Compositions and methods for antigen-specific T cell expansion

Abstract

The present disclosure provides improved methods for producing tumor antigen-specific T cells, including methods that shorten procedures and enhance certain aspects of tumor antigen-specific T cells. The present disclosure also provides various compositions and products related to the improved production methods. The present disclosure also provides tumor antigen-specific T cell products.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 310,500, filed February 15, 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] The present disclosure relates to methods for generating tumor antigen-specific T cells, populations, compositions, and products derived therefrom, as well as therapeutic methods thereof.

[0003] Adoptive transfer therapy of expanded tumor antigen-specific T cells is an attractive approach for treating various cancers. The process of producing tumor antigen-specific T cells with desired activity and phenotype at the scale required for clinical use is challenging. In particular, the length of time required to produce tumor antigen-specific T cells for clinical use can adversely affect patient treatment. There is a need in the art for improved methods for producing tumor antigen-specific T cells that can be administered to patients for cancer treatment. There is also a need in the art for improved tumor antigen-specific T cell products that can be used to effectively treat patients. The present disclosure addresses these and other needs. Summary of the Invention

[0004] In one embodiment, the disclosure provides a method for generating tumor antigen-specific T cells, comprising: (i) placing a population of mononuclear cells obtained from a subject into a container comprising at least one gas permeable surface, wherein the gas permeable surface comprises at least one gas permeable surface square centimeter (cm 2 ) approximately 0.1 × 10 6 ~Approx. 1×10 7Disclosed is a method comprising: (i) adding mononuclear cells to a container at a cell seeding density of 100-1500 μg / ml to form a cell culture; and (ii) adding one or more peptides from one or more tumor antigens to the cell culture, and culturing the one or more peptides with the mononuclear cells in the container at about 37° C. in the absence of exogenous cytokines. In some embodiments, the method further comprises adding one or more exogenous cytokines to the cell culture. In a further embodiment, the method comprises harvesting the tumor antigen-specific T cells, e.g., for autologous transplantation or for cryopreservation.

[0005] In one embodiment, each peptide of the one or more peptides is added to the culture at a concentration of about 0.1 μg / mL to about 0.5 μg / mL. In one embodiment, each peptide of the one or more peptides is added to the culture at a concentration of about 0.1 μg / mL, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, or about 0.5 μg / mL. In an embodiment, the one or more peptides include one or more peptides derived from one or more tumor antigens. In some embodiments, the one or more peptides include one or more libraries of peptides derived from one or more tumor antigens. In an embodiment, each library of peptides includes multiple peptides, and in some embodiments, the multiple peptides span the sequence of the target tumor antigen. In an embodiment, each library of peptides is added to the culture at a concentration of about 0.1 to about 0.5 μg / mL for each peptide. In embodiments, the one or more peptides are added to the culture at a concentration of about 0.1 μg / mL, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, or about 0.5 μg / mL for each peptide.

[0006] In one embodiment, the mononuclear cells and one or more peptides are cultured in a container at about 37° C. prior to the addition of one or more exogenous cytokines. In some embodiments, the mononuclear cells and one or more peptides are cultured in a container at about 37° C. for at least about 30 minutes prior to the addition of one or more exogenous cytokines. In some embodiments, the mononuclear cells and one or more peptides are cultured in a container at about 37° C. for at least about 30 minutes to about 30 hours prior to the addition of one or more exogenous cytokines. In an embodiment, the mononuclear cells and one or more peptides are cultured in a container at about 37° C. for about 8 to about 24 hours prior to the addition of one or more exogenous cytokines. In one embodiment, the mononuclear cells and one or more peptides are cultured in a container at about 37° C. overnight prior to the addition of one or more exogenous cytokines.

[0007] In one embodiment, mononuclear cells are present at a density of about 0.1 x 10 per square centimeter of gas permeable surface. 6 ~Approx. 1×10 7 Cells are added to the vessel at a seeding density of approximately 0.1 x 10. For example, mononuclear cells are added at a seeding density of approximately 0.1 x 10. 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 , about 1×10 6 cells / cm 2 , about 2×10 6 cells / cm 2 , about 3×10 6 cells / cm 2 , about 4×10 6 cells / cm 2 , about 5×10 6 cells / cm 2 , about 6×10 6 cells / cm 2 , about 7×10 6 cells / cm 2 , about 8×10 6 cells / cm 2 , about 9×10 6 cells / cm 2 , or about 1 × 10 7The mononuclear cells are added to the vessel at a seeding density of about 0.1×10 cells / mL. In one embodiment, the mononuclear cells are added at a seeding density of about 0.1×10 6 cells / mL ~ approx. 1×10 7 The cells are cultured with one or more peptides at a cell concentration of about 0.1×10 cells / mL. For example, mononuclear cells are cultured at a cell concentration of about 0.1×10 6 cells / mL, approximately 0.5×10 6 cells / mL, approximately 1×10 6 cells / mL, approximately 2×10 6 cells / mL, approximately 3×10 6 cells / mL, approximately 4×10 6 cells / mL, approximately 5×10 6 cells / mL, approximately 6×10 6 cells / mL, approximately 7×10 6 cells / mL, approximately 8×10 6 cells / mL, approximately 9×10 6 cells / mL, or approximately 1 x 10 7 The mononuclear cells are added to the container at a cell concentration of 10000000000000000 cells / mL. In one embodiment, the mononuclear cells are peripheral blood mononuclear cells (PBMCs) or leukocytes. In one embodiment, the mononuclear cells are obtained from a subject (e.g., a donor) by leukapheresis. In one embodiment, the donor is a healthy donor. In another embodiment, the donor is a patient to whom T cells with specificity for multiple tumor-associated antigens (MultiTAA-specific T cells) are generated and then administered.

[0008] In one embodiment, the container comprises at least one gas permeable surface. In one embodiment, the container comprises 1, 2, 3, 4, 5, or 6 gas permeable surfaces. In one embodiment, the container comprising at least one gas permeable surface is a G-Rex device. For example, in one embodiment, the container is a 100M G-Rex device or a 500M G-Rex device.

[0009] In one embodiment, each library of peptides comprises peptides spanning the sequence of said tumor antigen. In one embodiment, the method further comprises adding one or more peptides from at least four tumor antigens, optionally a library of one or more peptides, prior to adding one or more exogenous cytokines. In one embodiment, the method comprises adding one or more peptides from at least six tumor antigens, optionally a library of one or more peptides, prior to adding one or more exogenous cytokines. In one embodiment, the method comprises adding one or more peptides from 4, 6, 10, or 12 different tumor antigens, optionally a library of one or more peptides, prior to adding one or more exogenous cytokines.In one embodiment, the tumor antigen is CEA, MHC, CTLA-4, gp100, mesothelin, PRAME (OIP3) PD-L1, TRP1, CD40, EGFP, Her2, TCR alpha, trp2, TCR, MUC1, cdr2, ras, 4-1BB, CT26, GITR, OX40, TGF-α. WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, Melan A / MART1, Ras mutant, gp 100, p53 mutant, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, Myc, MYCN, RhoC, TRP-2, GD3, fucosyl-GM1, mesothelin, PSCA, MAGE A1, MAGE A4, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, mucin-1, BRAF, CCNA1, and Fos-related antigen 1.

[0010] In one embodiment, the one or more tumor antigens are selected from PRAME, NY-ESO-1, survivin, and WT1. In one embodiment, the tumor antigens are PRAME, NY-ESO-1, survivin, and WT1, and the total concentration of peptides added to the container is about 78 μg / mL. In a further embodiment, the container is a 100M G-Rex device, and the total amount of peptides (corresponding to PRAME, NY-ESO-1, survivin, and WT1 antigens) added to the container is about 7.775 mg. In an additional embodiment, the container is a 500M G-Rex device, and the total amount of peptides (corresponding to PRAME, NY-ESO-1, survivin, and WT1 antigens) added to the container is about 38.875 mg. In one embodiment, the method disclosed herein comprises generating tumor antigen-specific T cells specific for each of PRAME, NY-ESO-1, survivin, and WT1.

[0011] In one embodiment, the one or more tumor antigens are selected from PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2. In one embodiment, the tumor antigens are PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2, and the total concentration of peptides added to the container is about 108 μg / mL. In a further embodiment, the container is a 100M G-Rex device, and the total amount of peptides (corresponding to PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2 antigens) added to the container is about 10.825 mg. In an additional embodiment, the container is a 500M G-Rex device, and the total amount of peptides (corresponding to PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2 antigens) added to the container is about 54.125 mg. In one embodiment, the methods disclosed herein comprise generating tumor antigen-specific T cells specific for each of PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2.

[0012] In one embodiment, 1, 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, 29, 30, or more tumor antigens are represented in one or more peptides cultured with mononuclear cells. In one embodiment, the tumor antigen-specific T cell product generated by the method herein exhibits antigen specificity for at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the tumor antigens represented by the one or more peptides used in the initial culture. In one embodiment, the tumor antigen-specific T cell product generated by the method herein exhibits antigen specificity for each of the tumor antigens represented by the one or more peptides. In one embodiment, increasing the number of tumor antigens cultured with mononuclear cells surprisingly increases the antigen specificity as well as the antigen diversity of the tumor antigen-specific T cell product generated.

[0013] In one embodiment, mononuclear cells are obtained from a subject by leukocyte therapy. The subject may be a healthy donor or a patient diagnosed with cancer. In one embodiment, the mononuclear cells are white blood cells or peripheral blood mononuclear cells (PBMCs). In one embodiment, the method of generating tumor antigen-specific T cells disclosed herein comprises depleting CD56+ cells from a population of mononuclear cells obtained from a subject. In one embodiment, depletion is performed by one or more conventional methods, such as, but not limited to, Dynabeads, EasySep® cell separation beads, RosetteSep® immunodensity cell separation, or the Cloudz Human Cell Expansion platform. In one embodiment, the population of mononuclear cells placed in a container for culturing with one or more peptides comprises about 0.05% to about 2.5% CD56+ cells. In one embodiment, the population of mononuclear cells placed in a container for culture with one or more peptides comprises about 5% or less, about 2.5% or less, or about 2% or less CD56+ cells. In one embodiment, the population of mononuclear cells placed in a container for culture with one or more peptides comprises about 35% to about 85% T cells. In one embodiment, the population of T cells comprises both CD4+ T cells and CD8+ T cells.

[0014] In one embodiment, the method for generating tumor antigen-specific T cells disclosed herein comprises depleting CD19+ cells from a population of mononuclear cells obtained from a subject. In one embodiment, depletion of CD19+ cells is performed by one or more conventional methods, such as, but not limited to, magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), Dynabeads, EasySep® cell separation beads, RosetteSep® immunodensity cell separation, or the Cloudz Human Cell Expansion platform.

[0015] In one embodiment, the method of generating tumor antigen-specific T cells disclosed herein comprises depleting CD56+ and CD19+ cells from a population of mononuclear cells obtained from a subject. In one embodiment, depletion of both CD56+ and CD19+ cells is performed by one or more conventional methods, including but not limited to magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), Dynabeads, EasySep® cell separation beads, RosetteSep® immunodensity cell separation, or Cloudz Human Cell Expansion platform. In one embodiment, the tumor antigen-specific T cell product disclosed herein generated by the method comprising depletion of CD19+ T cells is suitable for use in treating patients with B cell cancer.

[0016] In one embodiment, the population of mononuclear cells placed in a vessel for culture with one or more peptides comprises from about 0.01% to about 2% CD56+ cells, from about 0.5% to about 50% monocytes, from about 0.01% to about 1.5% CD19+ B cells, and / or from about 40% to about 99% T cells.

[0017] In one embodiment, the method of generating tumor antigen-specific T cells disclosed herein comprises culturing mononuclear cells and peptides together in the absence of exogenous cytokines, followed by adding one or more exogenous cytokines to the culture. In one embodiment, the method of generating tumor antigen-specific T cells disclosed herein comprises culturing mononuclear cells and peptides together in the absence of exogenous cytokines for at least 8 hours, followed by adding one or more exogenous cytokines to the culture. In one embodiment, the one or more exogenous cytokines added to the culture are selected from IL-6, IL-7, IL-15, IL-12, and any combination thereof. In a further embodiment, the exogenous cytokines added to the culture include IL-6, IL-7, IL-15, and IL-12.

[0018] In one embodiment, the mononuclear cells are cultured in the presence of one or more peptides and one or more exogenous cytokines for about 5 to about 14 days. Thus, in one embodiment, the method for generating tumor antigen-specific T cells disclosed herein comprises culturing mononuclear cells with one or more peptides in the absence of exogenous cytokines in a container comprising at least one gas permeable surface for about 8 to about 30 hours at about 37° C., followed by adding one or more exogenous cytokines to the culture, wherein the mononuclear cells, one or more peptides, and one or more exogenous cytokines are then cultured together in the container at about 37° C. for about 5 to about 14 days. In one embodiment, the method does not comprise a washing step. In one embodiment, the mononuclear cells, one or more peptides, and exogenous cytokines are cultured for about 5 to about 14 days without disturbance, e.g., without intervention such as addition or replacement of medium, addition of peptides, addition of cytokines, or addition of other components.

[0019] In some embodiments, the method comprises, after culturing the mononuclear cells with one or more peptides, splitting the culture into two or more separate cultures prior to adding one or more exogenous cytokines. For example, in some embodiments, splitting the initial culture into two or more cultures allows for further expansion of tumor antigen-specific T cells in the culture in the presence of one or more exogenous cytokines.

[0020] In one embodiment, the method further comprises the step of cryopreserving the mononuclear cells and then thawing the cryopreserved cells prior to culturing with the one or more peptides. In one embodiment, the thawed mononuclear cells are allowed to rest overnight prior to culturing with the one or more peptides. In one embodiment, the thawed mononuclear cells are allowed to rest for about 4 hours to about 48 hours prior to culturing with the one or more peptides. In one embodiment, the method further comprises the step of counting the cells remaining after the resting period prior to initiating culturing with the one or more peptides. In these embodiments, in this way, the seeding density for culturing the mononuclear cells with the one or more peptides can be accurately calculated, e.g., about 0.1×10 6 cells / cm 2 ~Approx. 1×10 7 cells / cm 2 (For example, about 2×10 6 cells / cm 2 , or about 3 × 10 6 cells / cm 2 , or about 4 × 10 6 cells / cm 2 ) and / or approximately 0.1 x 10 6 cells / mL ~ approx. 1×10 7 cells / mL (e.g., approximately 1 x 10 6 cells / mL, or approximately 2 x 10 6 cells / mL, or approximately 3 x 10 6 Place mononuclear cells in a container at a concentration of 1000 x g (cells / mL).

[0021] In one embodiment, the tumor antigen-specific T cells disclosed herein are cryopreserved after collection. In one embodiment, the mononuclear cells and tumor antigen-specific T cells are cryopreserved in CryoStor® CS10 or a derivative thereof.

[0022] In one embodiment, the method disclosed herein further comprises HLA typing the donor from whom the population of mononuclear cells was obtained. In one embodiment, the method disclosed herein comprises storing HLA typing information for the donor and maintaining a bank of one or more tumor antigen-specific T cell products available as off-the-shelf products. In one embodiment, the off-the-shelf products are suitable for use in patients who have a desired level of HLA match, e.g., at least one HLA match, with the donor of the tumor antigen-specific T cell product.

[0023] In one embodiment, the method of generating tumor antigen-specific T cells disclosed herein comprises harvesting the tumor antigen-specific T cells after first culturing with one or more peptides and then one or more exogenous cytokines. In some embodiments, harvesting the tumor antigen-specific T cells does not include purification or enrichment based on antigen specificity. In a further embodiment, the method comprises harvesting the tumor antigen-specific T cells for cryopreservation and / or storage, e.g., as an off-the-shelf product, optionally as part of a cell bank or inventory.

[0024] In one embodiment, the present disclosure provides a population of tumor antigen-specific T cells obtained by the methods disclosed herein. In one embodiment, the present disclosure provides a composition comprising a population of tumor antigen-specific T cells obtained by the methods disclosed herein. In one embodiment, the population of tumor antigen-specific T cells exhibits superior antigen specificity compared to tumor antigen-specific T cells generated by prior methods. In one embodiment, the degree of specificity of the tumor antigen-specific T cells for a tumor antigen is at least about 500 SFU / 2×10 as measured by IFN-γ ELISpot. 5 At least about 700SFU / 2×10 5 At least about 1000SFU / 2×10 5 or at least about 1200 SFU / 2×10 5 There are 10.

