Process for the production of tumor infiltrating lymphocytes (TIL) and methods of using same

Abstract

The present invention relates to a method for expanding TILs from tumor tissue using a long first expansion process and a shorter second expansion process, the method comprising: obtaining a first population of TILs from a tumor resected from a subject; culturing the first population of TILs in a cell culture medium containing a 4-1BB agonist, IL-2, and OKT-3 to perform the first expansion for a period of about 21 to about 35 days to produce a second population of TILs; and supplementing the cell culture medium of the second population of TILs with antigen-presenting cells (APCs) and additional 4-1BB agonist, IL-2, and OKT-3, and culturing the second population of TILs to perform the second expansion for a period of about 7 to about 10 days to produce a third population of TILs, wherein the third population of TILs is a therapeutic TIL population.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 946,620, filed December 11, 2019, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION This invention relates to methods for producing tumor-infiltrating lymphocytes (TILs) from tumors and methods for using such TILs in the treatment of cancer. [Background technology]

[0003] Background of the Invention

[0003] Adoptive cell therapy using TILs cultured ex vivo by rapid expansion protocols (REP) has led to successful adoptive cell therapy in melanoma patients after host immunosuppression. Current infusion eligibility parameters depend on readings of TIL composition (e.g., CD28, CD8, or CD4 positivity) and numerical expansion and viability of the REP products.

[0004] Current REP protocols provide little insight into the health of the TILs that will be infused into patients. T cells undergo a significant metabolic shift during their maturation from naive to effector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364, expressly incorporated herein in its entirety, and particularly for a discussion and markers of anaerobic and aerobic metabolism). For example, naive T cells rely on mitochondrial respiration for ATP production, whereas mature, healthy effector T cells, such as TILs, are highly glycolytic and rely on aerobic glycolysis to provide the bioenergetic substrates they require for proliferation, migration, activation, and antitumor efficacy.

[0005]

[0005] Previous studies have reported that cells highly dependent on glycolysis suffer from nutrient deprivation during adoptive transfer, resulting in the death of a large proportion of the transferred cells. Therefore, it is desirable to limit glycolysis and promote mitochondrial metabolism in TILs before transfer. Therefore, the art teaches that promoting mitochondrial metabolism may promote in vivo longevity, and indeed, the use of glycolysis inhibitors before the induction of an immune response has been proposed. See Chang et al. (Chang, et al., Nat. Immunol. 2016, 17(364), 574-582). Summary of the Invention [Means for solving the problem]

[0006] In a preferred embodiment, the present invention surprisingly demonstrates that central memory (CD45RA) TILs express a CD45-dependent cytotoxicity factor (CDE) when compared to freshly harvested TILs or thawed cryopreserved TILs for restimulation (sometimes referred to herein as "reTILs"). - CCR7 + ) or Effector Memory (CD45RA - CCR7 - The present invention relates to a novel method of enhancing REP with an additional restimulation protocol, sometimes referred to herein as a "re-stimulation rapid expansion protocol" or "reREP," which leads to the expansion of memory T cell subsets comprising a phenotype and / or leads to a significant enhancement of glycolytic respiration. That is, by using the reREP procedure (i.e., a procedure including a first expansion and a second expansion) on cryopreserved TILs, patients can receive highly metabolically active and healthy TILs, which can lead to better outcomes.

[0007]

[0007] In some embodiments, the present invention further relates to methods for determining and quantifying this increase in metabolic health. Accordingly, the present invention provides methods for assaying the relative health of a TIL population using one or more common metabolic determinations, including, but not limited to, the rate and amount of glycolysis, oxidative phosphorylation, spare respiratory capacity (SRC), and glycolytic reserve. [Brief explanation of the drawings]

[0008] Brief description of the diagram [Figure 1]

[0008] A flowchart of the expansion culture method according to the present invention is shown. [Figure 2]

[0009] 1 shows successful TIL cell expansion of endometrial and thyroid cancer samples. [Figure 3A]

[0010] Figure 1 shows the percentage of CD45+ cells in normal, tumor, and frozen pre-REP TILs in endometrial cancer tumor samples. The percentage of CD45+ cells is increased in pre-REP TILs compared to normal and tumor tissues. [Figure 3B]

[0010] Figure 1 shows the percentage of CD45+CD3+ cells in normal, tumor, and frozen pre-REP TILs in endometrial cancer tumor samples. The percentage of CD45+CD3+ cells is increased in pre-REP TILs compared to normal and tumor tissues. [Figure 4A]

[0011] Figure 1 shows the percentage of CD45+CD3+ cells in CD4+ and CD8+ cells from normal endometrial tissue and endometrial cancer tumor samples. Normal and tumor samples show similar results. [Figure 4B]

[0011] Figure 1 shows the percentage of CD45+CD3+ in CD4+ and CD8+ cells from pre-REP TILs in endometrial cancer tumor samples and frozen samples. The percentage of CD45+CD3+ is decreased in CD4+ cells and increased in CD8+ cells in pre-REP TILs compared to tumor tissue. [Figure 5A]

[0012] Figure 1 shows CD3+CD8+ TIL subset phenotypes in endometrial cancer samples. The percentages of CD3+CD8+ TILs in normal (left) and tumor (right) tissues are shown for the following markers: BTLA, CTLA-4, ICOS, Ki67, LAG3, PD-1, CD103+CD69+, CD103+CD69-, TIGIT, and TIM3. Wherever possible, comparative samples were from the same patient; i.e., each patient provided frozen samples of normal, tumor, and pre-REP TILs. [Figure 5B]

[0012] Figure 1 shows CD3+CD8+ TIL subset phenotypes in endometrial cancer samples. The percentages of CD3+CD8+ TILs (left) and frozen pre-REP TILs (right) in tumors for the same markers are shown. Whenever possible, comparison samples were from the same patient, i.e., each patient provided normal, tumor, and pre-REP TILs in frozen samples. [Figure 6]

[0013] CD3+CD8+CD103+CD69+ subset phenotyping is shown in normal (left) and tumor (right) endometrial tissue samples for the following markers: BTLA, CTLA-4, ICOS, Ki67, LAG3, PD-1, TIGIT, and TIM3. Wherever possible, comparison samples were from the same patient, i.e., each patient provided tumor and pre-REP TILs in frozen samples. [Figure 7A]

[0014] Figure 1 shows CD3+CD4+ TIL subset phenotypes in endometrial cancer samples. The percentages of CD3+CD4+ TILs in normal (left) and tumor (right) tissues are shown for the following markers: BTLA, CTLA-4, ICOS, Ki67, LAG3, PD-1, CD103+CD69+, CD103+CD69-, TIGIT, TIM3, and Treg. Wherever possible, comparison samples were from the same patient; i.e., each patient provided frozen samples of normal, tumor, and pre-REP TILs. [Figure 7B]

[0014] Figure 1 shows CD3+CD4+ TIL subset phenotypes in endometrial cancer samples. The percentages of CD3+CD4+ TILs (left) and frozen pre-REP TILs (right) in tumors for the same markers are shown. Wherever possible, comparison samples were from the same patient, i.e., each patient provided normal, tumor, and pre-REP TILs in frozen samples. [Figure 8]

[0015] CD3+CD4+CD103+CD69+ subset phenotyping is shown in normal (left) and tumor (right) endometrial tissue samples for the following markers: BTLA, CTLA-4, ICOS, Ki67, LAG3, PD-1, TIGIT, and TIM3. Wherever possible, comparison samples were from the same patient, i.e., each patient provided tumor and pre-REP TILs in frozen samples. [Figure 9A]

[0016] Figure 1 shows the percentage of CD45+ cells in normal, tumor, and frozen pre-REP TILs in anaplastic thyroid cancer samples. The percentage of CD45+ cells is increased in pre-REP TILs compared to normal and tumor tissues. [Figure 9B]

[0016] Figure 1 shows the percentage of CD45+CD3+ cells in normal, tumor, and frozen pre-REP TILs in anaplastic thyroid cancer samples. The percentage of CD45+CD3+ cells is increased in pre-REP TILs compared to normal and tumor tissues. [Figure 10A]

[0017] Figure 1 shows the percentage of CD45+CD3+ cells in CD4+ and CD8+ cells from normal endometrial tissue and anaplastic thyroid carcinoma samples. Normal and tumor samples show similar results. [Figure 10B]

[0017] Figure 1 shows the percentage of CD45+CD3+ in CD4+ and CD8+ cells from pre-REP TILs in anaplastic thyroid carcinoma samples and frozen samples. The percentage of CD45+CD3+ appears similar in both the CD4+ and CD8+ subsets of tumor and pre-REP TILs. [Figure 11A]

[0018] Figure 1 shows CD3+CD8+ TIL subset phenotypes in anaplastic thyroid cancer samples. The percentages of CD3+CD8+ TILs in normal (left) and tumor (right) tissues are shown for the following markers: BTLA, CTLA-4, ICOS, Ki67, LAG3, PD-1, CD103+CD69+, CD103+CD69-, TIGIT, and TIM3. Wherever possible, comparative samples were from the same patient; i.e., each patient provided frozen samples of normal, tumor, and pre-REP TILs. [Figure 11B]

[0018] Figure 1 shows CD3+CD8+ TIL subset phenotypes in anaplastic thyroid carcinoma samples. The percentages of CD3+CD8+ TILs (left) and frozen pre-REP TILs (right) in tumors for the same markers are shown. Whenever possible, comparison samples were from the same patient; i.e., each patient provided normal, tumor, and pre-REP TILs in frozen samples. [Figure 12]

[0019] CD3+CD8+CD103+CD69+ subset phenotyping is shown in normal (left) and tumor (right) endometrial tissue samples for the following markers: BTLA, CTLA-4, ICOS, Ki67, LAG3, PD-1, TIGIT, and TIM3. Wherever possible, comparison samples were from the same patient, i.e., each patient provided tumor and pre-REP TILs in frozen samples. [Figure 13A]

[0020] Figure 1 shows CD3+CD4+ TIL subset phenotypes in anaplastic thyroid cancer samples. The percentages of CD3+CD4+ TILs in normal (left) and tumor (right) tissues are shown for the following markers: BTLA, CTLA-4, ICOS, Ki67, LAG3, PD-1, CD103+CD69+, CD103+CD69-, TIGIT, TIM3, and Treg. Wherever possible, comparative samples were from the same patient; i.e., each patient provided frozen samples of normal, tumor, and pre-REP TILs. [Figure 13B]

[0020] Figure 1 shows CD3+CD4+ TIL subset phenotypes in anaplastic thyroid cancer samples. The percentages of CD3+CD4+ TILs (left) and frozen pre-REP TILs (right) in tumors for the same markers are shown. Wherever possible, comparison samples were from the same patient, i.e., each patient provided normal, tumor, and pre-REP TILs in frozen samples. [Figure 14]

[0021] CD3+CD4+CD103+CD69+ subset phenotyping is shown in normal (left) and tumor (right) endometrial tissue samples for the following markers: BTLA, CTLA-4, ICOS, Ki67, LAG3, PD-1, TIGIT, and TIM3. Wherever possible, comparison samples were from the same patient, i.e., each patient provided tumor and pre-REP TILs in frozen samples. [Figure 15]

[0022] Structures IA and IB are shown, with the cylinders indicating the 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

[0009] Summary of the Invention

[0023] The present invention describes a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: obtaining a first TIL population from a tumor resected from a subject; performing the first expansion culture for a period of about 21 days to about 35 days by culturing the first TIL population in a cell culture medium comprising 4-1BB, IL-2, and OKT-3 to produce a second TIL population; and performing the second expansion culture for a period of about 6 days to about 12 days by supplementing the cell culture medium of the second TIL population with antigen-presenting cells (APCs) and additional 4-1BB, IL-2, and OKT-3, and culturing the second TIL population to produce a third TIL population, which is a therapeutic TIL population.

[0010]

[0024] In one embodiment of the present invention, the method further comprises harvesting the therapeutic TIL population obtained from the second expansion step; and transferring the harvested TIL population to an infusion bag.

[0011]

[0025] In some embodiments, the method includes performing a first expansion step in the presence of antigen-presenting cells (APCs). In one embodiment, the APCs are peripheral blood mononuclear cells (PBMCs). In some embodiments, the ratio of the number of APCs in the second expansion culture to the number of APCs in the first expansion culture ranges from about 1.5:1 to about 20:1. In some embodiments, the ratio is about 2:1.

[0012]

[0026] In one embodiment, the TILs may be cryopreserved at some point during the first or second expansion process. In one embodiment, the second TIL population is cryopreserved.

[0013]

[0027] In some embodiments of the present invention, the first expansion culture is carried out for a period of about 21 days, and the second expansion culture is carried out for a period of about 6 to 10 days. In some embodiments, the first expansion culture is carried out for a period of about 35 days, and the second expansion culture is carried out for a period of about 6 to 10 days. In some embodiments, the first expansion culture is carried out for a period of about 28 days, and the second expansion culture is carried out for a period of about 7 to 10 days.

[0014]

[0028] In some embodiments, the second or third TIL population comprises subpopulations of cells with increased expression of certain markers indicative of T cell function and decreased expression of certain exhaustion markers. In one embodiment, one or both of the second or third TIL populations comprises an expanded subpopulation of effector T cells and / or central memory T cells relative to the first or second TIL population. In another embodiment, one or both of the second or third TIL populations comprises an expanded subpopulation of cells expressing one or more of BTLA, Ki67, LAG3, TIGIT, and TIM3. In another embodiment, one or both of the second or third TIL populations comprises a decreased subpopulation of cells expressing one or more of CTLA-4, ICOS, PD-1, CD103+CD69+, and CD103+CD69-. In one embodiment, one or both of the second or third TIL populations comprises an expanded subpopulation of CD45+ cells. In one embodiment, one or both of the second or third TIL populations comprises an expanded subpopulation of CD45+CD3+ cells. In one embodiment, one or both of the second or third TIL populations comprises an expanded subpopulation of CD8+ cells. In one embodiment, one or both of the second or third TIL populations comprises a depleted subpopulation of CD4+ cells.

[0015]

[0029] In some embodiments, the tumor is of a cancer type selected from the group consisting of thyroid cancer, melanoma (including uveal melanoma and cutaneous melanoma), cervical cancer, endometrial cancer, colon cancer, and colorectal cancer.

[0016]

[0030] In some embodiments, the second TIL population is at least 50 times greater in number than the first TIL population. In some embodiments, the second TIL population is at least 4×10 7 It is a cell.

[0017] Detailed Description definition

[0031] 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 herein by reference in their entirety.

[0018]

[0032] The term "in vitro" refers to an event that takes place outside a subject's body.

[0019]

[0033] The term "in vitro" refers to events that take place outside a subject's body. In vitro assays include cell-based assays, utilizing living or dead cells, and can also include cell-free assays, which do not utilize intact cells.

[0020]

[0034] 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.

[0021]

[0035] The term "secondary 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 secondary expansion protocols are outlined below.

[0022]

[0036] 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, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. TIL cell populations may include genetically modified TILs.

[0023]

[0037] 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, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can also be characterized by efficacy—for example, TILs can be considered efficacious if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. Interferons can include interferon gamma (IFNγ).

[0024]

[0038] 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, "cryopreserved TILs" are distinguishable from frozen tissue samples that may be used as a source of primary TILs.

[0025]

[0039] 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.

[0026]

[0040] As used herein, a "cell population" (including TILs) refers to a large number of cells that share a common trait. Generally, a population is generally between 1 x 10 6 ~1×10 10 The number of TILs ranges from 1 x 10 to 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 generally 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.

[0027]

[0041] Generally, TILs are first obtained from a patient tumor sample ("primary TILs"), 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.

[0028]

[0042] The harvested cell suspension is generally referred to as a "primary cell population" or a "freshly harvested" cell population.

[0029]

[0043] Generally, as discussed herein, TILs are prepared by first obtaining a primary TIL population from a tumor resected from a patient as discussed herein (the "primary cell population" or "first cell population"). This is followed by an initial bulk expansion using culturing the cells with IL-2 to form a second cell population (sometimes referred to herein as the "bulk TIL population" or "second population").

[0030]

[0044] The term "cytotoxic lymphocytes" includes cytotoxic T (CTL) cells (CD8 + Cytotoxic T lymphocytes and CD4 + Cytotoxic lymphocytes include T-helper lymphocytes, natural killer T (NKT) cells, and natural killer (NK) cells. Cytotoxic lymphocytes can include, for example, peripheral blood-derived α / β TCR-positive T cells or α / β TCR-positive T cells activated by tumor-associated antigens and / or transduced with tumor-specific chimeric antigen receptors or T cell receptors, and tumor-infiltrating lymphocytes (TILs).

[0031]

[0045] The term "central memory T cells" refers to a subset of T cells that are CD45RO+ and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi) in humans. 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 BMII. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells predominate in the CD4 compartment of the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0032]

[0046] 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 are predominant in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells have high amounts of perforin. 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. An example of a closed system is, but is not limited to, a closed G container. After tumor segments are added to the closed system, the system is not open to the external environment until immediately prior to administration of the TILs to a patient.

[0033]

[0047] 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, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.

[0034]

[0048] 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 described herein.

