Compositions and methods for antigen-specific t cell expansion

Abstract

The present disclosure provides improved methods for preparing tumor antigen-specific T cells, including shortened procedures and methods of enhancing certain aspects of tumor antigen-specific T cells. The present disclosure also provides various compositions and products related to the improved process of preparation. The present disclosure also provides tumor antigen specific T cell products.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 310,500, filed on February 15, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to methods for generating tumor antigen-specific T cells, populations, compositions and products derived therefrom, and methods of treating the same. Background Art

[0004] Adoptive transfer of expanded tumor antigen-specific T cells represents an attractive approach to treating a variety of cancers. Methods for generating tumor antigen-specific T cells with desired activity and phenotype on a scale required for clinical use are challenging. In particular, the length of time required to generate tumor antigen-specific T cells for clinical use may be detrimental to patient care. There is a need in the art for improved methods for generating tumor antigen-specific T cells that can be administered to patients to treat cancer. 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 needs and other needs.

[0005] BRIEF SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION

[0006] In an embodiment, the present disclosure provides a method for generating tumor antigen-specific T cells, comprising: (i) placing a population of monocytes obtained from a subject in a container comprising at least one gas-permeable surface, wherein the monocytes are present at a density of 100 μm / cm 2 The air permeable surface is about 0.1×10 6 cells per cm 2 Breathable surface about 1×10 7 (ii) adding one or more peptides from one or more tumor antigens to the cell culture; and incubating the one or more peptides with the monocytes 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 one embodiment. In further embodiments, the method comprises harvesting tumor antigen-specific T cells, for example, for autologous transfer or cryopreservation.

[0007] In embodiments, each of 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 embodiments, each of 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 embodiments, one or more peptides include one or more peptides derived from one or more tumor antigens. In some embodiments, one or more peptides include one or more libraries of peptides derived from one or more tumor antigens. In embodiments, each library of peptides includes a variety of peptides that cover the amino acid sequence of targeting tumor antigens in some embodiments. In embodiments, one or more libraries of peptides are added to the culture at a concentration of about 0.1 μg / mL to about 0.5 μg / mL for each peptide. In embodiments, one or more than one library of 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 per peptide.

[0008] In embodiments, prior to the addition of one or more exogenous cytokines, the monocytes are incubated with one or more peptides in a container at about 37°C. In some embodiments, prior to the addition of one or more exogenous cytokines, the monocytes are incubated with one or more peptides in a container at about 37°C for at least about 30 minutes. In some embodiments, prior to the addition of one or more exogenous cytokines, the monocytes are incubated with one or more peptides in a container at about 37°C for at least about 30 minutes to about 30 hours. In embodiments, prior to the addition of one or more exogenous cytokines, the monocytes are incubated with one or more peptides in a container at about 37°C for about 8 hours to about 24 hours. In embodiments, prior to the addition of one or more exogenous cytokines, the monocytes are incubated with one or more peptides in a container at about 37°C overnight.

[0009] In one embodiment, mononuclear cells are expressed as per cm 2 The air permeable surface is about 0.1×10 6 cells per cm 2 Breathable surface about 1×10 7 For example, mononuclear cells are seeded at a density of about 0.1 × 10 6 cells / cm 2 , about 0.5×10 6 cells / cm 2 , about 1×10 6cells / 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 7 In one embodiment, mononuclear cells are added to the container at a seeding density of about 0.1×10 6 cells / mL to about 1×10 7 Cells are incubated with one or more peptides at a concentration of about 0.1 × 10 cells / mL. 6 cells / mL, about 0.5×10 6 cells / mL, about 1×10 6 cells / mL, about 2×10 6 cells / mL, about 3×10 6 cells / mL, about 4×10 6 cells / mL, about 5×10 6 cells / mL, about 6×10 6 cells / mL, about 7×10 6 cells / mL, about 8×10 6 cells / mL, about 9×10 6 cells / mL or about 1×10 7 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 (such as a donor) by leukocyte apheresis. In one embodiment, the donor is a healthy donor. In other embodiments, the donor is a patient to whom T cells (MultiTAA-specific T cells) specific to multiple tumor-associated antigens are administered after they are generated.

[0010] In embodiments, the container comprises at least one gas permeable surface. In embodiments, the container comprises 1, 2, 3, 4, 5, or 6 gas permeable surfaces. In embodiments, the container comprising at least one gas permeable surface is a G-Rex device. For example, in embodiments, the container is a 100M G-Rex device or a 500M G-Rex device.

[0011] In embodiments, each library of peptides comprises peptides that encompass sequences of tumor antigens. In embodiments, the method further comprises, before adding one or more than one exogenous cytokines, adding one or more than one peptide, optionally adding a library of one or more than one peptide, the peptides being from at least 4 different tumor antigens. In embodiments, the method comprises, before adding one or more than one exogenous cytokines, adding one or more than one peptide, optionally adding a library of one or more than one peptide, the peptides being from 6 different tumor antigens. In embodiments, the method comprises, before adding one or more than one exogenous cytokines, adding one or more than one peptide, optionally adding a library of one or more than one peptide, the peptides being from 4, 6, 8, 10 or 12 different tumor antigens. In an embodiment, the tumor antigen is selected from CEA, MHC, CTLA-4, gp100, mesothelin, PRAME (OIP3) PD-L1, TRP1, CD40, EGFP, Her2, TCRα, 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, 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, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, mucin-1, BRAF, CCNA1, and Fos-related antigen 1.

[0012] In embodiments, one or more than one tumor antigen is selected from PRAME, NY-ESO-1, survivin and WT1. In embodiments, 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 other embodiments, the container is a 100M G-Rex device, and the total amount of peptides added to the container (corresponding to PRAME, NY-ESO-1, survivin and WT1 antigens) is about 7.775 mg. In other embodiments, the container is a 500MG-Rex device, and the total amount of peptides added to the container (corresponding to PRAME, NY-ESO-1, survivin and WT1 antigens) is about 38.875 mg. In embodiments, the methods provided herein include generating antigen-specific T cells that are specific to each of PRAME, NY-ESO-1, survivin and WT1.

[0013] In embodiments, one or more than one tumor antigen is selected from PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2. In embodiments, 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 further embodiments, the container is a 100M G-Rex device, and the total amount of peptides added to the container (corresponding to PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2 antigens) is about 10.825 mg. In other embodiments, the container is a 500M G-Rex device, and the total amount of peptides added to the container (corresponding to PRAME, NY-ESO-1, survivin, WT1, NY-ESO-1, and SSX2 antigens) is about 54.125 mg. In embodiments, the methods provided herein comprise generating antigen-specific T cells specific for each of PRAME, NY-ESO-1, survivin, WT1, NY-ESO-1, and SSX2.

[0014] In embodiments, 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 than 30 tumor antigens are represented in one or more than one peptide incubated with monocytes. In embodiments, the tumor antigen-specific T cell products 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 than one peptide used in the initial culture. In embodiments, the tumor antigen-specific T cell products generated by the methods herein exhibit antigen specificity for each of the tumor antigens represented by one or more than one peptide. In embodiments, increasing the amount of tumor antigen incubated with monocytes unexpectedly increases the antigen specificity and antigen diversity of the generated tumor antigen-specific T cell products.

[0015] In embodiments, monocytes are obtained from a subject by leukapheresis. The subject can be a healthy donor or a patient who has been diagnosed with cancer. In embodiments, monocytes are leukocytes or peripheral blood mononuclear cells (PBMCs). In embodiments, the methods provided herein for generating tumor antigen-specific T cells include removing CD56+ cells from a population of monocytes obtained from a subject. In embodiments, removal 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 TM Cell separation magnetic beads, RosetteSep TM Immunodensity cell separation or Cloudz human cell expansion platform. In embodiments, a population of monocytes placed in a container for incubation with one or more than one peptide comprises about 0.05% to about 2.5% CD56+ cells. In embodiments, a population of monocytes placed in a container for incubation with one or more than one peptide comprises no more than about 5%, no more than about 2.5%, or no more than about 2% CD56+ cells. In embodiments, a population of monocytes placed in a container for incubation with one or more than one peptide comprises about 35% to about 85% T cells. In embodiments, the T cell population comprises CD4+T cells and CD8+T cells.

[0016] In embodiments, the methods provided herein for generating tumor antigen-specific T cells include removing CD19+ cells from a mononuclear cell population obtained from a subject. In embodiments, the removal 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 TM Cell separation magnetic beads, RosetteSep TM Immunodensity cell isolation or Cloudz human cell expansion platform.

[0017] In embodiments, the methods provided herein for generating tumor antigen-specific T cells include removing CD56+ cells and CD19+ cells from a mononuclear cell population obtained from a subject. In embodiments, the removal of CD56+ cells and 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 TM Cell separation magnetic beads, RosetteSep TM Immunodensity cell separation or Cloudz human cell expansion platform. In embodiments, the tumor antigen-specific T cell products generated by the methods provided herein comprising the removal of CD19+ T cells are suitable for treating patients with B cell cancers.

[0018] In an embodiment, the population of monocytes placed in a container and incubated with one or more peptides comprises: about 0.01% to about 2% CD56+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.

[0019] In embodiments, the method for generating tumor antigen-specific T cells provided herein includes incubating monocytes and peptides together in the absence of exogenous cytokines, and then adding one or more exogenous cytokines to the culture. In embodiments, the method for generating tumor antigen-specific T cells provided herein includes incubating monocytes and peptides together in the absence of exogenous cytokines for at least 8 hours, and then adding one or more exogenous cytokines to the culture. In embodiments, 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 further embodiments, the exogenous cytokines added to the culture include IL-6, IL-7, IL-15, and IL-12.

[0020] In embodiments, monocytes are cultured for about 5 days to about 14 days in the presence of one or more peptides and one or more exogenous cytokines. Therefore, in embodiments, the methods provided herein for generating tumor antigen-specific T cells include incubating monocytes with one or more peptides at about 37°C for about 8 hours to about 30 hours in a container including at least one gas permeable surface in the absence of exogenous cytokines, followed by adding one or more exogenous cytokines to the culture, wherein monocytes, one or more peptides and one or more exogenous cytokines are then co-cultured in a container at about 37°C for about 5 days to about 14 days. In embodiments, the method does not include a washing step. In embodiments, monocytes, one or more peptides and exogenous cytokines are cultured for about 5 days to about 14 days without interference, such as without intervention such as adding culture medium or replacing culture medium, adding additional peptides, adding additional cytokines, or adding any other components.

[0021] In some embodiments, the method includes splitting the culture into two or more separate cultures after incubating the monocytes with one or more peptides and before adding one or more exogenous cytokines. For example, in some embodiments, the initial culture is split into two or more cultures to allow for further expansion of tumor antigen-specific T cells in the culture in the presence of one or more exogenous cytokines.

[0022] In embodiments, the method further comprises cryopreserving the monocytes, then thawing the cryopreserved cells, and then incubating with one or more peptides. In embodiments, the thawed monocytes are allowed to stand overnight before incubating with one or more peptides. In embodiments, the thawed monocytes are allowed to stand for about 4 hours to about 48 hours before incubating with one or more peptides. In embodiments, the method further comprises counting the cells remaining after a period of standing before starting to culture with one or more peptides. In embodiments, in this manner, the seeding density of monocytes cultured with one or more peptides will be accurately calculated, for example, monocytes are cultured at about 0.1×10 6 cells / cm 2 About 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 at a seeding density of about 0.1×106 cells / mL to about 1×10 7 cells / mL (e.g., about 1×10 6 cells / mL or about 2×10 6 cells / mL or about 3×10 6 The concentration of 10 cells / mL) was placed in a container.

[0023] In embodiments, the tumor antigen-specific T cells generated by the methods provided herein are cryopreserved after harvest. In embodiments, the monocytes and / or tumor antigen-specific T cells are cryopreserved after harvest. CS10 or its derivatives.

[0024] In embodiments, the methods provided herein also include HLA typing of the donor from which the mononuclear cell population is obtained. In embodiments, the methods provided herein include storing information about the HLA typing of the donor and maintaining a library of one or more than one tumor antigen-specific T cell products that can be used as a finished product (off the shelf product). In embodiments, the finished product is suitable for patients who have a desired level of HLA matching with the donor of the tumor antigen-specific T cell product, such as at least one HLA match.

[0025] In embodiments, the methods provided herein for generating tumor antigen-specific T cells include harvesting tumor antigen-specific T cells after first incubating with one or more than one peptide and then incubating with one or more than one exogenous cytokines. In some embodiments, harvesting tumor antigen-specific T cells does not include purification or enrichment based on antigen specificity. In another embodiment, the method includes harvesting tumor antigen-specific T cells for cryopreservation and / or storage, for example as a finished product, optionally as part of a cell bank or inventory.

[0026] In embodiments, the present disclosure provides a population of tumor antigen-specific T cells obtained by the methods provided herein. In embodiments, the present disclosure provides a composition comprising a population of tumor antigen-specific T cells obtained by the methods provided herein. In embodiments, the population of tumor antigen-specific T cells exhibits superior antigen specificity compared to tumor antigen-specific T cells generated by existing methods. In embodiments, the intensity of specificity of tumor antigen-specific T cells to tumor antigens is at least about 500 SFU / 2×10 5 cells, at least about 700 SFU / 2×10 5 cells, at least about 1000 SFU / 2×10 5 cells or at least about 1200 SFU / 2×105 cells.

[0027] In embodiments, the present disclosure provides an antigen-specific T cell population comprising about 80% to about 99% CD3+T cells, <1% CD14+monocytes, <10% CD19+B cells, and <10% CD3-CD56+NK cells, wherein the antigen-specific T cell population is active against one or more than one peptide of one or more than one antigen that has not been previously incubated with the T cell population. In embodiments, the antigen-specific T cell population comprises a specificity for at least one peptide of at least one antigen that has not been previously incubated with the T cell population as detected by IFN-γ ELISpot of at least about 100 SFU / 2×10 5 In embodiments, the specificity for at least one antigen not previously incubated with the population is at least about 30 SFU / 2×10 5 In embodiments, the population has been previously incubated with one or more peptides from one or more tumor antigens in the absence of exogenous cytokines.