[0025] In one embodiment, the disclosure provides a population of antigen-specific T cells comprising about 80% to about 99% CD3+ T cells, less than 1% CD14+ monocytes, less than 10% CD19+ B cells, and less than 10% CD3-CD56+ NK cells, wherein the population of antigen-specific T cells has activity against one or more peptides of one or more antigens with which the population of T cells has not been previously cultured. In one embodiment, the population of antigen-specific T cells has a degree of specificity for at least one peptide of at least one antigen with which the population of T cells has not been previously cultured, cumulatively at least about 100 SFU / 2×10 as measured by IFN-γ ELISpot. 5 In one embodiment, the degree of specificity for at least one antigen to which the population has not been previously exposed or cultured is at least about 30 SFU / 2×10 as measured by IFN-γ ELISpot. 5 In one embodiment, the population has been previously cultured with one or more peptides from one or more tumor antigens in the absence of exogenous cytokines.

[0026] In one embodiment, the tumor antigen-specific T cells are at least about 20% of central memory T cells (T CM In one embodiment, the tumor antigen-specific T cells comprise about 40% effector memory T cells (T EM In one embodiment, the tumor antigen-specific T cells include 10% or less of terminally differentiated effector memory cells that express CD45RA (T EMRA In one embodiment, the tumor antigen-specific T cells comprise at least about 20% of naive T cells. In one embodiment, the tumor antigen-specific T cells exhibit activity against at least 75% of the tumor antigens represented by one or more peptides. In one embodiment, the tumor antigen-specific T cells exhibit activity against each tumor antigen represented by one or more peptides. In one embodiment, activity against an antigen, or antigen specificity, is detected by IFNγ production. In one embodiment, IFNγ production is determined by an IFNγ ELISpot test.

[0027] In one embodiment, increasing the number of tumor antigens represented by one or more peptides, optionally a library of peptides, results in (a) increased specificity of antigen-specific T cells, and / or (b) increased specificity of antigen-specific T cells for tumor antigens.

[0028] In one embodiment, T cells present within the population exhibit activity against one or more peptides of one or more antigens with which the population has not been previously cultured (e.g., "non-target" antigens). In some embodiments, activity against one or more peptides of one or more antigens with which the population has not been previously cultured is the result of epitope plating.

[0029] In some embodiments, the degree of specificity for one or more peptides of one or more antigens with which the population has not been previously cultured is at least about 100 SFU / 2×10 cumulatively as measured by IFN-γ ELISpot. 5 In some embodiments, the degree of specificity for at least one antigen, where the population has not been previously cultured together, is at least about 30 SFU / 2×10 as measured by IFN-γ ELISpot. 5 It is a cell.

[0030] In one embodiment, the population of antigen-specific T cells is not subjected to purification or enrichment based on antigen specificity.

[0031] In one embodiment, the disclosure provides a composition comprising a population of ex vivo expanded tumor antigen-specific T cells comprising CD4+ T cells and CD8+ T cells with less than about 10% CD56+ NK cell component, wherein the degree of specificity for the tumor antigen is at least about 500 SFU / 2×10 as measured by IFN-γ ELISpot. 5In some embodiments, the degree of specificity for a tumor antigen is at least about 700 SFU / 2×10 as measured by IFN-γ ELISpot. 5 In one embodiment, the composition additionally comprises a pharma- ceutically acceptable buffer or excipient. In a further embodiment, the composition comprises less than about 10% CD19+ cells. In one embodiment, both the CD4+ and CD8+ T cell compartments show specificity for a tumor antigen.

[0032] In one embodiment, the tumor antigen-specific T cells are specific for PRAME, NY-ESO-1, survivin, and WT-1. In one embodiment, the tumor antigen-specific T cells are specific for PRAME, NY-ESO-1, survivin, WT-1, SSX2, and MAGEA4. In one embodiment, the tumor antigen-specific T cells are specific for PRAME, NY-ESO-1, survivin, WT-1, SSX2, and / or MAGEA4, and 1, 2, 3, 4, 5, 6, or more additional tumor associated antigens. In one embodiment, the tumor antigen-specific T cells are at least about 20% of central memory T cells (T CM In one embodiment, the tumor antigen-specific T cells comprise at least about 20% naive T cells. In one embodiment, the tumor antigen-specific T cells comprise at least about 40% effector memory T cells (T EM In one embodiment, the tumor antigen-specific T cells include 10% or less of terminally differentiated effector memory cells that express CD45RA (T EMRA ).

[0033] In one aspect, the disclosure provides a method for generating tumor antigen-specific T cells, comprising: (i) culturing 0.1-9x10 cells / mL of tumor antigen-specific T cells in a static cell culture device having at least one gas permeable surface with one or more peptides of one or more tumor associated antigens; 6 cells / cm 2wherein each peptide is present in the cell culture device at a concentration of about 0.1 μg / mL to about 0.5 μg / mL, and the white blood cells comprise about 1% to about 50% monocytes and about 40% to about 99% T cells; and (ii) culturing the cell culture device at about 37° C. and 5% CO. 2 (iii) after about 8 to about 30 hours of culturing, adding one or more exogenous cytokines; and (iv) culturing the cells, peptides, and exogenous cytokines for about 5 to about 14 days, wherein the culture is not disturbed after addition of the exogenous cytokines.

[0034] In one embodiment, the method additionally comprises the step of harvesting the tumor antigen-specific T cells after the culturing of step (iv). In one embodiment, the one or more peptides are from four tumor associated antigens and the total concentration of peptides present in the cell culture device in step (i) is about 78 μg / mL. In one embodiment, the one or more peptides are from six tumor associated antigens and the total concentration of peptides present in the cell culture device in step (i) is about 108 μg / mL. In one embodiment, the method does not comprise further purification or enrichment based on the antigen specificity of the T cells.

[0035] In one aspect, the disclosure provides T cells generated by the aforementioned methods. In one embodiment, the disclosure provides a tumor antigen-specific T cell product, comprising about 80% to about 99% CD3+ T cells, less than 1% CD14+ monocytes, less than 10% CD19+ B cells, and less than 10% CD3-CD56+ NK cells, and a CD3+ T cell compartment comprising about 12% to about 80% CD4+ T cells and about 17% to about 84% CD8+ T cells, wherein the CD3+ T compartment comprises at least about 20% central memory T cells (T CM In one embodiment, the tumor antigen-specific T cells comprise at least about 20% naive T cells. In one embodiment, the tumor antigen-specific T cells comprise at least about 40% effector memory T cells (TEM In one embodiment, the tumor antigen-specific T cells include 10% or less of terminally differentiated effector memory cells that express CD45RA (T EMRA ).

[0036] In one embodiment, the T cell product comprises cells capable of producing IFN-γ as determined by an enzyme-linked immunosorbent spot (ELISpot) test, e.g., the degree of specificity of the tumor antigen-specific T cells for a tumor antigen is at least about 500 SFU / 2×10 for all antigens combined. 5 In one embodiment, the degree of specificity for an antigen not represented by one or more peptides is at least about 100 SFU / 2×10 cumulatively as measured by IFN-γ ELISpot. 5 In one embodiment, the degree of specificity for an antigen not represented by one or more peptides is at least about 30 SFU / 2×10 cumulatively as measured by IFN-γ ELISpot. 5 In one embodiment, the T cell product is cryopreserved. In one embodiment, the T cell product is suitable for use in autologous cell therapy for the treatment of cancer, and / or allogeneic cell therapy for the treatment of cancer.

[0037] In one embodiment, the disclosure provides a method for treating a patient comprising administering to the patient a composition and / or a population of antigen-specific T cells disclosed herein. The population of antigen-specific T cells is as defined herein and / or a population of antigen-specific T cells generated by the methods provided herein. In one embodiment, the patient has cancer. In one embodiment, the cancer is a leukemia, lymphoma, or solid tumor. In one embodiment, the cancer is selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), multiple myeloma, non-Hodgkin's lymphoma, sarcoma, breast cancer, and pancreatic cancer. In one embodiment, the patient has undergone an allogeneic hematopoietic stem cell transplant (HSCT). In some embodiments, the patient is immunocompromised. In other embodiments, the patient is not immunocompromised. In some embodiments, the patient is the same individual as the subject from whom the mononuclear cells were obtained, i.e., the treatment method is autologous. In other embodiments, the patient is a different individual, i.e., the treatment method is allogeneic. In some embodiments, the patient is partially HLA-matched with the subject from whom the mononuclear cells were obtained. In some embodiments, the patient has at least one HLA match with the subject from whom the mononuclear cells were obtained. In some embodiments, the method comprises HLA typing the patient prior to treatment. In some embodiments, the method comprises selecting a tumor antigen-specific T cell product from a bank of products based on the HLA type of the product and the patient.

[0038] In one embodiment, the method of treatment disclosed herein further comprises administering to the patient one or more additional populations of antigen-specific T cells (e.g., a second population, optionally a third population, optionally a fourth population, optionally a fifth population, and optionally a sixth population), wherein the one or more additional populations of antigen-specific T cells are antigen-specific T cells or antigen-specific T cell products described herein and / or obtained by the methods provided herein, the method comprising treating the patient with the one or more additional populations of antigen-specific T cells at least about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after treatment with the previously administered population. In one embodiment, the one or more additional populations (e.g., second and / or third) of antigen-specific T cells may be generated after obtaining one or more additional (e.g., second and / or third) populations of mononuclear cells from the patient at least about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks after the previous (e.g., first and / or second) populations are administered to the patient in the case of autologous therapy. In one embodiment, the one or more additional populations may include an expanded number of antigen-specific cells and may include a more potent dose compared to the previous (e.g., first) population. In one embodiment, the one or more additional populations of antigen-specific T cells for allogeneic therapy are obtained from a bank of cryopreserved products or are generated after obtaining one or more additional populations of mononuclear cells from a subject (e.g., a donor) different from the patient.

[0039] In one embodiment, the method disclosed herein further comprises monitoring the progression of the patient's disease by measuring RNA expression of one or more tumor antigens. In one embodiment, the expression of one or more tumor antigens correlates with the patient's minimal residual disease (MRD). In one embodiment, the method further comprises measuring RNA expression of one or more additional tumor antigens that are not the tumor antigens corresponding to the one or more peptides. In one embodiment, the RNA expression of the one or more additional tumor antigens corresponds to the patient's MRD. In one embodiment, the method disclosed herein results in epitope plating against additional tumor antigens that are not the tumor antigens corresponding to the one or more peptides used to generate the antigen-specific T cell product (see, e.g., Figures 24A-24G).

[0040] In one embodiment, the present disclosure provides a cell composition as a starting material for generating an antigen-specific T cell product disclosed herein. In one embodiment, the composition comprises mononuclear cells depleted of CD56+ cells. In a further embodiment, the composition comprises about 40% to about 99% T cells and about 2% or less NK cells. In one embodiment, the composition further comprises one or more peptides from one or more tumor antigens. In one embodiment, the composition further comprises one or more libraries of peptides from one or more tumor antigens. In a further embodiment, each library of peptides comprises peptides spanning the sequence of a tumor antigen. Thus, each library of peptides comprises a plurality of different peptides, each corresponding to all or a portion of a tumor antigen. In one embodiment, each peptide in the composition is present at a concentration of about 0.1 μg / mL to about 0.5 μg / mL, for example, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, or about 0.5 μg / mL. In one embodiment, the composition comprises peptides, or optionally a library of peptides, from at least four different tumor antigens, e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 different tumor antigens. In one embodiment, the tumor antigens are selected from PRAME, NY-ESO-1, survivin, WT1, SSX2, and MAGEA4. In one embodiment, the tumor antigens are PRAME, NY-ESO-1, survivin, and WT1. In one embodiment, the composition is present in a container comprising at least one gas permeable surface, e.g., 1, 2, 3, 4, 5, or 6 gas permeable surfaces. In one embodiment, the composition is present in a container and the mononuclear cells are present in a concentration of about 0.1 x 10 per square centimeter of gas permeable surface. 6 pieces~approx. 9×10 6 The seeding density is 100 μg / ml. [Brief description of the drawings]

[0041] [Figure 1]FIG. 1 provides a schematic diagram of one embodiment of the method for generating tumor antigen-specific T cells disclosed herein. The procedure shown in FIG. 1A is referred to herein as "MAT110-CD56+Depl". FIG. 1B and FIG. 1C provide schematic diagrams of two prior art methods for generating tumor antigen-specific T cells. FIG. 1B provides a method referred to herein as "MAT101-4" which includes stimulating PBMCs with cytokines to generate dendritic cells (DCs), which are then pulsed with a peptide mix and cultured with cytokines, including two or more medium exchange and peptide pulsing steps, for a total culture period of about 31-36 days. FIG. 1C provides a method referred to herein as "MAT103-4" which includes culturing CD14+PBMCs in the presence of cytokines to generate mature DCs, which are then pulsed with peptides and cultured with CD56- cells and cytokines for a total culture period of about 15-20 days.

[0042] [Diagram 2] Figures 2A, 2B, and 2C provide details of the starting material for generating tumor antigen-specific T cells. Figure 2A shows the components of the PBMC starting material that was not depleted of any cell type. Figure 2B shows the components of the PBMC starting material that was depleted of CD56+ cells. Figure 2C shows the components of the PBMC starting material that was depleted of CD56+ and CD19+ cells.

[0043] [Diagram 3]Figure 3 provides details of tumor antigen-specific T cell products prepared according to method "MAT110-NoDepl" using non-depleted starting material. The first panel shows the % of viable cells that are monocytes, T cells, B cells, and NK cells. The second panel shows the percentage of CD4+ or CD8+ among the CD3+ T cells in the final product. The third panel shows the phenotype of CD3+ T cells in the final product (T naive, TCM, TEM, TEMRA). The fourth panel shows the fold expansion of cells in the final product. The fifth panel shows the combined antigen specificity (SFU / 2x105 cells, measured by IFNγ ELISpot). Statistical analysis is shown in the table below each panel. SFU = Spot Forming Units.

[0044] [Figure 4] Figure 4 provides details of tumor antigen-specific T cell products prepared starting from CD56+ depleted cells according to the method "MAT110-CD56+Depl". The first panel shows the % of viable cells that are monocytes, T cells, B cells and NK cells. The second panel shows the percentage of CD4+ or CD8+ among the CD3+ T cells in the final product. The third panel shows the phenotype (T naive, TCM, TEM, TEMRA) of the CD3+ T cells in the final product. The fourth panel shows the fold expansion of cells in the final product. The fifth panel shows the combined antigen specificity (SFU / 2x105 cells, measured by IFNγ ELISpot). Statistical analysis is shown in the table below each panel. SFU = spot forming units.

[0045] [Diagram 5]Figure 5 provides details of tumor antigen-specific T cell products using CD56+ and CD19+ depleted cells as starting material. The method used to generate these tumor antigen-specific T cells is called "MAT210" and is the same as the "MAT110-CD56+Depl" method shown in Figure 1A, except that mononuclear cells are depleted of CD19+ cells as well as CD56+ cells prior to peptide pulsing. The first panel shows the % of viable cells for monocytes, T cells, B cells, and NK cells. The second panel shows the percentage of CD4+ or CD8+ CD3+ T cells in the final product. The third panel shows the phenotype (T naive, TCM, TEM, TEMRA) of CD3+ T cells in the final product. The fourth panel shows the fold expansion of cells in the final product. The fifth panel shows the combined antigen specificity (SFU / 2x105 cells, measured by IFNγ ELISpot). Statistical analysis is shown in the table below each panel. SFU = spot forming units.

[0046] [Figure 6] Figures 6A and 6B show a comparison of the improved method disclosed herein (termed "MAT110-CD56+Depl" and shown diagrammatically in Figure 1A), MAT110-No Depl, with the prior method MAT103-4 (shown diagrammatically in Figure 1C). The addition of a CD56+ depletion step significantly improved the fold expansion of T cells generated using the MAT110 method.

[0047] [Figure 7] Figures 7A and 7B show the phenotype of T cells generated using the MAT110-CD56+Depl method, shown diagrammatically in Figure 1A, compared to MAT103-4 obtained using a previous method, shown diagrammatically in Figure 1C. The MAT110-CD56+Depl method resulted in a product with a comparable distribution of CD4+ and CD8+ T cells and a higher percentage of central memory T cells (TCM).