[0035]

[0049] In some embodiments, the disclosed methods further include a "pre-REP" stage in which tumor tissue or cells from tumor tissue are grown in standard laboratory medium (including, without limitation, RPMI) and treated with reagents such as irradiated feeder cells and anti-CD3 antibodies to achieve a desired effect, such as an increase in TIL numbers and / or enrichment of the population for cells containing desired cell surface markers or other structural, biochemical, or functional characteristics. The pre-REP stage may utilize laboratory-grade reagents (with the understanding that the laboratory-grade reagents will be diluted during the subsequent REP stage) to facilitate the incorporation of alternative strategies for improving TIL production. Thus, in some embodiments, the culture medium during the pre-REP stage may include the disclosed TLR agonists and / or peptides or peptidomimetics. The pre-REP culture may, in some embodiments, include IL-2.

[0036]

[0050] In a preferred embodiment, the present invention relates to a novel method of enhancing REP with an additional restimulation protocol, sometimes referred to herein as a "restimulation rapid expansion protocol" or "reREP," which unexpectedly leads to an expansion of memory T cell subsets, including memory effector T cell subsets, and / or leads to a significant enhancement of glycolytic respiration when compared to freshly harvested TILs or thawed cryopreserved TILs for restimulation (sometimes referred to herein as "reTILs"). That is, by using the reREP procedure on cryopreserved TILs, patients can receive highly metabolically active and healthy TILs, which may lead to better outcomes. Such restimulation protocols, also referred to herein as further "expansion" of the cell population, are described in further detail herein.

[0037]

[0051] The terms "fragmentation," "fragments," and "fragmented" as used herein to describe the process of disrupting tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as other methods that disrupt the physical structure of tumor tissue. The term "in vivo" refers to events that occur within a subject's body.

[0038]

[0052] The term "in vitro" refers to events that take place outside a subject's body. In vitro assays include cell-based assays, utilizing living or dead cells, and can also include cell-free assays, which do not utilize intact cells.

[0039]

[0053] 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, and UHCT-1. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0040]

[0054] 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.

[0041] [Table 1]

[0042]

[0055] 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.

[0043] [Table 2]

[0044]

[0056] 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).

[0045]

[0057] 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-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-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).

[0046]

[0058] 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).

[0047]

[0059] 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. 2014, 13, 379-95, 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).

[0048]

[0060] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in body weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally, pharmaceutical compositions comprising the genetically modified cytotoxic lymphocytes described herein are administered in an amount of 100 mg / kg or more. 4 ~10 11 cells / kg body weight (e.g., 10 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 ,10 7 ~10 11 , 10 7~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11 or 10 9 ~10 10 It can be said that the genetically modified cytotoxic lymphocyte compositions can be administered at dosages of 1000 to 10000 cells / kg body weight (including all integer values ​​within these ranges). The genetically modified cytotoxic lymphocyte compositions can also be administered multiple times at these dosages. The genetically modified cytotoxic lymphocytes can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the art of medicine by monitoring the patient for symptoms of disease and adjusting treatment accordingly.

[0049]

[0061] 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.

[0050]

[0062] 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.

[0051]

[0063] 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).

[0052]

[0064] 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.

[0053]

[0065] In certain embodiments, the present invention includes a method of treating cancer with a population of rTILs, wherein the patient is conditioned with non-myeloablative chemotherapy prior to infusion of the rTILs according to the present invention. In some embodiments, the rTIL population can be provided with a population of eTILs, wherein the patient is conditioned with non-myeloablative chemotherapy prior to infusion of the rTILs and eTILs according to the present invention. In one embodiment, the non-myeloablative chemotherapy comprises cyclophosphamide 60 mg / kg / day for two days (27 and 26 days before rTIL infusion) and fludarabine 25 mg / m for five days (27-23 days before rTIL infusion). 2 In one embodiment, after non-myeloablative chemotherapy and rTIL infusion according to the present invention (day 0), patients receive an intravenous infusion of 720,000 IU / kg of IL-2 intravenously every 8 hours to a physiologically tolerated dose.

[0054]

[0066] 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.

[0055]

[0067] As used herein, the terms "co-administration," "co-administering," "administered in combination with," "administering in combination with," "simultaneous," and "concurrent" encompass administration of two or more active pharmaceutical ingredients (e.g., at least one potassium channel agonist in combination with multiple TILs) to a subject such that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Concurrent administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0056]

[0068] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to achieve its intended use, including but not limited to disease treatment. A therapeutically effective amount may vary depending on the intended use (in vitro or in 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. 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 dosing regimen 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.

[0057]

[0069] The terms "treatment," "treating," "treat," and the like refer to achieving a desired pharmacological and / or physiological effect. The effect can be prophylactic, meaning that a disease or its symptoms are completely or partially prevented, and / or therapeutic, meaning that the disease and / or adverse effects resulting from the disease are partially or completely cured. "Treatment," as used herein, encompasses any treatment of disease in mammals, particularly humans, and includes (a) preventing the occurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., halting its development or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a disease state, e.g., in the case of a vaccine.

[0058]

[0070] The term "heterologous" when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, having two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0059]

[0071] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their variants, e.g., "99% identical"), 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 align amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information's BLAST website. 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.

[0060]

[0072] The term "variant" as used herein includes, but is not limited to, an antibody or fusion protein comprising an amino acid sequence that differs from the amino acid sequence 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 amino acids that are similarly charged or uncharged. A variant retains the ability of the reference antibody to specifically bind to an antigen. The term variant also includes pegylated antibodies or proteins.

[0061]

[0073] The term "in vivo" refers to an event that takes place inside a subject's body.

[0062]

[0074] The term "in vitro" refers to events that take place outside a subject's body. In vitro assays include cell-based assays, utilizing living or dead cells, and can also include cell-free assays, which do not utilize intact cells.

[0063]

[0075] 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.

[0064]

[0076] TIL Fabrication Process Embodiments

[0065]

[0077] The "step" designations A, B, C, etc. below are exemplary, and any combination or ordering of steps, as well as additional steps, repeated steps, and / or omission of steps, are contemplated by the present application and methods disclosed herein.

[0066] Step A: Obtain a patient tumor sample

[0078] Generally, TILs are first obtained from a patient tumor sample ("primary TILs"), 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.

[0067]

[0079] 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 cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, renal cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors, which have been reported to have particularly high levels of TILs.

[0068]

[0080] 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 3are 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.

[0069]

[0081] 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 patients.

[0070]

[0082] In some embodiments, if the tumor is a solid tumor, after obtaining a tumor sample, the tumor is subjected to physical fragmentation. In some embodiments, fragmentation is performed before cryopreservation. In some embodiments, fragmentation is performed after cryopreservation. In some embodiments, fragmentation is performed after obtaining the tumor without any cryopreservation. In some embodiments, the tumor is fragmented and 2, 3, or 4 fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 3 or 4 fragments or pieces are placed in each container for first expansion culture. In some embodiments, the tumor is fragmented and 4 fragments or pieces are placed in each container for first expansion culture.

[0071]

[0083] 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 is.

[0072] 1. Core / small biopsy-derived TILs

[0084] In some embodiments, TILs are initially obtained from patient tumor samples obtained by core biopsy or similar procedures ("primary TILs") and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, and optionally assessed for phenotypic and metabolic parameters.

[0073]

[0085] In some embodiments, patient tumor samples may be obtained using methods known in the art, typically by mini-biopsy, core biopsy, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples may be from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples may also be from liquid tumors, such as tumors obtained from hematological malignancies. In some embodiments, samples may be from multiple small tumor samples or biopsies. In some embodiments, samples may include multiple tumor samples from a single tumor from the same patient. In some embodiments, samples may include one, two, three, or four tumor samples from multiple tumors from the same patient. In some embodiments, samples may include multiple tumor samples from multiple tumors from the same patient. Solid tumors may be of any cancer type, including, but not limited to, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, renal cancer, gastric cancer, and skin cancer (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). 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 (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer (NSCLC). In some embodiments, useful TILs are obtained from malignant melanoma tumors, which have been reported to have particularly high levels of TILs.

[0074]

[0086] Generally, cell suspensions obtained from tumor cores or fragments are referred to as "primary cell populations" or "freshly obtained" or "freshly isolated" cell populations. In certain embodiments, a freshly obtained cell population of TILs is exposed to cell culture medium containing antigen-presenting cells, IL-2, and OKT-3.

[0075]

[0087] In some embodiments, if the tumor is metastatic and the primary lesion has been effectively treated / removed in the past, removal of one of the metastatic lesions may be necessary. In some embodiments, the least invasive approach is to remove a skin lesion or lymph nodes in the neck or axillary region, if available. In some embodiments, a skin lesion is removed or a small biopsy thereof is removed. In some embodiments, a lymph node or a small biopsy thereof is removed. In some embodiments, metastatic lesions in the lung or liver or intraperitoneal or thoracic lymph nodes or a small biopsy thereof may be utilized.

[0076]

[0088] In some embodiments, the tumor is a melanoma. In some embodiments, the minibiopsy of the melanoma includes a lentigo or part thereof.

[0077]

[0089] In some embodiments, the small biopsy is a punch biopsy. In some embodiments, the punch biopsy is taken with a circular blade pressed into the skin. In some embodiments, the punch biopsy is taken with a circular blade pressed into the skin around the suspicious mole. In some embodiments, the punch biopsy is taken with a circular blade pressed into the skin around the suspicious mole and a circular piece of skin is removed. In some embodiments, the small biopsy is a punch biopsy and a round portion of the tumor is removed.

[0078]

[0090] In some embodiments, the mini-biopsy is an excision biopsy. In some embodiments, the mini-biopsy is an excision biopsy, where the entire mole or tumor is removed. In some embodiments, the mini-biopsy is an excision biopsy, where the entire mole or tumor is removed with a small margin that has a normal appearance.

[0079]

[0091] In some embodiments, the mini-biopsy is an incisional biopsy. In some embodiments, the mini-biopsy is an incisional biopsy, where only the most irregular part of the mole or tumor is taken. In some embodiments, the mini-biopsy is an incisional biopsy, where an incisional biopsy is used when other techniques cannot be completed, such as when the suspicious mole is very large.

[0080]

[0092] In some embodiments, the small biopsy is a lung biopsy. In some embodiments, the small biopsy is obtained via bronchoscopy. Generally, with bronchoscopy, the patient is placed under anesthesia, and a small instrument is passed through the nose or mouth and down the throat to the bronchi, where it is used to remove some tissue. In some embodiments, if a tumor or growth cannot be reached via bronchoscopy, transthoracic needle biopsy can be utilized. Generally, with transthoracic needle biopsy, the patient is also under anesthesia, and a needle is inserted directly through the skin into the suspected area to remove a small sample of tissue. In some embodiments, transthoracic needle biopsy may require interventional imaging (e.g., the use of an X-ray or CT scan to guide the needle). In some embodiments, the small biopsy is obtained via needle biopsy. In some embodiments, the small biopsy is obtained with an ultrasound endoscope (e.g., an endoscope equipped with a light that is placed through the mouth and into the esophagus). In some embodiments, the small biopsy is obtained surgically.

[0081]

[0093] In some embodiments, the mini-biopsy is a head and neck biopsy. In some embodiments, the mini-biopsy is an incisional biopsy. In some embodiments, the mini-biopsy is an incisional biopsy, where a small piece of tissue is removed from an area that looks abnormal. In some embodiments, if the abnormal area is easily accessible, a sample can be taken without hospitalization. In some embodiments, if the tumor is located deeper inside the mouth or throat, the biopsy may need to be performed in an operating room with general anesthesia. In some embodiments, the mini-biopsy is an excisional biopsy. In some embodiments, the mini-biopsy is an excisional biopsy, where the entire area is removed. In some embodiments, the mini-biopsy is a fine needle aspiration (FNA). In some embodiments, the mini-biopsy is a fine needle aspiration (FNA), where a very thin needle attached to a syringe is used to extract (aspirate) cells from a tumor or lump. In some embodiments, the mini-biopsy is a punch biopsy. In some embodiments, the mini-biopsy is a punch biopsy, where a punch forceps is used to remove a piece of suspicious area.

[0082]

[0094] In some embodiments, the mini-biopsy is a cervical biopsy. In some embodiments, the mini-biopsy is obtained by colposcopy. Colposcopy generally employs the use of a lighted magnifying glass attached to magnifying binoculars (a colposcope) and is used to biopsy a small portion of the surface of the cervix. In some embodiments, the mini-biopsy is a cone biopsy. In some embodiments, the mini-biopsy is a cone biopsy, which may require outpatient surgery to remove larger pieces of tissue from the cervix. In some embodiments, in addition to helping confirm a diagnosis, a cone biopsy may serve as an initial procedure.

[0083]

[0095] In some embodiments, samples from tumors are obtained as fine needle aspirations (FNAs), core biopsies, or mini biopsies (including, for example, punch biopsies). In some embodiments, the samples are first placed in the G-Rex 10. In some embodiments, if there are one or two core biopsy and / or mini biopsy samples, the samples are first placed in the G-Rex 10. In some embodiments, if there are three, four, five, six, eight, nine, or ten core biopsy and / or mini biopsy samples, the samples are first placed in the G-Rex 100. In some embodiments, if there are three, four, five, six, eight, nine, or ten core biopsy and / or mini biopsy samples, the samples are first placed in the G-Rex 500.

[0084]

[0096] The FNA may be obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal, and sarcoma. In some embodiments, the FNA is obtained from a lung tumor, such as a lung tumor from a patient with non-small cell lung cancer (NSCLC). In some cases, the patient with NSCLC has previously undergone surgical treatment.

[0085]

[0097] The TILs described herein can be obtained from an FNA sample. In some cases, the FNA sample is obtained or isolated from a patient using a high-gauge needle, ranging from an 18-gauge needle to a 25-gauge needle. The high-gauge needle can be 18-gauge, 19-gauge, 20-gauge, 21-gauge, 22-gauge, 23-gauge, 24-gauge, or 25-gauge. In some embodiments, an FNA sample from a patient may contain at least 400,000 TILs, e.g., 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs or more.

[0086]

[0098] In some cases, the TILs described herein are obtained from a core biopsy sample. In some cases, the core biopsy sample is obtained or isolated from a patient using a surgical or medical needle ranging from an 11-gauge needle to a 16-gauge needle. The needle can be 11-gauge, 12-gauge, 13-gauge, 14-gauge, 15-gauge, or 16-gauge. In some embodiments, a core biopsy sample from a patient may contain at least 400,000 TILs, e.g., 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs or more.

[0087]

[0099] In some embodiments, TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated for 30 minutes at 37°C under 5% CO2, after which it may be mechanically disrupted again for approximately 1 minute. After another 30 minutes of 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 mechanical dissociations, with or without an additional 30-minute incubation at 37°C under 5% CO2. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using Ficoll may be performed to remove such cells.

[0088]

[0100] 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.

[0089]

[0101] In some embodiments, the cells may optionally be frozen after sample collection and stored frozen before proceeding to step B, described in more detail below.

[0090] B. Step B: First Expansion Culture

[0102] In some embodiments, the first expansion of TILs (also referred to as first expansion or first TIL expansion) may be performed using an initial bulk TIL expansion step (e.g., first expansion step; which may include an expansion step referred to as pre-REP) as described below and herein, followed by a second expansion step (e.g., referred to as a rapid expansion protocol (REP) step) as described below and herein, followed by optional cryopreservation, followed by a further second expansion step (e.g., sometimes referred to as a restimulation REP step) as described below and herein. TILs obtained by this process may optionally be characterized for phenotypic characteristics and metabolic parameters as described herein. In some embodiments, TILs are frozen (i.e., cryopreserved) after the first expansion and stored until phenotyped for selection, then thawed before proceeding to one or more second expansion steps.

[0091]

[0103] In some embodiments where the cells are frozen after being obtained from the tumor sample, the cells are thawed prior to the first expansion culture.

[0092]

[0104] 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). 6Tumor digest cells or 1 tumor fragment can be seeded. In some embodiments, tumor fragments are about 1 mm 3 ~10mm 3 is.

[0093]

[0105] After preparation of tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2, OKT-3, and 4-1BB agonists 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 21-35 days, to obtain a second TIL population. In some embodiments, the growth medium during the first expansion culture contains IL-2 or a variant thereof. In some embodiments, the IL-2 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 4. 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 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 comprises about 1,000 IU / mL, about 1,500 IU / mL, about 2,000 IU / mL, about 2,500 IU / mL, about 3,000 IU / mL, about 3,500 IU / mL, about 4,000 IU / mL, about 4,500 IU / mL, about 5,000 IU / mL, about 5,500 IU / mL, about 6,000 IU / mL, about 6,500 IU / mL, about 7,000 IU / mL, about 7,500 IU / mL, or about 8,000 IU / mL of IL-2. In one embodiment, the cell culture medium comprises between 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL or 8000 IU / mL of IL-2.

[0094]

[0106] In some embodiments, the culture medium for the first expansion culture comprises an anti-CD3 antibody, such as OKT-3, 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). In some embodiments, the anti-CD3 antibody, e.g., OKT-3, is present in an amount of about 20 ng / ml to about 80 ng / ml. In some embodiments, the anti-CD3 antibody is present in an amount of about 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, or 90 ng / ml. In some embodiments, the anti-CD3 antibody is present in an amount of about 30 ng / ml. In some embodiments, the anti-CD3 antibody is present in an amount of about 45 ng / ml. In some embodiments, the anti-CD3 antibody is present in an amount of about 60 ng / ml.