[0028] In embodiments, tumor antigen-specific T cells comprise at least about 20% of central memory T cells (T CM In an embodiment, the tumor antigen-specific T cells comprise about 40% of effector memory T cells (T EM In an embodiment, the tumor antigen-specific T cells comprise no more than 10% of terminally differentiated effector memory cells (T cells) expressing CD45RA. EMRA ). In embodiments, the tumor antigen-specific T cells comprise at least about 20% of naive T cells. In embodiments, the tumor antigen-specific T cells in the product exhibit activity against at least 75% of the tumor antigens represented by one or more than one peptide. In embodiments, the tumor antigen-specific T cells in the product exhibit activity against each tumor antigen represented by one or more than one peptide. In embodiments, activity against an antigen or antigen specificity is detected by the production of IFNγ. In embodiments, the production of IFNγ is determined by an IFNγ ELISpot assay.

[0029] In some embodiments, an increase in the number of tumor antigens represented by one or more than one peptide, optionally represented by a library of peptides, results in (a) an increase in the specificity of antigen-specific T cells and / or (b) an increase in the specificity of antigen-specific T cells for tumor antigens.

[0030] In some embodiments, T cells present in the cell population exhibit activity against one or more peptides of one or more antigens (e.g., "non-targeted" antigens) that have not been previously incubated with the cell population. In some embodiments, the activity against one or more peptides of one or more antigens that have not been previously incubated with the cell population is the result of epitope spreading.

[0031] In some embodiments, the accumulation of the specificity of one or more peptides for one or more antigens not previously incubated with the population is at least about 100 SFU / 2×10 5 In some embodiments, the specificity for at least one antigen not previously incubated with the population is at least about 30 SFU / 2×10 5 cells.

[0032] In embodiments, the population of antigen-specific T cells has not been purified or enriched based on antigen specificity.

[0033] In an embodiment, the present disclosure provides a composition comprising a tumor antigen-specific T cell population comprising CD4+ T cells and CD8+ T cells expanded ex vivo, and comprising less than about 10% of a CD56+ NK cell component, wherein the strength of specificity for the tumor antigen as detected by IFN-γ ELISpot is at least about 500 SFU / 2×10 5 In some embodiments, the specificity for the tumor antigen is at least about 700 SFU / 2×10 5 In embodiments, the composition further comprises a pharmaceutically acceptable buffer or excipient. In other embodiments, the composition comprises less than about 10% of the CD19+ component. In embodiments, both the CD4+ T cell and CD8+ T cell compartments exhibit specificity for tumor antigens.

[0034] In embodiments, tumor antigen-specific T cells are specific for PRAME, NY-ESO-1, survivin, and WT-1. In embodiments, tumor antigen-specific T cells are specific for PRAME, NY-ESO-1, survivin, WT-1, SSX2, and MAGEA4. In embodiments, tumor antigen-specific T cells are specific for PRAME, NY-ESO-1, survivin, WT1, SSX2, and / or MAGEA4, and are specific for 1, 2, 3, 4, 5, 6, or more than 6 other tumor-associated antigens. In embodiments, tumor antigen-specific T cells comprise at least about 20% of central memory T cells (T CMIn embodiments, the tumor antigen-specific T cells comprise at least about 20% of naive T cells. In embodiments, the tumor antigen-specific T cells comprise about 40% of effector memory T cells (T EM In an embodiment, the tumor antigen-specific T cells comprise no more than 10% of terminally differentiated effector memory cells (T cells) expressing CD45RA. EMRA ).

[0035] In one aspect, the present disclosure provides a method for generating tumor antigen-specific T cells, comprising:

[0036] (i) The white blood cell population was divided into 0.1×10 6 cells / cm 2 Up to 9×10 6 cells / cm 2 A method of treating a tumor in a static cell culture device having at least one gas permeable surface comprising: placing the peptides in a static cell culture device having at least one gas permeable surface at a density of about 0.1 μg / mL to about 0.5 μg / mL, wherein 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 wherein the white blood cells comprise about 1% to about 50% monocytes and about 40% to about 99% T cells;

[0037] (ii) incubating the cells and peptide of step (i) at about 37° C. and 5% CO 2 for about 8 hours to about 30 hours;

[0038] (iii) adding one or more than one exogenous cytokine after about 8 hours to about 30 hours of incubation;

[0039] (iv) culturing the cells, peptide and exogenous cytokine for about 5 days to about 14 days, wherein the culture is not disturbed after the addition of the exogenous cytokine.

[0040] In embodiments, the method further comprises, after the culture of (iv), harvesting tumor antigen-specific T cells. In embodiments, one or more than one 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 embodiments, one or more than one 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 embodiments, the method does not include purification or enrichment based on the antigen specificity of T cells.

[0041] In one aspect, the present disclosure provides a T cell product generated by the above method. In an embodiment, the present disclosure provides a tumor antigen-specific T cell product, which comprises: about 80% to about 99% CD3+T cells, <1% CD14+ monocytes, <10% CD19+B cells and <10% CD3-CD56+NK cells, and a CD3+T cell compartment, wherein the CD3+T cell compartment comprises about 12% to about 80% CD4+T cells and about 17% to about 84% CD8+T cells, wherein the CD3+T cell compartment comprises at least about 20% central memory T cells (T CM In embodiments, the tumor antigen-specific T cells comprise at least about 20% of naive T cells. In embodiments, the tumor antigen-specific T cells comprise about 40% of effector memory T cells (T EM In an embodiment, the tumor antigen-specific T cells comprise no more than 10% of terminally differentiated effector memory cells (T cells) expressing CD45RA. EMRA ).

[0042] In embodiments, the T cell product comprises cells capable of producing IFN-γ as determined by an ELISpot assay, e.g., wherein the strength of the specificity of the tumor antigen-specific T cells for the tumor antigen is at least about 500 SFU / 2×10 5 In an embodiment, the intensity of antigen-specific T cell products specific for an antigen not represented by one or more than one peptide is cumulatively at least about 100 SFU / 2×10 5 In an embodiment, the intensity of antigen-specific T cell products specific for an antigen not represented by one or more than one peptide is accumulated to at least about 30 SFU / 2×10 5 In embodiments, the T cell product is cryopreserved. In embodiments, the T cell product is suitable for use in autologous cell therapy for treating cancer and / or allogeneic cell therapy for treating cancer.

[0043] In embodiments, the present disclosure provides a method for treating a patient, the method comprising administering a composition and / or a cell colony of antigen-specific T cells provided herein to the patient. The antigen-specific T cell colony is defined herein, and / or an antigen-specific T cell colony generated by a method provided herein. In embodiments, the patient suffers from cancer. In embodiments, cancer is leukemia, lymphoma or solid tumor. In embodiments, cancer is selected from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), multiple myeloma, non-Hodgkin's lymphoma, sarcoma, breast cancer and pancreatic cancer. In embodiments, the patient has undergone allogeneic hematopoietic stem cell transplantation (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 object from which mononuclear cells are obtained, i.e., the method of treatment is autologous. In other embodiments, the patient is a different individual, i.e., the method of treatment is allogeneic. In some embodiments, the patient is partially HLA matched with the object from which mononuclear cells are obtained. In some embodiments, the patient has at least one HLA that matches the subject from which the monocytes were obtained. In some embodiments, the method includes performing HLA typing on the patient prior to treatment. In some embodiments, the method includes selecting a tumor antigen-specific T cell product from a product library based on the HLA type of the product and the patient.

[0044] In embodiments, the treatment methods provided herein further include administering to the patient one or more additional antigen-specific T cell populations (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 antigen-specific T cell populations are antigen-specific T cells or antigen-specific T cell products described herein and / or obtained by the methods provided herein, and wherein the method comprises treating the patient with one or more additional antigen-specific T cell populations 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 embodiments, one or more additional antigen-specific T cell populations (e.g., a second and / or third population) for autologous treatment can be generated after obtaining one or more additional (e.g., a second and / or third) monocyte populations 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) population is administered to the patient. In embodiments, one or more additional cell populations may comprise a greater number of antigen-specific cells than a previous (e.g., first) cell population, and may comprise a more effective dose. In embodiments, one or more additional antigen-specific T cell populations for allogeneic therapy are obtained from a library of cryopreserved products, or are generated after obtaining one or more additional monocyte populations from a subject (e.g., a donor) different from the patient.

[0045] In embodiments, the methods provided herein also include monitoring the patient's disease progression by detecting RNA expression of one or more tumor antigens. In embodiments, the expression of one or more tumor antigens is associated with minimal residual disease (MRD) in the patient. In embodiments, the method also includes detecting RNA expression of one or more additional tumor antigens, and one or more additional tumor antigens do not correspond to one or more peptides. In embodiments, one or more additional tumor antigens correspond to MRD in the patient. In embodiments, the methods provided herein result in epitope expansion to additional tumor antigens, and the additional tumor antigens do not correspond to one or more peptides used to generate antigen-specific T cell products (see, e.g., Figures 24A to 24B). Figure 24G ).

[0046] In embodiments, the present disclosure provides a cell composition as a raw material for generating an antigen-specific T cell product provided herein. In embodiments, the composition comprises mononuclear cells from which CD56+ cells have been removed. In other embodiments, the composition comprises about 40% to about 99% of T cells and no more than about 2% of NK cells. In embodiments, the composition further comprises one or more than one peptide from one or more than one tumor antigen. In embodiments, the composition further comprises one or more than one library of peptides from one or more than one tumor antigen. In other embodiments, each library of peptides comprises peptides covering the sequence of tumor antigens. Therefore, each library of peptides comprises a variety of different peptides, each of which corresponds to all or part of a tumor antigen. In embodiments, the concentration of each peptide present in the composition is 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 embodiments, the composition comprises peptides, optionally a library of peptides, from at least 4 different tumor antigens, e.g., from 4, 5, 6, 7, 8, 9, 10, 11, or 12 different tumor antigens. In embodiments, the tumor antigens are selected from PRAME, NY-ESO-1, survivin, WT1, SSX2, and MAGEA4. In embodiments, the tumor antigens are PRAME, NY-ESO-1, survivin, and WT1. In embodiments, the composition is present in a container comprising at least one gas permeable surface, e.g., a container comprising 1, 2, 3, 4, 5, or 6 gas permeable surfaces. In embodiments, the composition is in a container, and the monocytes are present at a density of 100 μg / cm2. 2 The air permeable surface is about 0.1×10 6 cells per cm 2 Breathable surface approx. 9×10 6 The seeding density of cells exists. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG1 provides a schematic diagram of a method for generating tumor antigen-specific T cells according to one embodiment. Figure 1A The procedure shown is referred to herein as "MAT110-CD56+ depletion". Figure 1B and Figure 1C A schematic diagram of two existing methods for generating tumor antigen-specific T cells is provided. Figure 1B A method referred to herein as "MAT101-4" is provided, which includes stimulating PBMCs with cytokines to generate dendritic cells (DCs), followed by pulsing the DCs with a peptide mixture and incubating with the cytokines, which includes two or more steps of changing the culture medium and the step of peptide pulsing, and the total culture period is about 31 days to 36 days. Figure 1C A method referred to herein as "MAT103-4" is provided, wherein CD14+ PBMCs are cultured in the presence of cytokines to generate mature DCs, which are then pulsed with peptides and incubated with CD56- cells and cytokines for a total of 15 to 20 days.

[0048] Figure 2A , Figure 2B and Figure 2C Details of the materials used to generate tumor antigen-specific T cells are provided. Figure 2A The composition of the PBMC stock without depletion of any cell type is shown. Figure 2B Fractions of the PBMC stock depleted of CD56+ cells are shown. Figure 2C Fractions of CD56+ cell-depleted and CD19+ cell-depleted PBMC stocks are shown.

[0049] Figure 3 Details of tumor antigen-specific T cell products prepared according to the "MAT110-Non-depleted" method using non-depleted raw materials are provided. The first panel shows the percentage of monocytes, T cells, B cells, and NK cells in the viable cells. The second panel shows the percentage of CD4+ or CD8+ in the CD3+ T cells in the final product. The third panel shows the phenotype of CD3+ T cells in the final product (naive T cells, T CM , T EM and T EMRA The fourth panel shows the expansion fold of cells in the final product. The fifth panel shows the combined antigen specificity (SFU / 2×10 5 cells, detected by IFNγ ELISpot). The table below each plate shows the statistical analysis. SFU = spot forming unit.

[0050] Figure 4 Provides detailed information on tumor antigen-specific T cell products prepared according to the "MAT110-CD56+ depleted" method using CD56+ depleted cells as raw materials. The first panel shows the percentage of monocytes, T cells, B cells, and NK cells in the living cells. The second panel shows the percentage of CD4+ or CD8+ in the CD3+ T cells in the final product. The third panel shows the phenotype of CD3+ T cells in the final product (naive T cells, T CM , T EM and T EMRA The fourth panel shows the expansion fold of cells in the final product. The fifth panel shows the combined antigen specificity (SFU / 2×10 5cells, detected by IFNγ ELISpot). The table below each plate shows the statistical analysis. SFU = spot forming unit.

[0051] Figure 5 Details of the production of tumor antigen-specific T cells using CD56+-depleted and CD19+-depleted cells as starting materials are provided. The procedure used to generate these tumor antigen-specific T cells is called "MAT210" and is the same as that used in the previous study, except that the monocytes were CD19+-depleted and CD56+-depleted before peptide pulsing. Figure 1A The same procedure as shown in the "MAT110-CD56+ depletion" is shown in the figure. The first panel shows the percentage of monocytes, T cells, B cells and NK cells in the viable cells. The second panel shows the percentage of CD4+ or CD8+ in the CD3+ T cells in the final product. The third panel shows the phenotype of CD3+ T cells in the final product (naive T cells, T CM , T EM and T EMRA The fourth panel shows the expansion fold of cells in the final product. The fifth panel shows the combined antigen specificity (SFU / 2×10 5 cells, detected by IFNγ ELISpot). The table below each plate shows the statistical analysis. SFU = spot forming unit.

[0052] Fig. 6A and Figure 6B The improved method provided herein (called "MAT110-depleted CD56+" and in Figure 1A ), MAT110-without removal and prior art method MAT103-4 (in Figure 1C The addition of a CD56+ removal step significantly increased the expansion fold of T cells generated using the MAT110 method.