[0048] [Figure 8]Figures 8A and 8B show the antigen specificity of MAT110-CD56+Depl cells obtained using the improved method disclosed herein compared to MAT103-4 and MAT110-No-Depl cells obtained using the previous method, respectively. Figure 8A shows the antigen specificity in SFU / 2x105 cells measured by IFNγ ELISpot for cells from individual donors. Each panel shows the results for cells obtained from an individual donor. Figure 8B shows the total degree of antigen specificity (combined reactivity against each target antigen, when donor data are pooled) and shows that the increased degree of response achieved with the MAT110-CD56+Depl method is statistically significant. SFU = spot forming units.

[0049] [Figure 9] Figure 9A shows the total response (sum of all antigens) in SFU / 2x105 cells as measured by IFNγ ELISpot. The product produced with MAT110-CD56 Depl had more antigen-specific T cells. Figure 9B shows the spectrum of antigen specificity of tumor antigen-specific T cell products produced with the prior method and MAT110-CD56+Depl. The manufacturing process of MAT110-CD56 Depl increased the percentage of multi-target T cells. Figure 9C shows the increase in the number of products with specificity for multiple tumor antigens achieved with MAT110-CD56+Depl cells. "Run" indicates a manufacturing run using cells to produce T cell products. Figure 9D shows the linear correlation between antitumor activity of the T cell products (measured in in vitro co-culture experiments) and antigen specificity (measured by IFNγ ELISpot, SFU / 2x105 cells, x-axis) Furthermore, FIG. 9D shows that T cell products generated using the MAT110-CD56+Depl method have higher antigen specificity and antitumor activity compared to those generated using the MAT103-4 method.

[0050] [Figure 10]FIG. 10 shows exemplary calculations of peptide amounts for the 4 antigen pool (PRAME, NY-ESO-1, survivin, and WT1) and the 6 antigen pool (PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2), as well as the total peptide amount used on the 100M G-Rex or 500M G-Rex instruments.

[0051] [Figure 11] Figure 11 shows the antigen specificity of the MultiTAA-specific T cell product after an initial peptide pulsing step in the presence (+cytokines) and absence (-cytokines) of cytokines. To generate MultiTAA-specific T cells, PBMCs from healthy donors were pulsed overnight with peptides from six different antigens (WT1, survivin, NY-ESO-1, PRAME, SSX2, MAGEA4) in the presence or absence of cytokines (IL-6, IL-12, IL-7, and IL-15). MultiTAA-specific T cells were subjected to IFN-γ ELISpot analysis as a readout of antigen specificity. SFU = spot forming units.

[0052] [Figure 12] Figure 12 shows the antigen specificity of the MultiTAA-specific T cell products generated by using high and low volumes for an overnight peptide pulsing step. To generate MultiTAA-specific T cells, PBMCs from healthy donors were pulsed overnight with peptides from four different antigens (WT1, survivin, NY-ESO-1, and PRAME) in either a low volume (10 mL) or a high volume (100 mL) (n=3). MultiTAA-specific T cells were subjected to IFN-γ ELISpot analysis as a readout of antigen specificity. SFU = spot forming units.

[0053] [Figure 13]Figure 13 shows the fold expansion (bar graph, left), % viability (line graph, top), and antigen specificity (bar graph, right) of MultiTAA-specific T cell products generated using positively and negatively selected cell populations as starting material for culture. PBMCs were either positively selected for T cells and monocytes (CD8+CD4+CD14+) or depleted of CD56+ NK cells (CD56posDepl) before cells were placed in culture. MultiTAA-specific T cells were subjected to IFN-γ ELISpot analysis as a readout for antigen specificity.

[0054] [Figure 14] Figure 14 shows the correlation between T cell specificity for target antigen and minimal residual disease (MRD). MRD (left Y-axis and solid line) and antigen specificity (right Y-axis and bar graph) were measured before and 8, 12, 18, 24, and 32 weeks after MT101-4 cell infusion. Target antigen-specific T cells are present in the patient's peripheral blood and show an inverse correlation with MRD, suggesting antitumor activity.

[0055] [Figure 15] Figure 15 shows the correlation between T cell specificity to non-target antigens (bar graph) and MRD (solid line). T cells specific for the indicated additional antigens (i.e., "non-target antigens" that were not used to generate the antigen-specific T cell product) present in patient peripheral blood following treatment are inversely correlated with MRD, indicating epitope spreading.

[0056] [Figure 16] Figure 16 shows T cell gene expression in the bone marrow of patients pre-infusion and at 8, 12, 18, 24, and 32 weeks after treatment. MRD is also shown. Gene expression was determined by next generation sequencing (NGS).

[0057] [Figure 17]Figure 17 shows the T cell composition and phenotype of the patient before infusion and at 8, 12, and 24 weeks after treatment. CD4 and CD8 populations are shown on the top panel. The bottom panel shows CD197 and C45RA staining, indicating naive (CD197+CD45RA+), central memory (TCM, CD197+CD45RA-), effector memory (TEM, CD197-CD45RA-), and terminally differentiated (TEMRA, CD197-CD45RA+) populations.

[0058] [Figure 18] FIG. 18 shows that both the patient's CD4+ and CD8+ T cells are functional (expressing IFNγ as shown in the bar graph) and that function correlates with MRD (solid line).

[0059] [Figure 19] FIG. 19 shows IFNγ expression by Lag3− and Lag3+ T cells in patients after infusion.

[0060] [Figure 20] FIG. 20 shows the expansion of peripheral T cell clones in patients after infusion.

[0061] [Figure 21] Figure 21 shows target antigen RNA expression in patient bone marrow before administration and at weeks 8, 12, 18, 24, and 32. The upper dotted line shows survivin RNA expression, and the lower dotted line shows WT1 RNA expression. The solid line is % MRD. Bar graphs show specificity for the indicated antigens. * means PRAME and NY-ESO-1 transcripts were not detected.

[0062] [Figure 22] Figure 22 shows RNA expression of the non-target antigen proteinase-3 in the patient's bone marrow (dotted line) before infusion and at weeks 8, 12, 18, 24, and 32. The solid line is % MRD. Also shown is the specificity of patient cells for non-target antigens, including proteinase-3 (bar graph), for each time point.

[0063] [Diagram 23] Figure 23 shows RNA expression of additional non-targeted antigens in the patient's bone marrow before infusion and at weeks 8, 12, 18, 24, and 32 (dotted lines; week 18, from top to bottom: BRAF, TERT, Mucin-1, Myc, CCNA1). Solid lines are % MRD. Specificity of patient cells for non-targeted antigens is also shown (bar graphs).

[0064] [Figure 24] Figures 24A-G show that injecting MultiTAA-specific T cells further generates an immune response against "non-targeted" TAAs, representing epitope plating. Figure 24A is a schematic of the kinetics of the immune response. Figure 24B shows complete responses (CRs) in pancreatic cancer patients after MultiTAA-specific T cell therapy, which correlate with epitope plating as shown in Figure 24D (peak post column bars from top to bottom are WT1, AFP, MART1, MC1, MA3 [largest part of the bar], MA2B, MA1). The antigen specificity of the infused formulation for the patient is shown in Figure 24C (peak post column bars from top to bottom are Survivin, NYESO1 [largest part of the bar], MAGEA4, SSX2, and Prame). Figure 24E shows partial responses (PRs) in pancreatic cancer patients after MultiTAA-specific T cell therapy, which correlate with epitope spreading in patients as shown in Figure 24G (peak post column bars, from top to bottom, are WT1, AFP, MART1, MC1, MA3 [largest part of the bar], MA2B, MA1). The antigen specificity of the infusion formulation for patients is shown in Figure 24F (peak post column bars, from top to bottom, are Survivin, NYESO1, MAGEA4, SSX2 [largest part of the bar], and Prame).

[0065] [Diagram 25]Figure 25A shows images of tumor size in a patient injected with T cells generated using the MAT101 method. Images show tumors before and 9 months after injection of this treatment. Figure 25B shows antigen specificity measured by IFN-γ ELISpot analysis against targeted and non-targeted antigens 3 and 9 months after cell injection in a patient compared to the antigen specificity of the patient before injection (bars in the Mth3 and Mth9 columns, from top to bottom, are Survivin, NYESO1, MAGEA4, SSX2, and Prame).

[0066] [Figure 26] Figure 26 is a graph measuring T cell proliferation in responding and non-responding lymphoma patients infused with MultiTAA-specific T cells (T cell populations generated with peptides of five tumor-associated antigens [TAAs]). Proliferation of T cells specific for "target" epitopes (left) and "non-target" epitopes (right) showing epitope splatting from the peptides used to generate the antigen-specific T cell product was measured 6 weeks after the first infusion of MultiTAA-specific T cells (Figure 26).

[0067] [Figure 27] Figures 27A-D are schematic diagrams of in vitro experimental assays assessing epitope plating (Figure 27A), associated cell proliferation (Figure 27B), and antigen specificity (Figures 27C-D). Figure 27B shows a graphical (left) and visual representation (right, bioluminescence indicates cell density) of cell proliferation of PANC1 cells (pancreatic cancer cell line) cultured in vitro alone or with non-specific T cells activated with anti-CD3 / CD28 antibodies or MT-601 antigen-specific cells. Antigen specificity was measured by IFN-γ ELISpot analysis for target antigens (Figure 27C) and non-target antigens (Figure 27D).

[0068] [Figure 28]FIG. 28 is a graph showing the antigen specificity of the MT-401 cell population (a T cell population generated with peptides from the four TAAs) as measured by IFN-γ ELISpot analysis.

[0069] [Figure 29] FIG. 29 is a schematic showing bone marrow mononuclear cells (BM-MNC, target cells) cultured with control PBMC or MT-401 cells (effectors) and analysis of target cell killing during culture as measured by flow cytometry using the Tmem5 panel.

[0070] [Diagram 30] Figures 30A and 30B are graphs measuring glucose (Figure 30A) and lactate (Figure 30B) production on days 0, 3, and 4 of co-cultures of target cells alone (patient-derived AML), target cells and PBMCs, or target cells and MT-401.

[0071] [Diagram 31] Figures 31A-B are graphs showing target cell killing (measured by fold change in proliferation after 4 days of culture) at an effector to target (E:T) ratio of 4:1 using cells from a single patient (PT117-201). Changes in cell proliferation were measured for target cells (BM-MNC) alone, target cells cultured with control PBMC, and target cells cultured with MT-401. ****=p<0001; ns=not significant.

[0072] [Diagram 32] FIG. 32 is a graph showing the target antigen specificity of MT-401 cells (derived from one patient 0117-201 and prepared with peptides from four TAAs) before and after co-culture with target cells (BM-MNC) as measured by IFN-γ ELISpot analysis.

[0073] [Diagram 33]Figures 33A-B are graphs comparing antigen specificity between treatment groups of target cells (BM-MNC) alone, target cells with PBMC, or target cells cultured with MT-401 cells over a 4-day period. Target antigen specificity was observed in the MT-401 group (Figure 33A) (bars in the Target+PBMC column are, from top to bottom, WT1, Survivin [largest part of the bar], NY-ESO-1, and Prame; bars in the Target+MT-401 column are, from top to bottom, WT1, Survivin, NY-ESO-1, and Prame [largest part of the bar]). Further non-target antigen specificity was observed in the MT-401-treated group after co-culture with target cells (FIG. 33B) (bars in the target+PBMC column are, from top to bottom, WT1, survivin [largest part of the bar], NY-ESO-1, and Prame; bars in the target+MT-401 column are, from top to bottom, BRAF, mucin-1, TERT, Myc, MAGEA4, SXX2, WT1, survivin, NY-ESO-1, Prame [largest part of the bar]; CCNA1 and proteinase-3 are not visible).

[0074] [Diagram 34] FIG. 34 is a schematic diagram of an off-the-shelf program for donor selection, product manufacturing, cell banking, and patient treatment.

[0075] [Diagram 35] FIG. 35 shows the predicted number of cell lines required to achieve 90% coverage of a patient population.

[0076] [Diagram 36] FIG. 36 shows T cell fold expansion, viability, and total viable cells when MultiTAA-specific T cell products were prepared with G-Rex 10M and G-Rex-500M.

[0077] [Figure 37]Figure 37 shows the total antigen specificity and antigen specificity diversity in cells obtained from three different donors and stimulated with four or six peptide libraries. As shown in the fourth panel, cells from the fourth donor were stimulated with 12 peptide libraries corresponding to the 12 indicated tumor antigens (bars in the four antigen column are, from top to bottom, WT1, survivin, NY-ESO-1, PRAME; bars in the six antigen column are, from top to bottom, MAGEA4, SSX2, WT1, survivin, NY-ESO-1, PRAME; bars in the twelve antigen column are, from top to bottom, proteinase-3, CCNA1, BRAF, mucin-1, TERT, Myc, MAGEA4, SSX2, WT1, survivin, NY-ESO-1, PRAME). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] In one aspect, the present disclosure provides an improved method for generating tumor antigen-specific T cells. The tumor antigen-specific T cells described herein and generated by the methods provided herein have superior properties compared to conventional tumor antigen-specific T cells, including improved sensitivity of the response to the target tumor antigen, increased specificity, increased survival rate, and desirable phenotypes that result in efficient killing of tumor cells upon administration to a patient. In some embodiments, the present disclosure provides a method for generating a tumor antigen-specific T cell product generated from mononuclear cells of a patient or subject (e.g., a donor). In some embodiments, the tumor antigen-specific T cell product is generated from the patient's mononuclear cells via the methods provided herein and then administered to the patient (autologous use). In some embodiments, the tumor antigen-specific T cell product is generated from the donor's mononuclear cells via the methods disclosed herein and maintained in a cryopreserved state until it is used in a patient in need thereof (which may be the donor's own or a different patient) (autologous or allogeneic use).

[0079] In one embodiment, the method disclosed herein comprises culturing mononuclear cells from a subject in a vessel containing a high concentration of peptide, and co-culturing the cells with the peptide (also referred to herein as "pulsing" the peptide) prior to adding one or more exogenous cytokines to the culture. The inventors have surprisingly found that pre-culturing the cells with the peptide prior to adding one or more exogenous cytokines significantly improves various aspects of the tumor antigen-specific T cells generated by this process, including antigen specificity and phenotype. Given the correlation between antigen specificity and anti-tumor activity disclosed herein, the tumor antigen-specific T cells of the present disclosure have significantly stronger anti-tumor activity. In addition, the process disclosed herein allows for a short (5-14 days) production process, which is beneficial for patient treatment. For example, when administering tumor antigen-specific T cells to a patient in need thereof, a shorter process time is highly desirable to avoid undue delays in treatment. Such a shortened processing time is an improvement over prior methods for generating tumor antigen-specific T cells that involve processing times of 14 days or more, such as 20 days, 30 days, 36 days, or more.

[0080] In one embodiment, the methods disclosed herein include a first culture step in which mononuclear cells (e.g., PBMCs or leukocytes) are cultured with a high concentration of one or more peptides in a low volume. In one embodiment, the delivery density of cells is 100% per square centimeter (cm) of gas permeable surface. 2 ) approximately 0.1 × 10 6 pieces~approx. 1×10 7 In one embodiment, the high concentration of peptide is about 0.1 μg / mL to about 0.5 μg / mL (e.g., about 0.1 μg / mL, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, or about 0.5 μg / mL) and / or the peptide is incubated with the cells in a low volume to promote interaction of the cells with the peptide. In some embodiments, the method comprises incubating mononuclear cells at a concentration of, for example, about 0.1×10 6 cells / cm 2 ~Approx. 1×10 7 cells / cm 2In another embodiment, the method comprises placing the peptide mix in the container or device, followed by adding mononuclear cells at a seeding density of, for example, about 0.1×10 6 cells / cm 2 ~Approx. 1×10 7 cells / cm 2 In some embodiments, the method comprises mixing the mononuclear cells with the peptide mix and then placing the cells and peptides in a container or device. In some embodiments, the method comprises mixing a portion of the cells and / or a portion of the peptides together and then placing the mixture in a container or device and then adding the remaining cell and / or peptide mix to the container or device.

[0081] Mononuclear cells In one aspect, the disclosure provides a method, wherein mononuclear cells are obtained from a subject. In some embodiments, the methods disclosed herein comprise obtaining peripheral blood mononuclear cells (PBMCs) from the subject. In some embodiments, mononuclear cells are obtained from the subject via leukapheresis. In some embodiments, the mononuclear cells are PBMCs or leukocytes. In some embodiments, the subject is a donor. In some embodiments, the donor is a healthy donor. In some embodiments, the donor is a patient to whom MultiTAA-specific T cells are to be generated and then administered.