[0095]

[0107] In some embodiments, the first expansion cell culture medium comprises one or more tumor necrosis factor superfamily (TNFRSF) agonists. 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 in an amount 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 5 μg / mL to 40 μg / mL in the cell culture medium. In some embodiments, the TNFRSF agonist is present at a concentration of about 5 μg / mL. In some embodiments, the TNFRSF agonist is present at a concentration of about 10 μg / mL. In some embodiments, the TNFRSF agonist is present at a concentration of about 15 μg / mL. In some embodiments, the TNFRSF agonist is present at a concentration of about 20 μg / mL. In some embodiments, the TNFRSF agonist is present at a concentration of about 25 μg / mL. In some embodiments, the TNFRSF agonist is present at a concentration of about 30 μg / mL. In some embodiments, the TNFRSF agonist is present at a concentration of about 35 μg / mL. In some embodiments, the TNFRSF agonist is present at a concentration of about 40 μg / mL.

[0096]

[0108] In some embodiments, the first expansion may occur in the presence of feeder cells, also called antigen-presenting cells or APCs.

[0097]

[0109] In one embodiment, the first expansion procedure described herein requires feeder cells (also referred to herein as "antigen-presenting cells") at the initiation of TIL expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from an allogeneic healthy donor. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, 2.5 x 10 8 In some embodiments, 2.5 x 10 feeder cells per vessel are used during the first expansion culture. 8 In some embodiments, 2.5 x 10 feeder cells are used during the first expansion culture. 8 In some embodiments, 2.5 x 10 feeder cells per GREX-100 are used during the first expansion culture. 8 Feeder cells are used during the first expansion culture.

[0098]

[0110] 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.

[0099]

[0111] In some embodiments, if the total number of viable cells on day 14 is less than the initial number of viable cells placed in culture on day 0 of the first expansion culture, the PBMCs are considered non-replicating and are approved for use in the TIL expansion procedures described herein.

[0100]

[0112] 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 first 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 antigen-presenting feeder cells in the first expansion culture is 1:50 to 1:300. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the first expansion culture is 1:100 to 1:200.

[0101]

[0113] In one embodiment, the first expansion procedure described herein comprises culturing approximately 2.5 x 10 8 feeder cells and approximately 100 x 10 6 In another embodiment, the first expansion procedure described herein requires a ratio of about 2.5 x 10 TILs. 8 feeder cells and approximately 50 x 10 6 In yet another embodiment, the first expansion culture described herein requires a ratio of about 2.5 x 10 TILs. 8 feeder cells and approximately 25 x 10 6 In yet another embodiment, the first expansion culture described herein requires about 2.5 x 10 TILs. 8 In yet another embodiment, the first expansion culture requires one-quarter, one-third, five-twelfths, or one-half the number of feeder cells used in the second expansion culture.

[0102]

[0114] In one embodiment, the first expansion procedure described herein requires an excess of feeder cells relative to TILs during the first expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from an allogeneic 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.

[0103]

[0115] 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.

[0104]

[0116] In one embodiment, artificial antigen-presenting cells are used in the first expansion culture, either as a replacement for or in combination with PBMCs.

[0105]

[0117] In some embodiments, if the total number of viable cells on day 7 or 14 is less than the initial number of viable cells placed in culture on day 0 of the first expansion culture (i.e., the start day of the first expansion culture), the PBMCs are considered non-replicating and are approved for use in the TIL expansion procedures described herein.

[0106]

[0118] In some embodiments, if the total number of viable cells cultured in the presence of OKT3, a 4-1BB agonist, and IL-2 does not increase on days 7 and 14 from the initial number of viable cells entered into culture on day 0 of the first expansion culture (i.e., the start of the first expansion culture), the PBMCs are considered replication-incompetent and may be approved for use in the TIL expansion procedures described herein. In some embodiments, the PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody, 10 μg / ml anti-4-1BB antibody, and 3000 IU / ml IL-2. In some embodiments, the PBMCs are cultured in the presence of 60 ng / ml OKT3 antibody, 10 μg / ml anti-4-1BB antibody, and 6000 IU / ml IL-2. In some embodiments, the PBMCs are cultured in the presence of 60 ng / ml OKT3 antibody, 10 μg / ml anti-4-1BB antibody, and 3000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody, 10 ug / ml anti-4-1BB antibody, and 6000 IU / ml IL-2.

[0107]

[0119] In some embodiments, if the total number of viable cells cultured in the presence of OKT3, a 4-1BB agonist, 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 the first expansion culture (i.e., the start of the first expansion culture), the PBMCs are considered replication-incompetent and may be approved for use in the TIL expansion procedures described herein. In some embodiments, the PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 1000-6000 IU / ml of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 2000-5000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 2000-4000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 2500-3500 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 6000 IU / ml of IL-2.

[0108]

[0120] 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), each flask contains 10-40 x 10 cells in 10-40 mL of IL-2-containing CM. 6Load 50 live tumor digest cells or 5-30 tumor fragments. Both G-Rex10 and 24-well plates are incubated in a humidified incubator at 37°C under 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. 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, OKT-3, and a 4-1BB agonist.

[0109]

[0121] In some embodiments, the first TIL expansion culture may be continued for 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the first TIL expansion culture may be continued for 11 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 12 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 13 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 14 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 15 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 16 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 17 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 18 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 19 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 20 to 21 days. In some embodiments, the first TIL expansion culture may be continued for 21 days. In some embodiments, the first TIL expansion culture may be continued for up to 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days.

[0110]

[0122] In some embodiments, 4 or 5 days after the initiation of the first TIL expansion culture, the cultures are refed with additional culture medium supplemented with IL-2. In other embodiments, 4 or 5 days after the initiation of the first TIL expansion culture, the cultures are refed with additional culture medium supplemented with IL-2, and then half of the culture medium is replaced with an equal volume of fresh culture medium supplemented with IL-2 every 3 or 4 days until the end of the first expansion culture.

[0111]

[0123] In some embodiments, 4 or 5 days after the initiation of the first TIL expansion culture, the cultures are refed with additional culture medium supplemented with IL-2 and a 4-1BB agonist. In other embodiments, 4 or 5 days after the initiation of the first TIL expansion culture, the cultures are refed with additional culture medium supplemented with IL-2 and a 4-1BB agonist, and half of the culture medium is replaced with an equal volume of fresh culture medium supplemented with IL-2 every 3 or 4 days thereafter until the end of the first expansion culture. In other embodiments, 4 or 5 days after the initiation of the first TIL expansion culture, the cultures are refed with additional culture medium supplemented with IL-2 and a 4-1BB agonist, and half of the culture medium is replaced with an equal volume of fresh culture medium supplemented with IL-2 and a 4-1BB agonist every 3 or 4 days thereafter until the end of the first expansion culture.

[0112]

[0124] In some embodiments, 4 or 5 days after the initiation of the first TIL expansion culture, the cultures are refed with additional culture medium supplemented with IL-2, a 4-1BB agonist, and OKT-3. In other embodiments, 4 or 5 days after the initiation of the first TIL expansion culture, the cultures are refed with additional culture medium supplemented with IL-2, a 4-1BB agonist, and OKT-3, and half of the culture medium is replaced with an equal volume of fresh culture medium supplemented with IL-2 every 3 or 4 days thereafter until the end of the first expansion culture. In other embodiments, 4 or 5 days after the initiation of the first TIL expansion culture, the cultures are refed with additional culture medium supplemented with IL-2, a 4-1BB agonist, and OKT-3, and half of the culture medium is replaced with an equal volume of fresh culture medium supplemented with IL-2 every 3 or 4 days thereafter until the end of the first expansion culture. In other embodiments, 4 or 5 days after the initiation of the first TIL expansion culture, the cultures are refed with additional culture medium supplemented with IL-2, a 4-1BB agonist, and OKT-3, and then half of the culture medium is replaced with an equal volume of fresh culture medium supplemented with IL-2, a 4-1BB agonist, and OKT-3 every 3 or 4 days until the end of the first expansion culture.

[0113] C. Step C: Transition from the first expansion culture to the second expansion culture

[0125] In some embodiments, the TILs obtained from the first expansion culture are stored until phenotyping for selection. In some embodiments, the TILs obtained from the first expansion culture are cryopreserved after the first expansion culture and before the second expansion culture. In some embodiments, the TILs are cryopreserved as part of the transition from the first expansion culture to the second expansion culture. For example, in some embodiments, the TILs are cryopreserved after step B and before step D. In some embodiments, the TILs are cryopreserved and thawed as part of the transition from the first expansion culture to the second expansion culture. For example, in some embodiments, the TILs are cryopreserved after step B and then thawed before proceeding to step D. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 22, 23, 24, 25, 26, 27, 28, 29, or 30 days from the time tumor fragmentation was performed. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs at about 22 to 30 days from the time fragmentation was performed. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 24 to 30 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 26 to 30 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 28 to 30 days after fragmentation. In some embodiments, the transition from the first expansion culture to the second expansion culture occurs about 30 days after fragmentation.

[0114] D. Step D: Second Expansion Culture

[0126] In some embodiments, the TIL cell population is further expanded in number after the transfer, referred to as steps A and B, and step C, followed by recovery and first expansion. This further expansion is referred to herein as the second expansion, which may include an expansion process commonly referred to in the art as a rapid expansion method (rapid expansion protocol, or REP). The second expansion can generally be accomplished using culture medium in a gas-permeable vessel or other closed system containing one or more of several components, including feeder cells, a cytokine source, an anti-CD3 antibody, and a TNFRSF agonist. In some embodiments, 1, 2, 3, or 4 days after the initiation of the second expansion, the TILs are transferred to a vessel with a larger volume.

[0115]

[0127] In some embodiments, the second expansion culture of TILs (which may include expansion culture sometimes referred to as REP) 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 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 after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 1 day to about 9 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 2 days to about 9 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 3 days to about 9 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 4 days to about 9 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 5 to about 9 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 6 to about 9 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 7 to about 9 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 1 day after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 2 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 3 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 4 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 5 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 6 days after the initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 7 days after initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 8 days after initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 9 days after initiation of the second expansion culture.In some embodiments, the second TIL expansion culture may be continued for about 10 days after initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 11 days after initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 12 days after initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 13 days after initiation of the second expansion culture. In some embodiments, the second TIL expansion culture may be continued for about 14 days after initiation of the second expansion culture.

[0116]

[0128] In one embodiment of the invention, the second expansion step can be carried out in the presence of one or more of IL-2, OKT-3, and a 4-1BB agonist at the concentrations described above for the first expansion step. In one embodiment, the cell culture medium at the start of the second expansion step contains about 30 ng / ml OKT-3, 6000 IU / ml IL-2, and 10 μg / ml of a 4-1BB agonist.

[0117]

[0129] In certain embodiments, the second expansion culture can be performed in a gas-permeable container using the methods of the present disclosure (e.g., including expansion cultures referred to as REP). In some embodiments, TILs are expanded in the second expansion culture in the presence of feeder cells (also referred to herein as "antigen-presenting cells"). In some embodiments, TILs are expanded in the second expansion culture in the presence of feeder cells, which are added to a final concentration that is 2x, 2.4x, 2.5x, 3x, 3.5x, or 4x the concentration of feeder cells present in the first expansion culture. For example, TILs can be expanded using non-specific 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 of the TILs in vitro by including in the second expansion culture one or more antigens of the cancer, including an antigenic portion thereof, such as one or more epitopes of a human leukocyte antigen A2 (HLA-A2)-binding peptide, e.g., 0.3 μM MART-1:26-35 (27L) or gpl 00:209-217 (210M), optionally expressed from a vector, 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 cancer antigen or antigens pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.In some embodiments, restimulation occurs as part of a second expansion culture, hi some embodiments, the second expansion culture occurs in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0118]

[0130] 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 comprises between 1000-2000 IU / mL, 2000-3000 IU / mL, 3000-4000 IU / mL, 4000-5000 IU / mL, 5000-6000 IU / mL, 6000-7000 IU / mL, 7000-8000 IU / mL or 8000 IU / mL of IL-2.

[0119]

[0131] 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 comprises 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 one embodiment, the cell culture medium comprises 30 ng / mL to 60 ng / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises about 60 ng / mL of OKT-3. In some embodiments, the OKT-3 antibody is muromonab.

[0120]

[0132] In some embodiments, the medium for the second expansion culture comprises IL-2. In some embodiments, the medium comprises 6000 IU / mL of IL-2. In some embodiments, the medium for the second expansion culture comprises antigen-presenting feeder cells. In some embodiments, the medium for the second expansion culture comprises 7.5 x 10 cells per vessel. 8 In some embodiments, the medium for the second expansion culture comprises OKT-3. In some embodiments, the medium for the second expansion culture comprises 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium for the second expansion culture comprises 6000 IU / mL of IL-2, 60 ng / mL of OKT-3, and 7.5×10 8In some embodiments, the medium contains 500 mL of culture medium and 6000 IU / mL of IL-2, 30 μg of OKT-3, and 7.5×10 antigen-presenting feeder cells per container. 8 The cells contain antigen-presenting feeder cells.

[0121]

[0133] In some embodiments, the medium in the second expansion culture comprises IL-2. In some embodiments, the medium comprises 6000 IU / mL of IL-2. In some embodiments, the medium in the second expansion culture comprises antigen-presenting feeder cells. In some embodiments, the medium comprises 5 x 10 cells per vessel. 8 ~7.5×10 8 In some embodiments, the medium for the second expansion culture comprises OKT-3. In some embodiments, the medium for the second expansion culture comprises 500 mL of culture medium and 30 μg of OKT-3 per container. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium for the second expansion culture comprises 6000 IU / mL of IL-2, 60 ng / mL of OKT-3, and 5×10 8 ~7.5×10 8 In some embodiments, the medium for the second expansion culture contains 500 mL of culture medium, 6000 IU / mL of IL-2, 30 μg of OKT-3, and 5×10 antigen-presenting feeder cells per vessel. 8 ~7.5×10 8 The cells contain antigen-presenting feeder cells.

[0122]

[0134] 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.

[0123]

[0135] 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.

[0124]

[0136] 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 any combination thereof, may be included in the second expansion culture, including, for example, in a step D process 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 any combination thereof, may be included in a step D process as described herein.

[0125]

[0137] In some embodiments, the second expansion culture may be performed in supplemented cell culture medium containing IL-2, OKT-3, a 4-1BB agonist or other TNFRSF agonist and optionally antigen-presenting feeder cells.

[0126]

[0138] 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.

[0127]

[0139] 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.

[0128]

[0140] 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 expansion culture and / or second expansion culture is about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:75, 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 expansion culture and / or second expansion culture is 1:50 to 1:300. In one embodiment, the ratio of TILs to PBMCs in the expansion culture and / or the second expansion culture is 1:100 to 1:200.

[0129]

[0141] In one embodiment, the second expansion culture comprises culturing the second TIL population with a 100-fold or 200-fold excess of inactivated feeder cells in 150 ml of medium, wherein the concentration of feeder cells is at least 1.1 times (1.1×), 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.8×, 2×, 2.1×, 2.2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 21×, 22×, 23×, 24×, 25×, 26×, 27×, 28×, The incubation is performed in flasks containing 1000 μg / mL of ...

[0130]

[0142] In some embodiments, the second expansion culture (which may include a process called the REP process) is 7 to 9 days, as discussed in the Examples and Figures. In some embodiments, the second expansion culture is 7 days. In some embodiments, the second expansion culture is 8 days. In some embodiments, the second expansion culture is 9 days.

[0131]

[0143] In one embodiment, the second expansion culture (which may include the expansion culture referred to as REP; and referenced in step D) 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 x 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 6 The TILs may be cultured with PBMCs. The G-Rex 100 flask may be incubated at 37°C under 5% CO2. On day 5, 250 mL of supernatant may be removed and placed in a centrifuge bottle and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellet may be resuspended in 150 mL of fresh medium containing 5% human AB serum and 6000 IU / mL IL-2 and added back to the original GREX-100 flask. If TILs are continuously expanded in the GREX-100 flask, on day 10 or 11, the TILs may be transferred to a larger flask, such as a GREX-500. Cells may be harvested on day 14 of culture. Cells may be harvested on day 15 of culture. Cells may be harvested on day 16 of culture. 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 aspirating the spent medium and replacing it with an equal volume of fresh medium. In some embodiments, the separate growth chamber includes a GREX flask and a gas-permeable vessel, as discussed more fully below.

[0132]

[0144] In one embodiment, a second expansion (comprising an expansion referred to as REP) is performed, which further comprises selecting TILs for superior tumor response.

[0133]

[0145] In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises one or more of IL-2, OKT-3, a 4-1BB agonist, and antigen-presenting feeder cells (APCs), as discussed in more detail below. In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises 6000 IU / mL of IL-2, 30 μg / flask of OKT-3, 10 μg / mL of a 4-1BB agonist, and 7.5×10 8 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, a 4-1BB agonist, and antigen-presenting feeder cells (APCs), as discussed in more detail below. In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises 6000 IU / mL IL-2, 30 μg / flask OKT-3, 10 μg / mL 4-1BB agonist, and 5×10 8 Contains antigen-presenting feeder cells (APCs).

[0134]

[0146] In some embodiments, the second expansion, e.g., step D, is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion, as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as the vessel. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

[0135]

[0147] In one embodiment, a second expansion (comprising an expansion referred to as REP) is performed, which further comprises 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.