[0053] Fig. 7A and Figure 7B A comparison is shown between the phenotype of T cells generated using the MAT110-CD56+ depletion method and the phenotype of T cells obtained using the existing method MAT103-4. Figure 1A As schematically shown in FIG. 1 , the MAT103-4 method is Figure 1C The MAT110-CD56+ depleted approach generated products with similar distribution of CD4+ T cells and CD8+ T cells, as well as a higher proportion of central memory T cells (T CM ).

[0054] Fig. 8A and Figure 8B The antigen specificity of MAT110-depleted CD56+ cells obtained using the improved method provided herein is shown and compared with MAT103-4 cells and MAT110-non-depleted cells obtained by the existing method, respectively. Fig. 8A SFU / 2×10 detected by IFNγ ELISpot 5 Each panel provides results for cells obtained from an individual donor. Figure 8B The total intensity of antigen specificity is presented (when data from donors were pooled, responsiveness to each targeted antigen was combined), and the graph shows that the increase in intensity of the response achieved using the MAT110-depleted CD56+ approach was statistically significant. SFU = spot forming unit.

[0055] Fig.9A The SFU / 2×10 detected by IFNγ ELISpot is shown. 5 The total response strength of the cells (sum of all antigens) was higher using MAT110-depleted CD56+, resulting in a product with a greater number of antigen-specific T cells. Fig. 9B A comparison between the antigen specificity profiles of tumor antigen-specific T cell products generated by existing methods and those generated by MAT110-CD56+ depletion is shown. The MAT110-CD56+ depletion preparation method increases the proportion of multi-targeted T cells. Fig. 9C It was shown that MAT110-depleted CD56+ cells achieved an increase in the number of products specific for multiple tumor antigens. "Run" refers to a manufacturing run in which cells were used to generate T cell products. Fig.9D The percentage of antitumor activity of T cell products (detected in in vitro co-culture experiments) and antigen specificity (detected by IFNγ ELISpot, SFU / 2×10 5 cells, x-axis). In addition, Fig.9D showed that T cell products generated using the MAT110-CD56+ depletion approach had higher antigen specificity and antitumor activity than those generated using the MAT103-4 approach.

[0056] Fig.10 Exemplary calculations of peptide amounts for four antigen pools (PRAME, NY-ESO-1, survivin, and WT1) and six antigen pools (PRAME, NY-ESO-1, survivin, WT1, MAGEA4, and SSX2), as well as exemplary calculations of the total peptide amount used in a 100M G-Rex or 500MG-Rex device are provided.

[0057] Fig.11 A comparison of the antigen specificity of MultiTAA-specific T cell products generated by an initial peptide pulse step in the presence of cytokines (+cytokines) versus an initial peptide pulse step in the absence of cytokines (-cytokines) is provided. PBMCs from healthy donors were pulsed overnight with peptides from six different antigens (WT1, survivin, NY-ESO-1, PRAME, SSX2, and MAGEA4) and preparation of MultiTAA-specific T cells was performed in the presence or absence of cytokines (IL-6, IL-12, IL-7, and IL-15). IFN-γ ELISpot analysis was performed on MultiTAA-specific T cells as a readout of antigen specificity. SFU = spot forming unit.

[0058] Fig.12 A comparison of the antigen specificity of MultiTAA-specific T cell products generated using a large volume overnight peptide pulse step and a small volume overnight peptide pulse step is provided. PBMCs from healthy donors were pulsed overnight with peptides from four different antigens (WT1, survivin, NY-ESO-1, and PRAME) in a small volume (10 mL) or a large volume (100 mL) to prepare MultiTAA-specific T cells (n=3). IFN-γ ELISpot analysis was performed on MultiTAA-specific T cells as a readout of antigen specificity. SFU=spot forming unit.

[0059] Fig.13 A comparison of the expansion fold (histogram, left), survival rate (line graph, top) and antigen specificity (histogram, right) of MultiTAA-specific T cell products generated using positively selected cell populations as raw materials for culture and using negatively selected cell populations as raw materials for culture is shown. PBMCs were positively selected for T cells and monocytes (CD8+CD4+CD14+) before cells were placed in culture, or CD56+NK cells were removed (CD56+ removed) before cells were placed in culture. IFN-γ ELISpot analysis was performed on MultiTAA-specific T cells as a readout for antigen specificity.

[0060] Fig.14The correlation between T cell specificity and minimal residual disease (MRD) for the targeted antigen is shown. The MRD percentage (left y axis, solid line) and antigen specificity (right y axis, histogram) were determined before infusion and at weeks 8, 12, 18, 24, and 32 after administration of MT101-4 cells. Targeted antigen-specific T cells are present in the patient's peripheral blood and are negatively correlated with MRD, indicating anti-tumor activity.

[0061] Fig.15 The correlation between T cell specificity for non-targeted antigens (bars) and MRD (solid line) is shown. The presence of T cells specific for the additional antigens indicated (i.e., "non-targeted antigens" that were not used to generate antigen-specific T cell products) in the peripheral blood of patients after treatment is negatively correlated with MRD, representing epitope expansion.

[0062] Fig.16 Gene expression of T cells in the patient's bone marrow before infusion and at weeks 8, 12, 18, 24, and 32 after treatment is shown. MRD is also shown. Gene expression was determined by next-generation sequencing (NGS).

[0063] Fig.17 The composition and phenotype of T cells in patients before infusion and at weeks 8, 12, and 24 after treatment are shown. The upper row shows the CD4 population and the CD8 population. The lower row shows CD197 and CD45RA staining, and the initial (CD197+CD45RA+), central memory (T CM , CD197+CD45RA-), effector memory (T EM , CD197-CD45RA-) and terminally differentiated (T EMRA , CD197-CD45RA+) cell population.

[0064] Fig.18 It is shown that both CD4+ T cells and CD8+ T cells in the patient are functional (expressing IFNγ, as shown by the bars), and that function is correlated with MRD (solid line).

[0065] Fig.19 IFNγ expression by Lag3- and Lag3+ T cells in patients after infusion is shown.

[0066] Fig. 20 Shown is the expansion of peripheral T cell clones in patients following infusion.

[0067] Fig.21RNA expression of targeted antigens in patient bone marrow before infusion and at weeks 8, 12, 18, 24, and 32 is shown. The upper dotted line represents RNA expression of survivin; the lower dotted line represents RNA expression of WT1. The solid line is the MRD percentage. The bars show specificity for the indicated antigens. * = Transcripts for PRAME and NY-ESO-1 were not detected.

[0068] Fig. 22 RNA expression of non-targeted antigen proteinase-3 in patient bone marrow before infusion and at weeks 8, 12, 18, 24, and 32 is shown (dashed line). The solid line is the MRD percentage. The specificity of patient cells for non-targeted antigens including proteinase-3 at each time point is also shown (bars).

[0069] Fig.23 RNA expression of additional non-targeted antigens in patient bone marrow before infusion and at weeks 8, 12, 18, 24, and 32 is shown (dashed lines; at week 18, from top to bottom, BRAF, TERT, mucin-1, Myc, and CCNA1). The solid line is the MRD percentage. The specificity of patient cells for non-targeted antigens is also shown (bars).

[0070] Figure 24A to Figure 24G It is shown that the infusion of MultiTAA-specific T cells resulted in an immune response to additional "non-targeted" TAAs, which represents epitope expansion. Figure 24A is a schematic diagram of the dynamics of the immune response. Figure 24B shows the complete remission (CR) of pancreatic cancer patients after receiving MultiTAA-specific T cell therapy, which is associated with epitope expansion, as shown in Figure 24D (the bars in the "Peak post" column, from top to bottom: WT1, AFP, MART1, MC1, MA3 [largest part of the bar], MA2B, MA1). Figure 24C shows the antigenic specificity of the products infused by the patient (the bars in the "peak post" column, from top to bottom: survival protein, NYESO1 [largest part of the bar], MAGEA4, SSX2 and Prame). Figure 24E shows the partial remission (PR) of pancreatic cancer patients after receiving MultiTAA-specific T cell therapy, which is associated with epitope expansion in the patient, as shown in Figure 24D. Figure 24G FIG. 24F shows the antigenic specificity of the products infused into the patient (the bars in the "Post-Peak" column, from top to bottom: WT1, AFP, MART1, MC1, MA3 [largest part of the bar], MA2B, MA1). FIG. 24F shows the antigenic specificity of the products infused into the patient (the bars in the "Post-Peak" column, from top to bottom: Survivin, NYESO1, MAGEA4, SSX2 [largest part of the bar], and Prame).

[0071] Fig.25A Shown is an image of tumor size in a patient administered T cells prepared by the MAT101 method. The images show the tumor before infusion and 9 months after infusion treatment. Fig.25B A comparison of the antigen specificity for targeted and non-targeted antigens detected by IFNγ ELISpot at months 3 and 9 after cell administration in patients with the antigen specificity of patients before infusion is shown (bars in the "Mth3" and "Mth9" columns, from top to bottom: Survivin, NYESO1, MAGEA4, SSX2, and Prame).

[0072] Fig.26 Graph showing detection of T cell expansion in responding and non-responding lymphoma patients administered MultiTAA-specific T cells (T cell populations generated using peptides from five tumor-associated antigens [TAAs]). Six weeks after the initial infusion of MultiTAA-specific T cells, expansion of T cells specific for "targeted" epitopes from peptides used to generate antigen-specific T cell products was detected (left) and expansion of T cells specific for "non-targeted" epitopes (right), indicating epitope expansion ( Fig.26 ).

[0073] FIG. 27A to FIG. 27D Schematic diagram showing the evaluation of epitope spreading by in vitro assay ( Fig.27A ), and related cell growth ( Fig.27B ) and antigen specificity ( FIG. 27C to FIG. 27D ). Fig.27B Figure (left) and representative visualization (right, bioluminescence indicates cell density) of PANC1 cells (pancreatic cancer cell line) cultured alone or in vitro with nonspecific T cells activated with anti-CD3 / CD28 antibodies or MT-601 antigen-specific cells. Targeted antigen ( Fig.27C ) and non-targeted antigens ( Fig.27D ) antigen specificity.

[0074] Fig.28 is a graph showing the antigen specificity of MT-401 cell population (T cell population generated with peptides from four TAAs) detected by IFNγ ELISpot.

[0075] Fig.29is a schematic diagram showing that bone marrow mononuclear cells (BM-MNCs, target cells) were co-cultured with control PBMCs or MT-401 cells (effector cells) and the killing of target cells in culture was analyzed by flow cytometry using the Tmem5 colorimetric assay.

[0076] FIG. 30A to FIG. 30B Provided are glucose ( Fig. 30A ) and lactic acid ( Fig. 30B )’s output.

[0077] FIG. 31A to FIG. 31B Figure 2 shows target cell killing (measured by fold change in growth after 4 days of culture) using cells derived from a single patient (PT117-201) at a 4:1 effector cell:target cell (E:T) ratio. Changes in cell growth were measured for target cells (BM-MNC) cultured alone, co-cultured with control PBMCs, and co-cultured with MT-401. **** = p < 0001; ns = not significant.

[0078] Fig.32 Graph showing the targeting antigen specificity of MT-401 cells (derived from a single patient 0117-201, prepared using peptides from four TAAs) detected by IFNγ ELISpot before and after co-culture with target cells (BM-MNCs).

[0079] FIG. 33A to FIG. 33B 4 is a graph showing the comparison of antigen specificity between treatment groups in which target cells (BM-MNC) were cultured alone, co-cultured with PBMCs, or co-cultured with MT-401 cells over a 4-day period. Targeted antigen specificity ( Fig.33A ) (Histograms in the “Target cells + PBMC” column, from top to bottom: WT1, Survivin (largest part of the histogram), NY-ESO-1, and Prame; Histograms in the “Target cells + MT-401” column, from top to bottom: WT1, Survivin, NY-ESO-1, and Prame [largest part of the histogram]). Furthermore, additional non-target antigen specificity was observed in the MT-401-treated group after co-culture with target cells ( Fig.33B) (the histograms in the “target cells + PBMC” column, from top to bottom: WT1, survivin (largest part of the histogram), NY-ESO-1, and Prame; the histograms in the “target cells + MT-401” column, from top to bottom: BRAF, mucin-1, TERT, Myc, MAGEA4, SXX2, WT1, survivin, NY-ESO-1, and Prame [largest part of the histogram], while CCNA1 and proteinase-3 are not visible).

[0080] Fig.34 is a schematic diagram representing the finished product process for donor selection, product preparation, cell banking, and patient treatment.

[0081] Fig.35 The estimated number of cell lines required to achieve 90% coverage of the patient population is shown.

[0082] Fig.36 A comparison of the expansion fold, viability, and total viable cell number of T cells prepared in G-Rex 10M and G-Rex-500M is shown.

[0083] Fig.37 The sum and diversity of antigenic specificity of cells obtained from three different donors generated by stimulation with a library of 4 or 6 peptides are shown. As shown in the fourth panel, cells from four donors were stimulated with a library of 12 peptides corresponding to the 12 indicated tumor antigens (bars in the "4 antigens" column, from top to bottom: WT1, survivin, NY-ESO-1, PRAME; bars in the "6 antigens" column, from top to bottom: MAGEA4, SSX2, WT1, survivin, NY-ESO-1, and PRAME; bars in the "12 antigens" column, from top to bottom: proteinase-3, CCNA1, BRAF, mucin-1, TERT, Myc, MAGEA4, SSX2, WT1, survivin, NY-ESO-1, and PRAME).

[0084] Detailed Description

[0085] In one aspect, the present disclosure provides an improved method for generating tumor antigen-specific T cells. Relative to previously described tumor antigen-specific T cells, the tumor antigen-specific T cells described herein and generated by the method provided herein have excellent performance, including improved response intensity to targeted tumor antigens, increased specificity, increased survival rate, and the desired phenotype that enables it to effectively kill tumor cells when applied to patients. In some embodiments, the present disclosure provides a method for generating tumor antigen-specific T cell products, and the product is generated from the monocytes of a patient or object (such as a donor). In some embodiments, tumor antigen-specific T cell products are generated from the monocytes of the patient by the method provided herein, and then applied to the patient (autologous use). In some embodiments, tumor antigen-specific T cell products are generated from the monocytes of the donor by the method provided herein, and maintained in a frozen storage state until used when the patient needs, and the patient can be a donor or can be a different individual (autologous use or allogeneic use).

