T cell manufacturing compositions and methods

Abstract

The generation of antigen-specific T cells by controlled ex vivo induction or expansion can provide highly specific and beneficial T cell therapy. The present disclosure provides T cell manufacturing methods and therapeutic T cell compositions, as well as personalized antigen-specific T cell therapy, that can be used to treat subjects with cancer and other conditions, diseases and disorders. Tumor vaccines are typically composed of tumor antigens and immune stimulatory molecules (e.g., adjuvants, cytokines, or TLR ligands) that work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 256,427, filed October 15, 2021, and U.S. Provisional Patent Application No. 63 / 327,286, filed April 4, 2022, both of which are incorporated by reference herein in their entireties. [Background technology]

[0002] background Tumor vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., adjuvants, cytokines, or TLR ligands) that work in concert to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. Such vaccines contain either shared tissue-restricted tumor antigens or a mixture of shared and patient-specific antigens in the form of whole tumor cell preparations. Shared tissue-restricted tumor antigens are ideally immunogenic proteins selectively expressed in tumors across many individuals and are generally delivered to patients as synthetic peptides or recombinant proteins. In contrast, whole tumor cell preparations are delivered to patients as autologous irradiated cells, cell lysates, cell fusions, heat shock protein preparations, or total mRNA. Because whole tumor cells are isolated from autologous patients, the cells can contain both shared and patient-specific tumor antigens. Finally, there is a third class of tumor antigens, neoantigens, which consist of proteins with tumor-specific mutations (which may be patient-specific or shared) that result in altered amino acid sequences and have rarely been used in vaccines. Such mutated proteins (a) are unique to tumor cells (because the mutation and its corresponding protein are present only in tumors); (b) circumvent central tolerance and are therefore more likely to be immunogenic; and (c) provide excellent targets for immune recognition, including by both humoral and cellular immunity.

[0003] Adoptive immunotherapy, or adoptive cell therapy (ACT), is the transfer of lymphocytes into a subject for disease treatment. Adoptive immunotherapy has yet to realize its potential for treating a wide variety of diseases, including cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immune deficiencies. However, most, if not all, adoptive immunotherapy strategies require T cell activation and expansion steps to generate clinically effective, therapeutic doses of T cells. Due to the inherent complexities of live cell culture and patient-to-patient variability, current technologies for generating therapeutic doses of T cells, including engineered T cells, are still limited by cumbersome T cell manufacturing processes. Existing T cell manufacturing processes are not easily scalable, repeatable, reliable, or efficient, and often produce inferior T cell products that may be prone to exhaustion and loss of effector immune cell function. To date, engineered T cell adoptive immunotherapy has met with only limited success and routinely exhibits variable clinical activity. Therefore, such treatments are not suitable for widespread clinical use. Thus, there remains a need to develop compositions and methods for the expansion and induction of antigen-specific T cells with favorable phenotype and function. Summary of the Invention [Means for solving the problem]

[0004] overview While autologous T cell therapy is safe to use, some dramatic improvements are needed to meet the standard of care, and development in this field has been rapid and challenging.

[0003] An ex vivo method for preparing tumor antigen-specific T cells includes: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells; and (b) incubating the first population of APCs and T cells of step (a) for a first period of time in the presence of (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) a polynucleotide encoding (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polypeptide. (b) incubating the human subject with stimulated T cells, thereby forming a population of cells comprising stimulated T cells; and (c) expanding the population of cells comprising stimulated T cells, thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii), and at least 30% of the expanded population of cells comprising tumor antigen-specific T cells are effector memory T cells, and the human subject has ovarian cancer.

[0005] 1. A method of treating ovarian cancer in a human subject in need thereof, comprising: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells; and (b) treating the first population of APCs and T cells of step (a) for a first period of time with (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of (A) the human subject having cancer or (B). Also provided herein are methods comprising (a) incubating a human subject with a polynucleotide encoding the polypeptide, thereby forming a population of cells comprising stimulated T cells; (b) expanding the population of cells comprising stimulated T cells, thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii); and (d) administering the expanded population of cells of (c) to the human subject.

[0006] 1. An ex vivo method for preparing tumor antigen-specific T cells, comprising: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells; and (b) incubating the first population of APCs and T cells of step (a) for a first period of time in the presence of (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L) and (ii) mRNA encoding a polypeptide comprising at least two tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer, thereby producing stimulated tumor antigen-specific T cells. and (c) expanding the population of cells comprising stimulated T cells, thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) a tumor antigen epitope sequence of at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii), wherein at least 30% of the expanded population of cells comprising tumor antigen-specific T cells are effector memory T cells, and wherein the human subject has ovarian cancer.

[0007] 1. A method of treating ovarian cancer in a human subject in need thereof, comprising: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells; and (b) treating the first population of APCs and T cells of step (a) for a first period of time with a polypeptide encoding a polypeptide comprising (i) an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) and (ii) at least two tumor antigen epitope sequences expressed by cancer cells of the human subject with cancer. Also provided herein are methods comprising (a) incubating a human subject with an mRNA encoding a tumor antigen-specific T cell in the presence of an mRNA encoding a tumor antigen-specific T cell, thereby forming a population of cells comprising stimulated T cells; (b) expanding the population of cells comprising stimulated T cells, thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) a tumor antigen epitope sequence of at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii); and (d) administering the expanded population of cells of (c) to the human subject.

[0008] In some embodiments, the method further comprises administering the expanded population of cells of (c) to a human subject.

[0009] In some embodiments, steps (b) and (c) are carried out in less than 28 days.

[0010] In some embodiments, at least 30% of the expanded population of cells comprising tumor antigen-specific T cells are effector memory T cells.

[0011] In some embodiments, the percentage of IFNγ+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 15% of the tumor antigen-specific T cell population.

[0012] In some embodiments, the percentage of TNFα+ and IFNγ+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 2% of the tumor antigen-specific T cell population.

[0013] In some embodiments, the percentage of TNFα+ and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cell population.

[0014] In some embodiments, the percentage of IFNγ+ and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population.

[0015] In some embodiments, the percentage of TNFα+ and IFNγ+ and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 0.1% of the tumor antigen-specific T cell population.

[0016] In some embodiments, the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 15%.

[0017] In some embodiments, the percentage of CD4+ T cells in the expanded population of cells, including tumor antigen-specific T cells, that are effector memory T cells (CD62L- and CD45RA-) is at least 60%.

[0018] 1. A method of treating cancer in a subject in need thereof, comprising: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells; and (b) incubating the first population of APCs and T cells of step (a) for a first period of time in the presence of (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide. (c) expanding the stimulated T cells of step (b), thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence of step (b)(ii) and (ii) an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii); and (d) administering the expanded population of cells of (c) to the human subject, wherein the expanded population of cells of step (c) comprises 0.75 x 10^8 to 1.25 x 10^9 total cells.

[0019] 1. A method of treating cancer in a subject in need thereof, comprising: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells; and (b) incubating the first population of APCs and T cells of step (a) for a first period of time in the presence of (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide. (c) expanding the stimulated T cells of step (b), thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence of step (b)(ii) and (ii) an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii); and (d) administering the expanded population of cells of(c) to the human subject, wherein the expanded population of cells of step (c) comprises 1.5 x 10^9 to 1.25 x 10^10 total cells.