[0082] In some embodiments, the methods provided herein include a depletion step prior to placing the population of mononuclear cells in a vessel for culturing with one or more peptides. In some embodiments, the depletion step includes depleting CD56+ cells from the population of mononuclear cells. In some embodiments, the mononuclear cells are depleted of CD56+ cells and include CD3+ T cells. In some embodiments, the mononuclear cells are depleted of CD56+ cells and include CD3+ T cells and monocytes. In some embodiments, the cells in the vessel consist of CD3+ T cells and monocytes, and are depleted of CD56+ cells such that there are few contaminating CD56+ cells. In embodiments, the method including the depletion step is referred to herein as "MAT-110CD56+Depl". In some embodiments, the T cells generated by this process are referred to herein as "MAT110-CD56+Depl". In some embodiments, PBMCs are depleted of CD56+ cells using one or more methods known to those of skill in the art, including but not limited to magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS) Dynabeads, EasySep® cell separation beads, RosetteSep® immunodensity cell separation, or the Cloudz Human Cell Expansion platform.

[0083] In some embodiments, the methods provided herein further comprise depleting CD19+ cells from the population of mononuclear cells prior to placing the population of mononuclear cells in a vessel or device for culturing with one or more peptides. In some embodiments, the methods comprising depletion of CD19+ T cells are suitable for use in treating patients with B-cell cancer. In some embodiments, the depletion comprises depleting mononuclear cells of CD56+ and CD19+ cells. In embodiments, the methods comprising said depletion of both CD56+ and CD19+ cells are referred to herein as "MAT210" or "MAT210-CD56+Depl-CD19+Depl". In some embodiments, the T cells generated by the methods provided herein comprising depletion of both CD56+ and CD19+ cells are referred to herein as "MAT210" or "MAT210-CD56+Depl-CD19+Depl". In some embodiments, PBMCs are depleted of CD19+ cells using methods known to those of skill in the art, including but not limited to magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), Dynabeads, EasySep® cell separation beads, RosetteSep® immunodensity cell separation, or the Cloudz Human Cell Expansion platform.

[0084] In some embodiments, the mononuclear cells after depletion of CD56+ cells comprise about 0.05% to about 2.5% CD56+ cells. In some embodiments, the mononuclear cells after depletion of CD56+ cells comprise about 35% to about 85% T cells. In some embodiments, the mononuclear cells after depletion of CD56+ cells comprise about 0.01% to about 2% CD56+ cells, about 0.5% to about 50% monocytes, about 0.01% to about 1.5% CD19+ B cells, and / or about 40% to about 99% T cells. In some embodiments, the mononuclear cells after depletion of CD56+ cells comprise about 2.5% or less CD56+ cells. In some embodiments, the mononuclear cells after depletion of CD56+ cells comprise about 1% to about 50% monocytes and about 40% to about 99% T cells.

[0085] In some embodiments, the mononuclear cells after depletion of both CD56+ cells and CD19+ cells comprise about 0.01% to about 2% CD56+ cells. In some embodiments, the mononuclear cells after depletion of both CD56+ cells and CD19+ cells comprise about 40% to about 99% T cells. In some embodiments, the mononuclear cells after depletion of both CD56+ cells and CD19+ cells comprise about 0.01% to about 2% CD56+ cells, about 0.5% to about 50% monocytes, about 0.01% to about 1.5% CD19+ B cells, and / or about 40% to about 99% T cells. In some embodiments, the mononuclear cells after depletion of both CD56+ cells and toCD19+ cells comprise about 2.5% or less CD56+ cells. In some embodiments, the mononuclear cells after depletion of both CD56+ cells and toCD19+ cells comprise about 0.5% to about 50% monocytes and about 40% to about 99% T cells.

[0086] In one aspect, the disclosure provides methods, wherein the depletion step (e.g., depleting only CD56+ cells or depleting CD56+ and CD19+ cells) provides a population of mononuclear cells (also referred to herein as a "cell composition") having particular attributes or characteristics. In some embodiments, the cell composition may provide the starting material for generating an antigen-specific T cell product provided herein. In some embodiments, the population of mononuclear cells or cell composition is placed in a container or device for culture with one or more peptides from one or more tumor-associated antigens.

[0087] In some embodiments, the population of mononuclear cells placed in a container for culture with one or more peptides comprises about 0.05% to about 2.5% CD56+ cells. In some embodiments, the population of mononuclear cells placed in a container for culture with a peptide comprises about 35% to about 85% T cells, or about 40% to about 99% T cells. In some embodiments, the population of mononuclear cells placed in a container for culture with a peptide comprises about 0.01% to about 2% CD56+ NK and CD56+ T cells, about 0.5% to about 50% monocytes, about 0.01% to about 1.5% CD19+ B cells, and / or about 40% to about 99% T cells. In some embodiments, the population of mononuclear cells placed in a container with a peptide comprises about 2.5% or less CD56+ cells. In some embodiments, the population of mononuclear cells placed in a container for culture with a peptide comprises about 40% to about 99% T cells and about 2% or less NK cells.

[0088] peptide In one aspect, the present disclosure provides a method of culturing a population of mononuclear cells (referred to herein as "cells") with one or more peptides from one or more tumor antigens.

[0089] As used herein, peptide refers to a short polymer of amino acids linked by peptide bonds. The peptides in the methods provided herein are of sufficient length to constitute one or more linear epitopes, for example, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids in length.

[0090] In embodiments, the one or more peptides comprise peptides corresponding to 1, 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, 29, 30, or more tumor associated antigens (TAA). For example, in embodiments, the one or more peptides comprise peptides corresponding to at least 4, at least 6, at least 8, at least 12, at least 15, at least 20, at least 30, or at least 40 TAAs. In embodiments, the one or more peptides comprise one or more libraries of peptides derived from one or more TAAs. In embodiments, each library of peptides comprises peptides spanning a different portion of a TAA. In embodiments, the library of peptides corresponding to a given tumor antigen comprises multiple peptides, each spanning all or a portion of the amino acid sequence of the antigen. In an embodiment, the one or more peptides are cultured with the cells at a concentration of about 0.1 μg / mL to about 0.5 μg / mL, e.g., about 0.1 μg / mL, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, or about 0.5 μg / mL. In an embodiment, the total concentration of peptides present in the cell culture device during the culture period with the cells in the absence of exogenous cytokines is at least about 60 μg / mL, e.g., about 78 μg / mL for an exemplary library of peptides representing four different TAAs. In an embodiment, the total concentration of peptides present in the cell culture device during the culture period with the cells in the absence of exogenous cytokines is at least about 100 μg / mL, e.g., about 108 μg / mL for an exemplary library of peptides representing six different TAAs.

[0091] In embodiments, each peptide in the composition is present at a concentration of about 0.1 μg / mL to about 0.5 μg / mL, for example, about 0.1 μg / mL, about 0.2 μg / mL, about 0.25 μg / mL, about 0.3 μg / mL, about 0.4 μg / mL, or about 0.5 μg / mL. In some embodiments, each peptide in the composition is present at a concentration of about 0.1 μg / mL to about 0.5 μg / mL. In some embodiments, each peptide in the composition is present at a concentration of about 0.2 μg / mL to about 0.3 μg / mL. In some embodiments, each peptide in the composition is present at a concentration of about 0.1 μg / mL. In some embodiments, each peptide in the composition is present at a concentration of about 0.2 μg / mL. In some embodiments, each peptide in the composition is present at a concentration of about 0.25 μg / mL. In some embodiments, each peptide in the composition is present at a concentration of about 0.3 μg / mL. In some embodiments, each peptide in the composition is present at a concentration of about 0.4 μg / mL. In some embodiments, each peptide in the composition is present at a concentration of about 0.5 μg / mL.

[0092] In some embodiments, the concentration of the peptide in the cell culture (e.g., container) is about 50 μg / mL to about 100 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 50 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 60 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 70 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 75 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 80 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 85 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 90 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 95 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 100 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 105 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 110 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 78 μg / mL. In some embodiments, the concentration of the peptide in the cell culture is about 108 μg / mL.

[0093] In some embodiments, the total amount of peptide added to the container is about 6 mg to about 60 mg. In some embodiments, the total amount of peptide added to the container is about 6 mg. In some embodiments, the total amount of peptide added to the container is about 7 mg. In some embodiments, the total amount of peptide added to the container is about 8 mg. In some embodiments, the total amount of peptide added to the container is about 9 mg. In some embodiments, the total amount of peptide added to the container is about 10 mg. In some embodiments, the total amount of peptide added to the container is about 11 mg. In some embodiments, the total amount of peptide added to the container is about 12 mg. In some embodiments, the total amount of peptide added to the container is about 13 mg. In some embodiments, the total amount of peptide added to the container is about 14 mg. In some embodiments, the total amount of peptide added to the container is about 15 mg. In some embodiments, the total amount of peptide added to the container is about 20 mg. In some embodiments, the total amount of peptide added to the container is about 25 mg. In some embodiments, the total amount of peptide added to the container is about 30 mg. In some embodiments, the total amount of peptide added to the container is about 35 mg. In some embodiments, the total amount of peptide added to the container is about 40 mg. In some embodiments, the total amount of peptide added to the container is about 45 mg. In some embodiments, the total amount of peptide added to the container is about 50 mg. In some embodiments, the total amount of peptide added to the container is about 55 mg. In some embodiments, the total amount of peptide added to the container is about 60 mg. In some embodiments, the total amount of peptide added to the container is about 7.775 mg. In some embodiments, the total amount of peptide added to the container is about 10.825 mg. In some embodiments, the total amount of peptide added to the container is about 38.875 mg. In some embodiments, the total amount of peptide added to the container is about 54.125 mg.

[0094] In embodiments, the tumor antigen represented by one or more peptides is selected from the group consisting of CEA, MHC, CTLA-4, gp100, mesothelin, PRAME (OIP3) PD-L1, TRP1, CD40, EGFP, Her2, TCR alpha, trp2, TCR, MUC1, cdr2, ras, 4-1BB, CT26, GITR, OX40, TGF-alpha.WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, Melan A / MART1, Ras mutant, gp 100, p53 mutant, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, Myc, MYCN, RhoC, TRP-2, GD3, fucosyl-GM1, mesothelin, PSCA, MAGE A1, MAGEA4, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, mucin-1, BRAF, CCNA1, Fos-related antigen 1, and any combination thereof.In some embodiments, the tumor antigen represented by one or more peptides is selected from the group consisting of tumor antigens CEA, MHC, CTLA-4, gp100, mesothelin, PRAME (OIP3), PD-L1, TRP1, CD40, EGFP, Her2, TCR alpha, trp2, TCR, MUC1, cdr2, ras, 4-1BB, CT26, GITR, OX40, TGF-alpha.WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, Melan A / MART1, Ras mutant, gp 100, p53 mutant, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, Myc, MYCN, RhoC, TRP-2, GD3, fucosyl-GM1, mesothelin, PSCA, MAGE A1, MAGEA4, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, mucin-1, BRAF, CCNA1, Fos-related antigen 1, and combinations thereof.

[0095] In some embodiments, the one or more peptides comprise the tumor antigen PRAME. In some embodiments, the one or more peptides comprise the tumor antigen NY-ESO-1. In some embodiments, the one or more peptides comprise the tumor antigen survivin. In some embodiments, the one or more peptides comprise the tumor antigen WT1. In some embodiments, the one or more peptides comprise the tumor antigen MAGEA4. In some embodiments, the one or more peptides comprise the tumor antigen SSX2. In embodiments, the one or more peptides comprise peptides from the tumor antigens PRAME, NY-ESO-1, survivin, and WT1. In further embodiments, the one or more peptides further comprise peptides from MAGEA4 and SSX2. In some embodiments, the one or more peptides comprise peptides from the tumor antigens PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2.

[0096] In embodiments, a tumor antigen represented in one or more peptides used to generate MultiTAA-specific T cells is referred to herein as a "target antigen," "targeted antigen," "target tumor-associated antigen," "target TAA," etc. In embodiments, a tumor antigen not represented in one or more peptides used to generate MultiTAA-specific T cells is referred to herein as a "non-targeted antigen," etc.

[0097] Mononuclear cell culture method In one aspect, the disclosure provides a method of placing a population of mononuclear cells obtained from a subject into a container for culturing with one or more peptides. In embodiments, the population is about 0.1×10 per square centimeter of gas permeable surface. 6 pieces~approx. 9×10 6 In embodiments, the population is added to the vessel at a seeding density of about 0.1 x 10 6 cells / cm 2 ~Approx. 1×10 7 cells / cm 2In some embodiments, the population is added to the vessel at a seeding density of about 0.1 x 10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 0.5×10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 1 x 10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 2×10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 3×10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 4×10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 5×10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 6×10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 7×10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 8×10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 9×10 6 cells / cm 2 In some embodiments, the population is added to the vessel at a seeding density of about 1 x 10 7 cells / cm 2 The seeding density of the culture medium is then added to the vessel.

[0098] In embodiments, the population of mononuclear cells is added to a device or container comprising at least one gas permeable surface. In embodiments, the population of mononuclear cells is added to a device or container comprising at least one gas-permeable surface. In embodiments, the container to which the cells are added comprises 1, 2, 3, 4, 5, or 6 gas permeable surfaces. In some embodiments, the container comprises 1 gas permeable surface. In some embodiments, the container comprises 2 gas permeable surfaces. In some embodiments, the container comprises 3 gas permeable surfaces. In some embodiments, the container comprises 4 gas permeable surfaces. In some embodiments, the container comprises 5 gas permeable surfaces. In some embodiments, the container comprises 6 gas permeable surfaces. In some embodiments, the device or container comprising at least one gas permeable surface is a G-Rex device (available from Wilson Wolf Manufacturing). In some embodiments, the container is a 100M G-Rex device. In some embodiments, the container is a 500M G-Rex device. In other embodiments, the device or container comprising at least one gas permeable surface is a gas permeable bag. Devices and containers suitable for use in the methods disclosed herein are capable of, for example, expanding cell populations by about 10-fold, about 100-fold, about 1000-fold, or more.

[0099] In some embodiments, the cells are incubated in the vessel or device with one or more peptides at about 37° C. and 5% CO 2 In some embodiments, the method is performed at about 37° C. and 5% CO 2 In some embodiments, the method comprises culturing the cells with the peptide at about 37° C. and 5% CO for about 30 minutes to about 30 hours. 2The method includes culturing the cells with the peptide at 37° C. for about 8 hours to about 24 hours. In some embodiments, the cells are cultured with the peptide for about 30 minutes. In some embodiments, the cells are cultured with the peptide for about 1 hour. In some embodiments, the cells are cultured with the peptide for about 2 hours. In some embodiments, the cells are cultured with the peptide for several hours. In some embodiments, the cells are cultured with the peptide for about 4 hours. In some embodiments, the cells are cultured with the peptide for about 5 hours. In some embodiments, the cells are cultured with the peptide for about 6 hours. In some embodiments, the cells are cultured with the peptide for about 7 hours. In some embodiments, the cells are cultured with the peptide for about 8 hours. In some embodiments, the cells are cultured with the peptide for about 9 hours. In some embodiments, the cells are cultured with the peptide for about 10 hours. In some embodiments, the cells are cultured with the peptide for about 11 hours. In some embodiments, the cells are cultured with the peptide for about 12 hours. In some embodiments, the cells are cultured with the peptide for about 13 hours. In some embodiments, the cells are cultured with the peptide for about 14 hours. In some embodiments, the cells are cultured with the peptide for about 15 hours. In some embodiments, the cells are cultured with the peptide for about 16 hours. In some embodiments, the cells are cultured with the peptide for about 17 hours. In some embodiments, the cells are cultured with the peptide for about 18 hours. In some embodiments, the cells are cultured with the peptide for about 19 hours. In some embodiments, the cells are cultured with the peptide for about 20 hours. In some embodiments, the cells are cultured with the peptide for about 21 hours. In some embodiments, the cells are cultured with the peptide for about 22 hours. In some embodiments, the cells are cultured with the peptide for about 23 hours. In some embodiments, the cells are cultured with the peptide for about 24 hours. In some embodiments, the cells are cultured with the peptide for about 25 hours. In some embodiments, the cells are cultured with the peptide for about 26 hours. In some embodiments, the cells are cultured with the peptide for about 27 hours.In some embodiments, the cells are cultured with the peptide for about 28 hours. In some embodiments, the cells are cultured with the peptide for about 29 hours. In some embodiments, the cells are cultured with the peptide for about 30 hours. In some embodiments, the method is carried out at 37° C. and 5% CO. 2 In some embodiments, the method comprises culturing the cells with the peptide overnight at about 37° C. and 5% CO in the absence of exogenous cytokines. 2 In some embodiments, the method comprises culturing the cells with the peptide at about 37° C. and 5% CO in the absence of exogenous cytokines for about 30 minutes to about 30 hours. In some embodiments, the method does not comprise a washing step. In some embodiments, the method comprises culturing the cells with the peptide at about 37° C. and 5% CO in the absence of exogenous cytokines. 2 The method includes culturing the cells with the peptide at 37° C. for about 8 to about 30 hours and then splitting the culture into two or more individual cultures. In some embodiments, after adding one or more exogenous cytokines, the cells are cultured at about 37° C. and 5% CO2 for an additional 5 to 14 days.

