[0402] In one embodiment, REP and / or secondary expansion is performed in flasks by mixing bulk TILs with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml of medium. Media replenishment is performed (typically two-thirds via respiration with fresh medium) until the cells are transferred to a separate growth chamber. Separate growth chambers include G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0181]

[0403] In some embodiments, the second expansion culture (which may include a process called the REP process) is shortened to 7-14 days, as discussed in the Examples and Figures. In some embodiments, the second expansion culture is shortened to 11 days.

[0182]

[0404] In one embodiment, the REP and / or second expansion culture may be performed using T-175 flasks and gas permeable bags or gas permeable cultureware (G-Rex flasks) as previously described (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42). In some embodiments, the second expansion culture (including what are referred to as rapid expansion cultures) is performed in a T-175 flask and contains approximately 1 x 10 cells suspended in 150 mL of medium. 6 TILs can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. T-175 flasks can be incubated at 37°C under 5% CO2. Half of the medium can be replaced on day 5 using 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks can be combined into a 3 L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 is added to the 300 mL TIL suspension. The number of cells in each bag is counted daily or every two days, and fresh medium is added to keep the cell count at 0.5–2.0 × 10. 6 cells / mL.

[0183]

[0405] In one embodiment, the second expansion culture (which may include the expansion culture referred to as REP; and referenced in step D of FIG. 1 ) may be performed in a 100 cm gas-permeable silicone-bottomed, 500 mL capacity gas-permeable flask (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA) and cultured at 5×10 cells in 400 mL of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / mL IL-2, and 30 ng / mL anti-CD3 (OKT3). 6 or 10 x 10 6TILs can be cultured with PBMCs. The G-Rex 100 flasks can be incubated at 37°C under 5% CO2. On day 5, 250 mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet can be resuspended in 150 mL of fresh medium containing 5% human AB serum and 3000 IU / mL IL-2 and added back to the original G-Rex 100 flask. When continuously expanding TILs in G-Rex 100 flasks, on day 7, the TILs from each G-Rex 100 can be suspended in the 300 mL of medium present in each flask, and the cell suspension can be divided into three 100 mL aliquots that can be used to seed three G-Rex 100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 can be added to each flask. The G-Rex 100 flasks can be incubated at 37° C. under 5% CO2, and after 4 days, 150 mL of AIM-V containing 3000 IU / mL of IL-2 can be added to each G-Rex 100 flask. Cells can be harvested on day 14 of culture.

[0184]

[0406] In one embodiment, the second expansion culture (including the expansion culture referred to as REP) is performed in a flask by mixing bulk TILs with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml medium. In some embodiments, medium changes are performed until the cells are transferred to another growth chamber. In some embodiments, two-thirds of the medium is replaced by respiration with fresh medium. In some embodiments, the other growth chamber includes G-REX flasks and gas-permeable vessels, as discussed more fully below.

[0185]

[0407] In one embodiment, a second expansion culture (including an expansion culture called REP) is performed, which further includes selecting TILs for superior tumor responsiveness. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016 / 0010058A1, the disclosure of which is incorporated herein by reference, can be used to select TILs for superior tumor responsiveness.

[0186]

[0408] Optionally, after the second expansion (including the expansion referred to as the REP expansion), a cell viability assay can be performed using standard assays known in the art. For example, a sample of bulk TILs can be subjected to a trypan blue dye exclusion assay, which selectively labels dead cells and allows assessment of viability. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to a standard Cellometer K2 Image Cytometer automated cell counter protocol.

[0187]

[0409] In some embodiments, the second expansion of TILs (including expansions referred to as REPs) can be performed using T-175 flasks and gas-permeable bags as previously described (Tran KQ, Zhou J, Durflinger KH, et al., 2008, J Immunother., 31:742-751 and Dudley ME, Wunderlich JR, Shelton TE, et al. 2003, J Immunother., 26:332-342) or gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed using flasks. In some embodiments, the second expansion is performed using gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed in T-175 flasks and contains approximately 1 x 10 6TILs were suspended in approximately 150 mL of medium and added to each T-175 flask. The TILs were cultured at a 1:100 ratio with irradiated (50 Gy) allogeneic PBMCs as "feeder" cells, and the cells were cultured in a 1:1 mixture of CM and AIM-V medium (50 / 50 medium) supplemented with 3000 IU / mL IL-2 and 30 ng / mL anti-CD3. The T-175 flasks were incubated at 37°C under 5% CO. In some embodiments, the medium was changed on day 5 using 50 / 50 medium containing 3000 IU / mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks were combined into a 3 L bag, and 300 mL of the TIL suspension was added to 300 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2. The number of cells in each bag can be counted every day or every two days, and fresh medium can be added to keep the cell count at about 0.5 to about 2.0 × 10 6 cells / mL.

[0188]

[0410] In some embodiments, the second expansion culture (including the expansion culture referred to as REP) is 100 cm 2 The cells were cultured in a 500 mL volumetric flask (G-Rex 100, Wilson Wolf) with a gas-permeable silicone bottom (Figure 1) and approximately 5 × 10 cells were cultured in 400 mL of 50 / 50 medium supplemented with 3000 IU / mL of IL-2 and 30 ng / mL of anti-CD3. 6 or 10 x 10 6TILs are cultured with irradiated allogeneic PBMCs at a 1:100 ratio. G-Rex100 flasks are incubated at 37°C under 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is removed, placed in a centrifuge bottle, and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended in 150 mL of fresh 50 / 50 medium containing 3000 IU / mL of IL-2 and added back to the original G-Rex100 flask. In embodiments where TILs are serially expanded in G-Rex100 flasks, on day 7, the TILs in each G-Rex100 are suspended in the 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots, which are used to seed three G-Rex100 flasks. Next, 150 mL of AIM-V containing 5% human AB serum and 3000 IU / mL IL-2 was added to each flask. The G-Rex 100 flasks were incubated at 37°C under 5% CO2. After 4 days, 150 mL of AIM-V containing 3000 IU / mL IL-2 was added to each G-Rex 100 flask. Cells were harvested on day 14 of culture.

[0189]

[0411] Diverse antigen receptors for T and B lymphocytes are produced by somatic recombination of a limited but numerous gene segments. These gene segments, V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs that exhibit and increase T cell repertoire diversity. In some embodiments, TILs obtained by this method exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained in a second expansion culture exhibit increased T cell repertoire diversity. In some embodiments, the increased diversity is increased immunoglobulin diversity and / or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is in immunoglobulin heavy chains. In some embodiments, the immunoglobulin diversity is in immunoglobulin light chains. In some embodiments, the diversity is in the T cell receptor. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, expression of T cell receptor (TCR) alpha and / or beta is increased. In some embodiments, expression of T cell receptor (TCR) alpha is increased. In some embodiments, expression of T cell receptor (TCR) beta is increased. In some embodiments, expression of TCRab (i.e., TCRα / β) is increased.

[0190]

[0412] In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

[0191]

[0413] In some embodiments, the second expansion, e.g., step D according to FIG. 1, is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion, as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed bioreactor is a single bioreactor.

[0192] Feeder cells and antigen-presenting cells

[0414] In one embodiment, the second expansion procedure described herein (e.g., including the expansion described in step D of FIG. 1 and the expansion referred to as REP) requires an excess of feeder cells during the REP TIL expansion and / or during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy allogeneic donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. See, e.g., “Isolation of mononuclear cells: Methodology and Application,” GE Life Sciences technical publication 18-1152-69-AE, available at https: / / us.vwr.com / assetsvc / asset / en_US / id / 16286835 / contents.

[0193]

[0415] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment and used in the REP procedure, as described in the Examples, which provide an exemplary protocol for assessing the replicative capacity of allogeneic PBMCs.

[0194]

[0416] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells entered into culture on day 0 of REP and / or day 0 of the second expansion culture (i.e., the start day of the second expansion culture), the PBMCs are considered non-replicating and are approved for use in the TIL expansion culture procedures described herein.

[0195]

[0417] In some embodiments, PBMCs are considered non-replicating and approved for use in the TIL expansion procedures described herein if the total number of viable cells cultured in the presence of OKT3 and IL-2 has not increased on days 7 and 14 from the initial number of viable cells entered into culture on REP day 0 and / or second expansion day 0 (i.e., the start of the second expansion). In some embodiments, PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody and 3000 IU / ml IL-2.

[0196]

[0418] In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 does not increase on days 7 and 14 from the initial number of viable cells introduced into culture on day 0 of REP and / or day 0 of the second expansion culture (i.e., the start of the second expansion culture), the PBMCs are considered replication-incompetent and are approved for use in the TIL expansion procedures described herein. In some embodiments, the PBMCs are cultured in the presence of 5-60 ng / ml of OKT3 antibody and 1000-6000 IU / ml of IL-2. In some embodiments, the PBMCs are cultured in the presence of 10-50 ng / ml of OKT3 antibody and 2000-5000 IU / ml of IL-2. In some embodiments, the PBMCs are cultured in the presence of 20-40 ng / ml of OKT3 antibody and 2000-4000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng / ml of OKT3 antibody and 2500-3500 IU / ml of IL-2.

[0197]

[0419] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is about 1:10, about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is 1:50 to 1:300. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion culture is 1:100 to 1:200.

[0198]

[0420] In one embodiment, the second expansion procedure described herein comprises culturing approximately 2.5 x 10 9 feeder cells and approximately 100 x 10 6 In another embodiment, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9 feeder cells and approximately 50 x 10 6 In yet another embodiment, the second expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 9 feeder cells and approximately 25 x 10 6 TILs are required.

[0199]

[0421] In one embodiment, the second expansion procedure described herein requires an excess amount of feeder cells during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from a healthy donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen-presenting (aAPC) cells are used instead of PBMCs.

[0200]

[0422] Generally, allogeneic PBMCs are inactivated by either irradiation or heat treatment and used in the TIL expansion procedures described herein, including the exemplary procedures described in the figures and examples.

[0201]

[0423] In one embodiment, the artificial antigen presenting cells are used in the second expansion culture, either as a replacement for or in combination with PBMCs.

[0202] cytokines

[0424] The TIL expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as known in the art.

[0203]

[0425] Alternatively, it is further possible to use combinations of cytokines in the rapid and / or secondary expansion of TILs, such as combinations of two or more of IL-2, IL-15, and IL-21, generally as outlined in WO 2015 / 189356 and WO 2015 / 189357 (expressly incorporated herein by reference in their entireties). Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15, IL-21 and IL-2, and IL-15 and IL-21, the latter finding particular use in many embodiments. As described therein, the use of combinations of cytokines is particularly advantageous for the generation of lymphocytes, particularly T cells.

[0204] Step E: Harvesting TILs

[0426] After the second expansion step, the cells can be harvested. In some embodiments, the TILs are harvested after one, two, three, four, or more expansion steps, e.g., as provided in Figure 1. In some embodiments, the TILs are harvested after two expansion steps, e.g., as provided in Figure 1.

[0205]

[0427] The TILs can be collected by any suitable and sterile method, including, for example, by centrifugation. Methods for collecting TILs are well known in the art, and any such known method can be used with the present process. In some embodiments, the TILs are collected using an automated system.