[0136] E. Step E: Harvesting TILs

[0148] 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. In some embodiments, the TILs are harvested after two expansion steps. In some embodiments, the TILs are harvested after two expansion steps, one first expansion and one second expansion. In some embodiments, the TILs are harvested after one expansion step, the first expansion.

[0137]

[0149] 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.

[0138]

[0150] 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.

[0139]

[0151] In some embodiments, the second expansion, e.g., step D, is performed in a closed bioreactor. In some embodiments, a closed system is used for TIL expansion, as described herein. In some embodiments, a bioreactor is used. In some embodiments, a bioreactor is used as the vessel. In some embodiments, the bioreactor used is, for example, a G-REX-100 or a G-REX-500. In some embodiments, the bioreactor used is a G-REX-100. In some embodiments, the bioreactor used is a G-REX-500.

[0140]

[0152] In some embodiments, step E is performed according to a process described herein. 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 as described herein is used.

[0141]

[0153] In some embodiments, TILs are collected according to the methods described herein. In some embodiments, TILs are collected on days 14-16 using methods as described herein. In some embodiments, TILs are collected on day 14 using methods as described herein. In some embodiments, TILs are collected on day 15 using methods as described herein. In some embodiments, TILs are collected on day 16 using methods as described herein.

[0142] F. Step F: Final Formulation / Transfer to Infusion Bag

[0154] After steps A-E, outlined in detail above and herein and provided in an exemplary order, 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.

[0143]

[0155] In one embodiment, TILs expanded using the methods 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 as disclosed herein can be administered by any suitable route as known in the art. In some embodiments, TILs 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.

[0144] Feeder cells and antigen-presenting cells

[0156] 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 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.

[0145]

[0157] 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.

[0146]

[0158] 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.

[0147]

[0159] In some embodiments, if the total number of viable cells on day 7 or 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 acceptable for use in the TIL expansion culture procedures described herein.

[0148]

[0160] 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 entered 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 may be approved for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody and 3,000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng / ml OKT3 antibody and 6,000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng / ml OKT3 antibody and 3,000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody and 6,000 IU / ml IL-2.

[0149]

[0161] 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 entered 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 may be approved for use in the TIL expansion procedures described herein. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml OKT3 antibody and 1000-6000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml OKT3 antibody and 2000-5000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml OKT3 antibody and 2000-4000 IU / ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody and 2500-3500 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody and 6000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng / ml of OKT3 antibody, 10 μg / ml of anti-4-1BB antibody, and 3000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng / ml of OKT3 antibody, 10 μg / ml of anti-4-1BB antibody, and 6000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 60 ng / ml of OKT3 antibody, 10 μg / ml of anti-4-1BB antibody, and 3000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 30 ng / ml OKT3 antibody, 10 ug / ml anti-4-1BB antibody, and 6000 IU / ml IL-2.

[0150]

[0162] In some embodiments, if the total number of viable cells cultured in the presence of OKT3, a 4-1BB agonist, and IL-2 does not increase on days 7 and 14 from 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 replication-incompetent and may be approved for use in the TIL expansion procedures described herein. In some embodiments, the PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 1000-6000 IU / ml of IL-2. In some embodiments, the PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 2000-5000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 2000-4000 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 2500-3500 IU / ml of IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60 ng / ml of OKT3 antibody, 5-40 μg / ml of a 4-1BB agonist, and 6000 IU / ml of IL-2.

[0151]

[0163] 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.

[0152]

[0164] In one embodiment, the second expansion procedure described herein comprises culturing approximately 5×10 8 feeder cells and approximately 100 x 10 6 In one embodiment, the second expansion procedure described herein requires a ratio of about 7.5 x 10 TILs. 8 feeder cells and approximately 100 x 10 6 In another embodiment, the second expansion procedure described herein requires a ratio of about 5 x 10 TILs. 8 feeder cells and approximately 50 x 10 6 In another embodiment, the second expansion procedure described herein requires a ratio of about 7.5 x 10 TILs. 8 feeder cells and approximately 50 x 10 6 In yet another embodiment, the second expansion procedure described herein requires a ratio of about 5 x 10 TILs. 8 Approximately 25 x 10 feeder cells 6 In yet another embodiment, the second expansion procedure described herein requires about 7.5 x 10 TILs. 8 feeder cells and approximately 25 x 10 6 In yet another embodiment, the second expansion culture requires twice the number of feeder cells as the first expansion culture. In yet another embodiment, the first expansion culture described herein requires about 2.5 x 10 TILs. 8 If feeder cells are required, the second expansion culture should contain approximately 5 x 10 cells. 8 In yet another embodiment, the first expansion culture described herein requires about 2.5 x 10 feeder cells. 8 If feeder cells are required, the second expansion culture should contain approximately 7.5 x 10 cells. 8 In yet another embodiment, the second expansion culture requires twice (2.0x), 2.5x, 3.0x, 3.5x, or 4.0x the number of feeder cells as in the first expansion culture.

[0153]

[0165] In certain embodiments, the second expansion culture procedure described herein requires an excess amount of feeder cells during the second expansion culture. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit from an allogeneic 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. In some embodiments, PBMCs are added to the second expansion culture at twice the concentration of PBMCs added to the first expansion culture.

[0154]

[0166] 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.

[0155]

[0167] 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.

[0156] PBMC-feeder cell ratio

[0168] In one embodiment, the number of PBMC feeder layers is calculated as follows:

[0169] Volume of AT cell (diameter 10 μm): V = (4 / 3) πr 3 =523.6μm 3

[0170] B. G-Rex 100 (M) cylindrical flask with a height of 40 μm (4 cells): V = (4 / 3) πr 3 =4×10 12 μm 3

[0171] C. Number of cells needed to fill cylindrical flask B: 4 x 10 12 μm 3 / 523.6μm 3 =7.6×10 8 μm 3 *0.64=4.86×10 8

[0172] D. Number of cells that can be optimally activated in 4D space: 4.86 x 10 8 / 24=20.25×10 6

[0173] Number of feeders and TILs extrapolated to EG-Rex 500: TIL: 100 x 10 6 and feeder: 2.5 x 10 9

[0157]

[0174] In this calculation, 100 cm 2 A rough estimate of the number of mononuclear cells required to provide an icosahedral geometry for activating TILs within the base cylinder is used. This calculation yields a threshold activation of approximately 5 x 10 T cells, which closely reflects the NCI experimental data. 8 The experimental results are derived. (1) (C) The multiplier (0.64) is the random data density of the equivalent sphere calculated by Jaeger and Nagel in 1992. (2) (D) The divisor 24 is the "Newton number" in four-dimensional space. (3) is the number of equivalent spheres that may contact a similar object.

[0158]

[0175] (1) Jin, Jianjian, et.al., Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes (TIL) in Gas-Permeable Flasks to Numbers Needed for Patient Treatment.J Immunother.2012 Apr; 35(3):283- 292.

[0159]

[0176] (2) Jaeger HM, Nagel SR.Physics of the granular state.Science.1992 Mar 20;255(5051):1523-31.

[0160]

[0177] (3)ORMusin (2003). “The problem of the twenty-five spheres”. Russ.Math.Surv.58 (4):794-795.

[0161]

[0178] In one embodiment, the number of exogenously supplied antigen-presenting feeder cells during the first expansion culture is approximately half the number of exogenously supplied antigen-presenting feeder cells during the second expansion culture. In a specific embodiment, the method includes performing the first expansion culture in a cell culture medium containing approximately 50% fewer antigen-presenting cells compared to the cell culture medium of the second expansion culture.

[0162]

[0179] In another embodiment, the number of exogenously supplied antigen-presenting feeder cells (APCs) during the second expansion culture is greater than the number of exogenously supplied APCs during the first expansion culture.

[0163]

[0180] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 20:1 or about 20:1.

[0164]

[0181] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 10:1 or about 10:1.

[0165]

[0182] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 9:1 or about 9:1.

[0166]

[0183] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 8:1 or about 8:1.

[0167]

[0184] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 7:1 or about 7:1.

[0168]

[0185] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 6:1 or about 6:1.

[0169]

[0186] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 5:1 or about 5:1.

[0170]

[0187] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 4:1 or about 4:1.

[0171]

[0188] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 3:1 or about 3:1.

[0172]

[0189] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2.9:1 or about 2.9:1.

[0173]

[0190] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2.8:1 or about 2.8:1.

[0174]

[0191] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2.7:1 or about 2.7:1.

[0175]

[0192] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2.6:1 or about 2.6:1.

[0176]

[0193] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2.5:1 or about 2.5:1.

[0177]

[0194] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2.4:1 or about 2.4:1.

[0178]

[0195] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2.3:1 or about 2.3:1.

[0179]

[0196] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2.2:1 or about 2.2:1.

[0180]

[0197] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2.1:1 or about 2.1:1.

[0181]

[0198] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 1.1:1 or about 1.1:1 to 2:1 or about 2:1.

[0182]

[0199] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 10:1 or about 10:1.

[0183]

[0200] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 5:1 or about 5:1.

[0184]

[0201] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 4:1 or about 4:1.

[0185]

[0202] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 3:1 or about 3:1.

[0186]

[0203] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.9:1 or about 2.9:1.

[0187]

[0204] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.8:1 or about 2.8:1.

[0188]

[0205] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.7:1 or about 2.7:1.

[0189]

[0206] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.6:1 or about 2.6:1.

[0190]

[0207] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.5:1 or about 2.5:1.

[0191]

[0208] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.4:1 or about 2.4:1.

[0192]

[0209] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.3:1 or about 2.3:1.

[0193]

[0210] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.2:1 or about 2.2:1.

[0194]

[0211] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.1:1 or about 2.1:1.

[0195]

[0212] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is 2:1 or about 2:1.

[0196]

[0213] In another embodiment, the ratio of the number of exogenously supplied APCs during the second expansion culture to the number of exogenously supplied APCs during the first expansion culture is at or about 1.1:1, 1.2:1 or about 1.2:1, 1.3:1 or about 1.3:1, 1.4:1 or about 1.4:1, 1.5:1 or about 1.5:1, 1.6:1 or about 1.6:1, 1.7:1 or about 1.7:1, 1.8:1 or about 1.8:1, 1.9:1 or about 1.9:1, 2:1 or about 2:1, 2.1:1 or about 2.1:1, 2.2:1 or about 2.2:1, 2.3:1 or about 2.3:1, 2.4:1 or about 2.4:1, 2.5:1 or about 2.5:1, 2.6:1 or about 2.6:1, 2.7:1 or about 2.7:1, 2.8:1 or about 2.8: 1, 2.9:1 or about 2.9:1, 3:1 or about 3:1, 3.1:1 or about 3.1:1, 3.2:1 or about 3.2:1, 3.3:1 or about 3.3:1, 3.4:1 or about 3.4:1, 3.5:1 or about 3.5:1, 3.6:1 or about 3.6:1, 3.7:1 or about 3.7:1, 3.8:1 or about 3.8:1, 3.9:1 or about 3.9:1 , 4:1 or about 4:1, 4.1:1 or about 4.1:1, 4.2:1 or about 4.2:1, 4.3:1 or about 4.3:1, 4.4:1 or about 4.4:1, 4.5:1 or about 4.5:1, 4.6:1 or about 4.6:1, 4.7:1 or about 4.7:1, 4.8:1 or about 4.8:1, 4.9:1 or about 4.9:1, or 5:1 or about 5:1.

[0197]

[0214] In another embodiment, the number of exogenously supplied APCs during the first expansion culture is 1 x 10 8 or about 1 x 10 8 , 1.1×10 8 or approximately 1.1 x 10 8 , 1.2 × 10 8 or about 1.2 x 10 8 , 1.3 × 10 8 or about 1.3 x 10 8 , 1.4×10 8 or about 1.4 x 10 8 , 1.5×108 or about 1.5 x 10 8 , 1.6×10 8 or about 1.6 x 10 8 , 1.7×10 8 or about 1.7 x 10 8 , 1.8×10 8 or about 1.8 x 10 8 , 1.9×10 8 or about 1.9 x 10 8 , 2 × 10 8 or about 2 x 10 8 , 2.1×10 8 or approximately 2.1 x 10 8 , 2.2 × 10 8 or approximately 2.2 x 10 8 , 2.3 × 10 8 or approximately 2.3 x 10 8 , 2.4 × 10 8 or approximately 2.4 x 10 8 , 2.5×10 8 or about 2.5 x 10 8 , 2.6×10 8 or approximately 2.6 x 10 8 , 2.7 × 10 8 or approximately 2.7 x 10 8 , 2.8×10 8 or approximately 2.8 x 10 8 , 2.9 × 10 8 or approximately 2.9 x 10 8 , 3×10 8 or about 3 x 10 8 , 3.1 × 10 8 or approximately 3.1 x 10 8 , 3.2 × 10 8 or approximately 3.2 x 10 8 , 3.3 × 10 8 or approximately 3.3 x 10 8 , 3.4 × 10 8 or approximately 3.4 x 10 8 or 3.5 x 10 8 or about 3.5 x 10 8 APCs, and the number of exogenously supplied APCs during the second expansion culture was 3.5 x 10 8 or about 3.5 x 10 8 , 3.6×10 8or approximately 3.6 x 10 8 , 3.7 × 10 8 or approximately 3.7 x 10 8 , 3.8×10 8 or approximately 3.8 x 10 8 , 3.9 × 10 8 or approximately 3.9 x 10 8 , 4×10 8 or about 4 x 10 8 , 4.1×10 8 or approximately 4.1 x 10 8 , 4.2 × 10 8 or approximately 4.2 x 10 8 , 4.3 × 10 8 or approximately 4.3 x 10 8 , 4.4×10 8 or approximately 4.4 x 10 8 , 4.5×10 8 or about 4.5 x 10 8 , 4.6×10 8 or approximately 4.6 x 10 8 , 4.7×10 8 or about 4.7 x 10 8 , 4.8×10 8 or approximately 4.8 x 10 8 , 4.9 × 10 8 or approximately 4.9 x 10 8 , 5×10 8 Or about 5 x 10 8 , 5.1×10 8 or approximately 5.1 x 10 8 , 5.2 × 10 8 or approximately 5.2 x 10 8 , 5.3 × 10 8 or approximately 5.3 x 10 8 , 5.4×10 8 or approximately 5.4 x 10 8 , 5.5×10 8 or about 5.5 x 10 8 , 5.6×10 8 or approximately 5.6 x 10 8 , 5.7×10 8 or approximately 5.7 x 10 8 , 5.8×10 8 or approximately 5.8 x 10 8 , 5.9 × 108 or approximately 5.9 x 10 8 , 6×10 8 or about 6 x 10 8 , 6.1×10 8 or approximately 6.1 x 10 8 , 6.2 × 10 8 or approximately 6.2 x 10 8 , 6.3 × 10 8 or approximately 6.3 x 10 8 , 6.4×10 8 or approximately 6.4 x 10 8 , 6.5×10 8 or approximately 6.5 x 10 8 , 6.6×10 8 or approximately 6.6 x 10 8 , 6.7×10 8 or approximately 6.7 x 10 8 , 6.8×10 8 or approximately 6.8 x 10 8 , 6.9 × 10 8 or approximately 6.9 x 10 8 , 7×10 8 or about 7 x 10 8 , 7.1×10 8 or approximately 7.1 x 10 8 , 7.2 × 10 8 or approximately 7.2 x 10 8 , 7.3×10 8 or approximately 7.3 x 10 8 , 7.4×10 8 or approximately 7.4 x 10 8 , 7.5×10 8 or about 7.5 x 10 8 , 7.6×10 8 or approximately 7.6 x 10 8 , 7.7×10 8 or approximately 7.7 x 10 8 , 7.8×10 8 or approximately 7.8 x 10 8 , 7.9×10 8 or approximately 7.9 x 10 8 , 8×10 8 or about 8 x 10 8 , 8.1×10 8 or approximately 8.1 x 10 8 , 8.2 × 108 or approximately 8.2 x 10 8 , 8.3 × 10 8 or approximately 8.3 x 10 8 , 8.4×10 8 or approximately 8.4 x 10 8 , 8.5×10 8 or approximately 8.5 x 10 8 , 8.6×10 8 or approximately 8.6 x 10 8 , 8.7×10 8 or approximately 8.7 x 10 8 , 8.8×10 8 or approximately 8.8 x 10 8 , 8.9×10 8 or approximately 8.9 x 10 8 , 9×10 8 or about 9 x 10 8 , 9.1×10 8 or approximately 9.1 x 10 8 , 9.2 × 10 8 or approximately 9.2 x 10 8 , 9.3×10 8 or approximately 9.3 x 10 8 , 9.4×10 8 or approximately 9.4 x 10 8 , 9.5×10 8 or about 9.5 x 10 8 , 9.6×10 8 or approximately 9.6 x 10 8 , 9.7×10 8 or approximately 9.7 x 10 8 , 9.8×10 8 or approximately 9.8 x 10 8 , 9.9×10 8 or approximately 9.9 x 10 8 or 1 x 10 9 or about 1 x 10 9 It is an APC.

[0198]

[0215] In another embodiment, the number of exogenously supplied APCs during the first expansion culture is 1.5 x 10 8 or approximately 1.5 x 10 8 APC~3×10 8 or about 3 x 10 8The number of exogenously supplied APCs selected from a range of APCs during the second expansion culture was 4 x 10 8 or about 4 x 10 8 APC~7.5×10 8 or approximately 7.5 x 10 8 A range of APCs are selected.