[0020] 1. A method of treating cancer in a subject in need thereof, comprising: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells; and (b) treating the first population of APCs and T cells of step (a) for a first period of time with a polynucleotide encoding either (i) an FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer or (B) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of the human subject with cancer. (c) expanding the stimulated T cells of step (b), thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells that are specific for a complex comprising (i) at least one tumor antigen epitope sequence of step (b)(ii) and (ii) an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii); (d) administering the expanded population of cells of (c) to the human subject; and (e) administering an immune checkpoint inhibitor to the subject.

[0021] In some embodiments, the expanded population of cells in step (c) comprises between 0.75 x 10^8 and 1.25 x 10^10 total cells.

[0022] In some embodiments, the expanded population of cells administered in step (c) comprises 0.75x10^8 to 1x10^9 total cells, 0.75x10^8 to 0.75x10^9 total cells, 1x10^8 to 1.25x10^9 total cells, 1x10^8 to 1x10^9 total cells, 1x10^8 to 0.75x10^9 total cells, 1.25x10^8 to 1.25x10^9 total cells, 1.25x10^8 to 1x10^9 total cells, or 1.25x10^8 to 0.75x10^9 total cells.

[0023] In some embodiments, the expanded population of cells administered in step (c) comprises 1.5x10^9 to 1x10^10 total cells, 1.5x10^9 to 0.75x10^10 total cells, 2x10^9 to 1.25x10^10 total cells, 2x10^9 to 1x10^10 total cells, 2x10^9 to 0.75x10^10 total cells, 2.5x10^9 to 1.25x10^10 total cells, 2.5x10^9 to 1x10^10 total cells, or 2.5x10^9 to 0.75x10^10 total cells.

[0024] In some embodiments, the immune checkpoint inhibitor comprises an anti-PD1 agent.

[0025] In some embodiments, the immune checkpoint inhibitor comprises an anti-PD1 antibody.

[0026] In some embodiments, the immune checkpoint inhibitor comprises pembrolizumab or nivolumab.

[0027] In some embodiments, the immune checkpoint inhibitor is administered after the expanded population of cells of step (c) is administered.

[0028] In some embodiments, the immune checkpoint inhibitor is administered at a dose of 200-400 mg, 2 mg / kg-4 mg / kg, 200 mg, 2 mg / kg, 400 mg, or 4 mg / kg.

[0029] In some embodiments, the immune checkpoint inhibitor is administered Q3W or Q6W.

[0030] In some embodiments, the immune checkpoint inhibitor is administered Q6W.

[0031] In some embodiments, the immune checkpoint inhibitor is administered Q6W for up to 36 or 52 weeks after the expanded population of cells of step (c) is administered.

[0032] In some embodiments, the immune checkpoint inhibitor is not administered 36 or 52 weeks after the expanded population of cells of step (c) is administered.

[0033] In some embodiments, the immune checkpoint inhibitor further comprises an anti-CTLA4 agent.

[0034] In some embodiments, the anti-CTLA4 agent is an anti-CTLA4 antibody.

[0035] In some embodiments, the anti-CTLA4 antibody comprises ipilimumab.

[0036] In some embodiments, the human subject (i) has unresectable melanoma, (ii) has previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor and has disease progression, or (iii) has received or is currently receiving a PD-1 inhibitor or a PD-L1 inhibitor for at least 3 months and has stable disease or asymptomatic progressive disease.

[0037] In some embodiments, the cancer is melanoma.

[0038] In some embodiments, the cancer is ovarian cancer.

[0039] In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0040] In some embodiments, the polynucleotide encoding the polypeptide is mRNA.

[0041] In some embodiments, the polypeptide encoded by the mRNA comprises at least two tumor antigen epitope sequences.

[0042] In some embodiments, step (b) comprises incubating the APCs and the first population of T cells of step (a) in the presence of IL-21 for a first period of time.

[0043] In some embodiments, step (c) comprises expanding the population of cells comprising the stimulated T cells in the presence of IL-21.

[0044] In some embodiments, the population of immune cells is derived from a biological sample from a human subject of (b)(ii).

[0045] A composition comprising an expanded population of cells, including tumor antigen-specific T cells, produced according to the methods disclosed herein.

[0046] Also provided herein are compositions for use in treating ovarian cancer, the compositions comprising an expanded population of cells, including tumor antigen-specific T cells, produced according to the methods disclosed herein.

[0047] Also provided herein is a composition comprising an expanded population of cells comprising tumor antigen-specific T cells, wherein the expanded population of cells is derived from a population of immune cells comprising APCs and a first population of T cells that have been depleted of CD14+ and CD25+ cells and incubated for a first period of time in the presence of (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with ovarian cancer or (B) a polynucleotide encoding the polypeptide, wherein the tumor antigen-specific T cells comprise T cells that are specific for a complex comprising (i) the at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by cancer cells or APCs of the human subject with ovarian cancer.

[0048] In some embodiments, at least 30% of the expanded population of cells comprising tumor antigen-specific T cells are effector memory T cells.

[0049] In some embodiments, the polynucleotide encoding the polypeptide is mRNA.

[0050] In some embodiments, the polypeptide encoded by the mRNA comprises at least two tumor antigen epitope sequences.

[0051] In some embodiments, the expanded population of cells comprises between 0.75 x 10^8 and 1.25 x 10^10 total cells.

[0052] In some embodiments, the expanded population of cells comprises between 0.75x10^8 and 1.25x10^9 total cells or between 1.5x10^9 and 1.25x10^10 total cells.

[0053] In some embodiments, the percentage of IFNγ+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 15% of the tumor antigen-specific T cell population; the percentage of TNFα+ and IFNγ+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 2% of the tumor antigen-specific T cell population; the percentage of TNFα+ and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cell population; the percentage of IFNγ+ and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cell population. the percentage of TNFα+, IFNγ+ and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 0.1% of the tumor antigen-specific T cell population; the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 15%; and / or the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-) is at least 60%.

[0054] In some embodiments, the expanded population of cells comprising tumor antigen-specific T cells is (a) derived from a population of immune cells comprising a first population of APCs and T cells that has been depleted of CD14+ and CD25+ cells and incubated in the presence of IL-21 for a first period of time, and / or (b) expanded in the presence of IL-21. [Brief explanation of the drawings]

[0055] [Figure 1-1] FIG. 1A shows an exemplary schematic diagram of an antigen-specific T cell manufacturing protocol.

[0056] [Figure 1-2] FIG. 1B shows an exemplary schematic diagram of an antigen-specific T cell manufacturing protocol.

[0057] FIG. 1C shows an exemplary alternative schematic diagram of an antigen-specific T cell manufacturing protocol.

[0058] [Figure 2] Figure 2 shows exemplary results showing the percentage of antigen-specific CD8+ memory T cells induced by long or short peptides. "Bulk" indicates that the sample containing T cells used for induction was whole peripheral blood mononuclear cells (PBMCs). "Treg-" indicates that the sample containing T cells used for induction was PBMCs depleted of CD25-expressing cells.

[0059] [Figure 3] FIG. 3 shows an exemplary flow cytometry analysis showing the percentage of antigen-specific CD8+ naive T cells induced with GAS7 peptide.