[0206]

[0428] Cell harvesters and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based harvester can be used in the present methods. In some embodiments, the cell harvester and / or cell processing system is a membrane-based cell harvester. In some embodiments, cell collection is performed via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to an instrument or device manufactured by any vendor that can pump a cell-containing solution through a membrane or filter, such as a spinning membrane or spinning filter, in a sterile and / or closed environment, allowing for continuous flow and cell processing with removal of supernatant or cell culture medium without pelleting. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing, fluid exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0207]

[0429] In some embodiments, harvesting, e.g., step E according to Figure 1, is performed from a closed bioreactor. In some embodiments, a closed system is used for TIL expansion as described herein. In some embodiments, a single bioreactor is used. In some embodiments, the single bioreactor used is, for example, a G-REX-10 or G-REX-100. In some embodiments, the closed bioreactor is a single bioreactor.

[0208]

[0430] In some embodiments, step E according to Figure 1 is carried out according to the process described in Example 7. In some embodiments, the closed system is accessed via a syringe under sterile conditions to maintain the sterility and closure of the system. In some embodiments, a closed system such as that described in Example 7 is used.

[0209]

[0431] In some embodiments, TILs are harvested according to the method described in Example 7. In some embodiments, TILs are harvested on days 1-11 using a method as described herein (referred to in Example 7 as day 11 TIL harvest). In some embodiments, TILs are harvested on days 12-22 using a method as described herein (referred to in Example 7 as day 22 TIL harvest).

[0210] Step F: Final formulation and / or transfer to infusion bags

[0432] After steps A-E, as provided in the exemplary order in Figure 1 and as outlined above and in detail herein, are completed, the cells are transferred to a container for use in administration to a patient. In some embodiments, once a therapeutically sufficient number of TILs are obtained using the expansion methods described above, the TILs are transferred to a container for use in administration to a patient.

[0211]

[0433] In one embodiment, TILs expanded using the APCs of the present disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present disclosure can be administered by any suitable route as known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, preferably lasting approximately 30-60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic.

[0212] Anti-CD3 antibody, an optional media component

[0434] In some embodiments, the culture medium (including what is referred to as REP; see, e.g., FIG. 1) used in the expansion methods described herein also contains an anti-CD3 antibody. Anti-CD3 antibodies, when used in combination with IL-2, induce T cell activation and cell division in TIL populations. This effect can be seen with full-length antibodies as well as Fab and F(ab')2 fragments, the former being generally preferred; see, e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719 (incorporated herein by reference in its entirety).

[0213]

[0435] As one skilled in the art will appreciate, there are numerous suitable anti-human CD3 antibodies that find use in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies. In particular embodiments, the OKT3 anti-CD3 antibody muromonab (including the embodiments shown in Table 1) is used (commercially available from Ortho-McNeil, Raritan, NJ, or Miltenyi Biotech, Auburn, CA). Anti-CD3 antibodies also include UHCT1 clones, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0214] 4-1BB (CD137) agonist

[0436] In one embodiment, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist can be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1BB. The 4-1BB agonist or 4-1BB binding molecule can comprise an immunoglobulin heavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The 4-1BB agonist or 4-1BB binding molecule can have both a heavy chain and a light chain. As used herein, the term binding molecule includes antibodies (including full-length antibodies), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human, humanized, or chimeric antibodies, as well as antibody fragments, such as Fab fragments, F(ab') fragments, fragments produced by an Fab expression library, epitope-binding fragments of any of the above, and engineered forms of antibodies that bind to 4-1BB, such as scFv molecules. In one embodiment, the 4-1BB agonist is an antigen-binding protein that is a fully human antibody. In one embodiment, the 4-1BB agonist is an antigen-binding protein that is a humanized antibody. In some embodiments, 4-1BB agonists for use in the methods and compositions of the present disclosure include anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4-1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB Adnectins, anti-4-1BB domain antibodies, single-chain anti-4-1BB fragments, heavy-chain anti-4-1BB fragments, light-chain anti-4-1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives, conjugates, variants, or biosimilars thereof. Agonistic anti-4-1BB antibodies are known to induce strong immune responses. Lee, et al., PLOS One 2013, 8, e69677. In a preferred embodiment, the 4-1BB agonist is an agonistic anti-4-1BB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).In one embodiment, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utomilumab, or urelumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof. In a preferred embodiment, the 4-1BB agonist is utomilumab or urelumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.

[0215]

[0437] In a preferred embodiment, the 4-1BB agonist or 4-1BB binding molecule can also be a fusion protein. In a preferred embodiment, a multimeric 4-1BB agonist, such as a trimeric or hexameric 4-1BB agonist (having three or six ligand-binding domains), can induce superior receptor (4-1BBL) clustering and intracellular signaling complex formation compared to agonistic monoclonal antibodies, which typically have two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins containing three TNFRSF-binding domains and IgG1-Fc, optionally further linking two or more of these fusion proteins, are described, for example, in Gieffers, et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

[0216]

[0438] Agonistic 4-1BB antibodies and fusion proteins are known to induce strong immune responses. In a preferred embodiment, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that inhibits antibody-dependent cellular cytotoxicity (ADCC), e.g., NK cell cytotoxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that inhibits antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that inhibits complement-dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that inhibits Fc region functionality.

[0217]

[0439] In some embodiments, the 4-1BB agonist is characterized by binding to human 4-1BB (SEQ ID NO: 9) with high affinity and agonistic activity. In one embodiment, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 9). In one embodiment, the 4-1BB agonist is a binding molecule that binds to mouse 4-1BB (SEQ ID NO: 10). The amino acid sequences of the 4-1BB antigens to which the 4-1BB agonists or binding molecules bind are summarized in Table 6.

[0218] [Table 3]

[0219]

[0440] In some embodiments, the compositions, processes, and methods described have a K of about 100 pM or less. D binds to human or mouse 4-1BB with a K of approximately 90 pM or less D binds to human or mouse 4-1BB with a K of approximately 80 pM or less D binds to human or mouse 4-1BB at a K of approximately 70 pM or lessD binds to human or mouse 4-1BB with a K of approximately 60 pM or less D binds to human or mouse 4-1BB with a K of approximately 50 pM or less D binds to human or mouse 4-1BB with a K of approximately 40 pM or less D binds to human or mouse 4-1BB with a K of about 30 pM or less D The present invention includes 4-1BB agonists that bind to human or mouse 4-1BB at the agonist level.

[0220]

[0441] In some embodiments, the compositions, processes, and methods described provide a dose of about 7.5×10 5 k greater than 1 / M s assoc binds to human or mouse 4-1BB at approximately 7.5 × 10 5 k greater than 1 / M s assoc binds to human or mouse 4-1BB at approximately 8 × 10 5 k greater than 1 / M s assoc binds to human or mouse 4-1BB at approximately 8.5 × 10 5 k greater than 1 / M s assoc binds to human or mouse 4-1BB at approximately 9 × 10 5 k greater than 1 / M s assoc binds to human or mouse 4-1BB at approximately 9.5 × 10 5 k greater than 1 / M s assoc binds to human or mouse 4-1BB at approximately 1 × 10 6 k greater than 1 / M s assoc The present invention includes 4-1BB agonists that bind to human or mouse 4-1BB at the agonist level.

[0221]

[0442] In some embodiments, the compositions, processes and methods described provide a method for treating approximately 2×10 -5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 2.1 × 10 -5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 2.2 × 10 -5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 2.3 × 10-5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 2.4 × 10 -5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 2.5 × 10 -5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 2.6 × 10 -5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 2.7 x 10 -5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 2.8 × 10 -5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 2.9 × 10 -5 k less than 1 / s dissoc binds to human or mouse 4-1BB at approximately 3 x 10 -5 k less than 1 / s dissoc The present invention includes 4-1BB agonists that bind to human or mouse 4-1BB at the agonist level.

[0222]

[0443] In some embodiments, the compositions, processes and methods described have an IC of about 10 nM or less. 50 binds to human or mouse 4-1BB with an IC of approximately 9 nM or less 50 binds to human or mouse 4-1BB with an IC of approximately 8 nM or less 50 binds to human or mouse 4-1BB with an IC of approximately 7 nM or less 50 binds to human or mouse 4-1BB with an IC of approximately 6 nM or less 50 binds to human or mouse 4-1BB with an IC of approximately 5 nM or less 50 binds to human or mouse 4-1BB with an IC of approximately 4 nM or less 50 binds to human or mouse 4-1BB with an IC of approximately 3 nM or less 50 binds to human or mouse 4-1BB with an IC of approximately 2 nM or less 50 binds to human or mouse 4-1BB with an IC of about 1 nM or less 50 The present invention includes 4-1BB agonists that bind to human or mouse 4-1BB at the agonist level.

[0223]

[0444] In a preferred embodiment, the 4-1BB agonist is utomilumab, also known as PF-05082566 or MOR-7480, or a fragment, derivative, variant, or biosimilar thereof. Utomilumab is available from Pfizer, Inc. Utomilumab is an immunoglobulin G2-lambda, anti-[human (Homo sapiens) TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T-cell antigen ILA, CD137)], human (Homo sapiens) (fully human) monoclonal antibody. The amino acid sequence of utomilumab is shown in Table 7. Utomilumab has glycosylation sites at Asn59 and Asn292; positions 22-96 (V H -V L ), 143-199(C H 1-C L ), 256-316(C H 2) and 362-420(C H 3) intra-heavy chain disulfide bridge; positions 22'-87' (V H -V L ) and 136'-195'(C H 1-C L) intrachain light chain disulfide bridges at IgG2A isoform positions 218-218, 219-219, 222-222 and 225-225, at IgG2A / B isoform positions 218-130, 219-219, 222-222 and 225-225 and at IgG2B isoform positions 219-130(2), 222-222 and 225-225; and intrachain heavy chain-light chain disulfide bridges at IgG2A isoform position 130-213'(2), at IgG2A / B isoform positions 218-213' and 130-213' and at IgG2B isoform position 218-213'(2). The preparation and properties of utomilumab and its variants and fragments are described in U.S. Patent Nos. 8,821,867; 8,337,850; and 9,468,678 and International Publication No. WO 2012 / 032433 A1, the disclosures of each of which are incorporated herein by reference. Preclinical characterization of utomilumab is described in Fisher, et al., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinical trials of utomilumab in various hematological and solid tumor indications include the U.S. National Institutes of Health Clinicaltrials.gov identifiers NCT02444793, NCT01307267, NCT02315066, and NCT02554812.

[0224]

[0445] In one embodiment, the 4-1BB agonist comprises a heavy chain set forth in SEQ ID NO: 11 and a light chain set forth in SEQ ID NO: 12. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 99% identical to the sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 98% identical to the sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 97% identical to the sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 96% identical to the sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 95% identical to the sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0225]

[0446] In one embodiment, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of utomilumab. In one embodiment, the 4-1BB agonist heavy chain variable region (VR) H ) comprises the sequence shown in SEQ ID NO: 13, and the 4-1BB agonist light chain variable region (V L ) comprises the sequence set forth in SEQ ID NO: 14 and conservative amino acid substitutions thereof. In one embodiment, the 4-1BB agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L In one embodiment, the 4-1BB agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V LIn one embodiment, the 4-1BB agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L In one embodiment, the 4-1BB agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L In one embodiment, the 4-1BB agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L In one embodiment, the 4-1BB agonist comprises a V region that is at least 99% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L The present invention also includes scFv antibodies containing the domain.