[0199]

[0216] In another embodiment, the number of exogenously supplied APCs during the first expansion culture is 2 x 10 8 or about 2 x 10 8 APC~2.5×10 8 or approximately 2.5 x 10 8 The number of exogenously supplied APCs selected from a range of APCs during the second expansion culture was 4.5 x 10 8 or approximately 4.5 x 10 8 APC~5.5×10 8 or approximately 5.5 x 10 8 A range of APCs are selected.

[0200]

[0217] In another embodiment, the number of exogenously supplied APCs during the first expansion culture is 2.5 x 10 8 or approximately 2.5 x 10 8 APCs, and the number of exogenously supplied APCs during the second expansion culture was 5 x 10 8 or about 5 x 10 8 It is an APC.

[0201]

[0218] In another embodiment, the exogenously supplied APCs in the first expansion culture are 1.0 x 10 6 or approximately 1.0 x 10 6 APC / cm 2 ~4.5×10 6 or approximately 4.5 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density selected from the range of

[0202]

[0219] In another embodiment, the exogenously supplied APCs in the first expansion culture are 1.5 x 10 6 or approximately 1.5 x 10 6 APC / cm 2~3.5×10 6 or approximately 3.5 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density selected from the range of

[0203]

[0220] In another embodiment, the exogenously supplied APCs in the first expansion culture are 2×10 6 or about 2 x 10 6 APC / cm 2 ~3×10 6 or about 3 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density selected from the range of

[0204]

[0221] In another embodiment, the exogenously supplied APCs in the first expansion culture are 2×10 6 or about 2 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density of 1000 x g.

[0205]

[0222] In another embodiment, the exogenously supplied APCs in the first expansion culture are 1.0 x 10 6 or approximately 1.0 x 10 6 , 1.1×10 6 or approximately 1.1 x 10 6 , 1.2 × 10 6 or about 1.2 x 10 6 , 1.3 × 10 6 or about 1.3 x 10 6 , 1.4×10 6 or about 1.4 x 10 6 , 1.5×10 6 or about 1.5 x 10 6 , 1.6×10 6 or about 1.6 x 10 6 , 1.7×10 6 or about 1.7 x 10 6 , 1.8×10 6 or about 1.8 x 10 6 , 1.9×10 6 or about 1.9 x 10 6 , 2 × 10 6or about 2 x 10 6 , 2.1×10 6 or approximately 2.1 x 10 6 , 2.2 × 10 6 or approximately 2.2 x 10 6 , 2.3 × 10 6 or approximately 2.3 x 10 6 , 2.4 × 10 6 or approximately 2.4 x 10 6 , 2.5×10 6 or about 2.5 x 10 6 , 2.6×10 6 or approximately 2.6 x 10 6 , 2.7 × 10 6 or approximately 2.7 x 10 6 , 2.8×10 6 or approximately 2.8 x 10 6 , 2.9 × 10 6 or approximately 2.9 x 10 6 , 3×10 6 or about 3 x 10 6 , 3.1 × 10 6 or approximately 3.1 x 10 6 , 3.2 × 10 6 or approximately 3.2 x 10 6 , 3.3 × 10 6 or approximately 3.3 x 10 6 , 3.4 × 10 6 or approximately 3.4 x 10 6 , 3.5×10 6 or about 3.5 x 10 6 , 3.6×10 6 or approximately 3.6 x 10 6 , 3.7 × 10 6 or approximately 3.7 x 10 6 , 3.8×10 6 or approximately 3.8 x 10 6 , 3.9 × 10 6 or approximately 3.9 x 10 6 , 4×10 6 or about 4 x 10 6 , 4.1×10 6 or approximately 4.1 x 10 6 , 4.2 × 10 6 or approximately 4.2 x 10 6 , 4.3 × 10 6or approximately 4.3 x 10 6 , 4.4×10 6 or approximately 4.4 x 10 6 or 4.5 x 10 6 or about 4.5 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density of 1000 x g.

[0206]

[0223] In another embodiment, the exogenously supplied APCs in the second expansion culture are 2.5 x 10 6 or approximately 2.5 x 10 6 APC / cm 2 ~7.5×10 6 or approximately 7.5 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density selected from the range of

[0207]

[0224] In another embodiment, the exogenously supplied APCs in the second expansion culture are 3.5 x 10 6 or approximately 3.5 x 10 6 APC / cm 2 ~Approx. 6.0×10 6 APC / cm 2 The cells are seeded into culture flasks at a density selected from the range of

[0208]

[0225] In another embodiment, the exogenously supplied APCs in the second expansion culture are 4.0 x 10 6 or approximately 4.0 x 10 6 APC / cm 2 ~Approx. 5.5×10 6 APC / cm 2 The cells are seeded into culture flasks at a density selected from the range of

[0209]

[0226] In another embodiment, the exogenously supplied APCs in the second expansion culture are 4.0 x 10 6 or approximately 4.0 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density selected from the range of

[0210]

[0227] In another embodiment, the exogenously supplied APCs in the second expansion culture are 2.5 x 10 6 or about 2.5 x 10 6 APC / cm 2 , 2.6×10 6 or approximately 2.6 x 10 6 APC / cm 2 , 2.7 × 10 6 or approximately 2.7 x 10 6 APC / cm 2 , 2.8×10 6 or approximately 2.8 x 10 6 , 2.9 × 10 6 or approximately 2.9 x 10 6 , 3×10 6 or about 3 x 10 6 , 3.1 × 10 6 or approximately 3.1 x 10 6 , 3.2 × 10 6 or approximately 3.2 x 10 6 , 3.3 × 10 6 or approximately 3.3 x 10 6 , 3.4 × 10 6 or approximately 3.4 x 10 6 , 3.5×10 6 or about 3.5 x 10 6 , 3.6×10 6 or approximately 3.6 x 10 6 , 3.7 × 10 6 or approximately 3.7 x 10 6 , 3.8×10 6 or approximately 3.8 x 10 6 , 3.9 × 10 6 or approximately 3.9 x 10 6 , 4×10 6 or about 4 x 10 6 , 4.1×10 6 or approximately 4.1 x 10 6 , 4.2 × 10 6 or approximately 4.2 x 10 6 , 4.3 × 10 6 or approximately 4.3 x 10 6 , 4.4×10 6 or approximately 4.4 x 10 6 , 4.5×10 6 or about 4.5 x 106 , 4.6×10 6 or approximately 4.6 x 10 6 , 4.7×10 6 or about 4.7 x 10 6 , 4.8×10 6 or approximately 4.8 x 10 6 , 4.9 × 10 6 or approximately 4.9 x 10 6 , 5×10 6 Or about 5 x 10 6 , 5.1×10 6 or approximately 5.1 x 10 6 , 5.2 × 10 6 or approximately 5.2 x 10 6 , 5.3 × 10 6 or approximately 5.3 x 10 6 , 5.4×10 6 or approximately 5.4 x 10 6 , 5.5×10 6 or about 5.5 x 10 6 , 5.6×10 6 or approximately 5.6 x 10 6 , 5.7×10 6 or approximately 5.7 x 10 6 , 5.8×10 6 or approximately 5.8 x 10 6 , 5.9 × 10 6 or approximately 5.9 x 10 6 , 6×10 6 or about 6 x 10 6 , 6.1×10 6 or approximately 6.1 x 10 6 , 6.2 × 10 6 or approximately 6.2 x 10 6 , 6.3 × 10 6 or approximately 6.3 x 10 6 , 6.4×10 6 or approximately 6.4 x 10 6 , 6.5×10 6 or approximately 6.5 x 10 6 , 6.6×10 6 or approximately 6.6 x 10 6 , 6.7×10 6 or approximately 6.7 x 10 6 , 6.8×10 6or approximately 6.8 x 10 6 , 6.9 × 10 6 or approximately 6.9 x 10 6 , 7×10 6 or about 7 x 10 6 , 7.1×10 6 or approximately 7.1 x 10 6 , 7.2 × 10 6 or approximately 7.2 x 10 6 , 7.3×10 6 or approximately 7.3 x 10 6 , 7.4×10 6 or approximately 7.4 x 10 6 or 7.5 x 10 6 or about 7.5 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density of 1000 x g.

[0211]

[0228] In another embodiment, the exogenously supplied APCs in the first expansion culture are 1.0 x 10 6 or approximately 1.0 x 10 6 , 1.1×10 6 or approximately 1.1 x 10 6 , 1.2 × 10 6 or about 1.2 x 10 6 , 1.3 × 10 6 or about 1.3 x 10 6 , 1.4×10 6 or about 1.4 x 10 6 , 1.5×10 6 or about 1.5 x 10 6 , 1.6×10 6 or about 1.6 x 10 6 , 1.7×10 6 or about 1.7 x 10 6 , 1.8×10 6 or about 1.8 x 10 6 , 1.9×10 6 or about 1.9 x 10 6 , 2 × 10 6 or about 2 x 10 6 , 2.1×10 6 or approximately 2.1 x 10 6 , 2.2 × 10 6 or approximately 2.2 x 106 , 2.3 × 10 6 or approximately 2.3 x 10 6 , 2.4 × 10 6 or approximately 2.4 x 10 6 , 2.5×10 6 or about 2.5 x 10 6 , 2.6×10 6 or approximately 2.6 x 10 6 , 2.7 × 10 6 or approximately 2.7 x 10 6 , 2.8×10 6 or approximately 2.8 x 10 6 , 2.9 × 10 6 or approximately 2.9 x 10 6 , 3×10 6 or about 3 x 10 6 , 3.1 × 10 6 or approximately 3.1 x 10 6 , 3.2 × 10 6 or approximately 3.2 x 10 6 , 3.3 × 10 6 or approximately 3.3 x 10 6 , 3.4 × 10 6 or approximately 3.4 x 10 6 , 3.5×10 6 or about 3.5 x 10 6 , 3.6×10 6 or approximately 3.6 x 10 6 , 3.7 × 10 6 or approximately 3.7 x 10 6 , 3.8×10 6 or approximately 3.8 x 10 6 , 3.9 × 10 6 or approximately 3.9 x 10 6 , 4×10 6 or about 4 x 10 6 , 4.1×10 6 or approximately 4.1 x 10 6 , 4.2 × 10 6 or approximately 4.2 x 10 6 , 4.3 × 10 6 or approximately 4.3 x 10 6 , 4.4×10 6 or approximately 4.4 x 10 6 or 4.5 x 10 6or about 4.5 x 10 6 APC / cm 2 In the second expansion culture, exogenously supplied APCs were seeded in culture flasks at a density of 2.5 x 10 6 or about 2.5 x 10 6 APC / cm 2 , 2.6×10 6 or approximately 2.6 x 10 6 APC / cm 2 , 2.7 × 10 6 or approximately 2.7 x 10 6 APC / cm 2 , 2.8×10 6 or approximately 2.8 x 10 6 , 2.9 × 10 6 or approximately 2.9 x 10 6 , 3×10 6 or about 3 x 10 6 , 3.1 × 10 6 or approximately 3.1 x 10 6 , 3.2 × 10 6 or approximately 3.2 x 10 6 , 3.3 × 10 6 or approximately 3.3 x 10 6 , 3.4 × 10 6 or approximately 3.4 x 10 6 , 3.5×10 6 or about 3.5 x 10 6 , 3.6×10 6 or approximately 3.6 x 10 6 , 3.7 × 10 6 or approximately 3.7 x 10 6 , 3.8×10 6 or approximately 3.8 x 10 6 , 3.9 × 10 6 or approximately 3.9 x 10 6 , 4×10 6 or about 4 x 10 6 , 4.1×10 6 or approximately 4.1 x 10 6 , 4.2 × 10 6 or approximately 4.2 x 10 6 , 4.3 × 10 6 or approximately 4.3 x 10 6 , 4.4×10 6 or approximately 4.4 x 106 , 4.5×10 6 or about 4.5 x 10 6 , 4.6×10 6 or approximately 4.6 x 10 6 , 4.7 × 10 6 or approximately 4.7 x 10 6 , 4.8×10 6 or approximately 4.8 x 10 6 , 4.9 × 10 6 or approximately 4.9 x 10 6 , 5×10 6 Or about 5 x 10 6 , 5.1×10 6 or approximately 5.1 x 10 6 , 5.2 × 10 6 or approximately 5.2 x 10 6 , 5.3 × 10 6 or approximately 5.3 x 10 6 , 5.4×10 6 or approximately 5.4 x 10 6 , 5.5×10 6 or about 5.5 x 10 6 , 5.6×10 6 or approximately 5.6 x 10 6 , 5.7×10 6 or approximately 5.7 x 10 6 , 5.8×10 6 or approximately 5.8 x 10 6 , 5.9×10 6 or approximately 5.9 x 10 6 , 6×10 6 or about 6 x 10 6 , 6.1×10 6 or approximately 6.1 x 10 6 , 6.2 × 10 6 or approximately 6.2 x 10 6 , 6.3 × 10 6 or approximately 6.3 x 10 6 , 6.4×10 6 or approximately 6.4 x 10 6 , 6.5×10 6 or approximately 6.5 x 10 6 , 6.6×10 6 or approximately 6.6 x 10 6 , 6.7×10 6or approximately 6.7 x 10 6 , 6.8×10 6 or approximately 6.8 x 10 6 , 6.9 × 10 6 or approximately 6.9 x 10 6 , 7×10 6 or about 7 x 10 6 , 7.1×10 6 or approximately 7.1 x 10 6 , 7.2 × 10 6 or approximately 7.2 x 10 6 , 7.3×10 6 or approximately 7.3 x 10 6 , 7.4×10 6 or approximately 7.4 x 10 6 or 7.5 x 10 6 or about 7.5 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density of 1000 x g.

[0212]

[0229] In another embodiment, the exogenously supplied APCs in the first expansion culture are 1.0 x 10 6 or approximately 1.0 x 10 6 APC / cm 2 ~4.5×10 6 or approximately 4.5 x 10 6 APC / cm 2 In the second expansion culture, exogenously supplied APCs were seeded into culture flasks at densities ranging from 2.5 x 10 to 2.5 x 10. 6 or approximately 2.5 x 10 6 APC / cm 2 ~7.5×10 6 or approximately 7.5 x 10 6 APC / cm 2 The cells are seeded into culture flasks at densities ranging from 0.1 to 0.2 μg / ml.

[0213]

[0230] In another embodiment, the exogenously supplied APCs in the first expansion culture are 1.5 x 10 6 or approximately 1.5 x 10 6 APC / cm 2 ~3.5×10 6 or approximately 3.5 x 10 6 APC / cm 2In the second expansion culture, exogenously supplied APCs were seeded into culture flasks at densities ranging from 3.5 x 10 to 3.5 x 10. 6 or approximately 3.5 x 10 6 APC / cm 2 ~6×10 6 or about 6 x 10 6 APC / cm 2 The cells are seeded into culture flasks at densities ranging from 0.1 to 0.2 μg / ml.

[0214]

[0231] In another embodiment, the exogenously supplied APCs in the first expansion culture are 2×10 6 or about 2 x 10 6 APC / cm 2 ~3×10 6 or about 3 x 10 6 APC / cm 2 In the second expansion culture, exogenously supplied APCs were seeded into culture flasks at densities ranging from 4 x 10 to 4 x 10. 6 or about 4 x 10 6 APC / cm 2 ~5.5×10 6 or approximately 5.5 x 10 6 APC / cm 2 The cells are seeded into culture flasks at densities ranging from 0.1 to 0.2 μg / ml.

[0215]

[0232] In another embodiment, the exogenously supplied APCs in the first expansion culture are 2×10 6 or about 2 x 10 6 APC / cm 2 In the second expansion culture, exogenously supplied APCs were seeded in culture flasks at a density of 4 x 10 6 or about 4 x 10 6 APC / cm 2 The cells are seeded into culture flasks at a density of 1000 x g.

[0216]

[0233] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied PBMCs on day 0 (at the start) of the first expansion culture is in the range of at or about 1.1:1 to 20:1 or about 20:1.

[0217]

[0234] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied PBMCs on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 10:1 or about 10:1.

[0218]

[0235] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied PBMCs on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 9:1 or about 9:1.

[0219]

[0236] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 8:1 or about 8:1.

[0220]

[0237] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 7:1 or about 7:1.

[0221]

[0238] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 6:1 or about 6:1.

[0222]

[0239] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 5:1 or about 5:1.

[0223]

[0240] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 4:1 or about 4:1.

[0224]

[0241] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 3:1 or about 3:1.

[0225]

[0242] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2.9:1 or about 2.9:1.

[0226]

[0243] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2.8:1 or about 2.8:1.

[0227]

[0244] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2.7:1 or about 2.7:1.

[0228]

[0245] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2.6:1 or about 2.6:1.

[0229]

[0246] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2.5:1 or about 2.5:1.

[0230]

[0247] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2.4:1 or about 2.4:1.

[0231]

[0248] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2.3:1 or about 2.3:1.

[0232]

[0249] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2.2:1 or about 2.2:1.

[0233]

[0250] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2.1:1 or about 2.1:1.

[0234]

[0251] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 1.1:1 or about 1.1:1 to 2:1 or about 2:1.

[0235]

[0252] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1 or about 2:1 to 10:1 or about 10:1.