[0060] [Figure 4]Figure 4 shows exemplary results demonstrating antigen-specific CD8+ T cell responses to a peptide pool of HIV short peptides, short previously identified neoantigens (PINs), or long PINs. "Whole PBMC" indicates that the sample containing the T cells used for induction was whole PBMCs. "CD25-PBMC" indicates that the sample containing the T cells used for induction was depleted of CD25+ cells. Short, short peptide, or shortmer; long, long peptide, or longmer.

[0061] [Figure 5] Figure 5A shows an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to a single previously identified neoantigen (PIN) under the indicated conditions.

[0062] Figure 5B shows antigen-specific CD8 against a single previously identified neoantigen (PIN) under the indicated conditions. + 1 shows an exemplary flow cytometry analysis of naive T cell responses.

[0063] [Figure 6] FIG. 6 shows exemplary results demonstrating antigen-specific CD8+ T cell responses to the indicated peptides using PBMC samples from two human donors.

[0064] [Figure 7] FIG. 7 shows exemplary flow cytometry plots of antigen-specific CD8+ T cell responses to the indicated mutated epitopes in healthy donors before and after up to three rounds of stimulation.

[0065] [Figure 8A] Figure 8A shows an exemplary bar graph depicting the results of antigen-specific memory CD8+ T cell responses to viral antigens. After up to three rounds of stimulation, approximately 50% of all CD8+ T cells were specific for the indicated viral epitopes (CMV pp65, EBV YVL, EBV BMLF1, and Mart-1).

[0066] [Figure 8B] Figure 8B shows exemplary results of a recall assay of antigen-specific memory CD8+ T cell responses to peptide-loaded antigen-presenting cells, followed by incubation with viral antigen-loaded and unloaded APCs. The percentages of CD8+ T cells from the two time points releasing the indicated cytokines are shown in the chart.

[0067] [Figure 9] Figure 9 shows exemplary results of a cytotoxicity assay used to assess whether induced T cell cultures can kill antigen-expressing tumor lines. The percentage of live and dead caspase-3-positive tumor cells relative to total tumor cells is shown. Live caspase-3-positive tumor cells indicate cells undergoing early cell death.

[0068] [Figure 10] Figure 10 shows an exemplary flow cytometry analysis of antigen-specific CD4+ T cell responses to peptide-loaded antigen-presenting cells, followed by incubation with PIN-loaded and unloaded APCs. The percentage of CD4+ T cells releasing IFNγ is shown.

[0069] [Figure 11] FIG. 11 shows exemplary results of the percentage of antigen-specific CD4+ T cells releasing IFNγ after restimulation with mutant or wild-type peptides.

[0070] [Figure 12] Figure 12 shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to short HIV5 peptides. Both short-term and long-term induction is shown.

[0071] [Figure 13]FIG. 13 shows an exemplary flow cytometry analysis showing the percentage of antigen-specific CD8+ naive T cell responses to short ME1 peptides using whole PBMC samples from human donors.

[0072] [Figure 14] FIG. 14 shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to short HIV3 peptides using whole PBMC samples from human donors.

[0073] [Figure 15] FIG. 15 shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to long CSNK1A1 peptides using whole PBMC samples from human donors.

[0074] [Figure 16] FIG. 16 shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to long CSNK1A1 peptides using a PBMC sample from a human donor that was depleted of CD25+ cells.

[0075] [Figure 17] FIG. 17 shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to short GAS7 peptides using a PBMC sample from a human donor that was depleted of CD25+ cells.

[0076] [Figure 18] FIG. 18 shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to short ACTN4 peptides using a PBMC sample from a human donor that was depleted of CD25+ cells.

[0077] [Figure 19]Figure 19A shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to short ACTN4 peptides using a PBMC sample from a human donor that was depleted of CD25+ cells. Short-term induction is shown.

[0078] Figure 19B shows CD25 + Antigen-specific CD8 expression against short HIV3 peptides using PBMC samples from cell-depleted human donors + 1 shows an exemplary flow cytometry analysis showing naive T cell responses. Long-term induction is shown.

[0079] [Figure 20] Figure 20 shows an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to short HIV5 peptides using whole PBMC samples from human donors. Both short-term and long-term induction is shown.

[0080] [Figure 21] 21 shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to short HIV-3 peptides using a whole PBMC sample from a human donor. Short-term induction is shown.

[0081] [Figure 22] Figure 22 shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to short PRDX5 peptides using a PBMC sample from a human donor that had been depleted of CD25+ cells. Both very short-term and long-term induction is shown.

[0082] [Figure 23] Figure 23 shows an exemplary flow cytometry analysis demonstrating antigen-specific CD8+ naive T cell responses to short HIV5 peptides using a PBMC sample from a human donor depleted of CD25+ cell tides. Both short-term and long-term induction is shown.

[0083] [Figure 24] FIG. 24 shows a schematic diagram of an example method for generating therapeutic T cell compositions, including expansion of memory T cells and induction of naive T cells.

[0084] [Figure 25] FIG. 25 shows an exemplary method for testing T cell functionality, phenotype and / or function and / or T cell responses.

[0085] [Figure 26] FIG. 26 shows an example of a recall assay for testing T cell functionality, phenotype and / or function and / or T cell responses.

[0086] [Figure 27A] Figure 27A shows an exemplary flow cytometry analysis demonstrating the ability to deconvolute multiplexed samples with labeled samples acquired separately or as a mixture in a recall assay. Uniquely labeled samples were resolved with minimal to no cross-contamination to other barcodes.

[0087] [Figure 27B] FIG. 27B shows an exemplary flow cytometry analysis demonstrating the detection of antigen-specific CD8+ T cells by multimer staining of a mixture of nine uniquely labeled samples in a recall assay.

[0088] [Figure 28A] FIG. 28A shows an exemplary flow cytometry analysis of a recall assay using six uniquely barcoded samples recalled with unloaded DCs and neoantigen-loaded DCs.

[0089] [Figure 28B]Figure 28B shows an exemplary bar graph of the percentage of CD4+ T cells with functional numbers incubated with DCs loaded with the indicated concentrations of peptide in a recall response assay. Samples of two induced cultures containing de novo CD4+ T cell responses were analyzed either alone without barcoding or mixed with an unrelated sample. Barcoding did not alter the detectable functionality. The functional numbers and magnitude of the responses elicited from the cells did not change significantly with sample barcoding.

[0090] [Figure 29] Figure 29A shows an exemplary bar graph depicting the results of antigen-specific memory CD8+ T cell responses to viral antigens. CD8+ memory responses to CMV pp65, MART-1, and EBV BRLF1 and BMLF1 epitopes can be increased from 0.23% of CD8+ T cells in healthy donor starting material to over 60%.

[0091] Figure 29B shows antigen-specific memory CD8 cells against viral antigens and then recalled with viral antigen-loaded and non-loaded DCs. + Exemplary results of a recall assay of T cell responses are shown. CD8 T cells from two time points releasing the indicated cytokines. + The percentage of T cells is shown in the chart.

[0092] [Figure 30-1] Figure 30A shows exemplary results of hit identification by detection and functional characterization of de novo induced CD4+ responses with multiple specificities in the same culture. In the example shown, induction was performed in four replicate cultures targeting 10 HIV-derived epitopes that are naive targets in HIV-negative healthy donors. Antigen-specific responses were detected in 4 / 4 biological replicates, with responses of varying magnitude.