[0226]

[0447] In one embodiment, the 4-1BB agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and their conservative amino acid substitutions, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and their conservative amino acid substitutions.

[0227]

[0448] In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by a drug regulatory agency for utomilumab. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and including one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is utomilumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is a 4-1BB agonist antibody that has been approved or submitted for approval, where the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and the reference drug or reference biological product is utomilumab. 4-1BB agonist antibodies may be approved by drug regulatory authorities, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, a biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is utomilumab. In some embodiments, a biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is utomilumab.

[0228] [Table 4]

[0229]

[0449] In a preferred embodiment, the 4-1BB agonist is the monoclonal antibody urelumab, also known as BMS-663513 and 20H4.9.h4a, or a fragment, derivative, variant, or biosimilar thereof. Urelumab is available from Bristol-Myers Squibb, Inc. and Creative Biolabs, Inc. Urelumab is an immunoglobulin G4-kappa, anti-[human (Homo sapiens) TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T cell antigen ILA, CD137)], human (Homo sapiens) (fully human) monoclonal antibody. The amino acid sequence of urelumab is shown in Table 7. Urelumab has an N-glycosylation site at position 298 (and 298''); positions 22-95 (V H -V L ), 148-204(C H 1-C L ), 262-322(C H 2) and 368-426(C H 3) (and positions 22'-95', 148'-204', 262'-322', and 368'-426') intra-heavy chain disulfide bridges; positions 23'-88' (V H -V L ) and 136'-196'(C H 1-C L) (and intra-light chain disulfide bridges at positions 23'''-88''' and 136'''-196'''); intra-heavy chain-heavy chain disulfide bridges at positions 227-227'' and 230-230''; and intra-heavy chain-light chain disulfide bridges at positions 135-216' and 135''-216'''. The preparation and properties of urelumab and variants and fragments thereof are described in U.S. Pat. Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. Preclinical and clinical characteristics of urelumab are described in Segal, et al., Clin. Cancer Res. 2016, available at http: / / dx.doi.org / 10.1158 / 1078-0432.CCR-16-1272. Current clinical trials of urelumab in various hematological and solid tumor indications include National Institutes of Health Clinicaltrials.gov identifiers NCT01775631, NCT02110082, NCT02253992, and NCT01471210.

[0230]

[0450] In one embodiment, the 4-1BB agonist comprises a heavy chain set forth in SEQ ID NO:21 and a light chain set forth in SEQ ID NO:22. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 99% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 98% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 97% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 96% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain that are at least 95% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively.

[0231]

[0451] In one embodiment, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of urelumab. In one embodiment, the 4-1BB agonist heavy chain variable region (VR) H ) comprises the sequence set forth in SEQ ID NO: 23, and the 4-1BB agonist light chain variable region (V L ) comprises the sequence set forth in SEQ ID NO: 24 and conservative amino acid substitutions thereof. In one embodiment, the 4-1BB agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L In one embodiment, the 4-1BB agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V LIn one embodiment, the 4-1BB agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L In one embodiment, the 4-1BB agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L In one embodiment, the 4-1BB agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L In one embodiment, the 4-1BB agonist comprises a V region that is at least 99% identical to the sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. H and V L The present invention also includes scFv antibodies containing the domain.

[0232]

[0452] In one embodiment, the 4-1BB agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and conservative amino acid substitutions thereof.

[0233]

[0453] In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by a drug regulatory agency for urelumab. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and including one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is urelumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is a 4-1BB agonist antibody that has been approved or submitted for approval, where the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and the reference drug or reference biological product is urelumab. 4-1BB agonist antibodies may be approved by drug regulatory authorities, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is urelumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is urelumab.

[0234] [Table 5]

[0235]

[0454] In one embodiment, the 4-1BB agonist is selected from the group consisting of 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), the antibody produced by the cell line deposited under ATCC number HB-11248 and disclosed in U.S. Pat. No. 6,974,863, 5F4 (BioLegend 31 1503), C65-485 (BD Pharmingen 559446), antibodies disclosed in U.S. Patent Application Publication No. 2005 / 0095244, antibodies disclosed in U.S. Patent No. 7,288,638 (such as 20H4.9-IgG1 (BMS-663031)), antibodies disclosed in U.S. Patent No. 6,887,673 (such as 4E9 or BMS-554271), antibodies disclosed in U.S. Patent No. 7,214,493, antibodies disclosed in U.S. Patent No. 6,303,121, antibodies disclosed in U.S. Patent No. 6,569,997, antibodies disclosed in U.S. Patent No. 6,905,685 (such as 4E9 or BMS-554271), antibodies disclosed in U.S. Patent No. 6,362,325 (1D8 or BMS-469492; 3H3 or BMS-4694 97; or 3El), antibodies disclosed in US Pat. No. 6,974,863 (such as 53A2); antibodies disclosed in US Pat. No. 6,210,669 (such as 1D8, 3B8 or 3E1), antibodies described in US Pat. No. 5,928,893, antibodies disclosed in US Pat. No. 6,303,121, antibodies disclosed in US Pat. No. 6,569,997, antibodies disclosed in WO 2012 / 177788, WO 2015 / 119923 and WO 2010 / 042433, and fragments, derivatives, conjugates, variants or biosimilars thereof, wherein the disclosure of each of the foregoing patents or patent application publications is incorporated herein by reference.

[0236]

[0455] In one embodiment, the 4-1BB agonist is selected from the group consisting of compounds disclosed in WO 2008 / 025516 A1, WO 2009 / 007120 A1, WO 2010 / 003766 A1, WO 2010 / 010051 A1, and WO 2010 / 078966 A1; U.S. Patent Application Publication Nos. 2011 / 0027218 A1, 2015 / 0126709 A1, 2011 / 0111494 A1, 2015 / 0110734 A1, and 2015 / 0126710 A1; and 4-1BB agonistic fusion proteins described in U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

[0237]

[0456] In one embodiment, the 4-1BB agonist is a 4-1BB agonistic fusion protein shown in Structure IA (C-terminal Fc antibody fragment fusion protein) or Structure IB (N-terminal Fc antibody fragment fusion protein) in Figure 50, or a fragment, derivative, conjugate, variant, or biosimilar thereof. In structures IA and IB, the cylinders refer to individual polypeptide binding domains. Structures IA and IB contain three linearly linked TNFRSF binding domains, e.g., from antibodies that bind to 4-1BBL or 4-1BB, which fold to form a trivalent protein, which then binds to IgG1-Fc (C H 3 and C H The TNFRSF-binding domain, shown as a cylinder, is linked to a second trivalent protein via a V domain (comprising a V domain), which is then used to link the two trivalent proteins via a disulfide bond (small elongated oval), stabilizing the structure and providing an agonist that can combine the six intracellular signaling domains of the receptor and the signaling protein to form a signaling complex. The TNFRSF-binding domain, shown as a cylinder, is connected by a V domain connected by a linker that may contain, for example, hydrophilic residues and Gly and Ser sequences for flexibility and Glu and Lys for solubility. H and V LThe scFv domain may be an scFv domain containing a chain. Any scFv domain design may be used, such as those described in de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad, et al., Clin. & Dev. Immunol. 2012, 980250; Monnier, et al., Antibodies, 2013, 2, 193-208; or references incorporated elsewhere herein. Fusion protein structures of this type are described in U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

[0238]

[0457] The amino acid sequences of other polypeptide domains of Structure IA are shown in Table 9. The Fc domain preferably comprises the complete constant domain (amino acids 17-230 of SEQ ID NO:31), the complete hinge domain (amino acids 1-16 of SEQ ID NO:31), or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO:31). Preferred linkers for connecting the C-terminal Fc antibody may be selected from the embodiments shown in SEQ ID NOs:32-41, including linkers suitable for fusing additional polypeptides.

[0239] [Table 6]

[0240]

[0458] The amino acid sequences of the other polypeptide domains of Structure IB are shown in Table 10. When an Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein, as in Structure IB, the sequence of the Fc module is preferably that shown in SEQ ID NO: 42, and the linker sequence is preferably selected from the embodiments shown in SEQ ID NOs: 43 to 45.

[0241] [Table 7]

[0242]

[0459] In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains selected from the group consisting of the variable heavy and variable light chains of utomilumab, the variable heavy and variable light chains of urelumab, the variable heavy and variable light chains of utomilumab, a variable heavy and variable light chain selected from the variable heavy and variable light chains set forth in Table 9, any combination of the above variable heavy and variable light chains, and fragments, derivatives, conjugates, variants, and biosimilars thereof.

[0243]

[0460] In one embodiment, a 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains comprising a 4-1BBL sequence. In one embodiment, a 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO: 46. In one embodiment, a 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains comprising a soluble 4-1BBL sequence. In one embodiment, a 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO: 47.

[0244]

[0461] In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB comprises a V that is at least 95% identical to the sequence set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L one or more 4-1BB binding domains that are scFv domains comprising a region, wherein V H and V L The domains are connected by a linker. In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB comprises a V domain that is at least 95% identical to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively. H and V L one or more 4-1BB binding domains that are scFv domains comprising a region, wherein V Hand V L The domains are connected by a linker. In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB comprises a V domain as shown in Table 11. H and V L V, each of which is at least 95% identical to the sequence H and V L one or more 4-1BB binding domains that are scFv domains comprising a region, wherein V H and V L The domains are connected by linkers.

[0245] [Table 8]

[0246]

[0462] In one embodiment, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising an additional domain at the N-terminus and / or C-terminus, wherein the additional domain is a Fab or Fc fragment domain. In one embodiment, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, and further comprising an additional domain at the N-terminus and / or C-terminus, wherein the additional domain is a Fab or Fc fragment domain, each of the soluble 4-1BB domains lacks a stalk region (which contributes to trimerization and provides a certain distance to the cell membrane, but is not part of the 4-1BB binding domain), and the first and second peptide linkers are independently 3 to 8 amino acids in length.

[0247]

[0463] In one embodiment, the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising: (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain; (ii) a first peptide linker; (iii) a second soluble TNF superfamily cytokine domain; (iv) a second peptide linker; and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stalk region, the first and second peptide linkers independently have a length of 3 to 8 amino acids, and each TNF superfamily cytokine domain is a 4-1BB binding domain.

[0248]

[0464] In one embodiment, the 4-1BB agonist is selected from the group consisting of the aforementioned V L The aforementioned V linked to one of the domains H It is a 4-1BB agonistic scFv antibody containing either one of the domains.