[0236]

[0253] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1 or about 2:1 to 5:1 or about 5:1.

[0237]

[0254] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1 or about 2:1 to 4:1 or about 4:1.

[0238]

[0255] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1 or about 2:1 to 3:1 or about 3:1.

[0239]

[0256] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1, or about 2:1 to 2.9:1, or about 2.9:1.

[0240]

[0257] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1 or about 2:1 to 2.8:1 or about 2.8:1.

[0241]

[0258] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1, or about 2:1 to 2.7:1, or about 2.7:1.

[0242]

[0259] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is selected from the range of 2:1 or about 2:1 to 2.6:1 or about 2.6:1.

[0243]

[0260] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1, or about 2:1 to 2.5:1, or about 2.5:1.

[0244]

[0261] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1, or about 2:1 to 2.4:1, or about 2.4:1.

[0245]

[0262] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1, or about 2:1 to 2.3:1, or about 2.3:1.

[0246]

[0263] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1, or about 2:1 to 2.2:1, or about 2.2:1.

[0247]

[0264] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is in the range of 2:1, or about 2:1 to 2.1:1, or about 2.1:1.

[0248]

[0265] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture is 2:1 or approximately 2:1.

[0249]

[0266] In another embodiment, the ratio of the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the beginning) of the second expansion culture to the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the beginning) of the first expansion culture is at or about 1.1:1, 1.2:1 or about 1.2:1, 1.3:1 or about 1.3:1, 1.4:1 or about 1.4:1, 1.5:1 or about 1.5:1, or :1, 1.6:1 at or about 1.6:1, 1.7:1 at or about 1.7:1, 1.8:1 at or about 1.8:1, 1.9:1 at or about 1.9:1, 2:1 at or about 2:1, 2.1:1 at or about 2.1:1, 2.2:1 at or about 2.2:1, 2.3:1 at or about 2.3:1, 2.4:1 at or about 2.4:1, 2.5:1 at or about 2.5:1, 2.6:1 at or about 2.6:1, 2.7:1 or about 2.7:1, 2.8:1 or about 2.8:1, 2.9:1 or about 2.9:1, 3:1 or about 3:1, 3.1:1 or about 3.1:1, 3.2:1 or about 3.2:1, 3.3:1 or about 3.3:1, 3.4:1 or about 3.4:1, 3.5:1 or about 3.5:1, 3.6:1 or about 3.6:1, 3.7:1 or about 3.7:1, 3.8:1 or about 3.8:1, 3.9: 1 or about 3.9:1, 4:1 or about 4:1, 4.1:1 or about 4.1:1, 4.2:1 or about 4.2:1, 4.3:1 or about 4.3:1, 4.4:1 or about 4.4:1, 4.5:1 or about 4.5:1, 4.6:1 or about 4.6:1, 4.7:1 or about 4.7:1, 4.8:1 or about 4.8:1, 4.9:1 or about 4.9:1, or 5:1 or about 5:1.

[0250]

[0267] In another embodiment, the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (the beginning) of the first expansion culture is 1×10 8 or about 1 x 10 8 , 1.1×10 8 or approximately 1.1 x 10 8 , 1.2 × 10 8 or about 1.2 x 10 8 , 1.3 × 10 8 or about 1.3 x 108 , 1.4×10 8 or about 1.4 x 10 8 , 1.5×10 8 or about 1.5 x 10 8 , 1.6×10 8 or about 1.6 x 10 8 , 1.7×10 8 or about 1.7 x 10 8 , 1.8×10 8 or about 1.8 x 10 8 , 1.9×10 8 or about 1.9 x 10 8 , 2 × 10 8 or about 2 x 10 8 , 2.1×10 8 or approximately 2.1 x 10 8 , 2.2 × 10 8 or approximately 2.2 x 10 8 , 2.3 × 10 8 or approximately 2.3 x 10 8 , 2.4 × 10 8 or approximately 2.4 x 10 8 , 2.5×10 8 or about 2.5 x 10 8 , 2.6×10 8 or approximately 2.6 x 10 8 , 2.7 × 10 8 or approximately 2.7 x 10 8 , 2.8×10 8 or approximately 2.8 x 10 8 , 2.9 × 10 8 or approximately 2.9 x 10 8 , 3×10 8 or about 3 x 10 8 , 3.1 × 10 8 or approximately 3.1 x 10 8 , 3.2 × 10 8 or approximately 3.2 x 10 8 , 3.3 × 10 8 or approximately 3.3 x 10 8 , 3.4 × 10 8 or approximately 3.4 x 10 8 or 3.5 x 10 8 or about 3.5 x 10 8APCs (e.g., including PBMCs), and the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (the beginning) of the second expansion culture was 3.5 x 10 8 or about 3.5 x 10 8 , 3.6×10 8 or approximately 3.6 x 10 8 , 3.7 × 10 8 or approximately 3.7 x 10 8 , 3.8×10 8 or approximately 3.8 x 10 8 , 3.9 × 10 8 or approximately 3.9 x 10 8 , 4×10 8 or about 4 x 10 8 , 4.1×10 8 or approximately 4.1 x 10 8 , 4.2 × 10 8 or approximately 4.2 x 10 8 , 4.3 × 10 8 or approximately 4.3 x 10 8 , 4.4×10 8 or approximately 4.4 x 10 8 , 4.5×10 8 or about 4.5 x 10 8 , 4.6×10 8 or approximately 4.6 x 10 8 , 4.7×10 8 or about 4.7 x 10 8 , 4.8×10 8 or approximately 4.8 x 10 8 , 4.9 × 10 8 or approximately 4.9 x 10 8 , 5×10 8 Or about 5 x 10 8 , 5.1×10 8 or approximately 5.1 x 10 8 , 5.2 × 10 8 or approximately 5.2 x 10 8 , 5.3 × 10 8 or approximately 5.3 x 10 8 , 5.4×10 8 or approximately 5.4 x 10 8 , 5.5×10 8 or about 5.5 x 10 8 , 5.6×10 8or approximately 5.6 x 10 8 , 5.7×10 8 or approximately 5.7 x 10 8 , 5.8×10 8 or approximately 5.8 x 10 8 , 5.9×10 8 or approximately 5.9 x 10 8 , 6×10 8 or about 6 x 10 8 , 6.1×10 8 or approximately 6.1 x 10 8 , 6.2 × 10 8 or approximately 6.2 x 10 8 , 6.3 × 10 8 or approximately 6.3 x 10 8 , 6.4×10 8 or approximately 6.4 x 10 8 , 6.5×10 8 or approximately 6.5 x 10 8 , 6.6×10 8 or approximately 6.6 x 10 8 , 6.7×10 8 or approximately 6.7 x 10 8 , 6.8×10 8 or approximately 6.8 x 10 8 , 6.9×10 8 or approximately 6.9 x 10 8 , 7×10 8 or about 7 x 10 8 , 7.1×10 8 or approximately 7.1 x 10 8 , 7.2 × 10 8 or approximately 7.2 x 10 8 , 7.3×10 8 or approximately 7.3 x 10 8 , 7.4×10 8 or approximately 7.4 x 10 8 , 7.5×10 8 or about 7.5 x 10 8 , 7.6×10 8 or approximately 7.6 x 10 8 , 7.7×10 8 or approximately 7.7 x 10 8 , 7.8×10 8 or approximately 7.8 x 10 8 , 7.9×108 or approximately 7.9 x 10 8 , 8×10 8 or about 8 x 10 8 , 8.1×10 8 or approximately 8.1 x 10 8 , 8.2 × 10 8 or approximately 8.2 x 10 8 , 8.3 × 10 8 or approximately 8.3 x 10 8 , 8.4×10 8 or approximately 8.4 x 10 8 , 8.5×10 8 or approximately 8.5 x 10 8 , 8.6×10 8 or approximately 8.6 x 10 8 , 8.7×10 8 or approximately 8.7 x 10 8 , 8.8×10 8 or approximately 8.8 x 10 8 , 8.9×10 8 or approximately 8.9 x 10 8 , 9×10 8 or about 9 x 10 8 , 9.1×10 8 or approximately 9.1 x 10 8 , 9.2 × 10 8 or approximately 9.2 x 10 8 , 9.3×10 8 or approximately 9.3 x 10 8 , 9.4×10 8 or approximately 9.4 x 10 8 , 9.5×10 8 or about 9.5 x 10 8 , 9.6×10 8 or approximately 9.6 x 10 8 , 9.7×10 8 or approximately 9.7 x 10 8 , 9.8×10 8 or approximately 9.8 x 10 8 , 9.9×10 8 or approximately 9.9 x 10 8 or 1 x 10 9 or about 1 x 10 9 APCs (including, for example, PBMCs).

[0251]

[0268] In another embodiment, the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (the beginning) of the first expansion culture is 1×10 8 or approximately 1 x 10 8 APCs (e.g., including PBMCs) ~3.5 x 10 8 or approximately 3.5 x 10 8 The number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (start) of the second expansion culture ranged from 3.5 x 10 8 or approximately 3.5 x 10 8 APCs (e.g., including PBMCs) ~1 x 10 9 or approximately 1 x 10 9 A range of APCs (including, for example, PBMCs).

[0252]

[0269] In another embodiment, the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (the beginning) of the first expansion culture is 1.5 x 10 8 or approximately 1.5 x 10 8 APC~3×10 8 or about 3 x 10 8 The number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (the start) of the second expansion culture ranged from 4 x 10 8 or about 4 x 10 8 APCs (e.g., including PBMCs) ~7.5 x 10 8 or approximately 7.5 x 10 8 A range of APCs (including, for example, PBMCs).

[0253]

[0270] In another embodiment, the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (the beginning) of the first expansion culture is 2×10 8 or about 2 x 10 8 APCs (e.g., including PBMCs) ~2.5 x 10 8 or approximately 2.5 x 10 8 The number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (start) of the second expansion culture ranged from 4.5 x 108 or approximately 4.5 x 10 8 APCs (e.g., including PBMCs) ~5.5 x 10 8 or approximately 5.5 x 10 8 A range of APCs (including, for example, PBMCs).

[0254]

[0271] In another embodiment, the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (the beginning) of the first expansion culture is 2.5 x 10 8 or approximately 2.5 x 10 8 APCs (e.g., including PBMCs), and the number of exogenously supplied APCs (e.g., including PBMCs) on day 0 (the beginning) of the second expansion culture was 5 x 10 8 or about 5 x 10 8 APCs (including, for example, PBMCs).

[0255]

[0272] In one embodiment, the number of layers of APCs (e.g., including PBMCs) added on day 0 (at the beginning) of the first expansion culture is approximately half the number of layers of APCs (e.g., including PBMCs) added on day 0 (at the beginning) of the second expansion culture. In certain embodiments, the method includes adding an antigen-presenting cell layer to the first TIL population on day 0 (at the beginning) of the first expansion culture and adding an antigen-presenting cell layer to the second TIL population on day 0 (at the beginning) of the second expansion culture, wherein the number of antigen-presenting cell layers added to the first TIL population is approximately 50% of the number of antigen-presenting cell layers added to the second TIL population.

[0256]

[0273] In another embodiment, the number of layers of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the second expansion culture is greater than the number of layers of exogenously supplied APCs (e.g., including PBMCs) on day 0 (at the start) of the first expansion culture.

[0257]

[0274] In another embodiment, day 0 (starting) of the first expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 2 or about 2 cell layers, and day 0 (starting) of the second expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 4 or about 4 cell layers.

[0258]

[0275] In another embodiment, day 0 (initiation) of the first expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 1 or about 1 cell layer, and day 0 (initiation) of the second expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 3 or about 3 cell layers.

[0259]

[0276] In another embodiment, day 0 (initiation) of the first expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 1.5 or about 1.5 cell layers to 2.5 or about 2.5 cell layers, and day 0 (initiation) of the second expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 3 or about 3 cell layers.

[0260]

[0277] In another embodiment, day 0 (initiation) of the first expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 1 or about 1 cell layer, and day 0 (initiation) of the second expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 2 or about 2 cell layers.

[0261]

[0278] In another embodiment, on day 0 (start) of the first expansion culture, the cell number is 1 or about 1, 1.1 or about 1.1, 1.2 or about 1.2, 1.3 or about 1.3, 1.4 or about 1.4, 1.5 or about 1.5, 1.6 or about 1.6, 1.7 or about 1.7, 1.8 or about 1.8, 1.9 or about 1.9, 2 or about 2, 2.1 or about 2.1, 2.2 or about 2.2, 2.3 or about 2.3, 2.4 or about 2.4, 2.5 or about 2.5, 2.6 or about 2.6, 2.7 or about 2.7 , 2.8 or about 2.8, 2.9 or about 2.9, or 3 or about 3 cell layers thick, and day 0 (starting) of the second expansion culture is 3.1 or about 3.1, 3.2 or about 3.2, 3.3 or about 3.3, 3.4 or about 3.4, 3.5 or about 3.5, 3.6 or about 3.6, 3.7 or about 3.7, 3.8 or about 3.8, 3.9 or about 3.9, 4 or about 4, 4.1 or about 4.1, 4.2 or about 4.2, 4.3 or about 4.4, 4.4 or about 4.5, 4.5 or about 4.6, 4.6 or about 4.7, 4.7 or about 4.8, 4.8 or about 4.9, 4.9 or about 5.9, 5 ... about 4.3, 4.4 or about 4.4, 4.5 or about 4.5, 4.6 or about 4.6, 4.7 or about 4.7, 4.8 or about 4.8, 4.9 or about 4.9, 5 or about 5, 5.1 or about 5.1, 5.2 or about 5.2, 5.3 or about 5.3, 5.4 or about 5.4, 5.5 or about 5.5, 5.6 or about 5.6, 5.7 or about 5.7, 5.8 or about 5.8, 5.9 or about 5.9, 6 or about 6, 6.1 or about 6.1, 6.2 or about 6.2, 6.3 or about 6.3 , 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.5, 7.6, 7.7, 7.8, 7.9, or 7.9, or 8, or 8 cell layers thick.

[0262]

[0279] In another embodiment, day 0 (initiation) of the first expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 1 or about 1 cell layer to 2 or about 2 cell layers, and day 0 (initiation) of the second expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 3 or about 3 cell layers to 10 or about 10 cell layers.

[0263]

[0280] In another embodiment, day 0 (initiation) of the first expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 2 or about 2 cell layers to 3 or about 3 cell layers, and day 0 (initiation) of the second expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 4 or about 4 cell layers to 8 or about 8 cell layers.

[0264]

[0281] In another embodiment, day 0 (starting) of the first expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 2 or about 2 cell layers, and day 0 (starting) of the second expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 4 or about 4 cell layers to 8 or about 8 cell layers.

[0265]

[0282] In another embodiment, day 0 (starting) of the first expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 1 or about 1, 2 or about 2, or 3 or about 3 cell layers, and day 0 (starting) of the second expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having an average thickness of 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, or 10 or about 10 cell layers.

[0266]

[0283] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.1 or about 1:1.1 to 1:10 or about 1:10.

[0267]

[0284] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.1 or about 1:1.1 to 1:8 or about 1:8.

[0268]

[0285] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.1 or about 1:1.1 to 1:7 or about 1:7.

[0269]

[0286] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.1 or about 1:1.1 to 1:6 or about 1:6.

[0270]

[0287] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.1 or about 1:1.1 to 1:5 or about 1:5.

[0271]

[0288] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.1 or about 1:1.1 to 1:4 or about 1:4.

[0272]

[0289] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.1 or about 1:1.1 to 1:3 or about 1:3.

[0273]

[0290] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.1 or about 1:1.1 to 1:2 or about 1:2.

[0274]

[0291] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.2 or about 1:1.2 to 1:8 or about 1:8.

[0275]

[0292] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.3 or about 1:1.3 to 1:7 or about 1:7.

[0276]

[0293] In another embodiment, day 0 (the beginning) of the first expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., including PBMCs), and day 7 (the beginning) of the second expansion culture is carried out in the presence of layered APCs (e.g., including PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., including PBMCs), and the ratio of the first number of layers of APCs (e.g., including PBMCs) to the second number of layers of APCs (e.g., including PBMCs) is in the range of 1:1.4 or about 1:1.4 to 1:6 or about 1:6.

[0277]

[0294] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.5 or about 1:1.5 to 1:5 or about 1:5.

[0278]

[0295] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.6 or about 1:1.6 to 1:4 or about 1:4.

[0279]

[0296] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.7 or about 1:1.7 to 1:3.5 or about 1:3.5.

[0280]

[0297] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.8 or about 1:1.8 to 1:3 or about 1:3.

[0281]

[0298] In another embodiment, day 0 (starting point) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to the first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting point) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to the second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is in the range of 1:1.9 or about 1:1.9 to 1:2.5 or about 1:2.5.

[0282]

[0299] In another embodiment, day 0 (starting) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to a first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to a second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is 1:2 or approximately 1:2.