[0093] Figure 30B shows de novo induced CD4 with multiple specificities in the same culture.+ Exemplary results of pooled deconvolution with response detection and functional characterization are shown. Multiple responses were detected in each replicate tested, with the same two epitopes (HIV#5 and HIV#7) producing the highest magnitude responses in each case.

[0094] [Figure 30-2] Figure 30C shows exemplary results of sensitivity determination by detection and functional characterization of de novo induced CD4+ responses with multiple specificities in the same culture. Similar magnitudes were observed for each response in the pooled deconvolution assay. Responses to HIV#5, HIV#6, and HIV#4 demonstrated EC50s of 0.45 μM, 0.43 μM, and 9.1 μM, respectively.

[0095] [Figure 31] FIG. 31 shows an exemplary schematic diagram of an antigen-specific T cell manufacturing protocol.

[0096] [Figure 32] FIG. 32 shows an exemplary schematic of a T cell induction protocol.

[0097] [Figure 33] FIG. 33 shows an exemplary schematic diagram of a dendritic cell generation protocol.

[0098] [Figure 34]Figure 34 shows exemplary pMHC multimer plots showing CD8+ T cell responses induced in leukapheresis material from a melanoma patient targeting patient-specific epitopes: SRSF1E>K, ARAP1Y>H, and PKDREJG>R, and in leukapheresis material from a melanoma patient targeting patient-specific epitopes (AASDH neoORF and seven model neoantigens: ACTN4K>N, CSNK1A1S>L, DHX40neoORF, GLI3P>L QARSR>W, FAM178BP>L, and RPS26P>L). The first panel plot in the first and second columns shows memory responses, and the remaining plots show de novo responses.

[0099] [Figure 35] Figure 35 shows exemplary data (left panel) of pMHC multimer plots of SRSF1E>K and ARAP1Y>H before and after peptide stimulation, and the pie charts show the functionality of neoantigen-specific T cells upon rechallenge with neoantigen-loaded DCs; gated on pMHC multimer+CD8+ or CD4+ T cells. The polyfunctional profile of CD8+ memory, CD8+ de novo, and CD4+ de novo responses induced in patients with melanoma is demonstrated by a combination of one, two, or three functions (e.g., one or more functions is the production of one or more factors selected from IFNγ, TNFα, CD107a, and 4-1BB).

[0100] [Figure 36]Figure 36 shows the specificity of memory and de novo responses induced in melanoma patients against mutated and wild-type peptides. SRSF1E>K and ARAP1Y>H-specific T cell responses were measured by challenging DCs loaded with mutant or wild-type neoantigen peptides at different concentrations (X-axis: 0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM), and measuring the IFN-γ and / or TNFα and / or CD107a (Y-axis) levels of total CD8+ T cells in the samples; both responses showed significant differences relative to the 0 μM concentration, which was not responsive to the wild-type neoantigen peptide. Statistical analysis: FDR for adjusted p-values, P-values: *≦0.05, ***≦0.001, ****≦0.0001.

[0101] [Figure 37A] Figure 37A shows the cytotoxicity profile of memory responses induced in patients with melanoma, quantified by the frequency of CD8+CD107a+ T cells. It also shows target cell killing by these T cell responses, quantified by the frequency of aCAS3+ tumor cells. The cytotoxic potential of the induced CD8+ T cell responses was assessed by rechallenging tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The constructs contained either mutant or wild-type sequences, with a central mutation. Upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells was measured upon coculture. Target ratio: 3.3:1 (SRSF1E>K).

[0102] [Figure 37B]Figure 37B shows another example of the cytotoxicity profile of a memory response induced in a patient with melanoma, quantified by the frequency of CD8+CD107a+ T cells. It also shows target cell killing by these T cell responses, quantified by the frequency of aCAS3+ tumor cells. The cytotoxic potential of the induced CD8+ T cell response was assessed by rechallenging tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The constructs contained either mutant or wild-type sequences, with a central mutation. Upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells was measured during coculture. Red circles highlight the pMHC+ ratio. Effector:target ratio: 5:1 (SRSF1E>K). Statistical analysis: unpaired t-test, P-value **≦0.01, ****≦0.0001.

[0103] [Figure 37C] Figure 37C shows the cytotoxicity profile of de novo responses induced in melanoma patients, quantified by the frequency of CD8+CD107a+ T cells. It also shows target cell killing by these T cell responses, quantified by the frequency of aCAS3+ tumor cells. The cytotoxic potential of the induced CD8+ T cell responses was assessed by rechallenging tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The constructs contained either mutant or wild-type sequences, with a central mutation. Upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells was measured upon coculture. Circles highlight the pMHC+ ratio. Effector:target ratio: 0.66:1 (ARAP1Y>H). Statistical analysis: unpaired t-test, P-value **≦0.01, ****≦0.0001.

[0104] [Figure 38A]Figure 38A shows the identification of neoantigen-specific CD4+ T cell responses in melanoma patients. Responses are identified based on IFN-γ and TNFα production (Y-axis) upon re-challenge with mutant neoantigen peptide-loaded DCs (0.8 μM). MKRN1S>L, CREBBPS>L, and TPCN1K>E were identified as positive responses.

[0105] [Figure 38B] Figure 38B shows the specificity of the CD4+ T cell responses shown in Figure 38A to the indicated mutated and wild-type peptides. In a validation study, the CD4 T cell responses shown in Figure 38A were challenged with different concentrations (X-axis - 0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM) of mutant and wild-type neo-antigen peptides, and the IFNγ+ and / or TNFα+ (Y-axis) of total CD4+ T cell responses in the samples was measured. Two of the CD4+ T cell responses (MKRN1S>L and CREEBPS>L) showed significant differences relative to the 0 μM concentration and were not responsive to the wild-type neo-antigen peptide, while the TPCN1K>E response was reactive to both the mutant and wild-type neo-antigen peptides. Statistical analysis: FDR for adjusted p-values, P-value < 0.05.

[0106] [Figure 38C] Figure 38C shows the polyfunctional profiles of these CD4+ T cell responses, as indicated by a combination of one, two, three, or four functions (e.g., one or more functions are the production of one or more factors selected from IFNγ, TNFα, CD107a, and 4-1BB). The polyfunctionality of the identified CD4+ T cell responses was assessed by rechallenge with DCs (0.8 μm) loaded with mutant neoantigen peptides. The percentages in the pie chart indicate the percentage functional CD4+ T cells (one, two, and / or three functions). Representative data shown are generated from post-stimulation CD4+ T cell responses induced in patients.

[0107] [Figure 39]Figure 39 shows the functionality of memory responses induced in two healthy donors with or without the addition of epacadostat, as indicated by a combination of one, two, or three functions (e.g., the one or more functions are the production of one or more factors selected from IFNγ, TNFα, and CD107a).

[0108] [Figure 40] FIG. 40 shows the percent induction of de novo CD8+ T cell responses ("hit rate," averaged over four healthy donors) across six replicate inductions with and without the addition of epacadostat.

[0109] [Figure 41] Figure 41A shows the absolute numbers of antigen-specific cells from healthy donors after induction by the T cell manufacturing protocol provided herein with or without the addition of a PD-1 blocking antibody.

[0110] Figure 41B shows the absolute numbers of antigen-specific cells from healthy donors after induction by the T cell manufacturing protocol provided herein, with or without the addition of a PD-1 blocking antibody.