[0100] Cytokines In embodiments, the methods presented herein comprise culturing with an exogenous cytokine suitable for stimulating and / or activating T cell proliferation and / or differentiation. A surprising aspect of the present invention is that pre-culturing the cells with one or more peptides prior to the addition of one or more exogenous cytokines significantly improves various aspects of the tumor antigen-specific T cells generated by this process, including antigen specificity and phenotype. In one aspect, the disclosure provides a method of culturing a population of mononuclear cells with one or more peptides from one or more tumor antigens in the absence of exogenous cytokines. In some embodiments, the population is cultured in the absence of exogenous cytokines for about 30 minutes to about 30 hours. In some embodiments, the population is cultured in the absence of exogenous cytokines for about 8 hours to about 24 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 30 minutes. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 1 hour. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 2 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 3 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 4 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 5 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 5 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 7 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 8 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 9 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 10 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 11 hours.In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 12 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 13 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 14 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 15 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 16 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 17 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 18 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 19 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 20 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 21 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 22 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 23 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 24 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 25 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 26 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 27 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 28 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 29 hours. In some embodiments, the cells are cultured in the absence of exogenous cytokines for at least about 30 hours.

[0101] In an embodiment, after culturing the cells and peptides in the absence of exogenous cytokines, one or more exogenous cytokines are added to the culture. In an embodiment, the exogenous cytokines include IL-6, IL-7, IL-15, IL-12, or any combination thereof. In an embodiment, the exogenous cytokines added to the culture consist of IL-6, IL-7, IL-15, and IL-12. In an embodiment, IL-6 is added to the culture at a concentration of about 10 ng / mL to about 100 ng / mL, for example, about 10 ng / mL to about 50 ng / mL, or about 25 ng / mL to about 75 ng / mL, or about 50 ng / mL to about 100 ng / mL. In an embodiment, IL-7 is added to the culture at a concentration of about 1 ng / mL to about 10 ng / mL, for example, about 1 ng / mL to about 5 ng / mL, or about 2.5 ng / mL to about 7.5 ng / mL, or about 5 ng / mL to about 10 ng / mL. In an embodiment, IL-15 is added to the culture at a concentration of about 1 ng / mL to about 5 ng / mL, for example, about 1 ng / mL to about 2.5 ng / mL, or about 2.5 ng / mL to about 5 ng / mL. In an embodiment, IL-12 is added to the culture at a concentration of about 10 to about 100 IU / mL, for example, about 10 IU / mL to about 55 IU / mL, or about 50 IU / mL to about 100 IU / mL, or about 25 IU / mL to about 75 IU / mL. In an embodiment, once the cytokines are added to the culture, the culture is not disturbed until the MultiTAA-specific T cells are harvested. In an embodiment, the cells are harvested after a culture period of about 5 to about 14 days, for example, about 5 to about 10 days, for example, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days.

[0102] MultiTAA-specific T cell populations and their composition In one aspect, the disclosure provides a population of antigen-specific T cells (also referred to as tumor antigen-specific T cells, MultiTAA-specific T cells) generated by the methods described herein. In one aspect, the disclosure provides a population of antigen-specific T cells comprising between about 80% and about 99% CD3+ T cells, <1% CD14+ monocytes, <10% CD19+ B cells, and <10% CD3-CD56+ NK cells.

[0103] T cells are composed of different subsets that arise through differentiation and may differ in their activity, life span, and proliferation kinetics. T cell subsets include CD4+ and CD8+ T cells, naive T cells, and central memory (T CM ), and Effector Memory (T EM T cell subsets may be detected and measured by methods known to those of skill in the art, such as by flow cytometry following labeling of identifiable phenotypic markers (e.g., CD4, CD8, CD45RA, CD45RO, etc.) with fluorescently labeled antibodies.

[0104] In embodiments, the present disclosure provides a method for the treatment of cancer by inhibiting at least about 20% of central memory T cells (T CM In an embodiment, the tumor antigen-specific T cells comprise at least about 20% naive T cells. In an embodiment, the tumor antigen-specific T cells comprise at least about 40% effector memory T cells (T EM In embodiments, the tumor antigen-specific T cells include 10% or less of terminally differentiated effector memory cells that express CD45RA (T EMRA ).

[0105] In embodiments, the present disclosure provides a population of antigen-specific T cells that exhibits a higher degree of antigen-specific response compared to a population of antigen-specific T cells generated by prior methods, for example, a population of antigen-specific T cells that exhibits at least about 500 SFU / 2×10 as measured by IFNγ ELISpot. 5T cells exhibit individual specificity (combined reactivity to each target antigen). T cell functionality and antigen specificity can be determined by methods known in the art, including methods that measure proliferation and / or cytokine expression, such as IFNγ expression. IFNγ expression can be assessed, for example, by ELISA, ELISpot, or flow cytometry analysis.

[0106] The T cell population may be characterized by the diversity of antigen specificity (e.g., the number of tumor antigens that the T cell product has activity against among those represented by one or more peptides cultured with mononuclear cells). A high diversity of antigen specificity may be a desirable property of a T cell population. In some embodiments, the tumor antigen-specific T cells generated by the methods herein exhibit antigen specificity for at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the tumor antigens represented by one or more peptides used in the initial culture. In some embodiments, the tumor antigen-specific T cells generated by the methods herein exhibit antigen specificity for at least 75% of the tumor antigens represented by one or more peptides used in the initial culture. In some embodiments, the tumor antigen-specific T cells generated by the methods herein exhibit antigen specificity for each of the tumor antigens represented by one or more peptides. In embodiments, increasing the number of tumor antigens cultured with mononuclear cells surprisingly increases not only the antigen specificity but also the antigen diversity of the generated tumor antigen-specific T cells.

[0107] In some embodiments, the population comprises T cells that exhibit activity against one or more peptides of one or more antigens with which the population has not been previously cultured. In some embodiments, said activity against one or more peptides of one or more antigens with which the population has not been previously cultured is the result of epitope splatting. In some embodiments, the population has a cumulative degree of specificity for one or more peptides of one or more antigens with which the population has not been previously cultured, as measured by IFN-γ ELISpot, of at least about 100 SFU / 2×10 5 In some embodiments, the population has a cumulative total of at least about 30 SFU / 2×10 cells, as measured by IFNγ ELISpot, for a degree of specificity for one or more peptides of one or more antigens with which the population has not been previously cultured. 5 This includes those that are individual cells.

[0108] In an embodiment, the present disclosure provides a composition comprising a population of MultiTAA-specific T cells obtained by the methods provided herein. In an embodiment, the composition further comprises a pharma- ceutically acceptable buffer or excipient.

[0109] T cell products In one aspect, the disclosure provides a MultiTAA-specific T cell product (also referred to herein as a T cell product) generated by the methods described herein. In one aspect, the disclosure provides a T cell product generated by the methods described herein that comprises about 80% to about 99% CD3+ T cells, <1% CD14+ monocytes, <10% CD19+ B cells, and <10% CD3-CD56+ NK cells.

[0110] In embodiments, the present disclosure provides a method for the treatment of cancer by inhibiting at least about 20% of central memory T cells (T CMIn an embodiment, the tumor antigen-specific T cells comprise at least about 20% naive T cells. In an embodiment, the tumor antigen-specific T cells comprise at least about 40% effector memory T cells (T EM In embodiments, the tumor antigen-specific T cells comprise 10% or less of terminally differentiated effector memory cells expressing CD45RA (T EMRA ). CM are generally believed to have a greater potential for expansion and persistence in vivo compared to terminally differentiated T cells, and therefore the T cell products described herein are likely to exhibit advantageous therapeutic properties.

[0111] In embodiments, the present disclosure provides a MultiTAA-specific T cell product that exhibits a higher degree of antigen-specific response compared to a MultiTAA-specific T cell product generated by prior methods. For example, the MultiTAA-specific T cell product exhibits a higher degree of antigen-specific response as measured by IFNγ ELISpot than at least about 500 SFU / 2×10 5 For example, in an embodiment, the MultiTAA-specific T cell product exhibits at least about 700 SFU / 2×10 as measured by IFNγ ELISpot. 5 cells, at least about 1000 SFU / 2 x 10 5 cells, or at least about 1200 SFU / 2×10 5 This indicates the degree of specificity (combined reactivity to each target antigen) of individual cells.

[0112] In some embodiments, the T cell product generated by the methods herein exhibits antigen specificity for at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the tumor antigens represented by the peptide or peptides used in the initial culture. In some embodiments, the T cell product generated by the methods herein exhibits antigen specificity for at least 75% of the tumor antigens represented by the peptide or peptides used in the initial culture. In some embodiments, the T cell product generated by the methods herein exhibits antigen specificity for each of the tumor antigens represented by the peptide or peptides. In embodiments, increasing the number of tumor antigens cultured with mononuclear cells surprisingly increases the antigen specificity as well as the antigen diversity of the tumor antigen-specific T cells generated.

[0113] In some embodiments, the T cell product comprises T cells that exhibit activity against one or more peptides of one or more antigens that were not used to generate the T cell product. In some embodiments, the T cell product cumulatively exhibits at least about 100 SFU / 2×10 as measured by IFN-γ ELISpot. 5 In some embodiments, the T cell product comprises a degree of specificity for one or more peptides of one or more antigens that were not used to generate the T cell product, which are T cell. In some embodiments, the T cell product cumulatively has at least about 30 SFU / 2×10 as measured by IFN-γ ELISpot. 5 The T cell product is a single cell that has a degree of specificity for one or more peptides of one or more antigens that were not used to generate the T cell product.

[0114] In some embodiments, said activity against one or more peptides of one or more antigens with which the population has not previously been cultured is the result of epitope plating.

[0115] Epitope spreading occurs when T cells targeting a specific antigen destroy the antigen and expose new "non-target" epitopes / antigens. Epitope spreading in this context was previously thought to be the result of the endogenous immune system from the patient. Surprisingly, we discovered that epitope spreading results from the spread of the MultiTAA-specific T cell product itself. Without wishing to be bound by theory, it is highly likely that cells specific for non-target antigens within the final drug product contribute to the epitope spreading that eliminates tumors in-vivo.

[0116] cryopreservation In some embodiments, the method further comprises cryopreserving the mononuclear cells and then thawing the cryopreserved cells prior to placement in the container. In some embodiments, the method further comprises cryopreserving the MultiTAA-specific T cell product. Cryopreservation may be performed by placing the mononuclear cells and / or MultiTAA-specific T cells in a cryopreservation medium or freezing solution, such as, for example, CryoStor® CS10 or a derivative thereof, Hyperthermasol, or any freezing solution containing about 5 to about 15% DMSO. Thus, in some embodiments, the present disclosure provides the mononuclear cells and / or MultiTAA-specific T cells described herein in a cryopreserved state. To utilize the mononuclear cells and / or MultiTAA-specific T cells after cryopreservation, for example, for use of the mononuclear cells for culturing with a peptide or for use of the MultiTAA-specific T cells in the treatment of a patient, the cells are removed from the freezer and thawed in a water bath at about 37° C. until most of the solution has thawed. The cells are then resuspended in medium and optionally washed prior to use. In some embodiments, the thawed mononuclear cells are allowed to rest for about 4 to 48 hours before being placed in a container. In some embodiments, mononuclear cells and / or thawed MultiTAA-specific T cells are prepared for use by counting and determining cell viability.

[0117] In embodiments, the present disclosure provides a pre-made tumor antigen-specific T cell product. Such a product eliminates the need for donor identification, cell procurement, T cell product manufacturing, and T cell product analysis steps that must occur between patient identification / enrollment and patient treatment, thus significantly shortening the time to treatment for a patient. Instead of a time to treatment of 14, 21, 28, 35, or more hours (for example), a patient can be treated with a pre-made tumor antigen-specific T cell product within about 72 hours of patient identification. In embodiments, the method for generating a MultiTAA-specific T cell product disclosed herein further comprises HLA typing a donor of mononuclear cells used to generate the MultiTAA-specific T cell product.

[0118] Treatment method In one aspect, the disclosure provides a method for treating a patient with cancer. In some embodiments, the method disclosed herein comprises administering to a patient in need thereof a pharmaceutical composition comprising the MultiTAA-specific T cells disclosed herein. In one embodiment, the pharmaceutical composition is a suspension of MultiTAA-specific T cells in a pharma- ceutically acceptable buffer or excipient. In some embodiments, the method comprises administering to a patient a tumor antigen-specific T cell product disclosed herein, the product comprising about 50 x 10 per treatment. 6 Pieces ~ approx. 300×10 6 For example, in one embodiment, the product is administered to a patient in a single dose of about 100×10 6 pieces~approx. 200×10 6 In one embodiment, the product is administered to a patient in doses of about 100×10 6 Or about 200 x 10 6In embodiments, the product is administered to the patient in a single dose. In embodiments, the product is administered to the patient by any suitable route, in particular intravenous or intra-arterial infusion. In embodiments, the infusion may last for about 15 minutes, 30 minutes, 45 minutes, 60 minutes, or 90 minutes per dose. In embodiments, the MultiTAA-specific T cell product or pharmaceutical composition is administered in a single dose or multiple doses.

[0119] In embodiments, the cancer is a lymphoma or leukemia. In embodiments, the cancer is a solid tumor. In embodiments, the patient has a cancer selected from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), multiple myeloma, non-Hodgkin's lymphoma, sarcoma, breast cancer, and pancreatic cancer.

[0120] Thus, in an embodiment, the present disclosure provides a method for treating a patient with cancer with a pre-made MultiTAA-specific T cell product disclosed herein, where the patient has been HLA typed or the patient's HLA type is known, and the MultiTAA-specific T cell product is selected from a bank of previously generated MultiTAA-specific T cell products to achieve a desired level of HLA match between the donor and the recipient. In an embodiment, the donor and the recipient have at least one HLA match, at least two HLA matches, or at least three HLA matches. In an embodiment, the method comprises administering to the patient a pre-made MultiTAA-specific T cell product disclosed herein, and further comprises treating the patient with one or more additional pre-made MultiTAA-specific T cell products. In an embodiment, the one or more additional T cell products (e.g., a second T cell product) may be administered to the patient at least about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after the previous T cell product treatment has been administered. In embodiments, the one or more additional treatments may be treatments with the same pre-made MultiTAA-specific T cell product as the previous one or more treatments (e.g., a MultiTAA-specific T cell product derived from cells of the same donor) or may be treatments with a pre-made MultiTAA-specific T cell product different from the previous one or more treatments (e.g., the one or more additional treatments may be treatments with one or more MultiTAA-specific T cell products generated from cells of one or more different donors). In embodiments, the selection of a different MultiTAA-specific T cell product for the second or further treatment may be suitable for use in patients who have mounted an immune response against the first MultiTAA-specific T cell product and / or have rejected the first MultiTAA-specific T cell product or are suspected of rejecting the first MultiTAA-specific T cell product.

[0121] In embodiments, a second population of antigen-specific T cells (second treatment) in an autologous setting is generated by: (i) obtaining a first population of mononuclear cells from a patient; (ii) generating the first population of antigen-specific T cells according to the methods described herein; (iii) administering the first population of antigen-specific T cells to the patient; (iv) obtaining a second population of mononuclear cells from the patient at least about 4 weeks, about 5 weeks, or about 6 weeks after administration of the first population of antigen-specific T cells; (v) generating the second population of antigen-specific T cells according to the methods described herein; and (vi) administering the second population of antigen-specific T cells to the patient. This second antigen-specific T cell product may have an expanded number of antigen-specific cells compared to the first antigen-specific T cell product, providing a more potent second treatment.