[0283]

[0300] In another embodiment, day 0 (starting) of the first expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a first average thickness equal to a first number of layers of APCs (e.g., comprising PBMCs), and day 0 (starting) of the second expansion culture is carried out in the presence of layered APCs (e.g., comprising PBMCs) having a second average thickness equal to a second number of layers of APCs (e.g., comprising PBMCs), and the ratio of the first number of layers of APCs (e.g., comprising PBMCs) to the second number of layers of APCs (e.g., comprising PBMCs) is at or about 1:1.1, 1:1.2 or less. or about 1:1.2, 1:1.3 or about 1:1.3, 1:1.4 or about 1:1.4, 1:1.5 or about 1:1.5, 1:1.6 or about 1:1.6, 1:1.7 or about 1:1.7, 1:1.8 or about 1:1.8, 1:1.9 or about 1:1.9, 1:2 or about 1:2, 1:2.1 or about 1:2.1, 1:2.2 or about 1:2.2, 1:2.3 or about 1:2.3, 1:2.4 or about 1:2.4, 1:2.5 or about 1:2.5, 1:2.6 or about 1:2.6, 1:2.7 or about 1:2.7, 1: 1:2.8 or about 1:2.8, 1:2.9 or about 1:2.9, 1:3 or about 1:3, 1:3.1 or about 1:3.1, 1:3.2 or about 1:3.2, 1:3.3 or about 1:3.3, 1:3.4 or about 1:3.4, 1:3.5 or about 1:3.5, 1:3.6 or about 1:3.6, 1:3.7 or about 1:3.7, 1:3.8 or about 1:3.8, 1:3.9 or about 1:3.9, 1:4 or about 1:4, 1:4.1 or about 1:4.1, 1:4.2 or about 1:4.2, 1:4.3 or about 1:4.3, 1: 4.4 or about 1:4.4, 1:4.5 or about 1:4.5, 1:4.6 or about 1:4.6, 1:4.7 or about 1:4.7, 1:4.8 or about 1:4.8, 1:4.9 or about 1:4.9, 1:5 or about 1:5, 1:5.1 or about 1:5.1, 1:5.2 or about 1:5.2, 1:5.3 or about 1:5.3, 1:5.4 or about 1:5.4, 1:5.5 or about 1:5.5, 1:5.6 or about 1:5.6, 1:5.7 or about 1:5.7, 1:5.8 or about 1:5.8, 1:5.9 or about 1:5.1:6.9, 1:6 or about 1:6, 1:6.1 or about 1:6.1, 1:6.2 or about 1:6.2, 1:6.3 or about 1:6.3, 1:6.4 or about 1:6.4, 1:6.5 or about 1:6.5, 1:6.6 or about 1:6.6, 1:6.7 or about 1:6.7, 1:6.8 or about 1:6.8, 1:6.9 or about 1:6.9, 1:7 or or about 1:7, 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, or about 1: 8, 1:8.1 or about 1:8.1, 1:8.2 or about 1:8.2, 1:8.3 or about 1:8.3, 1:8.4 or about 1:8.4, 1:8.5 or about 1:8.5, 1:8.6 or about 1:8.6, 1:8.7 or about 1:8.7, 1:8.8 or about 1:8.8, 1:8.9 or about 1:8.9, 1:9 or about 1:9, 1:9. The ratio is selected from 1 or about 1:9.1, 1:9.2 or about 1:9.2, 1:9.3 or about 1:9.3, 1:9.4 or about 1:9.4, 1:9.5 or about 1:9.5, 1:9.6 or about 1:9.6, 1:9.7 or about 1:9.7, 1:9.8 or about 1:9.8, 1:9.9 or about 1:9.9, or 1:10 or about 1:10.

[0284]

[0301] In some embodiments, the number of APCs in the first expansion culture is about 1.0 x 10 6 APC / cm 2 ~Approx. 4.5×10 6 APC / cm 2 and the number of APCs in the second expansion culture was approximately 2.5 x 10 6 APC / cm 2 ~Approx. 7.5×10 6 APC / cm 2 The range is.

[0285]

[0302] In some embodiments, the number of APCs in the first expansion culture is about 1.5 x 106 APC / cm 2 ~Approx. 3.5×10 6 APC / cm 2 and the number of APCs in the second expansion culture was approximately 3.5 x 10 6 APC / cm 2 ~Approx. 6.0×10 6 APC / cm 2 The range is.

[0286]

[0303] In some embodiments, the number of APCs in the first expansion culture is about 2.0 x 10 6 APC / cm 2 ~Approx. 3.0×10 6 APC / cm 2 and the number of APCs in the second expansion culture was approximately 4.0 x 10 6 APC / cm 2 ~Approx. 5.5×10 6 APC / cm 2 The range is.

[0287]

[0304] In some embodiments, the TIL manufacturing process includes a first expansion step (or pre-REP) of at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days and a second expansion step (or REP) of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0288]

[0305] In some embodiments, the TIL manufacturing process comprises a first expansion step of about 21-35 days and a second expansion step of about 6-12 days, In some embodiments, the TIL manufacturing process comprises a first expansion step of about 21-25 days and a second expansion step of about 7-11 days.

[0289]

[0306] The following steps apply to any TIL fabrication embodiment disclosed herein.

[0290] G. Optional Cell Viability Analysis

[0307] Optionally, after the first or second expansion, a cell viability assay can be performed using standard assays known in the art. For example, a sample of TILs can be subjected to a trypan blue dye exclusion assay, which selectively labels dead cells and allows for assessment of viability. Other assays used to test viability include, but are not limited to, the Alamar Blue assay and the MTT assay. 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.

[0291]

[0308] In some embodiments, cell number and / or viability are measured. Expression of markers such as, but not limited to, CD3, CD4, CD8, CD45, 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, those commercially available from BD Biosciences (BD Biosciences, San Jose, CA) using a FACSCanto™ flow cytometer (BD Biosciences). 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.

[0292]

[0309] In some embodiments, B- and T-lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4; also known as CD152), inducible T-cell costimulator (ICOS), Ki67 (also known as MKI67), lymphocyte-activation gene 3 (LAG3; also known as CD223), programmed cell death protein 1 (PD1), integrin, alpha E (ITGAE; also known as CD103), CD69, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and T-cell immunoglobulin and mucin domain-containing-3 (TIM3; also known as hepatitis A virus cellular receptor 2 (HAVCR2)). Expression of these markers can be measured at any time during the processes disclosed herein, including after recovery of the first expansion, second expansion, or third TIL populations.

[0293]

[0310] In some cases, the second TIL population can be immediately cryopreserved using the protocol discussed below. Alternatively, the second 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 second or third TIL population can be subjected to genetic modification for the appropriate treatment.

[0294]

[0311] 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 reactivity.

[0295]

[0312] Diverse antigen receptors for T and B lymphocytes are generated by somatic recombination of a limited number of gene segments. These gene segments, V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). The present invention provides methods for generating TILs exhibiting increased T cell repertoire diversity. In some embodiments, TILs obtained by the methods exhibit increased T cell repertoire diversity. In some embodiments, TILs obtained in the first or 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 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., TCRα / β) is increased.

[0296] Cell culture materials and methods useful in the present invention

[0313] In one embodiment of the present invention, the method for expanding TILs 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 one embodiment, one or less types of cell culture medium are used to expand the number of TILs. Any suitable cell culture medium may be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, CA). In this regard, the method of the present invention advantageously reduces 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 may include adding fresh cell culture medium to the cells (also referred to as feeding the cells) no more frequently than every two or three days. Expanding the number of cells in a gas-permeable container simplifies the procedure required to expand the number of cells by reducing the feeding frequency required to expand the cells.

[0297]

[0314] In some embodiments, the culture medium used in the expansion culture processes disclosed herein is a serum-free or defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell culture medium and a serum supplement and / or serum replacement. In some embodiments, the serum-free or defined medium is used to prevent and / or reduce experimental variation due, in part, to lot-to-lot variation in serum-containing medium.

[0298]

[0315] In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and / or serum replacement. In some embodiments, the basal cell medium includes, but is not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle's (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0299]

[0316] In some embodiments, the serum supplement or serum replacement includes, but is not limited to, one or more of CTS™ OpTmizer T Cell Expansion Serum Supplement, CTS™ Immune Cell Serum Replacement, one or more albumin or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium includes albumin and glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag. + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3 ", Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb+ , Sn 2+ and Zr 4+ and one or more components selected from the group consisting of compounds comprising: In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and / or 2-mercaptoethanol.

[0300]

[0317] In some embodiments, CTS™ OpTmizer™ T Cell Immune Cell Serum Replacement is used with conventional growth media including, but not limited to, CTS™ OpTmizer™ T Cell Expansion Basal Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CTS™ AIM-V Medium, CST™ AIM-V SFM, LymphoONE™ T Cell Expansion Xeno-Free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle's (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, Iscove's Modified Dulbecco's Medium.

[0301]

[0318] In some embodiments, the total serum replacement concentration (volume %) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or more of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

[0302]

[0319] In some embodiments, the serum-free or defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement, mixed prior to use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific). In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with about 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0303]

[0320] In some embodiments, the defined medium is CTS™ OpTmizer™ T Cell Expansion SFM (ThermoFisher Scientific). Any formulation of CTS™ OpTmizer™ is useful in the present invention. CTS™ OpTmizer™ T Cell Expansion SFM is a combination of 1 L of CTS™ OpTmizer™ T Cell Expansion Basal Medium and 26 mL of CTS™ OpTmizer™ T Cell Expansion Supplement, mixed prior to use. In some embodiments, CTS™ OpTmizer™ T Cell Expansion SFM is supplemented with approximately 3% CTS™ Immune Cell Serum Replacement (SR) (ThermoFisher Scientific) along with 55 mM 2-mercaptoethanol. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further contains about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further comprises about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific), 55 mM 2-mercaptoethanol, and 2 mM L-glutamine, and further comprises about 6000 IU / mL of IL-2.In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific) and 55 mM 2-mercaptoethanol, and further comprises about 1000 IU / mL to about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 1000 IU / mL to about 8000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 3000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific) and about 2 mM glutamine, and further comprises about 6000 IU / mL of IL-2. In some embodiments, CTS™ OpTmizer™ T cell expansion SFM is supplemented with about 3% CTS™ immune cell serum replacement (SR) (ThermoFisher Scientific), and the final concentration of 2-mercaptoethanol in the medium is 55 μM.

[0304]

[0321] In some embodiments, the serum-free or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 0.1 mM to about 10 mM, 0.5 mM to about 9 mM, 1 mM to about 8 mM, 2 mM to about 7 mM, 3 mM to about 6 mM, or 4 mM to about 5 mM. In some embodiments, the serum-free or defined medium is supplemented with glutamine (i.e., GlutaMAX®) at a concentration of about 2 mM.

[0305]

[0322] In some embodiments, the serum-free or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 5 mM to about 150 mM, 10 mM to about 140 mM, 15 mM to about 130 mM, 20 mM to about 120 mM, 25 mM to about 110 mM, 30 mM to about 100 mM, 35 mM to about 95 mM, 40 mM to about 90 mM, 45 mM to about 85 mM, 50 mM to about 80 mM, 55 mM to about 75 mM, 60 mM to about 70 mM, or about 65 mM. In some embodiments, the serum-free or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

[0306]

[0323] In some embodiments, the defined media described in International Publication No. WO 1998 / 030679, incorporated herein by reference, are useful in the present invention. That publication describes serum-free eukaryotic cell culture media. The serum-free eukaryotic cell culture media include basal cell culture media supplemented with serum-free supplements capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement includes, or is obtained by combining, one or more components selected from the group consisting of one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the defined media further includes L-glutamine, sodium bicarbonate, and / or β-mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, and the trace element moiety Ag. + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3 ", Ge 4+ , Se 4+ , Br, T, Mn 2+ , P, Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn2+ and Zr 4+ and one or more components selected from the group consisting of compounds comprising: In some embodiments, the basal cell culture medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Medium Eagle's (BME), RPMI 1640, F-10, F-12, Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (G-MEM), RPMI Growth Medium, and Iscove's Modified Dulbecco's Medium.

[0307]

[0324] In some embodiments, the concentration of glycine in the defined medium ranges from about 5 to 200 mg / L, the concentration of L-histidine from about 5 to 250 mg / L, the concentration of L-isoleucine from about 5 to 300 mg / L, the concentration of L-methionine from about 5 to 200 mg / L, the concentration of L-phenylalanine from about 5 to 400 mg / L, the concentration of L-proline from about 1 to 1000 mg / L, the concentration of L-hydroxyproline from about 1 to 45 mg / L, the concentration of L-serine from about 1 to 250 mg / L, the concentration of L-threonine from about 10 to 500 mg / L, and the concentration of L-tryptophan from about 2 to 110 mg / L. g / L, the concentration of L-tyrosine is about 3-175 mg / L, the concentration of L-valine is about 5-500 mg / L, the concentration of thiamine is about 1-20 mg / L, the concentration of reduced glutathione is about 1-20 mg / L, the concentration of L-ascorbic acid-2-phosphate is about 1-200 mg / L, the concentration of iron-saturated transferrin is about 1-50 mg / L, the concentration of insulin is about 1-100 mg / L, the concentration of sodium selenite is about 0.000001-0.0001 mg / L, and the concentration of albumin (e.g., AlbuMAX® I) is about 5,000-50,000 mg / L.

[0308]

[0325] In some embodiments, the non-trace element components in the defined medium are present in the concentration ranges listed in the column under the heading "Concentration Ranges in 1x Medium" in Table A below. In other embodiments, the non-trace element components in the defined medium are present at the final concentrations listed in the column under the heading "Preferred Embodiments in 1x Medium" in Table A below. In other embodiments, the defined medium is a basal cell culture medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement comprises non-trace element components of the type and concentration listed in the column under the heading "Preferred Embodiments of Supplement" in Table A below.

[0309] [Table 3]

[0310]

[0326] In some embodiments, the osmolality of the defined medium is about 260-350 mOsmol. In some embodiments, the osmolality is about 280-310 mOsmol. In some embodiments, the defined medium is supplemented with sodium bicarbonate up to about 3.7 g / L or about 2.2 g / L. The defined medium can be further supplemented with L-glutamine (final concentration about 2 mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration about 100 μM), and 2-mercaptoethanol (final concentration about 100 μM).

[0311]

[0327] In some embodiments, the defined media described in Smith, et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement," Clin Transl Immunology, 4(1) 2015 (doi: 10.1038 / cti.2014.31) are useful in the present invention. Briefly, RPMI or CTS™ OpTmizer™ was used as the basal cell medium and supplemented with 0, 2%, 5%, or 10% CTS™ Immune Cell Serum Replacement.

[0312]

[0328] 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, CAS60-24-2).

[0313]

[0329] In one embodiment, the cell culture medium 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 lacks beta-mercaptoethanol (BME).

[0314]

[0330] In one embodiment, the duration of the method described herein is about 27 to about 50 days. In another embodiment, the duration is about 28 to about 46 days. In another embodiment, the duration is about 30 to about 42 days. In one embodiment of the present invention, the duration of the method from the start of the first expansion culture to the end of the second expansion culture is about 31 days. In one embodiment of the present invention, the duration of the method from the start of the first expansion culture to the end of the second expansion culture is about 35 days.

[0315]

[0331] In one embodiment, TILs are expanded in gas-permeable containers. Gas-permeable containers are used to expand TILs 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 gas-permeable bags. In one embodiment, TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion 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. In one embodiment, TILs can be expanded in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such an embodiment expands the cell population to about 5 x 10 5 cells / cm 2 From 10 x 10 6 ~30×10 6 cells / cm 2This allows cells to be expanded without adding fresh cell culture medium to the cells (also referred to as feeding the cells). In one embodiment, this is done without feeding, as long as there is about 10 cm of medium in the GRex flask. In one embodiment, this is without feeding, but with the addition of one or more cytokines. In one embodiment, the cytokines can be added as a bolus, without the need to mix them 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.

[0316] a. Anti-CD3 antibody

[0332] In some embodiments, the culture medium used in the expansion methods described herein contains an anti-CD3 antibody. When combined with IL-2, anti-CD3 antibodies 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, with the former generally being preferred; see, e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719 (incorporated herein by reference in its entirety).

[0317]

[0333] As one of skill 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 is used (commercially available from Ortho-McNeil, Raritan, NJ, or Miltenyi Biotech, Auburn, CA).

[0318] [Table 4]

[0319] b.TNFRSF agonist (4-1BB (CD137))

[0334] In one embodiment, the cell culture medium of the first expansion culture and / or the second expansion culture contains a TNFRSF agonist. 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.

[0320]

[0335] 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.

[0321]

[0336] 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.

[0322]

[0337] 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.

[0323] [Table 5]

[0324]

[0338] 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.

[0325]

[0339] 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.

[0326]

[0340] 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.

[0327]

[0341] 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.

[0328]

[0342] 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.

[0329]

[0343] 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 shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0330]

[0344] 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.

[0331]

[0345] 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.

[0332]

[0346] 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, where 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, the biosimilar is provided as a composition further comprising one or more excipients, which are the same as 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, the biosimilar is provided as a composition further comprising one or more excipients, which are the same as or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is utomilumab.

[0333] [Table 6]

[0334] [Table 7]

[0335] [Table 8]

[0336]

[0348] 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 EE. Urelumab has N-glycosylation sites at positions 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 light chain intrasulfide bridges at positions 23'''-88''' and 136'''-196'''); intrachain heavy-heavy chain disulfide bridges at positions 227-227'' and 230-230''; and intrachain heavy-light chain disulfide bridges at positions 135-216' and 135''-216'''. The preparation and properties of urelumab and its variants and fragments 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.

[0337]

[0349] 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.