[0086] In an embodiment, the method provided herein includes culturing monocytes from a subject with a high concentration of peptides in a container, and before adding one or more than one exogenous cytokines to the culture, the cells and peptides are incubated together (also referred to herein as peptide "pulses"). The inventors unexpectedly found that pre-incubating cells with peptides before adding one or more than one exogenous cytokines significantly improved various aspects of tumor antigen-specific T cells generated by the method, including antigen specificity and phenotype. Considering that there is a correlation between the total antigen specificity and anti-tumor activity provided herein, the tumor antigen-specific T cells disclosed herein have significantly more effective anti-tumor activity. In addition, the method provided herein allows for a rapid (5 to 14 days) preparation process, which is beneficial to the treatment of patients. For example, when tumor antigen-specific T cells are administered to patients in need, a shorter treatment time is particularly desired to avoid undue delays in treatment. This shorter treatment time is an improvement on existing methods for generating tumor antigen-specific T cells, which include treatment times of more than 14 days, such as 20 days, 30 days, 36 days, or more than 36 days.

[0087] In embodiments, the methods provided herein include a first step of incubating mononuclear cells (e.g., PBMCs or leukocytes) with a high concentration of one or more than one peptide in a low volume. In embodiments, the cells are seeded at a density of 100 μg / cm 2 The air permeable surface is about 0.1×10 6 cells per cm 2 Breathable surface about 1×10 7In embodiments, the high concentration of the 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, 0.3 μg / mL, about 0.4 μg / mL, or about 0.5 μg / mL), and / or the peptide and cells are incubated in a low volume to promote cell-peptide interaction. In some embodiments, the method includes, for example, incubating at about 0.1×10 6 cells / cm 2 About 1×10 7 cells / cm 2 The mononuclear cells are placed in a container or device at a seeding density of about 0.1×10 6 cells / cm 2 About 1×10 7 cells / cm 2 In some embodiments, the method comprises mixing the monocytes with the peptide mixture 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 and then placing the mixture in a container or device, and then adding the remaining cells and / or peptide mixture to the container or device.

[0088] Monocytes

[0089] In one aspect, the present disclosure provides a method for obtaining monocytes from a subject. In some embodiments, the method provided herein includes obtaining peripheral blood mononuclear cells (PBMC) from a subject. In some embodiments, the monocytes are obtained from a subject by leukapheresis. In some embodiments, the monocytes 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 these cells are administered after generating MultiTAA-specific T cells.

[0090] In some embodiments, the methods provided herein include a removal step before placing a monocyte population in a container and incubating with one or more than one peptide. In some embodiments, the removal step includes removing CD56+ cells from the monocyte population. In some embodiments, monocytes remove CD56+ cells and include CD3+T cells. In some embodiments, monocytes remove CD56+ cells and include CD3+T cells and monocytes. In some embodiments, cells have been removed from CD56+ cells so that the cells in the container are composed of CD3+T cells and monocytes, with a small amount of contaminated CD56+ cells. In embodiments, the method comprising the removal step is referred to herein as "MAT110-removal of CD56+". In some embodiments, T cells generated by this process are referred to herein as "MAT110-removal of CD56+". In some embodiments, removal of CD56+ cells in PBMCs is by using one or more methods known to those of ordinary skill in the art, such as (but not limited to) magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), Dynabeads, EasySep TM Cell separation magnetic beads, RosetteSep TM Immunodensity cell isolation or Cloudz human cell expansion platform.

[0091] In some embodiments, the method provided herein also includes removing CD19+ cells from the monocyte population before the monocyte population is placed in a container or device to be incubated with one or more than one peptide. In some embodiments, the method including the CD19+T cell removal step is suitable for treating patients with B cell cancer. In some embodiments, the removal step includes removing CD56+ cells and CD19+ cells in the monocytes. In embodiments, the method including removing CD56+ cells and removing CD19+ cell steps is referred to herein as "MAT210" or "MAT210-removes CD56+-removes CD19+". In some embodiments, the T cells generated by the method including removing CD56+ cells and removing CD19+ cell steps provided herein are referred to herein as "MAT210" or "MAT210-removes CD56+-removes CD19+". In some embodiments, the removal of CD19+ cells from PBMCs is performed by using methods known to those of ordinary skill in the art, such as, but not limited to, magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), Dynabeads, EasySep TM Cell separation magnetic beads, RosetteSep TM Immunodensity cell isolation or Cloudz human cell expansion platform.

[0092] In some embodiments, the monocytes after the removal of CD56+ cells contain about 0.05% to about 2.5% CD56+ cells. In some embodiments, the monocytes after the removal of CD56+ cells contain about 35% to about 85% T cells. In some embodiments, the monocytes after the removal of CD56+ cells contain 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 monocytes after the removal of CD56+ cells contain no more than about 2.5% CD56+ cells. In some embodiments, the monocytes after the removal of CD56+ cells contain about 1% to about 50% monocytes and about 40% to about 99% T cells.

[0093] In some embodiments, the monocytes after CD56+ cells and CD19+ cells are removed include about 0.01% to about 2% of CD56+ cells. In some embodiments, the monocytes after CD56+ cells and CD19+ cells are removed include about 40% to about 99% of T cells. In some embodiments, the monocytes after CD56+ cells and CD19+ cells are removed include about 0.01% to about 2% of CD56+ cells, about 0.5% to about 50% of monocytes, about 0.01% to about 1.5% of CD19+B cells and / or about 40% to about 99% of T cells. In some embodiments, the monocytes after CD56+ cells and CD19+ cells are removed include no more than about 2.5% of CD56+ cells. In some embodiments, the monocytes after CD56+ cells and CD19+ cells are removed include about 0.5% to about 50% of monocytes and about 40% to about 99% of T cells.

[0094] In one aspect, the present disclosure provides methods, wherein the removal step (e.g., removing CD56+ cells alone, or removing CD56+ cells and CD19+ cells) provides a monocyte population (also referred to herein as a "cell composition") having certain attributes or characteristics. In some embodiments, the cell composition can provide a raw material for generating antigen-specific T cell products provided herein. In some embodiments, the monocyte population or cell composition is placed in a container or device to be incubated with one or more than one peptide from one or more than one tumor-associated antigen.

[0095] In some embodiments, a monocyte population placed in a container and incubated with one or more than one peptide comprises about 0.05% to about 2.5% CD56+ cells. In some embodiments, a monocyte population placed in a container and incubated with a peptide comprises about 35% to about 85% T cells or about 40% to about 99% T cells. In some embodiments, a monocyte population placed in a container and incubated 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, a monocyte population placed in a container with a peptide comprises no more than about 2.5% CD56+ cells. In some embodiments, a monocyte population placed in a container and incubated with a peptide comprises about 40% to about 99% T cells and no more than about 2% NK cells.

[0096] Peptides

[0097] In one aspect, the present disclosure provides methods wherein a population of monocytes (referred to herein as "cells") are incubated with one or more than one peptides from one or more than one tumor antigen.

[0098] As used herein, a peptide refers to a short polymer of amino acids linked by peptide bonds. The peptides in the methods described herein are long enough to contain one or more than one linear epitope, 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.

[0099] In embodiments, one or more than one peptide comprises 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 than 30 tumor associated antigens (TAA). For example, in embodiments, one or more than one peptide comprises 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, one or more than one peptide comprises one or more than one library of peptides derived from one or more than one TAA. In embodiments, each library of peptides comprises peptides covering different portions of TAAs. In embodiments, the library of peptides corresponding to a given tumor antigen comprises a plurality of peptides, each of which covers all or part of the amino acid sequence of the antigen. In embodiments, one or more than one peptide is incubated with cells 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 embodiments, during incubation with cells in the absence of exogenous cytokines using an exemplary library representing four different TAAs, the total concentration of peptides present in the cell culture device is at least about 60 μg / mL, for example, about 78 μg / mL. In embodiments, during incubation with cells in the absence of exogenous cytokines using an exemplary library representing six different TAAs, the total concentration of peptides present in the cell culture device is at least about 100 μg / mL, for example, about 108 μg / mL.

[0100] In embodiments, the concentration of each peptide in the composition is about 0.1 μg / mL to about 0.5 μg / mL, for example, about 0.1 μg / mL, 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.

[0101] In some embodiments, the concentration of peptides in cell culture (e.g., container) is about 50 μg / mL to about 100 μg / mL. In some embodiments, the concentration of peptides in cell culture is about 50 μg / mL. In some embodiments, the concentration of peptides in cell culture is about 60 μg / mL. In some embodiments, the concentration of peptides in cell culture is about 70 μg / mL. In some embodiments, the concentration of peptides in cell culture is about 75 μg / mL. In some embodiments, the concentration of peptides in cell culture is about 80 μg / mL. In some embodiments, the concentration of peptides in cell culture is about 85 μg / mL. In some embodiments, the concentration of peptides in cell culture is about 90 μg / mL. In some embodiments, the concentration of peptides in cell culture is about 95 μg / mL. In some embodiments, the concentration of peptides in cell culture is about 100 μg / mL. In some embodiments, the concentration of peptides in 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.

[0102] 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.

[0103] In an embodiment, the tumor antigen represented in one or more than one peptide is a tumor antigen selected from CEA, MHC, CTLA-4, gp100, mesothelin, PRAME (OIP3) PD-L1, TRP1, CD40, EGFP, Her2, TCRα, trp2, TCR, MUC1, cdr2, ras, 4-1BB, CT26, GITR, OX40, and 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, gp100, 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 Tumor antigens of 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, legumin, 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 in one or more than one peptide is selected from CEA, MHC, CTLA-4, gp100, mesothelin, PRAME (OIP3) PD-L1, TRP1, CD40, EGFP, Her2, TCRα, 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 Tumor antigens of 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, legumin, 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.

[0104] In some embodiments, one or more than one peptide comprises the tumor antigen PRAME. In some embodiments, one or more than one peptide comprises the tumor antigen NY-ESO-1. In some embodiments, one or more than one peptide comprises the tumor antigen survival protein. In some embodiments, one or more than one peptide comprises the tumor antigen WT1. In some embodiments, one or more than one peptide comprises the tumor antigen MAGEA4. In some embodiments, one or more than one peptide comprises the tumor antigen SSX2. In embodiments, one or more than one peptide comprises peptides from tumor antigens PRAME, NY-ESO-1, survival protein and WT1. In other embodiments, one or more than one peptide also comprises peptides from MAGEA4 and SSX2. In some embodiments, one or more than one peptide comprises peptides from tumor antigens PRAME, NY-ESO-1, survival protein, WT1, MAGEA4 and SSX2.

[0105] In embodiments, tumor antigens represented in one or more than one peptide used to generate MultiTAA-specific T cells are referred to herein as "targeted antigens," "target antigens," "targeted tumor-associated antigens," "targeted TAAs," etc. In embodiments, tumor antigens not represented in one or more than one peptide used to generate MultiTAA-specific T cells are referred to herein as "non-targeted antigens," etc.

[0106] Monocyte culture method

[0107] In one aspect, the present disclosure provides a method wherein a population of mononuclear cells obtained from a subject is placed in a container and incubated with one or more than one peptide. In an embodiment, the cell population is placed in a container at a concentration of 100 μg / cm 2 The air permeable surface is about 0.1×10 6 cells per cm 2 Breathable surface approx. 9×10 6 In one embodiment, the cell population is seeded at a density of about 0.1×10 6 cells / cm 2 About 1×10 7 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 0.1×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 0.5×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 1×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 2×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 3×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 4×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 5×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 6×10 6 cells / cm 2In some embodiments, the cell population is seeded at a density of about 7×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 8×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 9×10 6 cells / cm 2 In some embodiments, the cell population is seeded at a density of about 1×10 7 cells / cm 2 The inoculum density was added to the container.

[0108] In embodiments, a monocyte colony is added to a device or container comprising at least one gas permeable surface. In embodiments, a monocyte colony is added to a device or container comprising at least one gas permeable surface. In embodiments, the container to which 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 (commercially available at 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 provided herein, for example, can allow for expansion of a cell population about 10-fold, about 100-fold, about 1000-fold, or more than 1000-fold.

[0109] In some embodiments, the cells are incubated with one or more than one peptide in the container or device at about 37°C and 5% CO2. In some embodiments, the method comprises incubating the cells with the peptide at about 37°C and 5% CO2 for about 30 minutes to about 30 hours. In some embodiments, the method comprises incubating the cells with the peptide at about 37°C and 5% CO2 for about 8 hours to about 24 hours. In some embodiments, the cells are incubated with the peptide for about 30 minutes. In some embodiments, the cells are incubated with the peptide for about 1 hour. In some embodiments, the cells are incubated with the peptide for about 2 hours. In some embodiments, the cells are incubated with the peptide for about several hours. In some embodiments, the cells are incubated with the peptide for about 4 hours. In some embodiments, the cells are incubated with the peptide for about 5 hours. In some embodiments, the cells are incubated with the peptide for about 6 hours. In some embodiments, the cells are incubated with the peptide for about 7 hours. In some embodiments, the cells are incubated with the peptide for about 8 hours. In some embodiments, the cells are incubated with the peptide for about 9 hours. In some embodiments, the cells are incubated with the peptide for about 10 hours. In some embodiments, the cells are incubated with the peptide for about 11 hours. In some embodiments, the cells are incubated with the peptide for about 12 hours. In some embodiments, the cells are incubated with the peptide for about 13 hours. In some embodiments, the cells are incubated with the peptide for about 14 hours. In some embodiments, the cells are incubated with the peptide for about 15 hours. In some embodiments, the cells are incubated with the peptide for about 16 hours. In some embodiments, the cells are incubated with the peptide for about 17 hours. In some embodiments, the cells are incubated with the peptide for about 18 hours. In some embodiments, the cells are incubated with the peptide for about 19 hours. In some embodiments, the cells are incubated with the peptide for about 20 hours. In some embodiments, the cells are incubated with the peptide for about 21 hours. In some embodiments, the cells are incubated with the peptide for about 22 hours. In some embodiments, the cells are incubated with the peptide for about 23 hours. In some embodiments, the cells are incubated with the peptide for about 24 hours. In some embodiments, the cells are incubated with the peptide for about 25 hours. In some embodiments, the cells are incubated with the peptide for about 26 hours. In some embodiments, the cells are incubated with the peptide for about 27 hours. In some embodiments, the cells are incubated with the peptide for about 28 hours. In some embodiments, the cells are incubated with the peptide for about 29 hours. In some embodiments, the cells are incubated with the peptide for about 30 hours. In some embodiments, the method comprises incubating the cells with the peptide at 37° C. and 5% CO2 overnight. In some embodiments, the method comprises incubating the cells with the peptide at about 37° C. and 5% CO2 for about 30 minutes to about 30 hours in the absence of exogenous cytokines. In some embodiments, the method does not include a washing step.In some embodiments, the method comprises incubating the cells with the peptide in the absence of exogenous cytokines at about 37° C. and 5% CO2 for about 8 hours to about 30 hours, after which the culture is divided into two or more separate cultures. In some embodiments, after adding one or more exogenous cytokines, the cells are further incubated at about 37° C. and 5% CO2 for an additional 5 to 14 days.