[0122] In one aspect, the present disclosure provides MultiTAA-specific T cells that are allogeneically transplanted into recipient patients and effectively treat cancer without significant effects of graft-versus-host disease (GVHD) responses. Generally, in patient populations receiving allogeneic tumor antigen-specific T cells for cancer treatment, GVHD responses are expected to occur because the patients are not immunocompromised. Without wishing to be bound by theory, the surprising lack of GVHD responses in recipients of the MultiTAA-specific T cells disclosed herein may be due to the improved tumor antigen-specific cells achieved by the compositions and methods disclosed herein.

[0123] definition The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that "including," "includes," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0124] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, for example within 5-fold or within 2-fold. When a particular value is described in the present specification and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0125] As used herein, "and / or" encompasses all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or"). The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0126] Any concentration range, percentage range, ratio range, or integer range herein is understood to include any integer value within the stated range, and fractions thereof (such as tenths and hundredths of integers) where appropriate, unless otherwise indicated. The terms "about" and "approximately" refer to a range of statistically meaningful values. Such ranges may be within an order of magnitude, preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. In certain embodiments, the term "about", when immediately preceding a numerical value or number, means that the numerical value or number is within a range of plus or minus 10%. The permitted variation encompassed by the terms "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely differing sizes, shapes, and dimensions may employ the described arrangements.

[0127] Unless otherwise specified, all terms used herein have the same meaning to those of ordinary skill in the art and the practice of the present invention employs conventional techniques of microbiology and recombinant DNA technology that are within the knowledge of those of ordinary skill in the art.

[0128] The term "mononuclear cells" refers to cells with a round nucleus, such as those isolated from peripheral blood of a patient or donor. Mononuclear cells may be a population of white blood cells or peripheral blood mononuclear cells (PBMCs) and may be obtained using known methods such as Ficoll-Hypaque density gradients. Mononuclear cells include monocytes, lymphocytes, T cells, B cells, and NK cells, among others.

[0129] The terms "subject," "individual," and "patient" are used interchangeably herein. In embodiments, the subject is a mammal, such as, for example, a mouse, rat, rabbit, cat, dog, pig, sheep, horse, non-human primate (e.g., cynomolgus monkey, chimpanzee), or human. The term "donor" refers to the subject, individual, or patient from which mononuclear cells are obtained.

[0130] As used herein, the term "exogenous" refers to a substance that is generated from outside a cell, cell culture, container, device, suspension, composition, etc. and introduced into a cell, cell culture, container, device, suspension, composition, etc. The term "endogenous" refers to a substance that is generated from within a cell, cell culture, container, device, suspension, composition, etc.

[0131] "T cells" is a term commonly used in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Th1) cells or T helper 2 (Th2) cells. T cells can be CD4+ T cells, CD8+ T cells, CD4+CD8+ T cells, CD4-CD8- T cells, or other subsets of T cells.

[0132] As used herein, the term "antigen-specific T cells" is intended to refer to T cells that recognize a particular antigen and respond, for example, by proliferating and / or producing cytokines in response thereto. Terms such as "tumor antigen-specific T cells" or "tumor antigen-specific T cell product" or "tumor antigen-specific T cell population" are used herein interchangeably with terms such as "MultiTAA-specific T cells" or "MultiTAA-specific T cell product". As used herein, the terms "tumor-specific antigen" and "tumor antigen" are used interchangeably with the term "tumor-associated antigen" or "TAA".

[0133] The term "MAT101-4" is used herein to refer to the MultiTAA-specific T cell manufacturing process as shown diagrammatically in FIG. 1B. The term "MAT103-4" is used herein to refer to the MultiTAA-specific T cell manufacturing process as shown diagrammatically in FIG. 1C. The term "MAT110-CD56+Depl" is used herein to refer to the MultiTAA-specific T cell manufacturing process as shown diagrammatically in FIG. 1A. The MAT110-CD56+Depl process involves direct stimulation of PBMCs with peptides in a vessel having at least one gas permeable surface. The term "MAT210-CD56+Depl-CD19+Depl" is used herein to refer to the same MultiTAA-specific T cell manufacturing process as MAT110-CD56+Depl, except that mononuclear cells are depleted of CD19+ cells as well as CD56+ cells prior to culture with peptides. Unlike the MAT101-4 and MAT110-No Depl processes, the MAT110-CD56+Depl and MAT210-CD56+Depl-CD19+Depl processes include culturing PBMCs and peptide mixes in the absence of exogenous cytokines prior to the addition of one or more exogenous cytokines. The MAT110-CD56+Depl and MAT210-CD56+Depl-CD19+Depl processes also require fewer total days, i.e., about 5 to about 14 days, compared to the MAT101-4 or MAT103-4 processes.

[0134] In embodiments, the identity and / or phenotype of cells are measured to determine the characteristics of the multiTAA-specific T cells provided herein or to determine the characteristics of cells used as starting material to generate the multiTAA-specific T cells provided herein. Expression markers such as, but not limited to, CD3, CD4, CD8, CD56, CD19, CD14, and any other markers disclosed herein or known in the art, can be measured by flow cytometry. For example, cells can be labeled with an antibody, e.g., a fluorescently labeled antibody, and analyzed by a flow cytometer. Furthermore, cell number and / or cell viability can be measured using hemocytometry, trypan blue staining, or other methods known in the art. In some embodiments, other cell markers, e.g., control markers, are measured to assess the functionality or exhaustion of the MultiTAA-specific T cells. Such markers may be selected from, for example, TCRαβ, CD45RA, CD45RO, CD25, CD28, CD127, CD95, CCR7, CD62L, Lag3, TIM-3, PD-1, TIGIT, etc.

[0135] The term "central memory T cells" is used herein to refer to CM " and refers to the subset of T cells that secrete IL-2 after activation. CM The cells were CD45RO+CD45RA lo , CD127+ (IL-7R+) and CCR7 (CCR7 hl ) and CD62L (CD62 hl The term "effector memory T cells" is used herein to refer to T EM " and are used interchangeably, and, like central memory T cells, are CD45RO+CD45RA lo , and CD127+ (IL-7R+) but CCR7 lo ) and CD62L lo Effector memory T cells (T EMRA CD45RO lo, CD45RA hi , CD62L lo , and CCR7 lo ) T EM and T EMRA is T CM They have more differentiated effector functions and generally have a higher proliferation capacity than T cells. As used herein, the term "epitope spreading" refers to the spreading or diversification of the epitope specificity of a T cell immune response. For example, in embodiments, epitope spreading refers to the spreading or diversification of an initial set of specificities (e.g., specificity for a tumor antigen used to generate or spread an antigen-specific T cell product) to additional specificities, such as specificity for other tumor antigens that were not present during the generation or initial spread of the antigen-specific T cell product.

[0136] As used herein, the term "harvest" means to collect, isolate, and / or remove from a culture vessel or device. Antigen-specific T cells may be harvested using methods known to those of skill in the art, including, but not limited to, removing from the culture vessel or device, optionally washing the cells using centrifugation or an automated cell processing device, and optionally resuspending in fresh medium, a solution suitable for counting, a solution suitable for analysis, a solution suitable for cryopreservation, or a solution suitable for administration to a patient.

[0137] As used herein, "treatment" or "treatment" or "palliation" or "amelioration" are used interchangeably. These terms refer to an approach to obtain a beneficial or desired result, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to a therapeutically relevant improvement or effect on one or more diseases, conditions, or symptoms under treatment, such as cancer. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject who reports physiological symptoms of one or more diseases, even if the disease, condition, or symptom has not yet manifested.

[0138] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve a result, e.g., to produce a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the method of administration, etc., which can be readily determined by one of ordinary skill in the art. The specific dosage may vary depending on one or more of the particular agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system by which it is delivered. In embodiments, an effective amount of multiTAA-specific T cells may be administered in either a single dose or multiple doses by any of the accepted modes of administration for such cell products, including intravenous injection, intraarterial injection, intraperitoneal injection, parenteral administration, intramuscular injection, intradermal injection, intralymphatic injection, or subcutaneous injection.

[0139] All articles, publications, and patents cited herein are incorporated by reference herein as if each individual article, publication, or patent was specifically and individually indicated to be incorporated by reference and are disclosed and described in connection with the methods and / or materials for which the publications are cited. However, reference to any article, article, publication, patent, patent publication, or patent application cited herein is not an admission or suggestion that they are valid prior art or form part of the general knowledge anywhere in the world.

[0140] It should be understood that the above description and the examples which follow are intended to be illustrative, not limiting, of the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0141] Working Example Example 1: Development of an improved manufacturing process

[0142] Known manufacturing processes for generating tumor antigen-specific T cells are costly, time-consuming, and difficult to scale up to large-scale manufacturing. The improved manufacturing process (MAT110-CD56+Depl shown in FIG. 1A) was developed to address these limitations of the previous processes (MAT101-4 shown in FIG. 1B; MAT110-No Depl or MAT103-4 shown in FIG. 1C).

[0143] Example 2: Comparison of starting material and developed product

[0144] To understand whether the starting cell population for generating tumor antigen-specific T cells affects the final cell phenotype, viability, and antigen specificity, three different starting cell populations of PBMCs were compared. PBMCs were obtained from donors and either not depleted, depleted of CD56+ cells using the MAT110-CD56+Depl method (removing NK cells) as shown in Figure 1A, or depleted of both CD56+ and CD19+ cells using the MAT210-CD56+Depl-CD19+Depl method (removing NK and B cells). The MAT210-CD56+Depl-CD19+Depl method is the same as the "MAT110-CD56+Depl" method, except that not only CD56+ cells but also CD19+ cells are removed from mononuclear cells prior to peptide pulsing. After cell populations were generated, they were analyzed by flow cytometry to determine the percent (%) of viable cells that were monocytes, T cells, B cells, or NK cells (Figures 2A-C). The proportions of CD4+ and CD8+ T cells in the CD3+ T cell compartment were also determined (Figures 2A-C).

[0145] Figures 3, 4, and 5 provide an analysis of tumor antigen-specific T cell products generated using the methods disclosed herein starting from cultures of non-depleted (Figure 3), CD56+ depleted (Figure 4), or CD56+ and CD19+ depleted (Figure 5) cells. The monocyte, T cell, B cell, and NK cell compartments are shown in the first bar of each figure, the percentage of CD3+ T cells that are CD4+ or CD8+ is shown in the second bar of each figure, and the phenotype of CD3+ T cells (Tナイーブ , T CM , T EM , or T EMRA ) is shown in the third bar of each figure, and the total response to tumor antigens measured by IFNγ ELISpot is shown in the fifth bar of each figure. Depletion of CD56+ or CD56+CD19+ was shown to improve T cell proliferation and antigen specificity compared to no depletion.

[0146] Example 3: Comparison of tumor antigen-specific T cells generated using an improved manufacturing process with tumor antigen-specific T cells generated using a prior process

[0147] To evaluate the quality of the T cell products generated using the MAT110-CD56+Depl method compared to those generated by the preceding process, the proliferation and viability of tumor antigen-specific T cells were investigated. Figure 6A shows the fold expansion of T cells in three of these methods. The MAT110-CD56+Depl method, an accelerated process compared to the MAT103-4 method, showed similar T cell proliferation fold levels to cells prepared by the MAT103-4 method. However, the MAT110-No Depl method showed significantly lower proliferation than the MAT103-4 and MAT110-CD56+Depl methods. The viability of cells was also similar in the three different methods (Figure 6B). Next, the phenotype of cells generated from each method was compared. Figure 7A shows that the percentage (%) of CD3+ cells and the ratio of CD4+ to CD8+ T cells were similar in the final products prepared by the three different methods. The T cell products obtained using MAT110-No Depl or MAT110-CD56+Depl were characterized as central memory (T CM) cells were significantly higher (Figure 7B). Thus, T cell products generated using MAT110-No Depl or MAT110-CD56+Depl may persist longer in vivo compared to T cell products generated using MAT103-4. Taken together, these data suggest that the MAT110-CD56+Depl method offers an accelerated process with favorable T cell expansion fold, viability, and potential for long-term persistence of the T cell product in vivo.

[0148] T cells obtained by the three different methods were also evaluated for antigen specificity by IFNγ ELISpot. PBMCs were collected from individual donors (i.e., donors D2009, D2021, D3205, D2957, D5145, D595, and D8966) and antigen-specific T cells were generated by the three methods described above. As shown in Figure 8A, cells generated using the MAT110-CD56+Depl method showed higher antigen specificity compared to cells from the same donor generated by the MAT103-4 or MAT110-No Depl methods (i.e., higher antigen specificity compared to MAT103-4 and MAT110-No Depl). The total degree of antigen specificity (combined reactivity of T cell products against each target antigen when donor data are pooled) is shown in Figure 8B. These data indicate a statistically significant increase in antigen specificity of T cell products generated using the MAT110-CD56+Depl method. The total degree of response is also shown in Figure 9A. As shown in Figure 9B, the MAT110-CD56+Depl method resulted in a higher number of T cell products with more antigen specificities, i.e., a higher degree of antigen diversity. Figure 9C further shows the increased number of products with specificity for multiple tumor antigens achieved with MAT110-CD56+Depl cells. Figure 9D shows the relationship between antitumor activity (measured as change in luminescence over 6 days in firefly luciferase-expressing THP-1 cells co-cultured with MultiTAA-specific T cells) and antigen specificity (IFNγ ELISpot, SFU / 2x10 5The results show that there is a linear correlation between the antigen specificity and the number of cells per cell (measured by 1000 cells per cell). Thus, the increase in antigen specificity achieved using the improved process disclosed herein is at least about 4-fold stronger than previous processes.

[0149] Thus, the MAT110-CD56+Depl manufacturing process significantly increased the proportion of multitarget T cells. For example, cells generated by the MAT110-CD56+Depl method had significantly greater antigen specificity as well as diversity compared to cells generated by the previous method.

[0150] These results demonstrated that the improved manufacturing process MAT110-CD56+Depl resulted in improved antigen specificity and diversity, higher expansion fold, higher cell viability, and a desirable T cell phenotype for antitumor activity.

[0151] Example 4: Analysis of the peptide pulsing process

[0152] A study was performed to evaluate pre-incubating high concentrations of peptides with cells prior to exogenous cytokine addition (using the MAT110-NoDepl method). PBMCs from healthy donors were pulsed overnight with peptides from six different antigens (WT1, survivin, NY-ESO-1, PRAME, SSX2, and MAGEA4) in the presence or absence of cytokines (IL-6, IL-12, IL-7, and IL-15) for the generation of MultiTAA-specific T cells. On day 1, cytokines were added to the "cytokine-free" cultures, while cytokines were maintained in the "cytokine-present" cultures, following the standard MAT110-NoDepl method. Figure 10 shows an exemplary series of calculations to determine peptide amounts and concentrations for the peptide pulsing step.

[0153] Multi-tumor antigen (MultiTAA)-specific T cells were subjected to IFN-γ ELISpot analysis as a readout of antigen specificity. As shown in Figure 11, the presence of cytokines during the peptide pulsing step significantly reduced antigen specificity. This indicates that culturing cells with peptides in the absence of cytokines for a period of time before adding exogenous cytokines significantly enhances the generation of MultiTAA-specific T cells.

[0154] Furthermore, a study was performed to evaluate the effect of the volume of the peptide pulsing step on antigen specificity (using the MAT110-NoDepl method). In this experiment, PBMCs from healthy donors were pulsed overnight with peptides from four different antigens (WT1, survivin, NY-ESO-1, PRAME) in either a low volume (10 mL) or a high volume (100 mL) to generate MultiTAA-specific T cells (n=3). MultiTAA-specific T cells were subjected to IFN-γ ELISpot analysis as a readout of antigen specificity. As shown in Figure 12, antigen specificity was significantly increased in T cells generated under low volume conditions. Without wishing to be bound by theory, it is believed that the increase in antigen specificity is due to the increased peptide-cell contact achieved in the low volume peptide pulsing culture step.

[0155] Taken together, these findings suggest that in the absence and / or low doses of exogenous cytokines (e.g., approximately 0.1 × 10 6 cells / cm 2 ~Approx. 1×10 7 cells / cm 2 It was shown that performing the peptide pulsing step at a low volume (which allows cells seeded at a density of 100 ng / mL to be contacted with a high concentration of peptide) significantly improves the antigen specificity of the resulting MultiTAA-specific T cells.