[0249]

[0465] In one embodiment, the 4-1BB agonist is BPS Bioscience 4-1BB agonist antibody catalog number 79097-2, which is commercially available from BPS Bioscience, San Diego, CA, USA. In one embodiment, the 4-1BB agonist is Creative Biolabs 4-1BB agonist antibody catalog number MOM-18179, which is commercially available from Creative Biolabs, Shirley, NY, USA.

[0250] OX40 (CD134) agonist as an optional media component

[0466] In one embodiment, the TNFRSF agonist is an OX40 (CD134) agonist. The OX40 agonist can be any OX40 binding molecule known in the art. The OX40 binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX40. The OX40 agonist or OX40 binding molecule can comprise an immunoglobulin heavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The OX40 agonist or OX40 binding molecule can have both a heavy chain and a light chain. As used herein, the term binding molecule includes antibodies (including full-length antibodies), monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human, humanized, or chimeric antibodies, as well as antibody fragments, e.g., Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the above, and engineered forms of antibodies that bind to OX40, such as scFv molecules. In one embodiment, the OX40 agonist is an antigen-binding protein that is a fully human antibody. In one embodiment, the OX40 agonist is an antigen-binding protein that is a humanized antibody. In some embodiments, OX40 agonists for use in the methods and compositions of the disclosure include anti-OX40 antibodies, human anti-OX40 antibodies, murine anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40 antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40 antibodies, anti-OX40 Adnectins, anti-OX40 domain antibodies, single-chain anti-OX40 fragments, heavy-chain anti-OX40 fragments, light-chain anti-OX40 fragments, anti-OX40 fusion proteins, and fragments, derivatives, conjugates, variants, or biosimilars thereof. In a preferred embodiment, the OX40 agonist is an agonistic, anti-OX40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).

[0251]

[0467] In a preferred embodiment, the OX40 agonist or OX40-binding molecule can also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun, et al., J. Immunother. 2009, 182, 1481-89. In a preferred embodiment, multimeric OX40 agonists, such as trimeric or hexameric OX40 agonists (having three or six ligand-binding domains), can induce superior receptor (OX40L) clustering and intracellular signaling complex formation compared to agonistic monoclonal antibodies, which typically possess two ligand-binding domains. Trimeric (trivalent) or hexameric (or hexavalent) or larger fusion proteins comprising three TNFRSF binding domains and IgG1-Fc, optionally further linking two or more of these fusion proteins, are described, for example, in Gieffers, et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

[0252]

[0468] Agonistic OX40 antibodies and fusion proteins are known to induce strong immune responses. Curti, et al., Cancer Res. 2013, 73, 7189-98. In a preferred embodiment, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to the OX40 antigen in a manner sufficient to reduce toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that inhibits antibody-dependent cellular cytotoxicity (ADCC), e.g., NK cell cytotoxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that inhibits antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that inhibits complement-dependent cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that inhibits Fc region functionality.

[0253]

[0469] In some embodiments, the OX40 agonist is characterized by binding to human OX40 (SEQ ID NO: 54) with high affinity and agonistic activity. In one embodiment, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 54). In one embodiment, the OX40 agonist is a binding molecule that binds to murine OX40 (SEQ ID NO: 55). The amino acid sequences of the OX40 antigens to which the OX40 agonists or binding molecules bind are summarized in Table 9.

[0254] [Table 9]

[0255]

[0470] In some embodiments, the compositions, processes, and methods described have a K of about 100 pM or less. D binds to human or mouse OX40 with a K of approximately 90 pM or less D binds to human or mouse OX40 with a K of approximately 80 pM or less D binds to human or mouse OX40 with a K of approximately 70 pM or less D binds to human or mouse OX40 with a K of approximately 60 pM or less D binds to human or mouse OX40 with a K of approximately 50 pM or less D binds to human or mouse OX40 with a K of approximately 40 pM or less D binds to human or mouse OX40 with a K of about 30 pM or less D The present invention includes an OX40 agonist that binds to human or mouse OX40 at a specific target site.

[0256]

[0471] In some embodiments, the compositions, processes, and methods described provide a dose of about 7.5×10 5 k greater than 1 / M s assoc binds to human or mouse OX40 at approximately 7.5 × 10 5 k greater than 1 / M s assoc binds to human or mouse OX40 at approximately 8 × 10 5 k greater than 1 / M s assocbinds to human or mouse OX40 at approximately 8.5 × 10 5 k greater than 1 / M s assoc binds to human or mouse OX40 at approximately 9 × 10 5 k greater than 1 / M s assoc binds to human or mouse OX40 at approximately 9.5 × 10 5 k greater than 1 / M s assoc binds to human or mouse OX40 at approximately 1 × 10 6 k greater than 1 / M s assoc The present invention includes an OX40 agonist that binds to human or mouse OX40 at a specific target site.

[0257]

[0472] In some embodiments, the compositions, processes and methods described provide a method for treating approximately 2×10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 2.1 × 10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 2.2 × 10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 2.3 × 10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 2.4 × 10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 2.5 × 10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 2.6 × 10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 2.7 x 10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 2.8 × 10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 2.9 × 10 -5 k less than 1 / s dissoc binds to human or mouse OX40 at approximately 3 x 10 -5 k less than 1 / s dissocThe present invention includes an OX40 agonist that binds to human or mouse OX40 at a specific target site.

[0258]

[0473] In some embodiments, the compositions, processes and methods described have an IC of about 10 nM or less. 50 binds to human or mouse OX40 with an IC of approximately 9 nM or less 50 binds to human or mouse OX40 with an IC of approximately 8 nM or less 50 binds to human or mouse OX40 with an IC of approximately 7 nM or less 50 binds to human or mouse OX40 with an IC of approximately 6 nM or less 50 binds to human or mouse OX40 with an IC of approximately 5 nM or less 50 binds to human or mouse OX40 with an IC of approximately 4 nM or less 50 binds to human or mouse OX40 with an IC of approximately 3 nM or less 50 binds to human or mouse OX40 with an IC of approximately 2 nM or less 50 binds to human or mouse OX40 with an IC of about 1 nM or less 50 The present invention includes an OX40 agonist that binds to human or mouse OX40 at a specific target site.

[0259]

[0474] In some embodiments, the OX40 agonist is taborixizumab, also known as MEDI0562 or MEDI-0562. Taborixizumab is available from Medimmune, a subsidiary of AstraZeneca, Inc. Taborixizumab is an immunoglobulin G1-kappa, anti-[human (Homo sapiens) TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)], humanized and chimeric monoclonal antibody. The amino acid sequence of taborixizumab is shown in Table 10. Taborixizumab has N-glycosylation sites at positions 301 and 301″; positions 22-95 (V H -V L ), 148-204(C H 1-C L ), 265-325(C H 2) and 371-429(CH 3) (and positions 22'-95', 148'-204', 265'-325', and 371'-429') intra-heavy chain disulfide bridges; positions 23'-88' (V H -V L ) and 134'-194'(C H 1-C L ) (and light chain intrasulfide bridges at positions 23'''-88''' and 134'''-194'''); intrachain heavy-heavy chain disulfide bridges at positions 230-230'' and 233-233''; and intrachain heavy-light chain disulfide bridges at positions 224-214' and 224''-214'''. Current clinical trials of taborixizumab in various solid tumor indications include the U.S. National Institutes of Health Clinicaltrials.gov identifiers NCT02318394 and NCT02705482.

[0260]

[0475] In one embodiment, the OX40 agonist comprises a heavy chain set forth in SEQ ID NO:56 and a light chain set forth in SEQ ID NO:57. In one embodiment, the OX40 agonist comprises heavy and light chains having the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 99% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 98% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 97% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 96% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain that are at least 95% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively.

[0261]

[0476] In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of taborixizumab. In one embodiment, the OX40 agonist heavy chain variable region (VR) H ) comprises the sequence set forth in SEQ ID NO: 58, and L ) comprises the sequence set forth in SEQ ID NO: 59 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 99% identical to the sequences set forth in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. H and V L The present invention also includes scFv antibodies containing the domain.

[0262]

[0477] In one embodiment, the OX40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, respectively, and conservative amino acid substitutions thereof.

[0263]

[0478] In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug regulatory agency for taborixizumab. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and including one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is taborixizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is an OX40 agonist antibody that has been approved or submitted for approval, where the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and the reference drug or reference biological product is taborixizumab. OX40 agonist antibodies may be approved by drug regulatory authorities, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is taborixizumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is taborixizumab.

[0264] [Table 10]

[0265]

[0479] In some embodiments, the OX40 agonist is 11D4, which is a fully human antibody available from Pfizer, Inc. The preparation and properties of 11D4 are described in U.S. Patent Nos. 7,960,515; 8,236,930; and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 11D4 is shown in Table 11.

[0266]

[0480] In one embodiment, the OX40 agonist comprises a heavy chain set forth in SEQ ID NO:66 and a light chain set forth in SEQ ID NO:67. In one embodiment, the OX40 agonist comprises heavy and light chains having the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 99% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 98% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 97% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 96% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain that are at least 95% identical to the sequences set forth in SEQ ID NO: 66 and SEQ ID NO: 67, respectively.

[0267]

[0481] In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4. In one embodiment, the OX40 agonist comprises the heavy chain variable region (VR) of 11D4. H) comprises the sequence set forth in SEQ ID NO: 68, and L ) comprises the sequence set forth in SEQ ID NO: 69 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. H and V L Includes the area.

[0268]

[0482] In one embodiment, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively, and conservative amino acid substitutions thereof.

[0269]

[0483] In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug regulatory agency for 11D4. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and including one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is 11D4. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is an OX40 agonist antibody that has been approved or submitted for approval, where the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and the reference drug or reference biological product is 11D4. OX40 agonist antibodies may be approved by drug regulatory authorities, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is 11D4. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is 11D4.

[0270] [Table 11]

[0271]

[0484] In some embodiments, the OX40 agonist is 18D8, a fully human antibody available from Pfizer, Inc. The preparation and properties of 18D8 are described in U.S. Patent Nos. 7,960,515; 8,236,930; and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 18D8 is shown in Table 12.

[0272]

[0485] In one embodiment, the OX40 agonist comprises a heavy chain set forth in SEQ ID NO:76 and a light chain set forth in SEQ ID NO:77. In one embodiment, the OX40 agonist comprises heavy and light chains having the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 99% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 98% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 97% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively. In one embodiment, the OX40 agonist comprises heavy and light chains that are at least 96% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain that are at least 95% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively.

[0273]

[0486] In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 18D8. In one embodiment, the OX40 agonist comprises the heavy chain variable region (VR) of 18D8. H ) comprises the sequence set forth in SEQ ID NO: 78, and L) comprises the sequence set forth in SEQ ID NO: 79 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. H and V L Includes the area.

[0274]

[0487] In one embodiment, the OX40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85, respectively, and conservative amino acid substitutions thereof.

[0275]

[0488] In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug regulatory agency for 18D8. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and including one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is 18D8. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is an OX40 agonist antibody that has been approved or submitted for approval, where the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and the reference drug or reference biological product is 18D8. OX40 agonist antibodies may be approved by drug regulatory authorities, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is 18D8. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is 18D8.