[0110] Cytokines

[0111] In embodiments, the methods provided herein include incubation with exogenous cytokines suitable for stimulating and / or activating T cell expansion and / or differentiation. The unexpected aspect of the present invention is that, before adding one or more than one exogenous cytokines, pre-incubating cells with one or more than one peptide significantly improves various aspects of tumor antigen-specific T cells generated by the method, including antigen specificity and phenotype. In one aspect, the present disclosure provides a method, wherein in the absence of exogenous cytokines, a mononuclear cell population is co-incubated with one or more than one peptide from one or more than one tumor antigen. In some embodiments, the cell population is cultured for about 30 minutes to about 30 hours in the absence of exogenous cytokines. In some embodiments, the cell population is cultured for about 8 hours to about 24 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 30 minutes in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 1 hour in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 2 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 3 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 4 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 5 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 5 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 7 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 8 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 9 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 10 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 11 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 12 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 13 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 14 hours in the absence of exogenous cytokines. 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 for at least about 20 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 21 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 22 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 23 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 24 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 25 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 26 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 27 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 28 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 29 hours in the absence of exogenous cytokines. In some embodiments, the cells are cultured for at least about 30 hours in the absence of exogenous cytokines.

[0112] In embodiments, after incubating cells and peptides in the absence of exogenous cytokines, one or more than one exogenous cytokines are added to the culture. In embodiments, exogenous cytokines include IL-6, IL-7, IL-15, IL-12, or any combination thereof. In embodiments, the exogenous cytokines added to the culture are composed of IL-6, IL-7, IL-15, and IL-12. In embodiments, the concentration of IL-6 added to the culture is about 10 ng / mL to about 100 ng / mL, such as 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 embodiments, the concentration of IL-7 added to the culture is about 1 ng / mL to about 10 ng / mL, such as 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 embodiments, the concentration of IL-15 added to the culture is 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 embodiments, the concentration of IL-12 added to the culture is about 10 IU / mL 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 embodiments, once the cytokine is added to the culture, the culture is no longer disturbed until the MultiTAA-specific T cells are harvested. In embodiments, the harvesting of cells is after incubation for about 5 days to about 14 days, for example, after incubation for about 5 days to about 10 days, for example, after incubation for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days or about 14 days.

[0113] MultiTAA-specific T cell populations and compositions

[0114] In one aspect, the present disclosure provides antigen-specific T cell populations (also referred to as tumor antigen-specific T cells, MultiTAA-specific T cells) generated by the methods herein. In one aspect, the present disclosure provides antigen-specific T cell populations comprising about 80% to about 99% CD3+T cells, <1% CD14+ monocytes, <10% CD19+B cells, and <10% CD3-CD56+NK cells.

[0115] T cells are composed of different subsets that can be generated by differentiation and have different activity, lifespan, and expansion kinetics. T cell subsets include CD4+ T cells and CD8+ T cells, naive T cells, central memory cells (TCM ) and effector memory cells (T EM ). T cell subsets can be detected and measured by methods known to those of ordinary skill in the art, such as flow cytometry after labeling markers of different phenotypes (e.g., CD4, CD8, CD45RA, CD45RO, etc.) with fluorescently labeled antibodies.

[0116] In embodiments, the present disclosure provides a population of antigen-specific T cells comprising at least about 20% central memory T cells (T CM In embodiments, the tumor antigen-specific T cells comprise at least about 20% of naive T cells. In embodiments, the tumor antigen-specific T cells comprise about 40% of effector memory T cells (T EM In an embodiment, the tumor antigen-specific T cells comprise no more than 10% of terminally differentiated effector memory cells (T cells) expressing CD45RA. EMRA ).

[0117] In an embodiment, the present disclosure provides an antigen-specific T cell population that exhibits a higher antigen-specific response intensity relative to an antigen-specific T cell population generated by an existing method. For example, as detected by IFN-γ ELISpot, the specific intensity exhibited by the antigen-specific T cell population (combined responsiveness to each targeted antigen) is at least about 500 SFU / 2×10 5 Cells. T cell functionality and antigen specificity can be determined by methods known in the art, including methods for detecting proliferation and / or expression of cytokines such as IFNγ. IFNγ expression can be assessed, for example, by ELISA, ELISpot or flow cytometry analysis.

[0118] T cell populations can be characterized by the diversity of antigen specificity (e.g., the number of tumor antigens to which T cell products are active in tumor antigens represented by one or more than one peptide incubated with monocytes). Higher diversity of antigen specificity can be a desired feature of T cell populations. In some embodiments, tumor antigen-specific T cells prepared by the methods herein show antigen specificity to 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 tumor antigens represented by one or more than one peptide used in the initial culture. In some embodiments, tumor antigen-specific T cells prepared by the methods herein show antigen specificity to at least 75% of tumor antigens represented by one or more than one peptide used in the initial culture. In some embodiments, tumor antigen-specific T cells prepared by the methods herein show antigen specificity to each of the tumor antigens represented by one or more than one peptide. In embodiments, increasing the number of tumor antigens incubated with monocytes unexpectedly increases the antigen specificity and antigen diversity of the prepared tumor antigen-specific T cells.

[0119] In some embodiments, the population comprises T cells that exhibit activity against one or more peptides of one or more antigens that have not been previously incubated with the population. In some embodiments, the activity against one or more peptides of one or more antigens that have not been previously incubated with the population is the result of epitope spreading. In some embodiments, the population comprises T cells that have a cumulative specificity for one or more peptides of one or more antigens that have not been previously incubated with the population of at least about 100 SFU / 2×10 5 In some embodiments, the population comprises at least about 30 SFU / 2×10 cells specific for one or more peptides of one or more antigens not previously incubated with the population as detected by IFN-γ ELISpot. 5 cells.

[0120] In an embodiment, the present disclosure provides a composition comprising a MultiTAA-specific T cell population obtained by the method provided herein. In an embodiment, the composition further comprises a pharmaceutically acceptable buffer or excipient. In a further embodiment.

[0121] T cell products

[0122] In one aspect, the present 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 present disclosure provides a T cell product generated by the methods described herein, comprising about 80% to about 99% CD3+T cells, <1% CD14+ monocytes, <10% CD19+B cells, and <10% CD3-CD56+NK cells.

[0123] In one embodiment, the invention provides a method comprising at least about 20% central memory T cells (T CM ) of a MultiTAA-specific T cell product. In embodiments, the tumor antigen-specific T cells comprise at least about 20% of naive T cells. In embodiments, the tumor antigen-specific T cells comprise about 40% of effector memory T cells (T EM In an embodiment, the tumor antigen-specific T cells comprise no more than 10% of terminally differentiated effector memory cells (T cells) expressing CD45RA. EMRA ). CM T cells are generally considered to have greater proliferation potential and persistence potential in vivo than terminally differentiated T cells; therefore, the T cell products described herein may exhibit characteristics that are therapeutically beneficial.

[0124] In an embodiment, the present disclosure provides a MultiTAA-specific T cell product that exhibits a higher antigen-specific response intensity than a MultiTAA-specific T cell product generated by an existing method. For example, as detected by IFN-γ ELISpot, the intensity of the specificity exhibited by the MultiTAA-specific T cell product (combined responsiveness to each targeted antigen) is at least about 500 SFU / 2×10 5 For example, in an embodiment, the intensity of specificity exhibited by the MultiTAA-specific T cell product (combined responsiveness to each targeted antigen) is at least about 700 SFU / 2×10 5 cells, at least about 1000 SFU / 2×10 5 cells or at least about 1200 SFU / 2×10 5 cells.

[0125] In some embodiments, the T cell products prepared by the methods herein show 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 than one peptide used in the initial culture. In some embodiments, the T cell products prepared by the methods herein show antigen specificity for at least 75% of the tumor antigens represented by one or more than one peptide used in the initial culture. In some embodiments, the T cell products prepared by the methods herein show antigen specificity for each of the tumor antigens represented by one or more than one peptide. In embodiments, increasing the number of tumor antigens incubated with monocytes unexpectedly increases the antigen specificity and antigen diversity of the prepared tumor antigen-specific T cells.

[0126] 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 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 with a cumulative intensity of at least about 100 SFU / 2×10 5 In some embodiments, the T cell product comprises one or more peptides specific for one or more antigens not used to generate the T cell product with a cumulative intensity of at least about 30 SFU / SFU / 2×10 5 cells.

[0127] In some embodiments, the activity of one or more peptides against one or more antigens not previously incubated with the population is the result of epitope spreading.

[0128] Epitope expansion occurs when T cells target specific antigens to destroy them, exposing new "non-targeted" epitopes / antigens, which recruit and amplify T cells specific for non-targeted antigens to help eliminate tumors, thereby limiting tumor escape. Epitope expansion in this case was previously thought to be a result from the patient's endogenous immune system. Surprisingly, the inventors discovered that epitope expansion comes from the expansion of the MultiTAA-specific T cell product itself. Without wishing to be bound by theory, cells specific for non-targeted antigens in the final drug product may contribute to epitope expansion to eliminate tumors in vivo.

[0129] Cryopreservation

[0130] In some embodiments, the method further comprises cryopreserving the monocytes, then thawing the cryopreserved cells, and then placing the cells in a container. In some embodiments, the method further comprises cryopreserving the MultiTAA-specific T cell product. Cryopreservation can be performed by placing the monocytes and / or MultiTAA-specific T cells in a cryopreservation medium or freezing solution, such as CS10 or its derivatives, Hyperthermasol or any freezing solution containing about 5% to about 15% DMSO. Therefore, in some embodiments, the present disclosure provides monocytes and / or MultiTAA-specific T cells herein in a cryopreserved state. In order to use monocytes and / or MultiTAA-specific T cells after cryopreservation, for example, using monocytes to incubate with peptides or using MultiTAA-specific T cells to treat patients, the cells are removed from the freezing device and thawed in a water bath at about 37°C until most of the solution is thawed. The cells can then be resuspended in culture medium, optionally washing the cells before use. In some embodiments, the thawed monocytes are left to stand for about 4 hours to about 48 hours before being placed in a container. In some embodiments, monocytes and / or thawed MultiTAA-specific T cells are prepared by counting and determining cell viability to prepare for use.

[0131] In the embodiments, the present disclosure provides finished tumor antigen-specific T cell products. Such products eliminate the need for steps such as donor identification, cell acquisition, T cell product preparation, and T cell product analysis, which usually must occur between patient identification / registration and patient treatment, thereby significantly shortening patient treatment time. It is not necessary to start treatment for (for example) 14 days, 21 days, 28 days, 35 days, or more than 35 days, and patients can receive treatment for finished tumor antigen-specific T cell products within about 72 hours after identification. In the embodiments, the method for generating MultiTAA-specific T cell products provided herein also includes the step of HLA typing of donors of mononuclear cells used to generate MultiTAA-specific T cell products.

[0132] Treatment

[0133] In one aspect, the present disclosure provides methods for treating cancer patients. In some embodiments, the methods provided herein include administering a pharmaceutical composition to a patient in need, the pharmaceutical composition comprising the MultiTAA-specific T cells provided herein. In one embodiment, the pharmaceutical composition is a suspension of MultiTAA-specific T cells, wherein the MultiTAA-specific T cells are in a pharmaceutically acceptable buffer or excipient. In some embodiments, the method includes administering a tumor antigen-specific T cell product provided herein to a patient, wherein the product is administered at a dose of about 50×10 per treatment. 6 cells to about 300×10 6 For example, in one embodiment, the product is administered to a patient at a dose of about 100×10 6 cells to about 200×10 6 In one embodiment, the product is administered to a patient at a dose of about 100×10 6 cells to about 200×10 6 In some embodiments, the product is administered to a patient by any suitable route, particularly by intravenous infusion or intra-arterial infusion. In some embodiments, the infusion may last for about 15 minutes, 30 minutes, 45 minutes, 60 minutes, or 90 minutes per dose. In some embodiments, the MultiTAA-specific T cell product or pharmaceutical composition is administered in a single dose or multiple doses.

[0134] In embodiments, the cancer is a lymphoma or leukemia. In embodiments, the cancer is a solid tumor. In embodiments, the cancer suffered by the patient is 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.