[0111] [Figure 42] FIG. 42A shows the multimer-positive frequency as a percentage of CD8+ T cells from the de novo CD8+ T cell compartment with or without the addition of IL-12.

[0112] FIG. 42B shows an exemplary graphical representation of the percentage of CD8+ T cells from the de novo CD8+ T cell compartment with or without the addition of IL-12.

[0113] [Figure 43]Figure 43 shows the exemplary graph representation of the percentage hit rate for the highly immunogenic and low immunogenic antigens that naive CD8 cells are responsive to after carrying out different antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors.Also shown is the exemplary graph representation of the absolute number of antigen-specific cells after carrying out different antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors, using Mart-1 peptide or highly immunogenic and low immunogenic antigens.

[0114] [Figure 44-1] FIG. 44A shows exemplary flow cytometry results of CD123-positive cells after performing the indicated antigen-presenting cell enrichment and antigen loading protocols using PBMCs from three different healthy donors. [Figure 44-2] Same as above.

[0115] [Figure 44-3] Figure 44B shows an exemplary graphical representation of the absolute numbers of the indicated CD11c+ cell subsets after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from healthy donors: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells.

[0116] [Figure 45] Figure 45 shows an exemplary graphical representation of the ratio of total CD8 T cells to the indicated cells after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from healthy donors. The treatments are: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells.

[0117] [Figure 46] FIG. 46 shows exemplary flow cytometry results of CD11b-positive cells after performing the indicated antigen-presenting cell enrichment and antigen loading protocols using PBMCs from three different healthy donors.

[0118] [Figure 47] FIG. 47 shows exemplary flow cytometry results of CD19-positive cells after performing the indicated antigen-presenting cell enrichment and antigen loading protocols using PBMCs from three different healthy donors.

[0119] [Figure 48] Figure 48 shows an exemplary graphical representation of the fold expansion of cells after three antigen-presenting cell enrichment and antigen-loading protocols: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment, and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment, and depletion of CD11b- and CD19-expressing cells.

[0120] [Figure 49A] Figure 49A shows exemplary data showing the number of specific antigens to which naive CD8 T cells were responsive after performing three antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors. Results were averaged across three healthy donors. Treatments were as follows: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment, and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment, and depletion of CD11b- and CD19-expressing cells. An exemplary graphical representation of the data is shown in the graph below.

[0121] [Figure 49B]Figure 49B shows an exemplary graphical representation of the percent hit rates for highly immunogenic antigens (left) and weakly immunogenic antigens (right) to which naive CD8 cells are responsive after performing three antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors. Results were averaged across three healthy donors. Treatments were as follows: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment, and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment, and depletion of CD11b- and CD19-expressing cells.

[0122] [Figure 50] Figure 50 shows an exemplary graphical representation of the number of antigen-specific cells in a population of cells activated by highly and poorly immunogenic antigens to which T cells are responsive, after performing three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from healthy donors. The treatments are: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment, and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment, and depletion of CD11b- and CD19-expressing cells.

[0123] [Figure 51-1] Figure 51A shows an exemplary graphical representation of the percentage of viable cells after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from a healthy donor. The treatments are as follows: Base, FLT3L treatment alone; Base + CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells; + APC, Base + CD11b- / CD19- plus an additional PBMC fraction, where the additional fraction was depleted of CD3, CD19, CD11b, CD25, and CD14-expressing cells.

[0124] Figure 51B shows an exemplary graphical representation of the percentage of viable cells after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs derived from a healthy donor. The treatments were as follows: Base, FLT3L treatment alone; Base + CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells; + APC, Base + CD11b- / CD19- plus an additional PBMC fraction, in which the additional fraction was depleted of CD3, CD19, CD11b, CD25, and CD14-expressing cells.

[0125] Figure 51C shows an exemplary graphical representation of the percentage of viable cells after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from a healthy donor. The treatments were as follows: Base, FLT3L treatment alone; Base + CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells; + APC, Base + CD11b- / CD19- plus an additional PBMC fraction, in which the additional fraction was depleted of CD3, CD19, CD11b, CD25, and CD14-expressing cells.

[0126] [Figure 51-2] FIG. 51D shows exemplary data showing the number of specific antigens to which CD8 cells are responsive per donor using an exemplary antigen-presenting cell enrichment protocol.

[0127] FIG. 51E shows an exemplary graphical representation of the percent hit rate for the indicated peptides to which CD8 cells were responsive, averaged across three healthy donors.

[0128] [Figure 52-1]Figure 52A shows exemplary flow cytometry analysis results from an experiment in which populations of cells added to the culture process at different time points were labeled with a membrane-permeable amine-reactive dye (e.g., carboxyfluorescein succinimidyl ester or TagIT Violet™) before stimulation with antigen-loaded APCs. When applied to the second stimulation, a population of cells already cultured for 14 days was labeled with one dye, while another population of cells containing antigen-loaded APCs and a new preparation of T cells was labeled with a different dye, and the two populations were mixed together for restimulation or expansion. The relative contribution of each of these populations to the overall antigen-specific T cell pool was indicated by the presence and dilution of each dye. In all examples, a population of cells was cultured for 14 days (first stimulation), labeled with one dye, and then added to another population of cells labeled with a different dye that had been stimulated with antigen one day (standard protocol), four days (starting five days earlier), or six days (starting seven days earlier) prior.

[0129] [Figure 52-2] Figure 52B shows an exemplary schematic representation of three different T cell expansion protocols, each with two stimuli including an early start for antigen-loaded APCs at 2, 5, or 7 days before contact with T cells.

[0130] Figure 52C shows an exemplary graph of the number of antigen-specific T cells over time using the three different T cell expansion protocols shown in Figure 52B: 1, standard protocol; 2, starting 5 days early; 3, starting 7 days early.

[0131] [Figure 53] Figure 53 shows an exemplary graph of fold expansion of cultures treated with the indicated neo-antigen peptides (peps) or neo-antigen RNA. After CD3 lymphocyte isolation or depletion, CD14 / CD25-depleted PBMC cells were stimulated with antigen (peptide or antigen-encoding mRNA). CD3 lymphocytes were reintroduced and stimulated for 14 days.

[0132] [Figure 54] Figure 54 shows an exemplary graph of the number of multimer-positive antigen-specific cells in cultures nucleofected with the indicated neo-antigen peptide (pep) or neo-antigen RNA. Cultures were nucleofected in the presence or absence of T cells (-CD3). Irr, irradiated.

[0133] [Figure 55] Figure 55 shows exemplary flow cytometry analyses demonstrating antigen-specific CD8+ memory responses using viral peptides or RNA encoding those peptides, and naive responses using neoantigen-encoding peptides or RNA in a short-term induction protocol.

[0134] [Figure 56A] FIG. 56A shows a schematic of an exemplary process for generating RNAs containing sequences encoding neo-antigens, loading them into PBMCs, and using them to activate T cells.

[0135] [Figure 56B] Figure 56B shows a schematic of an exemplary process for generating RNA containing sequences encoding neo-antigens, loading them into PBMCs, and using them to activate T cells.

[0136] [Figure 57] FIG. 57A shows a schematic of an exemplary RNA concatemer construct encoding a string of neo-antigens.