[0276] [Table 12]

[0277]

[0489] In some embodiments, the OX40 agonist is Hu119-122, a humanized antibody available from GlaxoSmithKline plc. The preparation and properties of Hu119-122 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and WO 2012 / 027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu119-122 is shown in Table 13.

[0278]

[0490] In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu119-122. H ) comprises the sequence set forth in SEQ ID NO: 86, and L ) comprises the sequence set forth in SEQ ID NO: 87 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. H and V L Includes the area.

[0279]

[0491] In one embodiment, the OX40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, respectively, and conservative amino acid substitutions thereof.

[0280]

[0492] In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug regulatory agency for Hu119-122. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and including one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is Hu119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is an OX40 agonist antibody that has been approved or submitted for approval, where the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and the reference drug or reference biological product is Hu119-122. OX40 agonist antibodies may be approved by drug regulatory authorities, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is Hu119-122. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is Hu119-122.

[0281] [Table 13]

[0282]

[0493] In some embodiments, the OX40 agonist is Hu106-222, a humanized antibody available from GlaxoSmithKline plc. The preparation and properties of Hu106-222 are described in U.S. Patent Nos. 9,006,399 and 9,163,085 and WO 2012 / 027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is shown in Table 14.

[0283]

[0494] In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hu106-222. H ) comprises the sequence set forth in SEQ ID NO: 94, and L ) comprises the sequence set forth in SEQ ID NO: 95 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 98% identical to the sequences set forth in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 97% identical to the sequences set forth in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 96% identical to the sequences set forth in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. H and V L In one embodiment, the OX40 agonist comprises a V region that is at least 95% identical to the sequences set forth in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. H and VL Includes the area.

[0284]

[0495] In one embodiment, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively, and conservative amino acid substitutions thereof.

[0285]

[0496] In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by a drug regulatory agency for Hu106-222. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity, to the amino acid sequence of a reference drug or reference biological product, and including one or more post-translational modifications compared to the reference drug or reference biological product, where the reference drug or reference biological product is Hu106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and cleavage. In some embodiments, the biosimilar is an OX40 agonist antibody that has been approved or submitted for approval, where the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and the reference drug or reference biological product is Hu106-222. OX40 agonist antibodies may be approved by drug regulatory authorities, such as the U.S. FDA and / or the European Union's EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is Hu106-222. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is Hu106-222.

[0286] [Table 14]

[0287]

[0497] In some embodiments, the OX40 agonist antibody is MEDI6469 (also referred to as 9B12). MEDI6469 is a murine monoclonal antibody. Weinberg, et al., J. Immunother. 2006, 29, 575-585. In some embodiments, the OX40 agonist is an antibody produced by the 9B12 hybridoma deposited with Biovest Inc. (Malvern, MA, USA), as described in Weinberg, et al., J. Immunother. 2006, 29, 575-585, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the antibody comprises the CDR sequences of MEDI6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and / or the light chain variable region sequence of MEDI6469.

[0288]

[0498] In one embodiment, the OX40 agonist is L106 BD (Pharmingen Product No. 340420). In some embodiments, the OX40 agonist comprises the CDRs of antibody L106 (BD Pharmingen Product No. 340420). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody L106 (BD Pharmingen Product No. 340420). In one embodiment, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, Catalog No. 20073). In some embodiments, the OX40 agonist comprises the CDRs of antibody ACT35 (Santa Cruz Biotechnology, Catalog No. 20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and / or the light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, Catalog No. 20073). In one embodiment, the OX40 agonist is the murine monoclonal antibody anti-mCD134 / mOX40 (clone OX86), commercially available from InVivoMAb, BioXcell Inc, West Lebanon, NH.

[0289]

[0499] In one embodiment, the OX40 agonist is a compound described in any of the following patent applications: WO 95 / 12673, WO 95 / 21925, WO 2006 / 121810, WO 2012 / 027328, WO 2013 / 028231, WO 2013 / 038191, and WO 2014 / 148895; EP 0672141; U.S. Patent Application Publication Nos. 2010 / 136030, 2014 / 377284, 2015 / 190506, and 2015 / 190617. 132288; and the OX40 agonists described in U.S. Patent Nos. 7,504,101, 7,550,140, ​​7,622,444, 7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085 (including 20E5 and 12H3), the disclosures of each of which are incorporated herein by reference in their entirety.

[0290]

[0500] In one embodiment, the OX40 agonist is an OX40 agonistic fusion protein shown in Structure IA (C-terminal Fc antibody fragment fusion protein) or Structure IB (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biosimilar thereof. The properties of Structures IA and IB are described above and in U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference. The amino acid sequence of the polypeptide domain of Structure IA is shown in Table 6. The Fc domain preferably includes the complete constant domain (amino acids 17-230 of SEQ ID NO:31), the complete hinge domain (amino acids 1-16 of SEQ ID NO:31), or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO:31). Preferred linkers for connecting the C-terminal Fc antibody may be selected from the embodiments set forth in SEQ ID NO: 32 to SEQ ID NO: 41, including linkers suitable for fusing additional polypeptides. Similarly, the amino acid sequences of the polypeptide domains of structure IB are shown in Table 7. When an Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein, as in structure IB, the sequence of the Fc module is preferably that set forth in SEQ ID NO: 42, and the linker sequence is preferably selected from the embodiments set forth in SEQ ID NO: 43 to SEQ ID NO: 45.

[0291]

[0501] In one embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains selected from the group consisting of the variable heavy and variable light chains of taborixizumab, the variable heavy and variable light chains of 11D4, the variable heavy and variable light chains of 18D8, the variable heavy and variable light chains of Hu119-122, the variable heavy and variable light chains of Hu106-222, a variable heavy and variable light chain selected from the variable heavy and variable light chains listed in Table 19, any combination of the above variable heavy and variable light chains, and fragments, derivatives, conjugates, variants, and biosimilars thereof.

[0292]

[0502] In one embodiment, an OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising an OX40L sequence. In one embodiment, an OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising a sequence according to SEQ ID NO: 102. In one embodiment, an OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising a soluble OX40L sequence. In one embodiment, an OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising a sequence according to SEQ ID NO: 103. In one embodiment, an OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40 binding domains comprising a sequence according to SEQ ID NO: 104.

[0293]

[0503] In one embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises a V sequence at least 95% identical to the sequence set forth in SEQ ID NO:58 and SEQ ID NO:59, respectively. H and V L one or more OX40-binding domains that are scFv domains comprising a region, wherein V H and V L The domains are connected by a linker. In one embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises a V domain that is at least 95% identical to the sequences set forth in SEQ ID NO:68 and SEQ ID NO:69, respectively. H and V L one or more OX40-binding domains that are scFv domains comprising a region, wherein V H and V L The domains are connected by a linker. In one embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises a V domain that is at least 95% identical to the sequences set forth in SEQ ID NO:78 and SEQ ID NO:79, respectively. H and V L one or more OX40-binding domains that are scFv domains comprising a region, wherein V H and VL The domains are connected by a linker. In one embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises a V domain that is at least 95% identical to the sequence set forth in SEQ ID NO:86 and SEQ ID NO:87, respectively. H and V L one or more OX40-binding domains that are scFv domains comprising a region, wherein V H and V L The domains are connected by a linker. In one embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises a V domain that is at least 95% identical to the sequence set forth in SEQ ID NO:94 and SEQ ID NO:95, respectively. H and V L one or more OX40-binding domains that are scFv domains comprising a region, wherein V H and V L The domains are connected by a linker. In one embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises one of the V domains shown in Table 15. H and V L V, each of which is at least 95% identical to the sequence H and V L one or more OX40-binding domains that are scFv domains comprising a region, wherein V H and V L The domains are connected by linkers.

[0294] [Table 15]

[0295] [Table 16]

[0296] [Table 17]

[0297]

[0504] In one embodiment, the OX40 agonist is an OX40 agonistic single-chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain, and further comprising an additional domain at the N-terminus and / or C-terminus, wherein the additional domain is a Fab or Fc fragment domain. In one embodiment, the OX40 agonist is an OX40 agonistic single-chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain, and further comprising additional domains at the N-terminus and / or C-terminus, wherein the additional domains are Fab or Fc fragment domains, each of the soluble OX40 binding domains lacks a stalk region (which contributes to trimerization and provides distance to the cell membrane, but is not part of the OX40 binding domain), and the first and second peptide linkers are independently 3 to 8 amino acids in length.

[0298]

[0505] In one embodiment, the OX40 agonist is an OX40 agonistic single-chain fusion polypeptide comprising: (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain; (ii) a first peptide linker; (iii) a second soluble TNF superfamily cytokine domain; (iv) a second peptide linker; and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stalk region; the first and second peptide linkers independently have a length of 3 to 8 amino acids; and the TNF superfamily cytokine domain is an OX40-binding domain.

[0299]

[0506] In some embodiments, the OX40 agonist is MEDI6383. MEDI6383 is an OX40 agonistic fusion protein and can be prepared as described in U.S. Patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

[0300]

[0507] In one embodiment, the OX40 agonist is selected from the group consisting of the aforementioned V L The aforementioned V linked to one of the domains H It is an OX40 agonistic scFv antibody containing either of the domains.

[0301]

[0508] In one embodiment, the OX40 agonist is Creative Biolabs OX40 agonist monoclonal antibody MOM-18455, which is commercially available from Creative Biolabs, Inc., Shirley, NY, USA.

[0302]

[0509] In one embodiment, the OX40 agonist is the OX40 agonistic antibody clone Ber-ACT35, commercially available from BioLegend, Inc., San Diego, CA, USA.

[0303] AKT pathway inhibitors as adjuncts to TIL production

[0510] In some embodiments, the AKT pathway inhibitor is added to the first culture medium together with the tumor fragments to form a closed system. In some embodiments, the AKT pathway inhibitor is an AKT inhibitor. In some embodiments, the AKT inhibitor is afuresertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is uprosertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is ipatasertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is AT7867 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is AT13148 or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is MK-2206 or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling.

[0304]

[0511] In some embodiments, the AKT pathway inhibitor is added to the first priming expansion step. In some embodiments, the AKT pathway inhibitor is an AKT inhibitor. In some embodiments, the AKT inhibitor is afuresertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is uprosertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is ipatasertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is AT7867 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is AT13148 or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT inhibitor is MK-2206 or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling.

[0305]

[0512] In various embodiments, the AKT pathway inhibitor is a PI3K inhibitor. In one embodiment, the PI3K inhibitor is LY294002 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to attenuate PI3K signaling.

[0306]

[0513] In some embodiments, the AKT pathway inhibitor comprises one or more AKT pathway inhibitors selected from the group consisting of PI3K, AKT, PDK1, mTORC2, GSK3, FOXO, TSC2, mTORC1, PTEN, BAD, ASK1, MERIT40, MDM2, CHK1, IKKα, p21CIP1, p27KIP1, RAF1, PFKFB2, PDE3B, PRAS40, AMPK, ACLY, PIKfyve, eNOS, WNK1, huntingtin, palladin, and TBC1D4.