[0135] Thus, in embodiments, the present disclosure provides methods for treating cancer patients with finished MultiTAA-specific T cell products provided herein, wherein the patient is HLA typed or wherein the patient's HLA type is known, and the MultiTAA-specific T cell products are selected from a library of previously generated MultiTAA-specific T cell products to achieve a desired level of HLA matching between the donor and the recipient. In embodiments, the donor and the recipient have at least one HLA match, at least two HLA matches, or at least 3 HLA matches. In embodiments, the method comprises administering to the patient a finished MultiTAA-specific T cell product provided herein, and further comprising treating the patient with one or more additional finished MultiTAA-specific T cell products. In embodiments, one or more additional T cell products (such as a second T cell product) can 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. In embodiments, one or more additional treatments may use the same finished MultiTAA-specific T cell product as the previous one or more treatments (e.g., a MultiTAA-specific T cell product derived from cells from the same donor), or may use a different finished MultiTAA-specific T cell product than the previous one or more treatments (e.g., one or more additional treatments may use one or more MultiTAA-specific T cell products generated from cells from one or more different donors). In embodiments, the option of a second or further treatment with a different MultiTAA-specific T cell product may be applicable to patients who have developed an immune response to a first MultiTAA-specific T cell product and / or have rejected the first MultiTAA-specific T cell product, or are suspected of having rejected the first MultiTAA-specific T cell product.

[0136] In embodiments, in an autologous setting, a second antigen-specific T cell population (second treatment) is generated by the following steps: (i) obtaining a first monocyte population from a patient; (ii) generating a first antigen-specific T cell population according to the methods herein; (iii) administering the first antigen-specific T cell population to a patient; (iv) obtaining a second monocyte population from a patient after at least about 4 weeks, about 5 weeks, or about 6 weeks of administering the first antigen-specific T cell population; (v) generating a second antigen-specific T cell population according to the methods herein, and (vi) administering the second antigen-specific T cell population to a patient. Compared to the first antigen-specific T cell product, the second antigen-specific T cell product can contain a greater number of antigen-specific cells to provide a more effective second administration.

[0137] In one aspect, the MultiTAA-specific T cells provided by the present disclosure are allogeneic to the recipient patient and are effective in treating cancer without producing a significant effect of graft-versus-host disease (GVHD) response. In general, a patient population receiving allogeneic tumor antigen-specific T cells for the treatment of cancer is expected to have a GVHD response because the patient is not immunocompromised. Without wishing to be bound by theory, the recipients receiving the MultiTAA-specific T cells provided herein unexpectedly had no GVHD response, which may be due to the enrichment of tumor antigen-specific cells achieved by the compositions and methods provided herein.

[0138] definition

[0139] The terms used herein are only used to describe specific embodiments and are not intended to limit the present invention. As used herein, plural references are not used unless the context indicates otherwise. In addition, with respect to the terms "include", "comprising", "having", "with" and variations thereof used in the detailed description and / or claims, these terms are intended to represent inclusion in a form similar to the terms "comprise" or "including".

[0140] 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 will depend in part on how the value is detected or determined, i.e., the limitations of the detection system. For example, "about" can mean a range of ±20%, ±10%, ±5%, or ±1% of a given value. Alternatively, particularly with respect to biological systems or methods, the term can mean within an order of magnitude, such as within 5 times or within 2 times of a value. When specific values ​​are described in the application and claims, the term "about" should be assumed to mean within an acceptable error range for the specific value unless otherwise indicated.

[0141] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more than one of the listed related items, as well as the lack of a combination when interpreted as an alternative ("or"). The use of an alternative (such as "or") should be understood to mean one, both, or any combination of the alternatives.

[0142] In this description, unless otherwise stated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range, and include its fractions (such as one tenth and one hundredth of an integer) where appropriate. The terms "about" and "approximately" represent a statistically significant range of a numerical value. Such a range can be within an order of magnitude of a given numerical value or range, preferably within 20%, more preferably within 10%, and even more preferably within 5%. In certain embodiments, when the term "about" is immediately before a number or value, it means that the range of the number or value is ± 10%. The allowed variation covered by the term "about" or "approximately" depends on the specific system under study and can be easily understood by a person of ordinary skill in the art. In addition, as used herein, the terms "about" and "approximately" mean that size, size, formula, parameter, shape and other quantities and characteristics are not and do not need to be accurate, but can be approximate and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art. Generally, 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 very different sizes, shapes and dimensions may employ the described arrangements.

[0143] Unless otherwise indicated, all terms used herein have the same meanings as understood by one skilled in the art, and the practice of the present invention will employ conventional techniques in the fields of microbiology and recombinant DNA technology, which are within the knowledge of those skilled in the art.

[0144] The term "monocyte" refers to a cell with a round nucleus, such as a cell isolated from the peripheral blood of a patient or donor. Monocytes can be a cell population of leukocytes or peripheral blood mononuclear cells (PBMCs), and can be obtained using known methods, such as the Ficoll-Hypaque density gradient method. Monocytes include monocytes, lymphocytes, T cells, B cells and NK cells.

[0145] The terms "subject," "individual," and "patient" are used interchangeably herein. In embodiments, the subject is a mammal, e.g., a mouse, rat, rabbit, cat, dog, pig, sheep, horse, non-human primate (e.g., cynomolgus monkey, chimpanzee), or human. The term "donor" refers to a subject, individual, or patient from which mononuclear cells are obtained.

[0146] As used herein, the term "exogenous" refers to a material introduced into a cell, cell culture, container, device, suspension, composition, etc., which is produced outside of the cell, cell culture, container, device, suspension, or composition. The term "endogenous" refers to any substance produced from within a cell, cell culture, container, device, suspension, or composition.

[0147] "T cell" is a commonly used term in the art, including thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. T cells can be helper T (Th) cells, such as type 1 helper T (Th1) or type 2 helper T (Th2) cells. T cells can be CD4+T cells, CD8+T cells, CD4+CD8+T cells, CD4-CD8-T cells or any other T cell subsets.

[0148] The term "antigen-specific T cell" as used herein is intended to refer to a T cell that recognizes a specific antigen and responds, for example, by proliferating and / or producing cytokines in response thereto. The terms "tumor antigen-specific T cell" or "tumor antigen-specific T cell product" or "tumor antigen-specific T cell population", etc., are used interchangeably herein with the terms "MultiTAA-specific T cell" or "MultiTAA-specific T cell product", etc. The terms "tumor-specific antigen" and "tumor antigen" are used interchangeably herein with the terms "tumor-associated antigen" or "TAA".

[0149] The term "MAT101-4" as used herein refers to Figure 1B The schematic diagram of the method for preparing MultiTAA-specific T cells is shown in FIG. 1 . The term "MAT103-4" as used herein refers to Figure 1C The term "MAT110-depleted CD56+" as used herein refers to Figure 1AA method for preparing MultiTAA-specific T cells as shown in a schematic diagram of the present invention. The MAT110-CD56+ removal method involves directly stimulating PBMCs with peptides in a container having at least one gas permeable surface. The term "MAT210-CD56+ removal-CD19+ removal" used herein refers to a method for preparing MultiTAA-specific T cells, which is the same as the MAT110-CD56+ removal except that CD19+ cells and CD56+ cells are removed from monocytes before incubation with peptides. Unlike the MAT101-4 method and the MAT110-unremoved method, the MAT110-CD56+ removal method and the MAT210-CD56+ removal-CD19+ removal method include a step of incubating the peptide mixture with PBMCs in the absence of exogenous cytokines before adding one or more exogenous cytokines. The total number of days for the MAT110-CD56+ depleted method and the MAT210-CD56+ depleted-CD19+ depleted method was also shorter, ie, from about 5 days to about 14 days, compared to the MAT101-4 method or the MAT103-4 method.

[0150] In an embodiment, the identity and / or phenotype of the cell is detected to determine the characteristics of the MultiTAA-specific T cells provided herein, or to determine the characteristics of the cells used as raw materials to generate the MultiTAA-specific T cells provided herein. Expression markers can be detected by flow cytometry, such as but not limited to CD3, CD4, CD8, CD56, CD19, CD14, and any other marker disclosed herein or known in the art. For example, antibodies, such as fluorescently labeled antibodies, can be used to mark cells, which can then be analyzed by flow cytometry. In addition, a hemocytometer, trypan blue staining, or any other method known in the art can be used to detect cell number and / or cell survival rate. In some embodiments, other cell markers, such as regulatory markers, are detected, for example, to assess the functionality or exhaustion of MultiTAA-specific T cells. These markers can be selected from, for example, TCRαβ, CD45RA, CD45RO, CD25, CD28, CD127, CD95, CCR7, CD62L, Lag3, TIM-3, PD-1, TIGIT, etc.

[0151] The term "central memory T cells" is used in this article in conjunction with "T CM " are used interchangeably to refer to the T cell subset that secretes IL-2 upon activation. CM Cells are CD45RO+CD45RA lo , CD127+(IL-7R+) and constitutively express CCR7(CCR7 hl ) and CD62L (CD62 hl). The term "effector memory T cells" is used herein in conjunction with "T EM " is used interchangeably to refer to a subset of T cells that, like central memory T cells, are CD45RO+CD45RA lo and CD127+(IL-7R+), but CCR7 lo and CD62L lo Re-expression of CD45RA in effector memory T cells (T EMRA ) is CD45RO lo 、CD45RA hi 、CD62L lo and CCR7 lo . T EM and T EMRA In terms of effector function, it is better than T CM More differentiated, T CM Typically have a higher proliferation potential. As used herein, the term "epitope expansion" refers to the expansion or diversification of the epitope specificity of a T cell immune response. For example, in embodiments, epitope expansion refers to expansion or diversification from an initial set of specificities (e.g., specificity for tumor antigens used to generate or amplify antigen-specific T cell products) to additional specificities, such as other tumor antigens that do not exist during the generation or initial amplification of antigen-specific T cell products.

[0152] As used herein, the term "harvest" refers to collection, separation and / or removal from a culture vessel or device. Antigen-specific T cells can be harvested using methods known to those of ordinary skill in the art, including but not limited to removal from a culture vessel or device, optionally centrifuged or washed using an automated cell processing device, and optionally resuspended in fresh culture 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.

[0153] As used herein, "treat," "treat," "alleviate," or "ameliorate" are used interchangeably. These terms refer to an approach to obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any treatment-related improvement or efficacy in the treatment of one or more diseases, disorders, or symptoms, such as cancer. For prophylactic benefit, the composition can be administered to a subject at risk for developing a particular disease, disorder, or symptom, or to a subject who has reported one or more physiological symptoms of a disease, even though the disease, disorder, or symptom may not yet be manifested.

[0154] The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent sufficient to achieve a certain result, such as a dose that can cause a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the following: the subject and the disease condition being treated, the subject's weight and age, the severity of the disease condition, the mode of administration, etc., which can be easily determined by a person of ordinary skill in the art. The specific dose may vary depending on one or more of the following: the specific agent selected, the dosing regimen followed, whether it is administered in combination with other compounds, the time of administration, the tissue to be imaged, and the physical delivery system to carry it. In an embodiment, an effective amount of MultiTAA-specific T cells can be administered in a single dose or multiple doses, by any acceptable mode of administration for such cell products, including intravenous injection, intraarterial injection, intraperitoneal injection, parenteral injection, intramuscular injection, intrathecal injection, intralymphatic injection, or subcutaneous injection.

[0155] All papers, publications and patents cited in this specification are incorporated herein by reference, just as if each individual paper, publication or patent was clearly and individually marked as being incorporated herein by reference, and the methods and / or materials associated with the cited publications are disclosed and described. However, the reference to any reference, article, publication, patent, patent publication and patent application cited herein is not, and should not be regarded as an admission or any form of suggestion that they constitute valid prior art or constitute part of the common general knowledge in any country in the world.

[0156] It should be understood that the above description and the following embodiments are intended to illustrate rather than limit the scope of the present invention. Other aspects, advantages and improvements within the scope of the present invention will be obvious to those skilled in the art to which the present invention belongs. Example

[0157] Example 1. Development of an improved preparation method

[0158] Known methods for generating tumor antigen-specific T cells are costly, time-consuming, and difficult to scale up. An improved method was developed (MAT110-depleted CD56+, e.g. Figure 1A ), to overcome the previous method (MAT101-4, as Figure 1B As shown; MAT110-not removed or MAT103-4, as Figure 1C limitations shown).

[0159] Example 2. Comparison of raw materials and amplified products

[0160] To understand whether the starting cell population for generating tumor antigen-specific T cells affects the final cell phenotype, survival, or antigen specificity, three PBMC starting cell populations were compared. PBMCs were obtained from donors and were either undepleted or depleted of CD56+ cells using the MAT110-CD56+ depletion method, as described in Figure 1A As indicated (to remove NK cells), either CD56+ cells and CD19+ cells were removed using the MAT210-CD56+-CD19+ removal method (to remove NK cells and B cells). The MAT210-CD56+-CD19+ removal process was identical to the “MAT110-CD56+ removal” process, except that CD19+ cells and CD56+ cells were removed from monocytes prior to peptide pulsing. After generating cell populations, they were analyzed by flow cytometry to determine the percentage (%) of live monocytes, T cells, B cells, or NK cells ( FIG. 2A to FIG. 2C The ratio of CD4+ T cells to CD8+ T cells in the CD3+ T cell compartment was also determined ( FIG. 2A to FIG. 2C ).

[0161] Figure 3 , Figure 4 and Figure 5 Provided are methods provided herein using cells ( Figure 3 ), removal of CD56+ cells ( Figure 4 ) or depletion of CD56+ and CD19+ cells ( Figure 5 ) as raw materials in culture. The first histogram of each figure shows the monocyte, T cell, B cell and NK cell compartments; the second histogram of each figure shows the percentage of CD4+ or CD8+ CD3+ T cells; the third histogram of each figure shows the phenotype of CD3+ T cells (naive T cells, T CM , T EM or T EMRA ); the fourth histogram of each figure shows the expansion fold achieved by the culture; and the fifth histogram of each figure shows the total response intensity to tumor antigens detected by IFNγELISpot. Studies have shown that depletion of CD56+ or depletion of CD56+CD19+ promotes the expansion and antigen specificity of T cells compared with non-depletion.