[0338]

[0350] 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 region.

[0339]

[0351] 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.

[0340]

[0352] 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, where 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, which are the same as 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, which are the same as or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is urelumab.

[0341] [Table 9]

[0342] [Table 10]

[0343]

[0354] 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-46 9497; 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, the disclosures of each of the foregoing patents or patent application publications are incorporated herein by reference.

[0344]

[0355] 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.

[0345]

[0356] 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 15, or a fragment, derivative, conjugate, variant, or biosimilar thereof.

[0346]

[0357] 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 and then bind 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 receptor intracellular signaling domains and signaling proteins 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 can 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.

[0347]

[0358] 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.

[0348] [Table 11]

[0349]

[0360] 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.

[0350] [Table 12]

[0351]

[0362] 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 10, any combination of the above variable heavy and variable light chains, and fragments, derivatives, conjugates, variants, and biosimilars thereof.

[0352]

[0363] 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.

[0353]

[0364] In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB comprises a V sequence at least 95% identical to the sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. H and V L one or more 4-1BB binding domains, which are scFv domains containing the 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, which are scFv domains containing the 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, which are scFv domains containing the V H and V L The domains are connected by linkers.

[0354] [Table 13]

[0355] [Table 14]

[0356]

[0366] 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 additional domains at the N-terminus and / or C-terminus, wherein the additional domains are Fab or Fc fragment domains, 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.

[0357]

[0367] 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 are independently 3 to 8 amino acids in length; and each TNF superfamily cytokine domain is a 4-1BB binding domain.

[0358]

[0368] 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.

[0359]

[0369] 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.

[0360] c.OX40 (CD134) agonist

[0370] 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).

[0361]

[0371] 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.

[0362]

[0372] 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.

[0363]

[0373] 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 12.

[0364] [Table 15]

[0365]

[0375] 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.

[0366]

[0376] 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.

[0367]

[0377] 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.

[0368]

[0378] 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.

[0369]

[0379] 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 13. 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.

[0370]

[0380] 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.

[0371]

[0381] 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 region.

[0372]

[0382] 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.

[0373]

[0383] 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, where 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, which are the same as 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, which are the same as or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is taborixizumab.

[0374] [Table 16]

[0375] [Table 17]

[0376] [Table 18]

[0377]

[0385] In some embodiments, the OX40 agonist is 11D4, 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 14.

[0378]

[0386] 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.

[0379]

[0387] 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 LIn 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.

[0380]

[0388] 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.

[0381]

[0389] 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, wherein 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, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and wherein 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 as 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 as or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is 11D4.

[0382] [Table 19]

[0383] [Table 20]

[0384] [Table 21]

[0385]

[0391] 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 15.

[0386]

[0392] 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.

[0387]

[0393] 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.

[0388]

[0394] 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.

[0389]

[0395] 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, wherein 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, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug or reference biological product, and wherein 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 as 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 as or different from the excipients contained in the reference drug or reference biological product, and the reference drug or reference biological product is 18D8.

[0390] [Table 22]

[0391] [Table 23]

[0392]

[0397] 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 16.

[0393]

[0398] 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.

[0394]

[0399] 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.

[0395]

[0400] 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, where 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, which are the same as 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, which are the same as 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.

[0396] [Table 24]

[0397]

[0402] 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 17.

[0398]

[0403] 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 V LIncludes the area.

[0399]

[0404] 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.

[0400]

[0405] 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, where 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, which are the same as 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, which are the same as 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.

[0401] [Table 25]

[0402] [Table 26]

[0403]

[0407] 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.

[0404]

[0408] 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.

[0405]

[0409] 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 / 190507. 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.

[0406]

[0410] 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 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 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 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 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.

[0407]

[0411] 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 17, any combination of the above variable heavy and variable light chains, and fragments, derivatives, conjugates, variants, and biosimilars thereof.

[0408]

[0412] 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.

[0409]

[0413] 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, which are scFv domains containing the 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, which are scFv domains containing the 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, which are scFv domains containing the V H and V LThe 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, which are scFv domains containing the 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, which are scFv domains containing the 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 the V domain shown in Table 14. 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, which are scFv domains containing the V H and V L The domains are connected by linkers.

[0410] [Table 27]

[0411] [Table 28]

[0412] [Table 29]

[0413] [Table 30]

[0414]

[0415] 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, 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.

[0415]

[0416] 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 are independently 3 to 8 amino acids in length; and the TNF superfamily cytokine domain is an OX40-binding domain.

[0416]

[0417] 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.

[0417]

[0418] 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.

[0418]

[0419] 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.

[0419]

[0420] In one embodiment, the OX40 agonist is the OX40 agonistic antibody clone Ber-ACT35, commercially available from BioLegend, Inc., San Diego, CA, USA.

[0420] d. cytokines

[0421] The first and second expansion methods described herein generally use culture media containing high doses of cytokines, particularly IL-2, as is known in the art.

[0421]

[0422] Alternatively, it is further possible to use a combination of cytokines in the secondary expansion of TILs, such as combinations of two or more of IL-2, IL-15, and IL-21, as generally 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 and IL-21, and IL-2, IL-15, and IL-21, the latter of which finds particular use in many embodiments. As described therein, the use of a combination of cytokines is particularly advantageous for the generation of lymphocytes, particularly T cells.

[0422] Phenotypic characteristics of expanded TILs

[0423] In some embodiments, the TILs are analyzed for expression of multiple phenotypic markers after expansion, including those described herein and in the Examples. In one embodiment, expression of one or more phenotypic markers is examined. In some embodiments, the phenotypic characteristics of the TILs are analyzed after the first expansion in step B. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transfer in step C. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transfer and after cryopreservation in step C. In some embodiments, the phenotypic characteristics of the TILs are analyzed after the second expansion in step D. In some embodiments, the phenotypic characteristics of the TILs are analyzed after two or more expansions in step D. In some embodiments, the markers are selected from the group consisting of TCRab, CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, the markers are selected from the group consisting of TCRab, CD57, CD28, CD4, CD27, CD56, and CD8a. In certain embodiments, the markers are selected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 markers is examined. In some embodiments, expression from one or more markers from each group is examined. In some embodiments, one or more of HLA-DR, CD38, and CD69 expression is maintained (i.e., does not exhibit a statistically significant difference) in fresh TILs compared to thawed TILs. In some embodiments, the activation state of TILs is maintained in thawed TILs.

[0423]

[0424] In one embodiment, the expression of one or more regulatory markers is measured. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, LAG3, CD4, CD3, PD1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, LAG3, CD4, CD3, PD1, and TIM-3. In some embodiments, the regulatory marker is selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, regulatory molecule expression is decreased in thawed TILs compared to fresh TILs. In some embodiments, expression of regulatory molecules LAG-3 and TIM-3 is decreased in thawed TILs compared to fresh TILs. In some embodiments, there is no significant difference in CD4, CD8, NK, or TCRαβ expression. In some embodiments, there is no significant difference in CD4, CD8, NK, TCRαβ expression and / or memory markers in fresh TILs compared to thawed TILs.

[0424]

[0425] In some embodiments, the memory marker is selected from the group consisting of CCR7 and CD62L.

[0425]

[0426] In some embodiments, the viability of fresh TILs compared to thawed TILs is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In some embodiments, the viability of both fresh and thawed TILs is greater than 70%, 75%, 80%, 85%, 90%, 95%, or 98%. In some embodiments, the viability of both fresh and thawed products is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%. In some embodiments, the viability of both fresh and thawed products is greater than 86%.

[0426]

[0427] In one embodiment, restimulated TILs can also be assessed for cytokine release using a cytokine release assay. In some embodiments, TILs can be assessed for interferon-7 (IFN-7) secretion in response to stimulation with either OKT3 or co-culture with autologous tumor digest. For example, in embodiments using OKT3 stimulation, TILs are washed extensively and plated at 1 x 10 in 0.2 mL CM onto a 96-well flat-bottom plate pre-coated with 0.1 or 1.0 μg / mL OKT3 diluted in phosphate-buffered saline. 5 Duplicate wells are prepared with cells. After overnight incubation, the supernatant is collected and IFN-gamma in the supernatant is measured by ELISA (Pierce / Endogen, Woburn, MA). For co-culture assays, 1 x 10 cells are used. 5 TIL cells are plated in 96-well plates together with autologous tumor cells (1:1 ratio). After 24 hours of incubation, supernatants can be collected and IFN-gamma release can be quantified, for example, by ELISA.

[0427]

[0428] Flow cytometry analysis of cell surface biomarkers: TIL samples were aliquoted for flow cytometry analysis of cell surface markers.

[0428]

[0429] In some embodiments, the TILs are assessed for various regulatory markers. In some embodiments, the regulatory marker is selected from the group consisting of TCRα / β, CD56, CD27, CD28, CD57, CD45RA, CD45RO, CD25, CD127, CD95, IL-2R, CCR7, CD62L, KLRG1, and CD122. In some embodiments, the regulatory marker is TCRα / β. In some embodiments, the regulatory marker is CD56. In some embodiments, the regulatory marker is CD27. In some embodiments, the regulatory marker is CD28. In some embodiments, the regulatory marker is CD57. In some embodiments, the regulatory marker is CD45RA. In some embodiments, the regulatory marker is CD45RO. In some embodiments, the regulatory marker is CD25. In some embodiments, the regulatory marker is CD127. In some embodiments, the regulatory marker is CD95. In some embodiments, the regulatory marker is IL-2R. In some embodiments, the regulatory marker is CCR7. In some embodiments, the regulatory marker is CD62L. In some embodiments, the regulatory marker is KLRG1. In some embodiments, the regulatory marker is CD122.

[0429]

[0430] Additional Process Embodiments

[0431] In some embodiments, the present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) performing the first expansion by culturing the first TIL population in a cell culture medium comprising IL-2, a 4-1BB agonist, and OKT-3, wherein the first expansion is performed for about 21 to 35 days to obtain a second TIL population; (c) performing a second expansion culture by contacting the second TIL population with a cell culture medium comprising IL-2, a 4-1BB agonist, OKT-3, and exogenous antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion culture is performed for about 6 to 10 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population; and (d) recovering the therapeutic TIL population obtained from step (c). In some embodiments, the second expansion step is divided into multiple steps to achieve culture scale-up by (1) performing the second expansion by culturing the second TIL population in a small-scale culture in a first vessel, e.g., a G-REX 100MCS container, for a period of about 2 to 4 days, and then (2) transferring the second TIL population from the small-scale culture to a second vessel larger than the first vessel, e.g., a G-REX 500MCS container, in which the second TIL population from the small-scale culture is cultured in a larger-scale culture in the second vessel for a period of about 4 to 8 days.In some embodiments, the expansion step is divided into multiple steps to achieve scale-out of the culture by (1) performing a second expansion culture by culturing the second TIL population in a first vessel, e.g., a G-REX 100MCS container, in a first small-scale culture for a period of about 2 to 4 days, and then (2) transferring and distributing the second TIL population from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels the same size as the first vessel, wherein in each second vessel, a portion of the second TIL population from the first small-scale culture transferred to such second vessel is cultured in a second small-scale culture for a period of about 4 to 8 days. In some embodiments, the second expansion step is divided into multiple steps to achieve scale-out and scale-up of the culture by (1) performing the second expansion by culturing the second TIL population in a first vessel, e.g., a G-REX 100MCS container, in a small-scale culture for a period of about 2 to 4 days, and then (2) transferring and allocating the second TIL population from the first small-scale culture to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels larger in size than the first vessel (e.g., a G-REX 500MCS container), wherein in each second vessel, a portion of the second TIL population transferred from the small-scale culture to such second vessel is cultured in a larger-scale culture for a period of about 4 to 8 days.In some embodiments, the second expansion step is divided into multiple steps to achieve scale-out and scale-up of the culture by (1) performing the second expansion by culturing the second TIL population in a first vessel, e.g., a G-REX 100MCS container, in a small-scale culture for a period of about 3 to 4 days, and then (2) transferring and distributing the second TIL population from the first small-scale culture to two, three, or four second vessels larger in size than the first vessel (e.g., a G-REX 500MCS container), and in each second vessel, a portion of the second TIL population transferred from the small-scale culture to such second vessel is cultured in a larger-scale culture for a period of about 5 to 7 days.

[0430]

[0432] In some embodiments, the present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) performing the first expansion by culturing the first TIL population in a cell culture medium comprising IL-2, a 4-1BB agonist, and OKT-3, wherein the first expansion is performed for about 21 to 35 days to obtain a second TIL population; (c) performing a second expansion culture by contacting the second TIL population with a cell culture medium comprising IL-2, a 4-1BB agonist, OKT-3, and exogenous antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion culture is performed for about 7 to 12 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population; and (d) recovering the therapeutic TIL population obtained from step (c). In some embodiments, the second expansion step is divided into multiple steps to achieve culture scale-up by (1) performing the second expansion by culturing the second TIL population in a small-scale culture in a first vessel, e.g., a G-REX 100MCS container, for a period of about 2 to 4 days, and then (2) transferring the second TIL population from the small-scale culture to a second vessel larger than the first vessel, e.g., a G-REX 500MCS container, in which the second TIL population from the small-scale culture is cultured in a larger-scale culture in the second vessel for a period of about 4 to 8 days.In some embodiments, the second expansion step is divided into multiple steps to achieve scale-out of the culture by (1) performing the second expansion by culturing the second TIL population in a first vessel, e.g., a G-REX 100MCS container, in a first small-scale culture for a period of about 2 to 4 days, and then (2) transferring and distributing the second TIL population from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels the same size as the first vessel, wherein in each second vessel, a portion of the second TIL population from the first small-scale culture transferred to such second vessel is cultured in a second small-scale culture for a period of about 4 to 8 days. In some embodiments, the second expansion step is divided into multiple steps to achieve scale-out and scale-up of the culture by (1) performing the second expansion by culturing the second TIL population in a first vessel, e.g., a G-REX 100MCS container, in a small-scale culture for a period of about 2 to 4 days, and then (2) transferring and allocating the second TIL population from the first small-scale culture to at leas...

Claims

1. 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 resected from a subject; (b) performing a first expansion culture by culturing the first TIL population in a cell culture medium comprising a 4-1BB agonist, IL-2, and OKT-3 for a period of 21 to 35 days to produce a second TIL population; and (c) performing a second expansion culture by supplementing the cell culture medium of the second TIL population with antigen presenting cells (APCs) and additional 4-1BB agonists, IL-2 and OKT-3, and culturing for a period of 7 to 10 days to produce a third TIL population, which is a therapeutic TIL population; A method comprising:

2. (d) recovering the therapeutic TIL population obtained from step (c); and (e) transferring the recovered TIL population from step (d) into an infusion bag. The method of claim 1 further comprising:

3. 10. The method of claim 1, further comprising carrying out said culturing step (b) in the presence of antigen-presenting cells (APCs).

4. The method according to any one of claims 1 to 3, wherein the APCs are peripheral blood mononuclear cells (PBMCs).

5. The method of claim 3, wherein the ratio of the number of APCs in the second expansion culture to the number of APCs in the first expansion culture is in the range of 1.5:1 to 20:

1.

6. 6. The method of claim 5, wherein the ratio is 2:

1.

7. The method of claim 1 , wherein the second population of TILs is cryopreserved.

8. 2. The method of claim 1, wherein the first expansion culture is carried out for a period of 21 to 28 days, and the second expansion culture is carried out for a period of 7 to 9 days.

9. 2. The method of claim 1, wherein the first expansion culture is carried out for a period of 28 to 35 days, and the second expansion culture is carried out for a period of 7 to 9 days.

10. 2. The method of claim 1, wherein the first expansion culture is carried out for a period of 21 days, and the second expansion culture is carried out for a period of 7 to 9 days.

11. 2. The method of claim 1, wherein one or both of the second or third TIL populations comprises an expanded subpopulation of effector T cells and / or central memory T cells relative to the first or second TIL population.

12. 2. The method of claim 1, wherein one or both of the second or third TIL populations comprises an expanded subpopulation of cells expressing one or more of BTLA, Ki67, LAG3, TIGIT, and TIM3.

13. 2. The method of claim 1, wherein one or both of the second or third TIL populations comprises a depleted subpopulation of cells expressing one or more of CTLA-4, ICOS, PD-1, CD103+CD69+, and CD103+CD69-.

14. 2. The method of claim 1, wherein one or both of the second or third TIL populations comprises an expanded subpopulation of CD45+ cells.

15. 2. The method of claim 1, wherein one or both of the second or third TIL populations comprises an expanded subpopulation of CD45+CD3+ cells.

16. 2. The method of claim 1, wherein one or both of the second or third TIL populations comprises an expanded subpopulation of CD8+ cells.

17. 2. The method of claim 1, wherein one or both of the second or third TIL populations comprises a depleted subpopulation of CD4+ cells.

18. 10. The method of claim 1, wherein the tumor is of a cancer type selected from the group consisting of thyroid cancer, melanoma, cervical cancer, endometrial cancer, colon cancer, and colorectal cancer.

19. 19. The method of any one of claims 1 to 18, wherein the second TIL population is at least 50 times greater in number than the first TIL population.

20. the second population of TILs is at least 4 x 10 ７ The method according to any one of claims 1 to 18, wherein the cell is a cell.

21. The method of any one of claims 1 to 18, wherein the 4-1BB agonist is utomilumab or urelumab.

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