[0156] Example 5: Comparison of positive T cell selection with NK cell depletion

[0157] A study was performed to compare the fold expansion, viability, and antigen specificity of MultiTAA-specific T cells starting from cells positively selected for T cell markers versus cells depleted of CD56 (NK cell marker) positive cells. Positively selected T cells and CD56+ depleted T cells were cultured with tumor antigen peptides (PRAME, NY-ESO-1, survivin, WT1) and harvested on day 9 to measure proliferation and viability. As shown in Figure 13, positively selected T cells (CD4+CD8+CD14+) showed reduced fold expansion and antigen specificity compared to CD56+ depleted cells (MAT110-CD56+Depl method). Without wishing to be bound by theory, positive selection of T cells via binding to CD4 and CD8 inhibits their ability to be stimulated by antigen presenting cells in the subsequent steps of the method.

[0158] Example 6: Characterization of antigen-specific T cells and disease progression in patients receiving MultiTAA-specific T cells

[0159] A study was conducted to perform various analyses of the efficacy of administering the MultiTAA-specific T cell product generated by the MAT103-4 method to cancer patients. In this study, the patient was diagnosed with acute myeloid leukemia (AML) and had failed five prior therapies (I+C [induction chemotherapy], FLAG-IDA [fludarabine, cytarabine (Ara-C), granulocyte colony-stimulating factor, idarubicin] + IC chemotherapy, MEC [mitoxantrone, etoposide, cytarabine]) + IC chemotherapy, azacitidine, decitabine). In addition, the patient expressed a c-KIT mutation and a genetic abnormality: t(8;21)(q22;q22.1) [RUNX1-RUNX1T1]. PBMCs from matched donors were collected by leukapheresis, and a MultiTAA-specific T cell product was generated using the MAT103-4 method described herein with a peptide library corresponding to WT1, NY-ESO-1, survivin, and PRAME. The MultiTAA-specific T cell products (5 × 10 per round) were then 6Patients were administered 100 mg of IFNγ ELISA kit (3 doses) and T cell specificity, composition, phenotype and functionality were assessed in peripheral blood samples obtained from patients pre-infusion and at weeks 8, 12, 18, 24 and 32 post-infusion. Tumor antigen expression in bone marrow of patients at the same time points was also assessed by next generation sequencing (NGS). Minimal residual disease levels (%MRD) were compared with various measures of T cell activity and tumor antigen expression. T cell specificity for target antigen was assessed by IFNγ ELISpot (SFU / 2×10 5 This was determined by the number of people.

[0160] The results are shown in Figures 14-23. Figure 14 shows that target antigen-specific T cells (i.e., T cells specific for the target antigens, WT1, survivin, NY-ESO-1, and PRAME, used to generate the MultiTAA-specific T cell product) were present in the patient's peripheral blood and inversely correlated with MRD, suggesting antitumor activity. In addition, T cells specific for additional tumor antigens not present in the set of target antigens used to generate the MultiTAA-specific T cell product were present in the patient's peripheral blood, which also inversely correlated with MRD (Figure 15). These data indicate that epitope spreading is occurring after infusion of the MultiTAA-specific T cell product, and further suggest a significant contribution of "non-targeted" immune responses to the antitumor response after infusion of the product. Figure 16 shows T cell expression in the patient's bone marrow as detected by next generation sequencing (NGS). T cell infiltration of the bone marrow (indicating lymphocytic infiltration) inversely correlated with disease (MRD), and increased tumor burden was noted following T cell infiltration.

[0161] Figure 17 shows that T cell composition and phenotype in patient PBMCs were relatively stable over time in this study. Functional studies showed that target antigen-specific IFNγ production was sustained in both CD4+ and CD8+ T cells (Figure 18). Furthermore, most antigen-specific T cells (i.e., IFNγ+) were Lag-3 negative, thus protecting the patient's antigen-specific T cells from exhaustion (Figure 19).

[0162] Figure 20 shows that a significant number of distinct T cell clones expanded in the patient after product infusion. Clonal expansion / contraction of T cells was observed using TCR monitoring.

[0163] Figure 21 shows that bone marrow expression of target tumor antigens survivin and WT1 correlates directly with MRD. Furthermore, the decrease in expression of survivin and WT1 correlated with the increase in target antigen-specific T cells detected in peripheral blood. Figure 22 shows that bone marrow expression of non-target antigens (e.g., proteinase-3) correlates with MRD at later time points detected after product infusion, indicating epitope spreading. Furthermore, the increase in the percentage of proteinase-3-specific T cells correlated inversely with proteinase-3 detected in the tumor. To understand whether additional non-target antigens were reduced, RNA expression of seven non-target antigens was measured in patients and antigen specificity from T cells was measured against the same non-target antigens. Figure 23 shows that additional non-target tumor antigens were significantly reduced after product infusion, with a trend toward reduction along with MRD. Taken together, these data demonstrate epitope spreading following administration of tumor antigen-specific T cells.

[0164] Example 7: Epitope plating after treatment with multiple tumor antigen-specific T cells Epitope spreading occurs when T cells targeting a specific antigen destroy the antigen, exposing new "non-target" epitopes / antigens. To further evaluate the observation of epitope spreading after administration of MultiTAA-specific T cells to patients, MultiTAA-specific T cells were tested in vitro and in vivo in different cancer types.

[0165] In a clinical trial for lymphoma, patients were administered MultiTAA-specific T cells to observe epitope plating. Specifically, patients were evaluated at 3 months (Mth3) and 9 months (Mth9) after infusion of MultiTAA-specific T cells generated using the MAT101-5 method. The MAT101-5 method is identical to the MAT101-4 method, except that the cells were cultured with peptides from five tumor-associated antigens (survivin, NY-ESO-1, MAGEA4, SSX2, PRAME). Nine months after the first dose, patients treated with MultiTAA-specific T cells had reduced tumor burden (Figure 25A), and antigen specificity was measured by IFN-γ ELISpot analysis. At 9 months, specificity for all tumor-associated target antigens was observed (Figure 25B), as well as increased specificity for the non-target antigen MAGEC1 (Figure 25B).

[0166] The proliferation of T cells specific for the "target" epitope (left) and the "non-target" epitope (right) showing epitope plaque splatting from the peptides used to generate the antigen-specific T cell product was measured in responder and non-responder lymphoma patients 6 weeks after infusion of MultiTAA-specific T cells (Figure 26B). Compared to non-responders, significantly greater proliferation of T cells specific for both the "target" and "non-target" epitopes was observed in responders, suggesting that epitope plaque splatting may be positively associated with responsiveness to therapy.

[0167] Preparation of TAA-specific cell products has generally focused on purifying and injecting a T cell population specific for a target antigen from a starting cell population (FIG. 27A, "PBMC"). Surprisingly, the examples presented herein show that the T cells in the cell product (including FIG. 27A, "Final Drug Product", MT-401, MT-601, or other described embodiments) have specificity for additional non-target antigens. Once the target antigen is destroyed, additional epitopes become available to expand the T cells in the final drug product specific for the "non-target" antigen. Furthermore, this "non-target" specificity is indicative of epitope spreading, which may be beneficial to the overall tumor response generated by the cell product.

[0168] To assess whether the "non-targeted" TAA responses within the T cell product are associated with suppression of cancer cell proliferation, MT-601 was further evaluated in a pancreatic cancer cell model. Using an in vitro test, target cells (i.e., pancreatic cancer cells) were co-cultured with MT-601 cells and non-specific T cells (anti-CD3 / CD28 activated). When PANC1 cells were co-cultured with anti-CD3+ / CD28+ activated non-specific T cells, suppression of cancer cell proliferation was not observed. However, co-culture with MT-601 cells resulted in a decrease in cancer cell proliferation (Figure 27B). Specificity for the target antigen was measured by IFN-γ ELISpot analysis, and specificity was observed after co-culture with MT-601 cells alone or with PANC1 cells, but no target antigen specificity was observed for anti-CD3+ / CD28+ activated non-specific T cells co-cultured with PANC1 cells (Figure 27C). Epitope blotting revealed specificity for non-target antigens in MT-601 cells co-cultured with PANC1 cells, but none in culture with anti-CD3+ / CD28+ activated T cells (Figure 27D). Together, these data indicate that T cells in the MT-601 cell product maintain specificity for target antigens and further increase specificity for non-target antigens, demonstrating epitope blotting.

[0169] An additional MultiTAA-specific product, MT-401, was generated using a peptide library with specificity for four antigens (PRAME, NY-ESO-1, survivin, and WT1) using the MAT110-CD56+ method (Figure 28). MT-401 was evaluated for its antitumor effect on cells from a patient with acute myeloid leukemia. The patient had previously received decitabine, venetoclax, and cytarabine for lymph node removal prior to hematopoietic stem cell transplantation, followed by fludarabine and cyclophosphamide, but relapsed prior to MT-401 infusion. Bone marrow (BM) mononuclear cells were isolated from the patient at relapse and co-cultured with control PBMCs or MT-401 cells (Figure 29) at an effector-to-target (E:T) ratio of 4:1. The cells were co-cultured for 4 days, and cell proliferation was monitored by measuring glucose and lactate production daily. A decrease in glucose production was observed in cells cultured with MT-401, indicating a decrease in target cell proliferation (Figure 30A). An increase in lactate production was observed in cells cultured with MT-401, which correlated with T cell proliferation (Figure 30B). Cancer cell growth was measured at the end of the co-culture, and target cancer cells cultured with MT-401 showed a decrease in cell growth (Figures 31A and 31B), indicating an improvement in target cell killing. The antigen specificity of MT-401 cells was compared before and after co-culture, and the specificity for PRAME and WT1 was increased after culture with cancer cells (Figure 32). Not only was the antigen specificity for the four target antigens from which MT-401 was generated maintained on T cells (Figure 33A), but specificity for non-target antigens was observed in MT-401 cells compared to PBMCs, which showed no specificity for non-target antigens (Figure 33B).

[0170] Example 8: Donor Selection and Manufacturing Scale-Up

[0171] Donors are screened for HLA diversity and selected based on their ability to match a broad set of HLA genotypes in a given sample population. After selection, cells are collected by leukapheresis and prepared as described herein. Antigen-specific T cells are frozen prior to administration to patients (Figure 34). From as few as 10 donors, a ready-to-go inventory of MultiTAAT cell preparations can be generated that can match over 90% of patients with 4 / 8 HLA alleles. Furthermore, from as few as five donors, a ready-to-go inventory of MultiTAAT cell preparations can be generated that can match nearly 100% of patients with 2 / 8 HLA alleles (Figure 35).

[0172] To evaluate whether the MAT110-CD56+Depl method can be scaled up for manufacturing, cells were generated on G-Rex 10M and G-Rex 500M. Figure 36 shows that scaling up from G-Rex 10M to G-Rex 500M device does not change the cell viability and T cell expansion fold of the final product. Therefore, the MAT110-CD56+Depl method provided herein is suitable for scaled up manufacturing process.

[0173] Example 9: Additional antigens enhance T cell specificity and diversity

[0174] Additional studies were performed to evaluate the effect of adding an additional peptide library of tumor antigens to the culture mix. Mononuclear cell populations from three donors were stimulated with a peptide library from four tumor antigens or six tumor antigens according to the MAT110-CD56+Depl method provided herein. Mononuclear cells from a fourth donor were stimulated with a peptide library from 12 tumor antigens. Surprisingly, as shown in Figure 37, the MultiTAA-specific T cell population generated using peptides from 12 tumor antigens produced 4,500 SFU / 2x10 6 T cells as measured by IFNγ ELISpot. 5 The results showed that the total number of responses (sum of responses to each target antigen) exceeded 100, indicating an increase in T cell diversity and specificity.

Claims

1. 1. A method for generating tumor antigen-specific T cells, comprising: (i) placing a population of mononuclear cells obtained from a subject into a container comprising at least one gas permeable surface, said mononuclear cells being present in an amount of about 0.1×10 per square centimeter of said gas permeable surface; ６ pieces ~ approx. 9×10 ６ adding the mononuclear cells to the vessel at a cell seeding density of 100 μg / ml to form a cell culture; (ii) adding one or more peptides from one or more tumor antigens to the cell culture; (iii) culturing said one or more peptides with said mononuclear cells in said vessel at about 37° C. in the absence of exogenous cytokines; A method comprising:

2. 2. The method of claim 1, further comprising the step of: (iv) adding one or more exogenous cytokines to the cell culture after the culturing of step (iii).

3. The method of claim 1 or 2, wherein the exogenous cytokine is added after about 30 minutes to about 30 hours of culture.

4. The method of any one of claims 1 to 3, wherein the mononuclear cells are cultured in the presence of the one or more peptides and exogenous cytokines for about 5 to about 14 days.

5. 5. The method of any one of claims 1 to 4, wherein each peptide of the one or more peptides of (ii) is added to the culture at a concentration of about 0.1 to about 0.5 μg / mL.

6. The method of any one of claims 1 to 5, wherein each peptide is added to the culture at a concentration of about 0.2 μg / mL to about 0.3 μg / mL.

7. 7. The method of any one of claims 1 to 6, wherein the one or more peptides are cultured with the mononuclear cells in the vessel at about 37°C for at least about 8 hours prior to the addition of exogenous cytokines.

8. 8. The method of claim 7, wherein the mononuclear cells and the one or more peptides are cultured in the vessel at about 37° C. for about 8 to about 30 hours prior to the addition of exogenous cytokines.

9. About 0.1×10 per square centimeter of the gas permeable surface ６ pieces ~ approx. 9×10 ６ 9. The method of claim 1, wherein the mononuclear cells are cultured with the one or more peptides at a cell concentration of 100-1500 μg / ml.

10. 10. The method of any one of claims 1 to 9, wherein the container comprises 1, 2, 3, 4, 5, or 6 gas permeable surfaces.

11. The method of any one of claims 1 to 10, wherein the container comprising at least one gas permeable surface is a G-Rex® device.

12. The method of claim 11, wherein the G-Rex device is a 100M G-Rex device.

13. The method of claim 11, wherein the G-Rex device is a 500M G-Rex device.

14. 13. The method of any one of claims 1 to 12, wherein the one or more peptides comprise one or more libraries of peptides derived from the one or more tumor antigens.

15. 15. The method of claim 1, wherein each library of peptides comprises peptides spanning the sequence of the tumor antigen.

16. 16. The method of any one of claims 1 to 15, comprising adding in step (ii) peptides from at least four different tumor antigens.

17. 16. The method of any one of claims 1 to 15, comprising adding peptides from 4, 5, 6, 7, 8, 9, 10, 11, or 12 different tumor antigens in step (ii).

18. The tumor antigens include CEA, MHC, CTLA-4, gp100, mesothelin, PRAME (OIP3) PD-L1, TRP1, CD40, EGFP, Her2, TCR alpha, trp2, TCR, MUC1, cdr2, ras, 4-1BB, CT26, GITR, OX40, TGF-α. WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, Melan A / MART1, Ras mutant, gp 100, p53 mutant, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, Myc, MYCN, RhoC, TRP-2, GD3, fucosyl GM1, mesothelin, PSCA, MAGE 16. The method of any one of claims 1 to 15, wherein the antigen is selected from the group consisting of: A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2, Page 4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, mucin-1, BRAF, CCNA1, and Fos-related antigen 1.

19. 19. The method of any one of claims 1 to 18, wherein the one or more tumor antigens are selected from PRAME, NY-ESO-1, survivin, and WT1.

20. the tumor antigens are PRAME, NY-ESO-1, survivin, and WT1; 20. The method of claim 19, wherein the total concentration of peptide added to the vessel is about 78 μg / mL.

21. the vessel is a 100M G-Rex device; 21. The method of claim 20, wherein the total amount of peptide added to the container is about 7.775 mg.

22. the vessel is a 500M G-Rex device; 21. The method of claim 20, wherein the total amount of peptide added to the container is about 38.875 mg.

23. 23. The method of any one of claims 1 to 22, comprising generating tumor antigen-specific T cells specific for each of PRAME, NY-ESO-1, survivin, and WT1.

24. 19. The method of any one of claims 1 to 18, wherein the one or more tumor antigens are selected from PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2.

25. the tumor antigens are PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2; 25. The method of claim 24, wherein the total concentration of peptide added to the vessel is about 108 μg / mL.

26. the vessel is a 100M G-Rex device; 26. The method of claim 25, wherein the total amount of peptide added to the container is about 10.825 mg.

27. the vessel is a 500M G-Rex device; 26. The method of claim 25, wherein the total amount of peptide added to the container is about 54.125 mg.

28. 19. The method of any one of claims 1 to 18, comprising generating tumor antigen-specific T cells specific for each of PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2.