[0162] Example 3. Comparison of tumor antigen-specific T cells generated using the improved preparation method and the previous method

[0163] To evaluate and compare the quality of T cell products generated using MAT110-CD56+ depletion with those generated using previous methods, the expansion and survival of tumor antigen-specific T cells were examined. Fig. 6AThe T cell expansion fold for three of these methods is shown: Compared with MAT103-4, the MAT110-CD56+ depletion method is an accelerated process, showing a similar level of T cell expansion fold as cells prepared by the MAT103-4 method. However, the expansion of the MAT110-undepleted method was significantly lower than that of the MAT103-4 and MAT110-CD56+ depletion methods. The cell survival rates of the three different methods were also similar ( Figure 6B ). Next, the phenotypes of cells generated by each method were compared. Fig. 7A The results showed that the percentage (%) of CD3+ cells and the ratio of CD4+ T cells to CD8+ T cells were similar in the final products prepared by the three different methods. The central memory (T) of T cell products obtained using MAT110-non-depleted or MAT110-depleted CD56+ CM ) cells were significantly higher ( Figure 7B ). Therefore, T cell products generated using MAT110-non-depleted or MAT110-depleted CD56+ may persist longer in vivo than T cell products generated using MAT103-4. In summary, these data suggest that the MAT110-depleted CD56+ approach provides an accelerated process with good T cell expansion fold, survival rate, and in vivo longevity potential of T cell products.

[0164] The antigen specificity of T cells obtained by three different methods was also evaluated 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 using the three methods described above. Fig. 8A As shown, cells generated using the MAT110-depleted CD56+ method exhibited higher antigen specificity compared to cells generated by either the MAT103-4 or MAT110-undepleted methods from the same donor (ie, higher antigen specificity compared to MAT103-4 and MAT110-undepleted). Figure 8B The strength of overall antigen specificity is shown (when data from donors are pooled, the responsiveness of T cell products to each targeted antigen is combined); these data show a statistically significant increase in antigen specificity of T cells generated using the MAT110-CD56+ depletion approach. Fig.9A The overall response strength is also provided. Fig. 9B As shown, the MAT110-depleted CD56+ method generated a greater number of T cell products, and the T cell products had a greater number of antigen specificities, i.e., a higher degree of antigen diversity. Fig. 9CIt is further described that MAT110-depletion of CD56+ cells achieves increased numbers of products specific for multiple tumor antigens. Fig.9D The antitumor activity (detected by fluorescence changes in THP-1 cells expressing firefly luciferase after 6 days of co-culture with MultiTAA-specific T cells) and antigen specificity (detected by IFNγ ELISpot, SFU / 2×10 5 Accordingly, the improved process provided herein obtains increased antigen specificity, which is at least about four times stronger than the existing process.

[0165] Accordingly, the MAT110-depleted CD56+ preparation process significantly increased the proportion of multi-target T cells. For example, compared with cells prepared by previous methods, cells prepared by the MAT110-depleted CD56+ method showed significantly higher antigen specificity and diversity.

[0166] In summary, our results demonstrate that the improved manufacturing process of MAT110-depleted CD56+ produced T cell phenotypes with increased antigen specificity and diversity, significant expansion fold, high levels of cell survival, and desirable antitumor activity.

[0167] Example 4. Analysis of the Peptide Pulse Step

[0168] Studies were conducted to evaluate the pre-incubation of peptides with cells at high concentrations prior to the addition of exogenous cytokines (using the MAT110-undepleted 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) to prepare MultiTAA-specific T cells. On day 1, cytokines were added to "no cytokine" cultures according to the standard MAT110-undepleted method, while cytokines were maintained in "with cytokine" cultures. Fig.10 A set of exemplary calculations for determining peptide amounts and concentrations for a peptide pulse step is provided.

[0169] Multi-tumor antigen-associated (MultiTAA)-specific T cells were then subjected to IFN-γ ELISpot analysis as a readout of antigen specificity. Fig.11 As shown, the presence of cytokines during the peptide pulsing step significantly reduced antigen specificity, indicating that the generation of MultiTAA-specific T cells was significantly improved when cells were incubated with peptides for a period of time in the absence of cytokines before the addition of exogenous cytokines.

[0170] Additional studies were performed to evaluate the effect of the volume of the peptide pulse step on antigen specificity (using the MAT110-undepleted method). In this experiment, PBMCs from healthy donors were pulsed overnight with peptides from four different antigens (WT1, survivin, NY-ESO-1, and PRAME) in either a small volume (10 mL) or a large volume (100 mL) to prepare MultiTAA-specific T cells (n=3). IFN-γ ELISpot analysis was performed on MultiTAA-specific T cells as a readout of antigen specificity. Fig.12 As shown, antigen specificity was significantly increased in T cells generated using low volume conditions. Without wishing to be bound by theory, the increase in antigen specificity may be due to the increase in peptide-cell contacts achieved during the low volume peptide pulse incubation step.

[0171] In summary, the results of this study suggest that in the absence of exogenous cytokines and / or at low volumes (e.g., which allow seeding densities of approximately 0.1 × 10 6个 Cells / cm 2 About 1×10 7 cells / cm 2 The peptide pulsing step performed under conditions of (100% WT cells, exposed to high concentrations of peptide) significantly improved the antigen specificity of the obtained MultiTAA-specific T cells.

[0172] Example 5. Comparison of positive T cell selection and NK cell removal

[0173] A study was conducted to compare the fold expansion, survival, and antigen specificity of MultiTAA-specific T cells when using starting cells that were positively selected for T cell markers versus cells that were depleted of CD56 (NK cell marker) positive cells. Positively selected T cells and CD56+ depleted T cells were incubated with tumor antigen peptides (PRAME, NY-ESO-1, survivin, WT1), harvested on day 9, and assayed for expansion and survival. Fig.13 As shown, the expansion fold and antigen specificity of positively selected T cells (CD4+CD8+CD14+) were reduced compared to cells with CD56+ removed (MAT110-CD56+ removal method). Without wishing to be bound by theory, the positive selection of T cells bound to CD4 and CD8 inhibits their ability to be stimulated by antigen presenting cells in the subsequent steps of the method.

[0174] Example 6. Characterization of Antigen-Specific T Cells and Disease Progression in Patient Recipients of MultiTAA-Specific T Cells

[0175] A study was conducted to provide various analyses of the efficacy of administering MultiTAA-specific T cell products prepared by the MAT103-4 method to cancer patients. In this study, patients were diagnosed with acute myeloid leukemia (AML) and had previously failed 5 treatments (I+C [induction chemotherapy], FLAG-IDA [fludarabine, cytarabine (Ara-C), granulocyte-colony stimulating factor, idarubicin] + IC chemotherapy, MEC [mitoxantrone, etoposide and cytarabine] + IC chemotherapy, azacitidine and decitabine), and had c-KIT mutations and genetic abnormalities: t(8; 21)(q22; q22.1) [RUNX1-RUNX1T1]. PBMCs from matched donors were collected by leukapheresis, and MultiTAA-specific T cell products were generated using the MAT103-4 method provided herein with peptide libraries corresponding to WT1, NY-ESO-1, survivin and PRAME. The patients were then administered the MultiTAA-specific T cell product (3 doses, 5 × 10 6 The specificity, composition, phenotype, and functionality of T cells were evaluated in peripheral blood samples obtained from patients before infusion and at weeks 8, 12, 18, 24, and 32 after infusion. Tumor antigen expression in the bone marrow of patients during the same period was also evaluated by next-generation sequencing (NGS). Minimal residual disease levels (MRD percentage) were compared with various readouts of T cell activity and tumor antigen expression. The specificity of T cells for targeted antigens was assessed by IFNγ ELISpot (SFU / 2×10 5 cells) to determine.

[0176] Figures 14 to 23 The results of this study are provided in . Fig.14 The results showed that T cells specific for the targeted antigens (i.e., T cells specific for the targeted antigens WT1, survivin, NY-ESO-1, and PRAME used to generate MultiTAA-specific T cell products) were present in the peripheral blood of patients and negatively correlated with MRD, indicating anti-tumor activity. In addition, T cells specific for additional tumor antigens were present in the peripheral blood of patients and were also negatively correlated with MRD, where the additional tumor antigens were not present in the set of targeted antigens used to generate MultiTAA-specific T cell products ( Fig.15 ). These data demonstrate that epitope spreading occurs after infusion of MultiTAA-specific T cell products and further suggest that “off-target” immune responses contribute significantly to the anti-tumor response after infusion of the product. Fig.16The expression of T cells in the patient's bone marrow detected by next-generation sequencing (NGS) is shown. T cell infiltration of the bone marrow (indicating lymphocyte infiltration) is negatively correlated with disease (MRD) and occurs after the tumor burden increases.

[0177] Fig.17 The results showed that the T cell composition and phenotype in the patient's PBMCs were relatively stable over time. Functional studies showed that the production of target antigen-specific IFNγ was sustained in both CD4+ T cells and CD8+ T cells ( Fig.18 In addition, most antigen-specific T cells (i.e., IFNγ+) are Lag-3 negative, thus avoiding exhaustion of antigen-specific T cells in patients ( Fig.19 ).

[0178] Fig. 20 The results showed that after the infusion of the product, a large number of different T cell clones were expanded in the patient's body. The clonal expansion / reduction of T cells was observed using TCR monitoring.

[0179] Fig.21 The results showed that the expression of targeted tumor antigens survivin and WT1 in the bone marrow was directly associated with MRD. In addition, the decrease in survivin and WT1 expression was associated with an increase in targeted antigen-specific T cells detected in the peripheral blood. Fig. 22 The results showed that expression of non-targeted antigens such as proteinase-3 was detected in the bone marrow after product infusion and was associated with MRD at late time points, demonstrating epitope expansion. In addition, the increase in the proportion of proteinase-3-specific T cells was negatively correlated with proteinase-3 detected in the tumor. To understand whether additional non-targeted antigens were reduced, RNA expression of seven non-targeted antigens was detected in patients, and the antigen specificity of T cells to the same non-targeted antigens was tested. Fig.23 The results showed that after infusion of the product, additional non-targeted tumor antigens were significantly reduced and showed a consistent trend with MRD. Together, these data demonstrate epitope expansion of tumor antigen-specific T cells after administration.

[0180] Example 7. Epitope expansion after multi-tumor antigen-specific T cell therapy

[0181] Epitope spreading occurs when T cells target a specific antigen for destruction, exposing new "off-target" epitopes / antigens, which recruit and expand T cells specific for the off-target antigen to help eliminate the tumor, thereby limiting tumor immune escape. 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.

[0182] In a lymphoma clinical trial, MultiTAA-specific T cells were administered to patients and epitope expansion was observed. Specifically, patients were evaluated at 3 months (Mth3) and 9 months (Mth9) after infusion of MultiTAA-specific T cells generated using the MAT101-5 approach. The MAT101-5 approach is identical to the MAT101-4 approach, except that the MAT101-5 approach includes incubation with peptides from 5 tumor-associated antigens (survivin, NY-ESO-1, MAGEA4, SSX2, and PRAME). Nine months after the first administration, patients treated with MultiTAA-specific T cells had a reduced tumor burden ( Fig.25A ) and antigen specificity was tested by IFN-γ ELISpot analysis. At the 9-month time point, specificity for all tumor-associated targeted antigens was observed ( Fig.25B ) and increased specificity for the non-targeted antigen MAGEC1 ( Fig.25B ).

[0183] Six weeks after the initial infusion of MultiTAA-specific T cells, expansion of T cells specific for “targeted” epitopes (left) and “non-targeted” epitopes (right) was detected in responding and non-responding lymphoma patients, where the “targeted” epitopes were derived from peptides used to generate antigen-specific T cell products, while the “non-targeted” epitopes indicated epitope expansion ( Fig.26 Significantly greater expansion of T cells specific for both “targeted” and “non-targeted” epitopes was observed in responders compared with non-responders, suggesting that epitope expansion may be positively correlated with treatment response.

[0184] The preparation of TAA-specific cell products usually focuses on the generation of Fig.27A Surprisingly, the examples given herein show that the cell products ( Fig.27A , "final drug product", including MT-401, MT-601 or other embodiments) has specificity for additional non-targeted antigens. Once the targeted antigen is destroyed, additional epitopes become accessible and expand T cells specific for the "non-targeted" antigen in the final drug product. In addition, this "non-targeted" specificity indicates epitope expansion and can benefit the overall tumor response generated by the cell product.

[0185] To evaluate whether the "off-target" TAA response in the T cell product was associated with the inhibition of cancer cell growth, MT-601 was further evaluated in a pancreatic cancer cell model. Using an in vitro assay, target cells (i.e., pancreatic cancer cells) were co-cultured with MT-601 cells and nonspecific T cells (anti-CD3 / CD28 activated). When PANC1 cells were co-cultured with nonspecific T cells activated with anti-CD3+ / CD28+, no inhibition of cancer cell growth was observed. However, co-culture with MT-601 cells resulted in a decrease in cancer cell growth ( Fig.27B ). The specificity for the target antigen was detected by IFN-γ ELISpot analysis, and the specificity of MT-601 cells alone or after co-culture with PANC1 cells was observed, while anti-CD3+ / CD28+ activated nonspecific T cells co-cultured with PANC1 cells had no target antigen specificity ( Fig.27C ). Due to epitope spreading, specificity for non-targeted antigens was found in MT-601 cells co-cultured with PANC1 cells, but no specificity for non-targeted antigens was observed when co-cultured with anti-CD3+ / CD28+ activated T cells ( Fig.27D Together, these data suggest that T cells in the MT-601 cell product maintain specificity for the targeted antigen and additionally gain specificity for non-targeted antigens, indicating epitope spreading.

[0186] Using the MAT110-CD56+ approach, another MultiTAA-specific product, MT-401 ( Fig.28 The antitumor effect of MT-401 on cells from patients with acute myeloid leukemia was evaluated. The patient had previously been treated with decitabine, venetoclax, and cytarabine, and then fludarabine and cyclophosphamide to deplete lymphocytes before HSCT, but relapsed before MT-401 infusion. Bone marrow (BM) mononuclear cells were isolated from the patient at relapse and cocultured with control PBMCs or MT-401 cells at an effector:target (E:T) ratio of 4:1 ( Fig.29 The cells were cultured together for 4 days, and glucose and lactate production was measured daily to monitor cell growth. A decrease in glucose production was observed in cells cultured with MT-401, indicating a decrease in the growth of the target cells ( Fig. 30A Increased lactate production was observed in cells cultured with MT-401 and correlated with T cell proliferation ( Fig. 30B ). Cancer cell growth was measured at the end of co-culture, and target cancer cells cultured with MT-401 showed reduced cell growth ( Fig.31A and 31B), indicating improved target cell killing. The antigen specificity of MT-401 cells was compared before and after co-culture, and the specificity of PRAME and WT1 increased after cancer cell culture ( Fig.32 Not only was the antigen specificity generated by MT-401 for the four targeted antigens maintained on T cells ( Fig.33A ), and the specificity of MT-401 cells for non-targeted antigens was observed compared with PBMCs that did not have specificity for non-targeted antigens ( Fig.33B ).