[0307]

[0514] In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 1000 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 500 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 100 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 50 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 50 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 25 μM.

[0308]

[0515] In one embodiment, the AKT pathway inhibitor is added to the first priming expansion step at a concentration of 0.01 μM to 1000 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 500 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 100 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 50 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 50 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 50 μM. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration of 0.01 μM to 25 μM.

[0309]

[0516] In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration such that the target enzyme is at least 95% occupied at steady state. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration such that the target enzyme is at least 85% occupied at steady state. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration such that the target enzyme is at least 75% occupied at steady state. In one embodiment, the AKT pathway inhibitor is added to the first culture medium at a concentration such that the target enzyme is at least 50% occupied at steady state.

[0310]

[0517] In one embodiment, the AKT pathway inhibitor is added to the first prime expansion step at a concentration that results in at least 95% occupancy of the target enzyme at steady state. In one embodiment, the AKT pathway inhibitor is added to the first prime expansion step at a concentration that results in at least 85% occupancy of the target enzyme at steady state. In one embodiment, the AKT pathway inhibitor is added to the first prime expansion step at a concentration that results in at least 75% occupancy of the target enzyme at steady state. In one embodiment, the AKT pathway inhibitor is added to the first prime expansion step at a concentration that results in at least 50% occupancy of the target enzyme at steady state.

[0311]

[0518] In some embodiments, the AKT pathway inhibitor is 10 nM, 20 nM, 25 nM, 30 nM, 50 nM, 60 nM, 75 nM, 80 nM, 90 nM, 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 375 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700 nM, 725 nM, 750 nM, 775 nM, 800 nM, 825 nM, 85 and 25 μM.

[0312]

[0519] In some embodiments, the AKT pathway inhibitor is 10 nM, 20 nM, 25 nM, 30 nM, 50 nM, 60 nM, 75 nM, 80 nM, 90 nM, 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 375 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700 nM, 725 nM, 750 nM, 775 nM, 800 nM, 825 nM, 850 nM, and 25 μM.

[0313]

[0520] In some embodiments, the first cell culture medium comprises at least two AKT pathway inhibitors. In a further embodiment, the first AKT pathway inhibitor is afuresertib or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof.

[0314]

[0521] In some embodiments, the first prime-expansion step comprises at least two AKT pathway inhibitors. In a further embodiment, the first AKT pathway inhibitor is afuresertib or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof.

[0315]

[0522] In some embodiments, the AKT pathway inhibitor is added to the second culture medium together with the tumor fragments to form a closed system. In one embodiment, the AKT pathway inhibitor is afuresertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT pathway inhibitor is uprosertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT pathway inhibitor is ipatasertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to block AKT signaling. In another embodiment, the AKT pathway inhibitor is AT7867 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to block AKT signaling. In another embodiment, the AKT pathway inhibitor is AT13148 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to block AKT signaling. In another embodiment, the AKT pathway inhibitor is MK-2206 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug, family member thereof, and is added at a concentration sufficient to block AKT signaling. In one embodiment, the AKT pathway inhibitor is LY294002 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug, family member thereof, and is added at a concentration sufficient to block AKT signaling.

[0316]

[0523] In some embodiments, an AKT pathway inhibitor is added to the second rapid expansion step. In one embodiment, the AKT pathway inhibitor is afuresertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT pathway inhibitor is uprosertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to attenuate AKT signaling. In another embodiment, the AKT pathway inhibitor is ipatasertib or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to block AKT signaling. In another embodiment, the AKT pathway inhibitor is AT7867 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to block AKT signaling. In another embodiment, the AKT pathway inhibitor is AT13148 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and is added at a concentration sufficient to block AKT signaling. In another embodiment, the AKT pathway inhibitor is MK-2206 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug, family member thereof, and is added at a concentration sufficient to block AKT signaling. In one embodiment, the AKT pathway inhibitor is LY294002 or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug, family member thereof, and is added at a concentration sufficient to block AKT signaling.

[0317]

[0524] In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration of 0.01 μM to 1000 μM. In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration of 0.01 μM to 500 μM. In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration of 0.01 μM to 100 μM. In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration of 0.01 μM to 50 μM. In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration of 0.01 μM to 50 μM. In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration of 0.01 μM to 25 μM.

[0318]

[0525] In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration of 0.01 μM to 1000 μM. In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration of 0.01 μM to 500 μM. In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration of 0.01 μM to 100 μM. In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration of 0.01 μM to 50 μM. In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration of 0.01 μM to 50 μM. In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration of 0.01 μM to 25 μM.

[0319]

[0526] In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration such that the target enzyme is at least 95% occupied at steady state. In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration such that the target enzyme is at least 85% occupied at steady state. In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration such that the target enzyme is at least 75% occupied at steady state. In one embodiment, the AKT pathway inhibitor is added to the second culture medium at a concentration such that the target enzyme is at least 50% occupied at steady state.

[0320]

[0527] In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration that results in at least 95% occupancy of the target enzyme at steady state. In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration that results in at least 85% occupancy of the target enzyme at steady state. In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration that results in at least 75% occupancy of the target enzyme at steady state. In one embodiment, the AKT pathway inhibitor is added to the second rapid expansion step at a concentration that results in at least 50% occupancy of the target enzyme at steady state.

[0321]

[0528] In some embodiments, the AKT pathway inhibitor is 10 nM, 20 nM, 25 nM, 30 nM, 50 nM, 60 nM, 75 nM, 80 nM, 90 nM, 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 375 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700 nM, 725 nM, 750 nM, 775 nM, 800 nM, 825 nM, 85 and 25 μM.

[0322]

[0529] In some embodiments, the AKT pathway inhibitor is 10 nM, 20 nM, 25 nM, 30 nM, 50 nM, 60 nM, 75 nM, 80 nM, 90 nM, 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 375 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700 nM, 725 nM, 750 nM, 775 nM, 800 nM, 825 nM, 850 nM , 875nM, 900nM, 925nM, 950nM, 975nM, 1000nM, 1100nM, 1200nM, 1300nM, 1400nM, 1500nM, 1600nM, 1700nM, 1800nM, 1900nM, 2000nM, 2.5µM, 3µM, 4µM, 5µM, 6µM, 7µM, 8µM, 9µM, 10µM, 12.5µM, 15µM, 18µM, 20µM and 25µM per 100,000 cells.

[0323]

[0530] In one embodiment, the AKT pathway inhibitor is added to the culture medium of the first expansion culture. In a further embodiment, the AKT pathway inhibitor is added to the culture medium of the first expansion culture and is present at a concentration sufficient to attenuate AKT pathway signaling. In one embodiment, the AKT pathway inhibitor is added to the first cell culture medium at intervals selected from the group consisting of daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, and every 2 weeks during the initial expansion culture.

[0324]

[0531] In another embodiment, the AKT pathway inhibitor is added to the culture medium of the second expansion culture. In a further embodiment, the AKT pathway inhibitor is added to the culture medium of the second expansion culture and is present at a concentration sufficient to attenuate AKT signaling. In a further embodiment, the AKT pathway inhibitor is added to the culture medium of the second expansion culture and is present at a concentration sufficient to attenuate AKT signaling.

[0325]

[0532] It is understood that the AKT pathway inhibitor is suitable for use in some TIL production methods known in the art.Furthermore, it is understood that the AKT pathway inhibitor is suitable for use in the TIL production methods described herein.For the avoidance of doubt, the AKT pathway inhibitor can comprise Gen3 process step B culture medium.For the avoidance of doubt, the AKT pathway inhibitor can comprise Gen3 process step D culture medium.

[0326] Optional cell viability analysis

[0533] Optionally, after the first expansion (sometimes referred to as initial bulk expansion), cell viability assays can be performed using standard assays known in the art. For example, a sample of bulk TILs can be subjected to a trypan blue dye exclusion assay, which selectively labels dead cells and allows viability to be assessed. Other assays used to test viability include, but are not limited to, the Alamar Blue assay and the MTT assay.

[0327] Cell counts, viability, and flow cytometry

[0534] In some embodiments, cell number and / or viability are measured. Expression of markers such as, but not limited to, CD3, CD4, CD8, and CD56, as well as any others disclosed or described herein, can be measured by antibody-based flow cytometry, such as, but not limited to, a FACSCanto™ flow cytometer (BD Biosciences) commercially available from BD Biosciences (BD Biosciences, San Jose, CA). Cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed using any method known in the art, including, but not limited to, trypan blue staining. Cell viability can also be assayed according to U.S. Patent No. 15 / 863,634, "Processes for Production of Tumor Infiltrating Lymphocytes and Uses of Same in Immunotherapy," which is incorporated herein by reference in its entirety.

[0328]

[0535] In some cases, the bulk TIL population can be immediately cryopreserved using the protocols discussed below. Alternatively, the bulk TIL population can be subjected to REP, as discussed below, and then cryopreserved. Similarly, if genetically modified TILs are to be used in therapy, the bulk or REP TIL population can be subjected to genetic modification for the appropriate treatment.

[0329]

[0536] According to the present disclosure, there is further provided a method for assaying the viability of TILs and / or administering the same to a subject. In some embodiments, the method for assaying tumor infiltrating lymphocytes (TILs) comprises: (i) Obtaining a first population of TILs; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally OKT-3 to generate a second TIL population; and (iii) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population, wherein the third TIL population is at least 50 times more numerous than the second TIL population; (iv) harvesting, washing, and cryopreserving the third population of TILs; (v) cryopreserved TILs are stored at cryogenic temperatures; (vi) thawing the third TIL population to provide a thawed third TIL population; and (vii) performing an additional second expansion of a portion of the thawed third TIL population for an additional expansion period (sometimes referred to as a reREP period) of at least 3 days by supplementing the cell culture medium of the third population with IL-2, OKT-3, and APC, wherein this third expansion is performed to obtain a fourth TIL population, and the number of TILs in the fourth TIL population is compared with the number of TILs in the third TIL population to determine a ratio; (viii) determining whether the thawed TIL population is suitable for administration to a patient based on the ratio of step (vii); (ix) administering a therapeutically effective dose of the thawed third TIL population to the patient when in step (viii), the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is determined to be greater than 5:1. Includes:

[0330]

[0537] In some embodiments, the TILs are assayed for viability after step (vii).

[0331]

[0538] The present disclosure also provides additional methods for assaying TILs. In some embodiments, the present disclosure provides methods for assaying TILs, the methods comprising: (i) obtaining a portion of the first cryopreserved TIL population; (ii) thawing a portion of the first cryopreserved TIL population; (iii) performing a first expansion by culturing a portion of the first TIL population in cell culture medium comprising IL-2, OKT-3, and antigen-presenting cells (APCs) for an additional expansion period (sometimes referred to as a reREP period) of at least 3 days to generate a second TIL population, wherein the portion from the first TIL population is compared with a second TIL population to determine a ratio, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 5:1; (iv) determining whether the first population of TILs is suitable for use in therapeutic administration to a patient based on the ratio of step (iii); (v) determining that the first TIL population is suitable for use in therapeutic administration when in step (iv) the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is determined to be greater than 5:1. Includes:

[0332]

[0539] In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the portion of the first TIL population is greater than 50:1.