29. 29. The method of any one of claims 1 to 28, wherein the mononuclear cells are obtained from the subject by leukapheresis.

30. 30. The method of any one of claims 1 to 29, wherein the mononuclear cells are leukocytes or peripheral blood mononuclear cells (PBMCs).

31. 31. The method of any one of claims 1 to 30, further comprising the step of depleting CD56+ cells from the population of mononuclear cells prior to (i).

32. The method of claim 31 , wherein the CD56+ cells are depleted.

33. 33. The method of claim 32, wherein the CD56+ cells are depleted using methods including magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), Dynabeads, EasySep® cell separation beads, RosetteSep® immunodensity cell separation, or the Cloudz Human Cell Expansion platform.

34. 34. The method of any one of claims 31 to 33, wherein the population of mononuclear cells placed in the container of (i) comprises about 0.05% to about 2.5% CD56+ cells.

35. 35. The method of any one of claims 31 to 34, wherein the population of mononuclear cells placed in the container of (i) comprises about 35% to about 85% T cells.

36. 36. The method of any one of claims 31 to 35, further comprising the step of depleting CD19+ cells from the population of mononuclear cells prior to (i).

37. 37. The method of claim 36, wherein the population of mononuclear cells is depleted of CD19+ cells.

38. 38. The method of claim 37, wherein the CD19+ cells are depleted using methods including magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), Dynabeads, EasySep® cell separation beads, RosetteSep® immunodensity cell separation, or the Cloudz Human Cell Expansion platform.

39. The population of mononuclear cells placed in the container of (i) is (a) about 0.01% to about 2% CD56+ cells; (b) about 0.5% to about 50% monocytes; (c) about 0.01% to about 1.5% CD19+ B cells, and / or (d) about 40% to about 99% T cells; 39. The method of any one of claims 31 to 38, comprising:

40. 40. The method of any one of claims 1 to 39, wherein the population of mononuclear cells placed in the container comprises about 2.5% or less CD56+ cells.

41. 41. The method of any one of claims 1 to 40, wherein the one or more exogenous cytokines are selected from IL-6, IL-7, IL-15, IL-12, and any combination thereof.

42. 42. The method of any one of claims 1 to 41, wherein the one or more exogenous cytokines comprise IL-6, IL-7, IL-15, and IL-12.

43. said mononuclear cells and said one or more peptides are cultured in said vessel in the absence of said exogenous cytokines at about 37° C. for about 8 to about 30 hours; 44. The method of any one of claims 1 to 43, wherein the mononuclear cells, the one or more peptides, and the one or more exogenous cytokines are then cultured together in the vessel at about 37°C for about 5 to about 14 days.

44. 44. The method of any one of claims 1 to 43, which does not include a washing step.

45. 45. The method of any one of claims 1 to 44, further comprising splitting the culture of step (ii) into two or more separate cultures after the culture period.

46. 46. ​​The method of any one of claims 1 to 45, further comprising the step of cryopreserving the mononuclear cells and then thawing the cryopreserved cells prior to step (i).

47. 47. The method of claim 46, wherein the thawed mononuclear cells are allowed to rest overnight prior to step (i).

48. 47. The method of claim 46, wherein the thawed mononuclear cells are rested for about 4 hours to about 48 hours prior to step (i).

49. 49. The method of claim 47 or 48, further comprising counting the remaining cells after the resting period prior to step (i).

50. 50. The method of any one of claims 2 to 49, further comprising, after adding exogenous cytokines to the culture as in (iv), (v) harvesting the tumor antigen-specific T cells.

51. 51. The method of any one of claims 1 to 50, wherein the tumor antigen-specific T cells are cryopreserved after harvesting.

52. 52. The method of claim 51, wherein the cells are cryopreserved in a CryoStor® CS10 or a derivative thereof.

53. 53. The method of any one of claims 1 to 52, further comprising the step of HLA typing the donor from which the population of mononuclear cells was obtained.

54. 54. The method of any one of claims 1 to 53, wherein the tumor antigen-specific T cells comprise polyclonal antigen-specific T cells that have not undergone further purification or enrichment based on antigen specificity.

55. 55. A population of antigen-specific T cells obtained by the method of any one of claims 1 to 54.

56. a population of antigen-specific T cells comprising about 80% to about 99% CD3+ T cells, less than 1% CD14+ monocytes, less than 10% CD19+ B cells, and less than 10% CD3-CD56+ NK cells; The population of antigen-specific T cells has activity against one or more peptides of one or more antigens with which the population of T cells has not been previously cultured.

57. The degree of specificity for said at least one peptide of at least one antigen with which said population of T cells has not been previously cultured is cumulatively at least about 100 SFU / 2×10 as measured by IFN-γ ELISpot. ５ 57. The population of claim 56,

58. 58. The population of claim 57, The degree of specificity for at least one antigen to which the population has not been previously exposed or cultured is at least about 30 SFU / 2×10 as measured by IFN-γ ELISpot. ５ An individual and a group.

59. 59. The population of any one of claims 55 to 58, wherein the population has been previously cultured with one or more peptides from one or more tumor antigens in the absence of exogenous cytokines.

60. The degree of specificity of the antigen-specific T cells for the one or more tumor antigens is at least about 500 SFU / 2×10 as measured by IFN-γ ELISpot. ５ 60. The population according to any one of claims 55 to 59,

61. The antigen-specific T cells are at least about 20% of central memory T cells (T ＣＭ 61. The population of any one of claims 55 to 60, comprising:

62. The antigen-specific T cells are approximately 40% effector memory T cells (T ＥＭ 62. The population of any one of claims 55 to 61, comprising:

63. The antigen-specific T cells include up to 10% terminally differentiated effector memory cells that express CD45RA (T ＥＭＲＡ ), A population according to any one of claims 55 to 62.

64. 64. The population of any one of claims 55 to 63, wherein the antigen-specific T cells comprise at least about 20% naive T cells.

65. 65. The population of any one of claims 55 to 64, wherein the antigen-specific T cells exhibit activity against at least 75% of the tumor antigens represented by one or more peptides.

66. 66. The population of any one of claims 54 to 65, wherein the antigen-specific T cells exhibit activity against each tumor antigen represented by one or more peptides.

67. 67. The population of claim 65 or 66, wherein activity against the antigen is detected by IFNγ production.

68. 68. The population of claim 67, wherein the IFNγ production is determined by an IFNγ ELISpot test.

69. Increasing the number of tumor antigens represented by the one or more peptides, (a) the specificity of the antigen-specific T cells is increased, and / or (b) the specificity of the antigen-specific T cells for the tumor antigen is increased.

70. 70. The population of any one of claims 55 to 69, wherein T cells within the population exhibit activity against one or more peptides of one or more antigens with which the population has not previously been cultured.

71. 71. The population of claim 70, wherein the activity of one or more antigens against the one or more peptides with which the population has not previously been cultured is the result of epitope plating.

72. The degree of specificity of one or more antigens with which the population has not been previously cultured for said one or more peptides is, cumulatively, at least about 100 SFU / 2×10 as measured by IFN-γ ELISpot. ５ 72. The population of claim 71 which is a cell.

73. The degree of specificity for at least one antigen with which the population has not been previously cultured is at least about 30 SFU / 2×10 as measured by IFN-γ ELISpot. ５ 73. The population of claim 72 which is a cell.

74. 74. The population of any one of claims 55 to 73, wherein the tumor antigen-specific T cells comprise polyclonal antigen-specific T cells that have not been subjected to further purification or enrichment based on antigen specificity.

75. 75. A composition comprising the population of any one of claims 55 to 74.

76. 76. The composition of claim 75, further comprising a pharma- ceutically acceptable buffer or excipient.

77. 1. A composition comprising a population of ex vivo expanded tumor antigen-specific T cells comprising CD4+ T cells and CD8+ T cells, with less than about 10% CD56+ NK cell component, comprising: The degree of specificity for tumor antigens is at least about 500 SFU / 2×10 as measured by IFN-γ ELISpot. ５ The composition is a cell.

78. 78. The composition of claim 77, further comprising less than about 10% CD19+ components.

79. 79. The composition of claim 77 or 78, wherein both the CD4+ and CD8+ T cell compartments exhibit specificity for the tumor antigen.

80. The composition of any one of claims 75 to 79, wherein the tumor antigen-specific T cells are specific for PRAME, NY-ESO-1, survivin, and WT-1.

81. The composition of any one of claims 75 to 80, wherein the tumor antigen-specific T cells are specific for PRAME, NY-ESO-1, survivin, WT-1, SSX2, and MAGEA4.

82. The tumor antigen-specific T cells are at least about 20% of central memory T cells (T ＣＭ 82. The composition of any one of claims 75 to 81, comprising:

83. 83. The composition of any one of claims 75 to 82, wherein the tumor antigen-specific T cells comprise at least about 20% of naive T cells.

84. The tumor antigen-specific T cells are approximately 40% effector memory T cells (T ＥＭ 84. The composition of any one of claims 75 to 83, comprising:

85. The tumor antigen-specific T cells include up to 10% terminally differentiated effector memory cells that express CD45RA (T ＥＭＲＡ ), A composition described in any one of claims 75 to 84.

86. 1. A method for generating tumor antigen-specific T cells, comprising: (i) injecting 0.1-9×10 IgG into a static cell culture device having at least one gas permeable surface with one or more peptides of one or more tumor associated antigens; ６ cells / cm ２ wherein each peptide is present in said cell culture device at a concentration of about 0.1 μg / mL to about 0.5 μg / mL, and the white blood cells comprise about 1% to about 50% monocytes and about 40% to about 99% T cells; (ii) The cells and peptides of step (i) are incubated at about 37° C. and 5% CO ２ for about 8 to about 30 hours; (iii) after about 8 to about 30 hours of culture, adding one or more exogenous cytokines; (iv) culturing the cells, the peptide, and the exogenous cytokine for about 5 to about 14 days, wherein the culture is not disturbed after addition of the exogenous cytokine; A method comprising:

87. 87. The method of claim 86, further comprising, after said culturing of step (iv), (v) harvesting the tumor antigen-specific T cells.

88. said one or more peptides are from four tumor-associated antigens; 88. The method of claim 87, wherein the total concentration of peptide present in the cell culture device in step (i) is about 78 μg / mL.

89. the one or more peptides are from six tumor-associated antigens; 89. The method of claim 88, wherein the total concentration of peptide present in the cell culture device in step (i) is about 108 μg / mL.

90. 90. The method of any one of claims 86 to 89, wherein the tumor antigen-specific T cells comprise polyclonal antigen-specific T cells that have not undergone further purification or enrichment based on antigen specificity.

91. 91. A T cell product produced by the method of any one of claims 86 to 90, comprising: (i) about 80% to about 99% CD3+ T cells, less than 1% CD14+ monocytes, less than 10% CD19+ B cells, and less than 10% CD3-CD56+ NK cells; (ii) a CD3+ T cell compartment comprising about 12% to about 80% CD4+ T cells and about 17% to about 84% CD8+ T cells; (iii) at least about 20% of central memory T cells (T ＣＭ ), at least about 20% naive T cells, about 40% effector memory T cells (T ＥＭ ), and 10% or less of terminally differentiated effector memory cells expressing CD45RA (T ＥＭＲＡ a CD3+ T compartment comprising 2. A T cell product comprising:

92. 92. The T cell product of claim 91, wherein the product comprises cells capable of producing IFN-γ as determined by an enzyme-linked immunosorbent spot (ELISpot) test.

93. The degree of specificity of the collected tumor antigen-specific T cells for the tumor antigen is at least about 500 SFU / 2×10 for the total of all antigens. ５ 93. The T cell product of claim 91 or 92 which is a cell.

94. The degree of specificity for an antigen not represented by the one or more peptides is at least about 100 SFU / 2×10 cumulatively as measured by IFN-γ ELISpot. ５ 94. A T cell product according to any one of claims 91 to 93 which is a cell.

95. The degree of specificity for at least one antigen to which the population has not previously been exposed or cultured is at least about 30 SFU / 2×10 as measured by IFN-γ ELISpot. ５ 95. The population of claim 94 which is a cell.

96. 96. The T cell product of any one of claims 86 to 95, wherein the T cell product is cryopreserved.

97. 97. A method of treating a patient comprising administering to the patient a population of antigen-specific T cells according to any one of claims 55 to 74, a composition according to any one of claims 75 to 85, and / or a T cell product according to any one of claims 91 to 96.

98. 98. The method of claim 97, wherein the patient is suffering from cancer.

99. 99. The method of claim 98, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), multiple myeloma, non-Hodgkin's lymphoma, sarcoma, breast cancer, and pancreatic cancer.

100. 100. The method of any one of claims 97 to 99, wherein the patient has undergone allogeneic hematopoietic stem cell transplantation (HSCT).

101. 101. The method of any one of claims 97 to 100, wherein the patient is the same individual as the subject (e.g., the treatment is autologous).

102. 102. The method of any one of claims 97 to 101, wherein the patient is a different individual than the subject (e.g., the treatment is allogeneic).

103. 103. The method of any one of claims 97 to 102, wherein the patient is partially HLA-matched to the subject.

104. The method of claim 103, wherein the patient has at least one HLA match with the subject.

105. 105. The method of any one of claims 86-90 or 97-104, further comprising the step of HLA typing the patient prior to treatment.

106. further comprising administering to said patient one or more additional populations of antigen-specific T cells in an allogeneic or autologous therapeutic setting; the one or more additional populations of antigen-specific T cells comprise the same T cell product as the previously administered population of antigen-specific T cells; 106. The method of any one of claims 86-90 or 97-105, comprising treating the patient with one or more additional populations of antigen-specific T cells at least about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after treatment with the previously administered population.

107. further comprising administering to said patient one or more additional populations of antigen-specific T cells in an allogeneic or autologous therapeutic setting; the one or more additional populations of antigen-specific T cells comprise a T cell product that is different from the previously administered population of antigen-specific T cells; 107. The method of any one of claims 86-90 or 97-106, comprising treating the patient with one or more additional populations of antigen-specific T cells at least about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after treatment with the previously administered population.

108. administering to the patient a second population of antigen-specific T cells in an autologous setting; The second population of antigen-specific T cells is obtained by the method of any one of claims 1 to 54 or 86 to 90, 108. The method of any one of claims 86-90 or 97-107, comprising treating the patient with a second population of antigen-specific T cells at least about 4 weeks, about 5 weeks, or about 6 weeks after treatment with the first population.

109. 109. The method of any one of claims 97 to 108, further comprising monitoring disease progression in the patient by measuring RNA expression of one or more of the tumor antigens.

110. 110. The method of claim 109, wherein the expression of the one or more tumor antigens correlates with minimal residual disease (MRD) of the patient.

111. 111. The method of any one of claims 97 to 110, further comprising measuring RNA expression of one or more additional tumor antigens that are not the tumor antigens corresponding to said one or more peptides.

112. 112. The method of claim 111, wherein the RNA expression of the one or more additional tumor antigens corresponds to MRD in the patient.

113. 113. The method of any one of claims 97 to 112, further comprising the step of HLA typing the patient.

114. 1. A composition comprising: Mononuclear cells, one or more peptides derived from one or more tumor antigens; Including, each of the one or more peptides in the composition is present at a concentration of about 0.1 to about 0.5 μg / mL; The population of mononuclear cells comprises about 40% to about 99% T cells and about 2% or less NK cells.

115. The composition of claim 114, wherein the one or more peptides comprise one or more libraries of peptides derived from the one or more tumor antigens.

116. The composition of claim 115, wherein each library of peptides comprises peptides spanning the sequence of the tumor antigen.

117. 117. The composition of any one of claims 114 to 116, wherein the population of mononuclear cells is depleted of NK cells.

118. The mononuclear cells are placed in a container having at least one gas permeable surface at a concentration of about 0.1 x 10 cells per square centimeter of gas permeable surface. ６ pieces ~ approx. 9 x 10 ６ 118. The composition of any one of claims 114 to 117, wherein the composition is present at a seeding density of 1.

119. 119. A composition according to any one of claims 114 to 118, comprising one or more peptides from at least four different tumor antigens.

120. 120. The composition of any one of claims 114 to 119, comprising one or more peptides from 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, 29, 30 or more different tumor antigens.

121. 121. The composition of any one of claims 114 to 120, comprising one or more peptides from PRAME, NY-ESO-1, survivin, and WT1.

122. 122. The composition of any one of claims 114 to 121, comprising one or more peptides from PRAME, NY-ESO-1, survivin, WT1, SSX2, and MAGEA4.

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