[0187] Example 8. Donor selection and scale-up of preparation

[0188] Donors are screened for HLA diversity and selected based on their ability to match a broad range of HLA genotypes in a given sample population. After selection, cells are collected by leukapheresis and prepared using the methods described herein. Antigen-specific T cells are then frozen prior to delivery and administration to the patient ( Fig.34 ). A finished inventory of MultiTAA T cell products can be generated from just 10 donors, matching 4 / 8 HLA alleles in more than 90% of patients. In addition, a finished inventory of MultiTAA T cell products can be generated from just 5 donors, matching 2 / 8 HLA alleles in almost 100% of patients ( Fig.35 ).

[0189] To evaluate whether the MAT110-depleted CD56+ approach could be scaled up, cells generated in G-Rex10M and G-Rex 500M were compared. Fig.36 It was shown that the scale-up from the G-Rex 10M to the G-Rex 500M device did not change the cell viability or T cell expansion fold of the final product. Therefore, the MAT110-depleted CD56+ method provided herein is suitable for scale-up production methods.

[0190] Example 9. Additional antigens increase T cell specificity and diversity

[0191] Another study was performed to evaluate the effect of adding peptide libraries of additional tumor antigens to the culture mixture. Three donor monocyte populations were stimulated with peptide libraries from four tumor antigens or from six tumor antigens according to the MAT110-depleted CD56+ method provided herein. Monocytes from a fourth donor were stimulated with peptide libraries from 12 tumor antigens. Fig.37As shown in Figure 2, surprisingly, the MultiTAA-specific T cell population generated using peptides from 12 tumor antigens showed increased T cell diversity and specificity, with the total response intensity (the sum of the responses to each targeted antigen) exceeding 4500 SFU / 2×10 5 cells.

Claims

1. A method for generating tumor antigen-specific T cells, comprising: (i) placing a population of monocytes obtained from a subject in a container comprising at least one gas permeable surface, wherein the monocytes are present at a density of 1 / cm 2 The air permeable surface is about 0.1×10 6 cells per cm 2 Breathable surface approximately 9×10 6 The seeding density of cells is added to the container to form a cell culture; (ii) adding one or more peptides from one or more tumor antigens to the cell culture; (iii) incubating one or more than one peptide with monocytes in a container at about 37°C in the absence of exogenous cytokines.

2. The method according to claim 1, further comprising: (iv) adding one or more than one exogenous cytokine to the cell culture after the incubation in step (iii).

3. The method of claim 1 or 2, wherein the exogenous cytokines are added after incubation for about 30 minutes to about 30 hours.

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

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

6. The method according to 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. The method of any one of claims 1 to 6, wherein the one or more than one peptides are incubated with the monocytes in a container at about 37°C for at least about 8 hours prior to the addition of exogenous cytokines.

8. The method of claim 7, wherein the monocytes are incubated with the one or more peptides in a container at about 37°C for about 8 hours to about 30 hours prior to the addition of exogenous cytokines.

9. The method according to any one of claims 1 to 8, wherein monocytes are treated with one or more than one peptide at a rate of 1:

1. 2 The air permeable surface is about 0.1×10 6 cells per cm 2 Breathable surface approximately 9×10 6 The cells were incubated at a concentration of 100 cells.

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. 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 one or more tumor antigens.

15. The method according to any one of claims 1 to 14, wherein each library of peptides comprises peptides covering the sequence of a tumor antigen.

16. The method according to any one of claims 1 to 15, comprising adding peptides from at least 4 different tumor antigens in step (ii).

17. The method according to 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 method according to any one of claims 1 to 15, wherein the tumor antigen is selected from CEA, MHC, CTLA-4, gp100, mesothelin, PRAME (OIP3) PD-L1, TRP1, CD40, EGFP, Her2, TCRα, 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, gp100, 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, 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, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, mucin-1, BRAF, CCNA1, and Fos-related antigen 1.

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

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

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

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

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

24. The method of any one of claims 1 to 18, wherein one or more than one tumor antigen is selected from PRAME, NY-ESO-1, Survivin, WT1, MAGEA4 and SSX2.

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

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

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

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

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

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

31. The method of any one of claims 1 to 30, wherein the method further comprises removing CD56+ cells from the monocyte population prior to (i).

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

33. The method of claim 32, wherein CD56+ cells are removed by magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), Dynabeads, EasySep TM Cell separation magnetic beads, RosetteSep TM Immunodensity cell isolation or Cloudz human cell expansion platform.

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

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

36. The method of any one of claims 31 to 35, wherein the method further comprises removing CD19+ cells from the monocyte population prior to (i).

37. The method of claim 36, wherein CD19+ cells are depleted from the monocyte population.

38. The method of claim 37, wherein CD19+ cells are removed by magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), Dynabeads, EasySep TM Cell separation magnetic beads, RosetteSep TM Immunodensity cell isolation or Cloudz human cell expansion platform.

39. The method of any one of claims 31 to 38, wherein the mononuclear cell population placed in the container of (i) comprises: (a) about 0.01% to about 2% CD56+ cells, (b) from 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% of T cells.

40. The method of any one of claims 1 to 39, wherein the population of monocytes disposed in the container comprises no more than about 2.5% CD56+ cells.

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

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

43. The method of any one of claims 1 to 43, wherein the monocytes are incubated with the one or more peptides in a container at about 37°C for about 8 hours to about 30 hours in the absence of exogenous cytokines, and wherein the monocytes, the one or more peptides and the one or more exogenous cytokines are then co-cultured in a container at about 37°C for about 5 days to about 14 days.

44. The method according to any one of claims 1 to 43, wherein the method does not comprise a washing step.

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

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

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

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

49. The method according to claim 47 or 48, further comprising counting the cells remaining after standing for a period of time before step (i).

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

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

52. The method of claim 51, wherein the cells are CS10 or its derivatives.

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

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 been further purified or enriched based on antigen specificity.

55. An antigen-specific T cell population obtained by the method according to any one of claims 1 to 54.

56. An antigen-specific T cell population comprising about 80% to about 99% CD3+ T cells, <1% CD14+ monocytes, <10% CD19+ B cells and <10% CD3-CD56+ NK cells, wherein the antigen-specific T cell population is active against one or more peptides of one or more antigens that have not been previously incubated with the T cell population.

57. A group according to claim 56, wherein The intensity of the specificity of at least one peptide for at least one antigen that has not been previously incubated with the T cell population is at least about 100 SFU / 2×10 5 cells.

58. A group according to claim 57, wherein The specificity for at least one antigen not previously exposed to or incubated with the population is at least about 30 SFU / 2×10 5 cells.

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

60. A population according to any one of claims 55 to 59, wherein The specificity of antigen-specific T cells for one or more tumor antigens is at least about 500 SFU / 2×10 5 cells.

61. A population according to any one of claims 55 to 60, wherein the antigen-specific T cells comprise at least about 20% of central memory T cells (T CM ).

62. A population according to any one of claims 55 to 61, wherein the antigen-specific T cells comprise about 40% effector memory T cells (T EM ).

63. A population according to any one of claims 55 to 62, wherein the antigen-specific T cells comprise no more than 10% of terminally differentiated effector memory T cells (T cells) expressing CD45RA. EMRA ).

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

65. A population according to any one of claims 55 to 64, wherein the antigen-specific T cells are active against at least 75% of the tumor antigens represented by one or more than one peptide.

66. A population according to any one of claims 54 to 65, wherein antigen-specific T cells are active against each tumor antigen represented by one or more than one peptide.

67. A population according to claim 65 or 66, wherein activity against the antigen is detected by production of IFNy.

68. A population according to claim 67, wherein the production of IFNγ is determined by an IFNγ ELISpot assay.

69. A population according to any one of claims 55 to 68, wherein an increase in the number of tumor antigens represented by one or more than one peptide results in (a) an increase in the specificity of antigen-specific T cells and / or (b) an increase in the specificity of antigen-specific T cells for tumor antigens.

70. A population according to any one of claims 55 to 69, wherein T cells in the population exhibit activity against one or more peptides of one or more antigens that have not been previously incubated with the population.

71. A population according to claim 70, wherein the activity against one or more peptides of one or more antigens not previously incubated with the population is the result of epitope spreading.

72. A group according to claim 71, wherein The accumulation of specificity for one or more peptides to one or more antigens not previously incubated with the population is at least about 100 SFU / 2×10 5 cells.

73. A group according to claim 72, wherein: The specificity for at least one antigen that has not been previously incubated with the population is at least about 30 SFU / 2×10 5 cells.

74. A population according to any one of claims 55 to 73, wherein the tumor antigen-specific T cells comprise polyclonal antigen-specific T cells that have not been further purified or enriched based on antigen specificity.

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

76. The composition of claim 75, further comprising a pharmaceutically acceptable buffer or excipient.

77. A composition comprising a population of tumor antigen-specific T cells comprising CD4+ T cells and CD8+ T cells expanded ex vivo and comprising less than about 10% of a CD56+ NK cell component, wherein: The specificity for tumor antigens was at least about 500 SFU / 2×10 5 cells.

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

79. The composition of claim 77 or 78, wherein both the CD4+ T cell compartment and the CD8+ T cell compartment are specific for a 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 composition of any one of claims 75 to 81, wherein the tumor antigen-specific T cells comprise at least about 20% of central memory T cells (T CM ).

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 composition of any one of claims 75 to 83, wherein the tumor antigen-specific T cells comprise about 40% of effector memory T cells (T EM ).

85. The composition of any one of claims 75 to 84, wherein the tumor antigen-specific T cells comprise no more than 10% of terminally differentiated effector memory cells (T cells) expressing CD45RA. EMRA ).

86. A method for generating tumor antigen-specific T cells, comprising: (i) The white blood cell population was divided into 0.1×10 6 cells / cm 2 Up to 9×10 6 cells / cm 2 The invention relates to a method for preparing a cell culture device having at least one gas permeable surface and wherein the cell culture device comprises one or more peptides of one or more tumor-associated antigens, wherein 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 wherein the leukocytes comprise about 1% to about 50% monocytes and about 40% to about 99% T cells; (ii) incubating the cells and peptide of step (i) at about 37° C. and 5% CO 2 for about 8 hours to about 30 hours; (iii) adding one or more than one exogenous cytokine after incubation for about 8 hours to about 30 hours; and (iv) culturing the cells, peptide, and exogenous cytokine for about 5 days to about 14 days, wherein the culture is not disturbed after the addition of the exogenous cytokine.

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

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

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

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 been further purified or enriched based on antigen specificity.

91. A T cell product produced by the method of any one of claims 86 to 90, wherein the product comprises: (i) about 80% to about 99% CD3+ T cells, <1% CD14+ monocytes, <10% CD19+ B cells, and <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; and (iii) a CD3+ T compartment comprising at least about 20% of central memory T cells (T CM ), at least about 20% of naive T cells, about 40% of effector memory T cells (T EM ) and no more than 10% of terminally differentiated effector memory cells (T EMRA ).

92. The T cell product of claim 91, wherein the product comprises cells capable of producing IFN-γ as determined by an ELISpot assay.

93. The T cell product according to claim 91 or 92, wherein The specificity of the harvested tumor antigen-specific T cells for tumor antigens is at least about 500 SFU / 2×10 5 cells.

94. The T cell product according to any one of claims 91 to 93, wherein As detected by IFN-γ ELISpot, the specificity for an antigen not represented by one or more than one peptide has a cumulative strength of at least about 100 SFU / 2×10 5 cells.

95. A group according to claim 94, wherein The specificity for at least one antigen not previously exposed to or incubated with the population is at least about 30 SFU / 2×10 5 cells.

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

97. A method for treating a patient, comprising administering to the patient an antigen-specific T cell population 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. The method of claim 97, wherein the patient has cancer.

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

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

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

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 to the subject.

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

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

107. The method of any one of claims 86 to 90 or 97 to 106, in the context of allogeneic therapy or autologous therapy, further comprising administering to the patient one or more additional antigen-specific T cell populations, wherein the one or more additional antigen-specific T cell populations comprise a different T cell product than a previously administered antigen-specific T cell population, and wherein the method comprises treating the patient with the one or more additional antigen-specific T cell populations after at least about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks of treatment with the previously administered cell population.

108. The method of any one of claims 86 to 90 or 97 to 107, in an autologous context, further comprising administering to the patient a second population of antigen-specific T cells, wherein the second population of antigen-specific T cells is obtained by a method according to any one of claims 1 to 54 or 86 to 90, and wherein the method comprises treating the patient with the 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. The method of any one of claims 97 to 108, further comprising monitoring the patient's disease progression by detecting RNA expression of one or more than one tumor antigen.

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

111. The method of any one of claims 97 to 110, further comprising detecting RNA expression of one or more additional tumor antigens, the one or more additional tumor antigens being other than the tumor antigen corresponding to the one or more peptides.

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

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

114. A composition comprising monocytes and one or more peptides derived from one or more tumor antigens, wherein each of the one or more peptides in the composition is present at a concentration of about 0.1 μg / mL to about 0.5 μg / mL, and wherein the monocyte population comprises about 40% to about 99% T cells and no more than about 2% NK cells.

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

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

117. A composition according to any one of claims 114 to 116, wherein the monocyte population has been depleted of NK cells.

118. according to the composition described in any one of claims 114 to 117, wherein monocytes are present at a rate of 1.5 to 2.0 cells per cm 2 The air permeable surface is about 0.1×10 6 cells per cm 2 Breathable surface approximately 9×10 6 The method comprises the step of: providing a seeding density of 10 cells per well in a container comprising at least one gas permeable surface.

119. A composition according to any one of claims 114 to 118, comprising one or more than one peptide from at least 4 different tumor antigens.

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 than 30 different tumor antigens.

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

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

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