[0137] FIG. 57B shows a schematic diagram of an exemplary arrangement of neo-antigen strings in a 5′-3′ orientation within the construct shown in FIG. 57A.

[0138] [Figure 58]Figure 58A shows a schematic diagram of an exemplary mRNA sequence for incorporating a 5'-CAP structure into an mRNA encoding a concatenated neo-antigen string for expression in PBMCs. The addition of an "A" nucleotide in the mRNA string was used for compatibility with CleanCap® Technology.

[0139] FIG. 58B shows an exemplary graphical representation of the percentage of viable cells 24 hours after expressing mRNA encoding concatenated neo-antigen strings with different 5′-CAP structures in PBMCs.

[0140] FIG. 58C shows an exemplary graphical representation of the total number of GFP-positive cells 24 hours after expressing mRNA encoding concatenated neoantigen strings with different 5′-CAP structures in PBMCs.

[0141] [Figure 59-1] Figure 59A shows exemplary results demonstrating the use of modified nucleotides to generate mRNA. The mRNA was modified by substituting either all (full) or some (partial) of the uridine (U) and cytidine (C) residues within the mRNA. For example, the partial C set contains 30% of the C residues replaced by methylcytidine. The results show the effect over time on the expression of mRNA-encoded peptides in transfected PBMCs.

[0142] [Figure 59-2] Figure 59B shows exemplary data comparing the effect of commercial and in-house preparations of mRNA containing substituted uridines and / or cytidines on generating multimer-specific T cells stimulated with mRNA-loaded PBMCs.

[0143] Figure 59C shows exemplary data comparing the expansion of stimulated T cells generated as described in Figure 59B.

[0144] [Figure 60]Figure 60A shows an exemplary schematic of mRNA constructs using shortmers (9-10 amino acids, top) and longmers (25 amino acids, bottom) used for expression in cells.

[0145] Figure 60B shows an exemplary graph of multimer-specific CD8+ cells as a percentage of total CD8+ cells. The antigens used in the multimer assay are indicated.

[0146] Figure 60C shows an exemplary flow cytometry analysis of the detection of multimer-positive CD8+ T cells comparing APCs stimulated with shortmer (9-10 amino acids) and longmer (25 amino acids) peptides with APCs containing coding for the same shortmer (9-10 amino acids) and longmer (25 amino acids) peptides.

[0147] [Figure 61-1] FIG. 61A shows a schematic diagram of exemplary RNA constructs transfecting cells for the experiments shown in FIGS. 61B-61D.

[0148] Figure 61B shows an exemplary graphical representation of results from the multimer assay. Under all three conditions of PBMC handling, RNA-transfected PBMCs were superior to peptide-loaded PBMCs in generating antigen-specific T cells. For the Gli3 antigen, a greater than 10-fold increase in multimer-positive cells is noted compared to peptide-loaded PBMCs.

[0149] Figure 61C shows exemplary flow cytometry data demonstrating the detection of Gli3 multimer-positive T cells in each indicated set, with and without depletion of CD3 cells. Transfection of CD25+ PBMCs directly results in increased multimer-positive cells over PBMCs depleted of CD14 and CD25 cells or PBMCs thawed from frozen stocks.

[0150] [Figure 61-2]Figure 61D shows an exemplary graphical representation of results from a multimer assay. PBMCs treated overnight with FTL3L cells or depleted of CD25 were electroporated with RNA encoding either 25 amino acid long neoantigen sequences (longmers) or epitope-length neoantigen sequences (shortmers). The percentage of neoantigen-positive cells in the cultures was assayed using multimer technology.

[0151] Figure 61E shows an exemplary graphical representation of the fold expansion results from the experiment described in Figure 61D. PBMCs treated overnight with FTL3L cells or CD25-depleted PBMCs were electroporated with RNA encoding either 25 amino acid long neo-antigen sequences (longmers) or epitope-length neo-antigen sequences (shortmers). The fold expansion of the cells after 26 days in culture and two stimulations is shown.

[0152] [Figure 62A] Figure 62A (top) depicts a schematic of exemplary RNA constructs used to transfect cells in the experiments shown in Figures 62A-C.

[0153] Figure 62A (bottom) depicts an exemplary graphical representation of the number of live ACTN4- and Gli3-responsive T cells from two donors at day 26 after maturation with the indicated combinations on the X-axis.

[0154] [Figure 62B] Figure 62B depicts exemplary data of the percentage of Gli3-responsive T cells from live cells grown in the presence of the indicated maturation mix.

[0155] [Figure 62C] Figure 62C depicts exemplary flow cytometry data showing detection of Gli3 multimer-positive T cells grown in the presence of the indicated maturation mixes.

[0156] [Figure 63A]Figure 63A shows representative mass spectrometry data demonstrating the detection of the presentation of the indicated Gli3 epitopes by PBMCs using radioisotope incorporation. PBMCs transfected with mRNA encoding multiple epitopes (including the Gli3 epitope), and peptide expression is detected using a reference peptide labeled with a heavier isotope.

[0157] [Figure 63B] Figure 63B shows an exemplary graphical representation of the percentage of maximum presentation of the indicated epitopes by HLA-A02:01 over time after transfection of PBMCs with mRNA encoding each of the epitopes. Each isotope-labeled epitope was detected by mass spectroscopy. Maximum surface presentation was observed 6 hours after transfection.

[0158] [Figure 64A] Figure 64A shows exemplary graphical representations from recall assays of the percentage change in TNFα and / or IFNγ production (left) or the percentage of CD107a positive cells (right) from neoantigen-specific CD8 T cells challenged with increasing concentrations of the indicated peptides used to load APCs.

[0159] [Figure 64B] Figure 64B shows exemplary graphical representations from multimer assays of the percentage change in TNFα and / or IFNγ production (left) or the percentage of CD107a positive cells (right) from neoantigen-specific CD8 T cells challenged with increasing concentrations of the indicated peptides used to load APCs.

[0160] [Figure 65] Figure 65 shows an exemplary Venn diagram of criteria considered to generate an optimal product, personalized T cell therapy, using mRNA as the immunogen.

[0161] [Figure 66] Figure 66 shows an exemplary flow diagram illustrating the steps for the selection of peptide sequences for preparing a patient-specific T cell product.

[0162] [Figure 67A] Figures 67A and 67B illustrate several aspects that are advantageous for a clinical approach using T cells produced by the process shown in Figures 1A and 67A. [Figure 67B] Same as above.

[0163] [Figure 68] Figure 68 shows exemplary representative flow cytometry data demonstrating the characterization of patient-specific T cell products prepared by multiple engineering runs. CD3+ as a percentage of live cells (upper panel) and CD8+ and CD4+ as a percentage of live CD3+ T cells (lower panel) are shown.

[0164] [Figure 69] Figure 69A shows an exemplary graphical representation of data showing the characterization of patient-specific T cell products prepared by multiple engineering runs. The percentage of multimer-positive CD8-positive cells is shown.

[0165] Figure 69B shows exemplary representative flow cytometry data demonstrating the characterization of patient-specific T cell products prepared by multiple engineering runs. The percentage of multimer A-positive and multimer B-positive CD8 cells is shown for the indicated epitopes.

[0166] Figure 69C shows the identified pMHC upon rechallenge with mutant neoantigen-loaded DCs compared to unloaded DCs. + CD8 + 1 shows an exemplary pie chart illustrating T cell polyfunctionality.