[0333]

[0540] In some embodiments, the method further comprises performing expansion of the entire first cryopreserved TIL population from step (i) by a method as described in any embodiment provided herein.

[0334]

[0541] In some embodiments, the method further comprises administering the entire first cryopreserved TIL population from step (i) to the patient.

[0335] Cell culture medium

[0542] In one embodiment, the method for expanding TILs, including those discussed above and illustrated in FIG. 1, may include using about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, the medium is serum-free. In some embodiments, the medium for the first expansion culture is serum-free. In some embodiments, the medium for the second expansion culture is serum-free. In some embodiments, both the medium for the first and second expansion cultures are serum-free. In one embodiment, no more than one type of cell culture medium is used to expand the number of TILs. Any suitable cell culture medium may be used, for example, AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad, CA). In this regard, the methods of the present invention advantageously reduce the amount of medium and the number of types of medium required to expand the number of TILs. In one embodiment, expanding the number of TILs can involve feeding the cells no more frequently than once every 3 or 4 days. Increasing the number of cells in a gas-permeable container simplifies the procedures required to expand the cell number by reducing the feeding frequency required to expand the cells.

[0336]

[0543] In one embodiment, the cell culture medium in the first and / or second gas-permeable containers is unfiltered. The use of unfiltered cell culture medium can simplify the procedures required to expand cell numbers. In one embodiment, the cell culture medium in the first and / or second gas-permeable containers lacks beta-mercaptoethanol (BME or βME, also known as 2-mercaptoethanol, CAS 60-24-2).

[0337]

[0544] In one embodiment, the duration of the method includes obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first gas-permeable container containing cell culture medium; obtaining TILs from the tumor tissue sample; and expanding the number of TILs in a second gas-permeable container containing cell culture medium for about 7-14 days, e.g., about 11 days. In some embodiments, the pre-REP is about 7-14 days, e.g., about 11 days. In some embodiments, the REP is about 7-14 days, e.g., about 11 days.

[0338]

[0545] In one embodiment, TILs are expanded in a gas-permeable container. Gas-permeable containers are used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005 / 0106717 A1 (the disclosure of which is incorporated herein by reference). In one embodiment, TILs are expanded in a gas-permeable bag. In one embodiment, TILs are expanded using a cell expansion system that expands TILs in a gas-permeable bag, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are expanded using a cell expansion system that expands TILs in a gas-permeable bag, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion culture system comprises a gas permeable cell bag having a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

[0339]

[0546] In one embodiment, TILs can be expanded in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such an embodiment involves expanding the cell population to approximately 5×105 cells / cm 2 From 10 x 10 6 ~30×10 6 cells / cm 2 In one embodiment, this is feed-free. In one embodiment, this is feed-free as long as there is about 10 cm of medium in the G-Rex flask. In one embodiment, this is feed-free, but one or more cytokines are added. In one embodiment, the cytokines can be added as a bolus without having to be mixed with the medium. Such vessels, devices and methods are known in the art and have been used for the expansion of TILs, including, for example, U.S. Patent Application Publication No. 2014 / 0377739 A1, WO 2014 / 210036 A1, U.S. Patent Application Publication No. 2013 / 0115617 A1, WO 2013 / 188427 A1, U.S. Patent Application Publication No. 2011 / 0136228 A1, U.S. Patent No. 8,809,050 B2, WO 2011 / 072088 A2, U.S. Patent Application Publication No. 2016 / 0208216 A1, U.S. Patent Application Publication No. 2012 / 0244133 A1, WO 2012 / 129201 A1, U.S. Patent Application Publication No. 2013 / 0102075 A1, U.S. Patent No. 8,956,860 B2, and U.S. Patent No. 8,956,860 B2. B2, WO 2013 / 173835 A1, and U.S. Patent Application Publication No. 2015 / 0175966 A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292.

[0340] TIL manufacturing method - Gen3 process

[0547] Without being limited to a particular theory, it is believed that a first priming expansion culture to prime T cell activation, followed by a second rapid expansion culture to boost T cell activation as described in the methods of the present invention, allows for the preparation of expanded T cells that retain a "more immature" phenotype; therefore, the expanded T cells of the present invention are expected to exhibit greater cytotoxicity against cancer cells than T cells expanded by other methods. In particular, activation of T cells primed by exposure to an anti-CD3 antibody (e.g., OKT-3), IL-2, and optionally antigen-presenting cells (APCs), followed by subsequent boosting by exposure to additional anti-CD3 antibody (e.g., OKT-3), IL-2, and APCs, as taught by the methods of the present invention, is believed to limit or avoid T cell maturation in culture, generating a T cell population with a less mature phenotype that is less depleted by expansion in culture and exhibits greater cytotoxicity against cancer cells.

[0341]

[0548] The Gen3 process is also described in U.S. Patent Application No. 62 / 755,954, filed November 5, 2018 (Attorney Docket No. 116983-5045-PR), which is incorporated herein by reference in its entirety.

[0342]

[0549] In some embodiments, a second rapid expansion culture is performed after the T cell activation resulting from the first priming expansion culture begins to decrease, attenuate, decay or subside.

[0343]

[0550] In some embodiments, the activation of T cells resulting from the first prime expansion culture is 1 or about 1, 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, 6 or about 6, 7 or about 7, 8 or about 8, 9 or about 9, 10 or about 10, 11 or about 11, 12 or about 12, 13 or about 13, 14 or about 14, 15 or about 15, 16 or about 16, 17 or about 17, 18 or about 18, 19 or about 19, 20 or about 20, 21 or about 21, 22 or about 22, 23 or or about 23, 24 or about 24, 25 or about 25, 26 or about 26, 27 or about 27, 28 or about 28, 29 or about 29, 30 or about 30, 31 or about 31, 32 or about 32, 33 or about 33, 34 or about 34, 35 or about 35, 36 or about 36, 37 or about 37, 38 or about 38, 39 or about 39, 40 or about 40, 41 or about 41, 42 or about 42, 43 or about 43, 44 or about 44, 45 or about 45, 46 or about 46, 47 or about 47, 48 or or about 48, 49 or about 49, 50 or about 50, 51 or about 51, 52 or about 52, 53 or about 53, 54 or about 54, 55 or about 55, 56 or about 56, 57 or about 57, 58 or about 58, 59 or about 59, 60 or about 60, 61 or about 61, 62 or about 62, 63 or about 63, 64 or about 64, 65 or about 65, 66 or about 66, 67 or about 67, 68 or about 68, 69 or about 69, 70 or about 70, 71 or about 71, 72 or about 72, 3 or about 73, 74 or about 74, 75 or about 75, 76 or about 76, 77 or about 77, 78 or about 78, 79 or about 79, 80 or about 80, 81 or about 81, 82 or about 82, 83 or about 83, 84 or about 84, 85 or about 85, 86 or about 86, 87 or about 87, 88 or about 88, 89 or about 89, 90 or about 90, 91 or about 91, 92 or about 92, 93 or about 93, 94 or about 94, 95 or about 95, 96 or about 96, 97 or about 97,After a 98 or about 98, 99 or about 99, or 100 or about 100% reduction, a second rapid expansion culture is performed.

[0344]

[0551] In some embodiments, a second rapid expansion culture is performed after the activation of T cells resulting from the first priming expansion culture has decreased by a percentage ranging from 1% or about 1% to 100% or about 100%.

[0345]

[0552] In some embodiments, the second rapid expansion culture is performed after the activation of T cells resulting from the first prime expansion culture has decreased by a percentage in the range of 1%, or about 1% to 10%, or about 10%, 10%, or about 10% to 20%, or about 20%, 20%, or about 20% to 30%, or about 30%, 30%, or about 30% to 40%, or about 40%, 40%, or about 40% to 50%, or about 50%, 50%, or about 50% to 60%, or about 60%, 60%, or about 60% to 70%, or about 70%, 70%, or about 70% to 80%, or about 80%, 80%, or about 80% to 90%, or about 90%, or 90%, or about 90% to 100%, or about 100%.

[0346]

[0553] In some embodiments, the activation of T cells resulting from the first prime-expansion culture is at least 1 or about 1, 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, 6 or about 6, 7 or about 7, 8 or about 8, 9 or about 9, 10 or about 10, 11 or about 11, 12 or about 12, 13 or about 13, 14 or about 14, 15 or about 15, 16 or about 16, 17 or about 17, 18 or about 18, 19 or about 19, 20 or about 20, 21 or about 21, 22 or about 22, 23 or about 23, 24 or about 24, 25 or about 25, 26 or about 26, 27 or about 27, 28 or about 28, 29 or about 29, 30 or about 30, 31 or about 31, 32 or about 32, 33 or about 33, 34 or about 34, 35 or about 35, 36 or about 36, 37 or about 37, 38 or about 38, 39 or about 39, 40 or about 40, 41 or about 41, 42 or about 42, 43 or about 43, 44 or about 44, 45 or about 45, 46 or about 46, 47 or about 47, 48 or about 48, 49 or about 49, 50 or about 50, 51 or about 51, 52 or about 52, 53 or about 53, 54 or about 54, 55 or about 55, 56 or about 56, 57 or about 57, 58 or about 58, 59 or about 59, 60 or about 60, 61 or about 61, 62 or about 62, 63 or about 63, 64 or about 64, 65 or about 65, 66 or about 66, 67 or about 67, 68 or about 68, 69 or about 69, 70 or about 70, 71 or about 71, 72 or about 72, 73 or about 73, 74 or about 74, 75 or about 75, 76 or about 76, 77 or about 77, 78 or about 78, 79 or about 79, 80 or about 80, 81 or about 81, 82 or about 82, 83 or about 83, 84 or about 84, 85 or about 85, 86 or about 86, 87 or about 87, 88 or about 88, 89 or about 89, 90 or about 90, 91 or about 91, 92 or about 92, 93 or about 93, 94 or about 94, 95 or about 95, 96 or about 96, 97 or about 97,After a 98 or about 98 or 99 or about 99% reduction, a second rapid expansion culture is performed.

[0347]

[0554] In some embodiments, the activation of T cells resulting from the first prime expansion culture is at most 1 or about 1, 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, 6 or about 6, 7 or about 7, 8 or about 8, 9 or about 9, 10 or about 10, 11 or about 11, 12 or about 12, 13 or about 13, 14 or about 14, 15 or about 15, 16 or about 16, 17 or about 17, 18 or about 18, 19 or about 19, 20 or about 20, 21 or about 21, 22 or about 22, 23 or more or about 23, 24 or about 24, 25 or about 25, 26 or about 26, 27 or about 27, 28 or about 28, 29 or about 29, 30 or about 30, 31 or about 31, 32 or about 32, 33 or about 33, 34 or about 34, 35 or about 35, 36 or about 36, 37 or about 37, 38 or about 38, 39 or about 39, 40 or about 40, 41 or about 41, 42 or about 42, 43 or about 43, 44 or about 44, 45 or about 45, 46 or about 46, 47 or about 47, 48 or about 48, 49 or about 49, 50 or about 50, 51 or about 51, 52 or about 52, 53 or about 53, 54 or about 54, 55 or about 55, 56 or about 56, 57 or about 57, 58 or about 58, 59 or about 59, 60 or about 60, 61 or about 61, 62 or about 62, 63 or about 63, 64 or about 64, 65 or about 65, 66 or about 66, 67 or about 67, 68 or about 68, 69 or about 69, 70 or about 70, 71 or about 71, 72 or about 72, 3 or about 73, 74 or about 74, 75 or about 75, 76 or about 76, 77 or about 77, 78 or about 78, 79 or about 79, 80 or about 80, 81 or about 81, 82 or about 82, 83 or about 83, 84 or about 84, 85 or about 85, 86 or about 86, 87 or about 87, 88 or about 88, 89 or about 89, 90 or about 90, 91 or about 91, 92 or about 92, 93 or about 93, 94 or about 94, 95 or about 95, 96 or about 96, 97 or about 97,After a 98 or about 98, 99 or about 99, or 100 or about 100% reduction, a second rapid expansion culture is performed.

[0348]

[0555] In some embodiments, the decrease in T cell activation resulting from the first prime expansion culture is determined by a decrease in the amount of interferon gamma released by the T cells in response to stimulation with an antigen.

[0349]

[0556] In some embodiments, the first prime expansion of the T cells is performed for a period of up to or about 7 days.

[0350]

[0557] In some embodiments, the first prime expansion of the T cells is carried out for a period of at most 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0351]

[0558] In some embodiments, the first prime expansion of the T cells is performed for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0352]

[0559] In some embodiments, the second rapid expansion of the T cells is carried out for a period of up to or about 11 days.

[0353]

[0560] In some embodiments, the second rapid expansion of the T cells is carried out for a period of at most 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0354]

[0561] In some embodiments, the second rapid expansion of the T cells is performed for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

[0355]

[0562] In some embodiments, the first priming expansion culture of T cells is performed for a period of at or about 1 day to at or about 7 days, and the second rapid expansion culture of T cells is performed for a period of at or about 1 day to at or about 11 days.

[0356]

[0563] In some embodiments, the first prime expansion culture of T cells is performed for a period of at most 1 day, 2 days or about 2 days, 3 days or about 3 days, 4 days or about 4 days, 5 days or about 5 days, 6 days or about 6 days, or 7 days or about 7 days, and the second rapid expansion culture of T cells is performed for a period of at most 1 day, 2 days or about 2 days, 3 days or about 3 days, 4 days or about 4 days, 5 days or about 5 days, 6 days or about 6 days, 7 days or about 7 days, 8 days or about 8 days, 9 days or about 9 days, 10 days or about 10 days, or 11 days or about 11 days.

[0357]

[0564] In some embodiments, the first priming expansion culture of T cells is performed for a period of at or about 1 day to at or about 7 days, and the second rapid expansion culture of T cells is performed for a period of at or about 1 day to at or about 9 days.

[0358]

[0565] In some embodiments, the first prime expansion of the T cells is performed over a 7 day period, and the second rapid expansion of the T cells is performed over a 9 day period.

[0359]

[0566] In some embodiments, the T cells are tumor-infiltrating lymphocytes (TILs).

[0360]

[0567] In some embodiments, the T cells are bone marrow infiltrating lymphocytes (MIL).

[0361]

[0568] In some embodiments, the T cells are peripheral blood lymphocytes (PBLs).

[0362]

[0569] In some embodiments, the T cells are obtained from a donor suffering from cancer.

[0363]

[0570] In some embodiments, the T cells are TILs obtained from a tumor resected from a patient suffering from cancer.

[0364]

[0571] In some embodiments, the T cells are MILs obtained from the bone marrow of a patient suffering from a hematological malignancy.

[0365]

[0572] In some embodiments, the T cells are PBLs obtained from peripheral blood mononuclear cells (PBMCs) from a donor. In some embodiments, the donor is afflicted with cancer. In some embodiments, the donor is afflicted with a hematological malignancy.

[0366]

[0573] In certain aspects...

Claims

1. 1. A method for preparing tumor infiltrating lymphocytes (TILs) for adoptive T cell therapy, comprising: (a) treating the tumor fragments with a first cell culture medium containing interleukin 2 (IL-2), an OKT-3 antibody, and one or more AKT pathway inhibitors (AKTi) to provide TILs; (b) expanding the TILs in a second cell culture medium comprising cell culture medium, irradiated feeder cells, OKT-3 antibody, and IL-2 to provide an expanded number of TILs; and (c) optionally, cryopreserving the expanded number of TILs. The method includes:

2. 2. The method of claim 1, wherein the one or more AKT pathway inhibitors are AKT kinase inhibitors.

3. 3. The method of claim 2, wherein the AKT kinase inhibitor is selected from the group consisting of afuresertib, uprosertib, ipatasertib, AT7867, AT13148, and pharmaceutically acceptable salts, solvates, hydrates, co-crystals, or prodrugs thereof.

4. 2. The method of claim 1, wherein the one or more AKT pathway inhibitors is an mTOR inhibitor.

5. 5. The method of claim 4, wherein the mTOR inhibitor is AZD8055 or a pharma- ceutically acceptable salt, solvate, hydrate, co-crystal or prodrug thereof.

6. 2. The method of claim 1, wherein the one or more AKT pathway inhibitors is a PI3K inhibitor.

7. 7. The method of claim 6, wherein the PI3K inhibitor is LY294002 or a pharma- ceutically acceptable salt, solvate, hydrate, co-crystal, or prodrug thereof.

8. 2. The method of claim 1, wherein the one or more AKT pathway inhibitors comprise two AKT pathway inhibitors, each of which targets a different AKT pathway component.

9. 9. The method of claim 8, wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets PI3K.

10. 9. The method of claim 8, wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets mTOR.

11. 9. The method of claim 8, wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets PDK1.

12. 9. The method of claim 8, wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets CHK1.

13. 9. The method of claim 8, wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets Raf1.

14. 9. The method of claim 8, wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets one or more of FOXO1, FOXO3A, and FOXO4.

15. 9. The method of claim 8, wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets GSK3.

16. 16. The method of any one of claims 1 to 15, wherein the tumor infiltrating lymphocytes (TILs) for adoptive T cell therapy are enriched for TILs expressing one or more selected from the group consisting of CD8, CD27, CD28, IL7R, CCR9, SELL (L-selectin or CD62L), FCER1G, SATB1, LEF1 and KLF2.

17. 16. The method of any one of claims 1 to 15, wherein the tumor infiltrating lymphocytes (TILs) for adoptive T cell therapy are enriched for TILs expressing memory subset T cell surface markers.

18. The method of any one of claims 1 to 15, wherein the tumor infiltrating lymphocytes (TILs) for adoptive T cell therapy are enriched for TILs expressing a juvenile TIL phenotype.

19. 16. The method of any one of claims 1-15, wherein the tumor infiltrating lymphocytes (TILs) for adoptive T cell therapy are enriched for TILs with long telomeres compared to TILs expanded without an AKT pathway inhibitor.

20. The method of any one of claims 1 to 19, wherein in step (a) the tumor fragments are cryopreserved.

21. 1. A method of treating a subject having cancer comprising administering expanded tumor infiltrating lymphocytes (TILs), (a) obtaining a first population of TILs from a tumor obtained from said subject; (b) performing a first expansion culture by culturing the first TIL population in cell culture medium comprising IL-2 and one or more AKT pathway inhibitors to give rise to a second TIL population, wherein the first expansion culture is performed in a closed system comprising a closed vessel providing a first gas permeable surface area, wherein the first expansion culture is performed for about 3-14 days to obtain the second TIL population, wherein the second TIL population is at least 50-fold more numerous than the first TIL population, and wherein the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen presenting cells (APCs) and culturing to give rise to a third TIL population, wherein the second expansion is performed for about 7-14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population comprising an expanded subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, wherein the second expansion is performed within the closed system in a second closed vessel providing a second gas permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic TIL population obtained from step (b), wherein the transition from step (c) to step (d) occurs without opening the system; (e) administering to the subject a therapeutically effective dose of the recovered therapeutic TIL population from step (d). The method comprises administering.

22. 22. The method of claim 21, further comprising the step of transferring the recovered therapeutic TIL population from step (d) to an infusion bag after step (d) and before step (e), wherein the transferring occurs without opening the system, and wherein step (e) is performed by administering a therapeutically effective dose of the recovered therapeutic TIL population from the infusion bag to the subject.

23. 23. The method of claim 21 or 22, further comprising, after the step of transferring the harvested TIL population from step (d) to an infusion bag, cryopreserving the infusion bag containing the harvested therapeutic TIL population using a cryopreservation process.

24. The method of any one of claims 21 to 23, further comprising administering to the subject a therapeutically effective dose of aldesleukin or a biosimilar thereof simultaneously with or following said administration of said therapeutically effective dose of said recovered therapeutic TIL population.

25. 25. The method of any one of claims 21-24, further comprising administering to the subject a therapeutically effective dose of a PD-1 / PD-L1 inhibitor concomitantly with, prior to, or following said administration of said therapeutically effective dose of said recovered therapeutic TIL population.

26. 26. The method of claim 25, wherein the PD-1 / PD-L1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, avelumab, durvalumab, atezolizumab, and biosimilars thereof.

27. 27. The method of any one of claims 21 to 26, wherein the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, bladder cancer, ovarian cancer, pancreatic cancer and sarcoma.

28. The method of any one of claims 2 to 27, wherein the culture medium in step (c) further comprises one or more AKT pathway inhibitors.

29. 29. The method of claim 28, wherein the AKT pathway inhibitor is selected from the group consisting of afuresertib, uprosertib, ipatasertib, AT7867, AT13148, AZD8055, MK-2206, LY294002, and pharma- ceutically acceptable salts, solvates, hydrates, co-crystals, or prodrugs thereof.

30. 1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) obtaining a first population of TILs from a tumor excised from a subject by processing a tumor sample obtained from said subject into a plurality of tumor fragments; (b) performing a first expansion culture by culturing the first TIL population in a cell culture medium comprising IL-2, OKT-3, one or more AKT pathway inhibitors (AKTi), and antigen presenting cells (APCs) to give rise to a second TIL population, wherein the first prime expansion culture is performed in a vessel comprising a first gas permeable surface area, wherein the first prime expansion culture is performed for a first period of about 1-7 days to obtain the second TIL population, wherein the second TIL population is more numerous than the first TIL population; (c) performing a second rapid-expansion culture by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APCs to give rise to a third TIL population, wherein the number of APCs added in the second rapid-expansion culture is at least twice the number of APCs added in step (b), and wherein the second rapid-expansion culture is performed for a second period of about 1-11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, and wherein the second rapid-expansion culture is performed in a vessel comprising a second gas permeable surface area; (d) harvesting the therapeutic TIL population obtained from step (c); and (e) transferring the harvested TIL population from step (d) to an infusion bag. The method includes:

31. 31. The method of claim 30, wherein the culture medium of step (c) is further supplemented with one or more AKT pathway inhibitors (AKTi).

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