[0167] [Figure 70]Figure 70 shows representative data demonstrating the changes in IFNγ and / or TNFα production by CD4+ cells in patient-specific T cell products prepared by multiple engineering runs. Exemplary representative data showing the characterization of IFNγ+ and / or TNFα+ and / or CD107a+ CD4+ cells in patient-specific T cell products prepared by multiple engineering runs are also shown.

[0168] [Figure 71] Figure 71 shows an exemplary graphical representation illustrating the percentage of central memory T cells (Tcm), effector memory T cells (Tem), effector T cells (Teff), and naive T cells (Tnaive) in patient-specific T cell products prepared by multiple engineering runs. Central memory T cells (Tcm): CD62L+CD45RA-, effector memory T cells (Tem): CD62L-CD45RA-, effector T cells (Teff): CD62L-CD45RA+, naive T cells (Tnaive): CD62L+CD45RA+.

[0169] [Figure 72] Figure 72 shows an exemplary graphical representation of data from a multimer assay showing the percentage of IFN-γ and / or TNFα and / or CD107a cells measured among total CD8+ cells (upper panel) or total CD4+ T cells (lower panel) in a sample upon challenge with various concentrations of peptide-loaded DCs. The peptide used is indicated on each graph.

[0170] [Figure 73] Figure 73 shows an exemplary graphical representation of data demonstrating upregulation of CD107a on CD8+ T cells (top row) and active caspase 3 on tumor cells (bottom row). Measurements were taken after co-culture with A375 tumor cell lines, or A375 tumor cell lines loaded or not with peptide, either untransduced or transduced with a 200 amino acid construct.

[0171] [Figure 74] Figure 74 shows an exemplary graphical representation of data demonstrating that induced T cells can kill antigen-expressing cells. Whether neoantigen-specific T cells recognize autologous tumors or peptide-loaded autologous tumors was tested via recall response assay. Readout: IFN-γ+ and / or TNFα+ and / or CD107a+ (Y-axis) of pMHC+ (% of CD8+) and pMHC- (% of CD8+) T cells. Significance was assigned using one-way ANOVA, P<0.05.

[0172] [Figure 75] Figure 75 shows an exemplary schematic of cohorts and doses for use in the clinical study (NEO-PTC-01).

[0173] [Figure 76A] FIG. 76A shows a schematic representation of the NEO-PTC-01 manufacturing process overview.

[0174] [Figure 76B] Figure 76B shows T cell product characteristics.

[0175] [Figure 77-1] Figure 77A shows the frequency of multimer-specific CD8+ T cells in each pool of either the NEO-PTC-01.pep or NEO-PTC-01.RNA process using patient PBMCs from patient 1.

[0176] Figure 77B shows the multimer-specific CD8 in each pool of either the NEO-PTC-01.pep or NEO-PTC-01.RNA process using patient PBMCs from patient 2. + The frequency of T cells is shown.

[0177] Figure 77C shows a summary of responses in each patient separated by NEO-PTC-01.pep and NEO-PTC-01.RNA processes. The denominator indicates the total number of neoantigen sequences used to induce a T cell response.

[0178] Figure 77D shows pMHC in the CD8+ population. + 1 shows an exemplary flow cytometry plot of T cells.

[0179] [Figure 77-2] Figure 77E shows the frequency of multimer-specific CD8+ T cells generated from NEO-PTC-01. The black circles represent pMHC+-specific responses from NEO-PTC-01.pep performed at large scale in melanoma patient samples; the red circles represent pMHC+-specific responses from NEO-PTC-01.pep performed at small scale using ovarian patient samples, and the green circles represent pMHC+-specific responses from NEO-PTC-01.RNA performed at small scale in ovarian patient samples.

[0180] [Figure 78] Figure 78A shows results demonstrating that neoantigen-specific CD8+ T cell responses from ovarian patient samples are polyfunctional. Representative data from the NEO-PTC-01.pep set. Data depicting the expression of functional markers (IFN-γ, TNF-α, and CD107a) of identified pMHC+CD8+ T cells after re-challenge with mutant neoantigen-loaded DCs compared to pMHC+CD8+ T cells challenged with DMSO-loaded DCs. Percentages above the bars represent the percentage of pMHC+CD8+ T cells present in the sample.

[0181] Figure 78B shows neoantigen-specific CD8 + Results are shown demonstrating that T cell responses are polyfunctional and representative data from the NEO-PTC-01.RNA set. pMHC challenged with DMSO-loaded DCs + CD8 +Identified pMHC after rechallenge with mutant neoantigen-loaded DCs compared with T cells + CD8 + Data depicting the expression of functional markers of T cells (IFN-γ, TNF-α, and CD107a). Percentages above the bars indicate the pMHC present in the sample. + CD8 + The percentage of T cells is expressed.

[0182] [Figure 79-1] Figure 79A shows the frequency of neoantigen-specific CD4+ T cells producing IFN-γ+ and / or TNF-α+ when co-cultured with their cognate peptide-loaded dendritic cells above a threshold (the amount of IFN-γ+ and / or TNF-α+ produced when co-cultured with dendritic cells alone). NEO-PTC-01.pep-treated ovarian patient sample 1.

[0183] Figure 79B shows the threshold (IFN-γ produced when co-cultured with dendritic cells alone). + and / or TNF-alpha + IFN-γ levels exceed those of dendritic cells loaded with their cognate peptides. + and / or TNF-alpha + Neoantigen-specific CD4 producing + The frequency of T cells is shown in NEO-PTC-01.RNA-derived ovarian patient sample 1.

[0184] Figure 79C shows the threshold (IFN-γ produced when co-cultured with dendritic cells alone). + and / or TNF-alpha + and / or CD107a) exceed the levels of IFN-γ when co-cultured with their cognate peptide-loaded dendritic cells. + and / or TNF-alpha + Neoantigen-specific CD4 producing + Shown are T cell frequencies in NEO-PTC-01.pep treated ovarian patient sample 2.

[0185] Figure 79D shows the threshold (IFN-γ produced when co-cultured with dendritic cells alone). + and / or TNF-alpha + and / or CD107a) exceed the levels of IFN-γ when co-cultured with their cognate peptide-loaded dendritic cells. + and / or TNF-alpha + Neoantigen-specific CD4 producing + The frequency of T cells is shown in NEO-PTC-01.RNA-derived ovarian patient sample 2.

[0186] [Figure 79-2] Figure 79E shows the total number of CD4+ T cell responses in each patient, separated by the NEO-PTC-01.pep and NEO-PTC-01.RNA processes. The data demonstrate the diversity of responses obtained in cells manufactured using peptide stimulation (left column) and RNA-mediated antigen expression (right column) processes from sample cells from ovarian cancer patients 1 and 2. The denominator represents the total number of neoantigens used to induce a response in each process.

[0187] Figure 79F shows CD4+ after restimulation with dendritic cells loaded with DMSO or their cognate neoantigens. + IFN-γ in the population + Flow cytometry plots of the cells are shown.

[0188] Figure 79G shows antigen-specific activation (IFN-γ) produced from NEO-PTC-01. + and / or TNF-α+)CD4 + The black circles represent the frequency of pMHC T cells from NEO-PTC-01.pep, which was performed on a large scale in melanoma patient samples. + Red circles represent specific responses; pMHC from NEO-PTC-01.pep, performed on a small scale using ovarian patient samples. + Green circles represent specific responses, and pMHC from NEO-PTC-01.RNA was performed on a small scale in ovarian patient samples. + Represents a specific response.

[0189] [Figure 80A] Figure 80A shows results demonstrating that neoantigen-specific CD4+ T cell responses from ovarian patient samples are polyfunctional. Polyfunctionality of antigen-specific CD4+ T cells in the NEO-PTC-01.pep process after re-challenge with neoantigen-loaded DCs compared to DMSO-loaded dendritic cells.

[0190] [Figure 80B] Figure 80B shows results demonstrating that neoantigen-specific CD4+ T cell responses from ovarian patient samples are polyfunctional. Polyfunctionality of antigen-specific CD4+ T cells in NEO-PTC-01.RNA-processed DCs after re-challenge with neoantigen-loaded DCs compared to DMSO-loaded DCs.

[0191] [Figure 81A] Figure 81A shows flow cytometry gating of multimer-responsive cell populations in the Neo-PTC-01.pep process. pMHC+ plot showing the frequency of multimer-positive cells.

[0192] [Figure 81B] Figure 81B shows the frequency of naive, central memory (CM), effector memory (EM) or effector T cells present in bulk CD4, bulk CD8 or multimer positive cells in the Neo-PTC-01.pep process.

[0193] [Figure 81C] Figure 81C shows representative data of cell populations in the Neo-PTC-01.pep process showing upregulation of CD107a neoantigen-specific CD8+ T cells measured 6 hours after co-culture of T cells with tumor cell lines loaded with mutant, wild-type, irrelevant peptide, or DMSO (no peptide).

[0194] [Figure 81D]Figure 81D shows flow cytometry gating of multimer-responsive cell populations in the Neo-PTC-01.RNA process. pMHC+ plot showing the frequency of multimer-positive cells.

[0195] [Figure 81E] Figure 81E shows the frequency of naive, central memory (CM), effector memory (EM) or effector T cells present in bulk CD4, bulk CD8 or multimer positive cells in the NEO-PTC-01.RNA process.

[0196] [Figure 81F] Figure 81F shows representative data of cell populations in the NEO-PTC-01.RNA process showing upregulation of CD107a neoantigen-specific CD8+ T cells measured 6 hours after co-culture of T cells with tumor cell lines loaded with mutant, wild-type, irrelevant peptide, or DMSO (no peptide).

[0197] [Figure 82]Figure 82 shows a schematic representation of the study design for a Phase 1, open-label, dose-finding and escalation study of the safety and activity of a personalized T-cell therapy. Part 1 of the study is designed for monotherapy with dose escalation, whereby patients who have progressed on anti-PD-1 therapy and received anti-CTLA4 therapy will receive ≥ 1 x 10 cells to ≤ 1 x 10 cells, + / - 25% (Dose 1) or 2 x 10 cells to ≤ 1 x 10 cells, + / - 25% (Dose 2). Next will come dose escalation in patients in the highest dose cohort deemed safe and tolerable. Part 2 of the study is designed for combination therapy, whereby patients deemed stable or asymptomatic progressors after 3 months on anti-PD-1 therapy (with or without anti-CTLA4 therapy) will receive personalized T-cell therapy at the dose determined to be safe in Part 1, plus anti-PD-1 therapy (with or without anti-CTLA4 therapy). Patients will receive anti-PD1 (+ / - anti-CTLA4) therapy after enrollment in the trial and production of their personalized T-cell therapy, and will continue anti-PD1 treatment throughout the follow-up period (up to EOS). This part of the study will measure the efficacy of the combination of anti-PD1 and personalized T-cell therapy.

[0198] [Figure 83] Figure 83 shows a schematic representation of the timeline of the study described in Figure 82. As depicted, the prescreening and screening phases are between days -20 and -16, respectively, relative to the administration of personalized T cell therapy on day 0. Selected subjects undergo leukapheresis on day -12, and the cells are processed for a subsequent period to produce personalized T cell therapy. On day -1, subjects receive their last dose of chemotherapy prior to personalized T cell therapy. Personalized T cell therapy is administered on day 0, followed by a 36- to 52-week follow-up period. Subjects will receive a dose of anti-PD-1 therapy (nivolumab) once every six weeks (Q6W).

[0199] [Figure 84]Figure 84 (top panel) shows an overview of small-scale induction performed on three ovarian cancer (OVC) patient samples. Patient samples (PBMCs) were obtained from subjects with ovarian cancer, and the small-scale manufacturing NEO-STIM process was used to generate T cell products, followed by analysis of product quality. The NEO-STIM manufacturing process was followed for antigen-specific T cell generation from ovarian cancer cells, including using peptides (peptide-loaded APCs) or APCs expressing RNA encoding the peptides to stimulate the cells, and the results were compared. Patient characteristics and mutated antigens (neo-antigens) identified from each patient are displayed in each box. Bottom panel: A graphical representation is provided showing the two RNA designs used for the process for CD8+ and CD4+ T cell stimulation.

[0200] [Figure 85] Figure 85 shows an overview of the assays and result highlights for the comparison and validation of peptide vs. RNA-mediated stimulation processes, demonstrating that both processes are suitable to meet manufactured product quality requirements.

[0201] [Figure 86] Figure 86 shows further details of the method for comparison and validation of peptide versus RNA-mediated stimulation processes in the NEOSTIM manufacturing process.

[0202] [Figure 87A] Figure 87A shows the results of the NEO-PTC-01.pep and NEO-PTC-01.RNA processes, both of which generate similar frequencies of neoantigen-specific CD8+ T cells (patient N16NEON-17, OVC#1).

[0203] [Figure 87B] Figure 87B shows the results of the NEO-PTC-01.pep and NEO-PTC-01.RNA processes, both of which generate similar frequencies of neoantigen-specific CD8+ T cells (patient N16NEON-19, OVC#2).

[0204] [Figure 87C] Figure 87C shows the results of the NEO-PTC-01.pep and NEO-PTC-01.RNA processes, both of which generate similar frequencies of neoantigen-specific CD8+ T cells (patient N16NEON-18, OVC#3).

[0205] [Figure 88] Figure 88 shows data demonstrating similar frequencies of neo-antigen-specific CD8+ T cells for the peptide and RNA protocols, consistent with observations in the engineering runs.

[0206] [Figure 89] Figure 89 shows data demonstrating successful generation of CD4+ responses in two OVC patients. Frequencies represent the (Δ) delta-change in CD4 positive samples from no peptide controls.

[0207] [Figure 90] Figure 90 shows data demonstrating that similar frequencies of neoantigen-specific CD4+ T cells are generated in OVC patient samples by both the NEO-PTC-01.pep and NEO-PTC-01.RNA protocols.

[0208] [Figure 91] Figure 91 presents data demonstrating that the predominant phenotype of the NEO-PTC-01.RNA and NEO-PTC-01.Peptide protocols is T effector memory cells.

[0209] [Figure 92] Figure 92 shows data demonstrating that the use of IL-21 in NEO-STIM increases the central memory phenotype in antigen-specific CD8+ T cells.

[0210] [Figure 93] Figure 93 shows data demonstrating that IL-21 addition improves expansion of NEO-STIM.RNA cultures.

[0211] [Figure 94] Figure 94 shows data demonstrating that IL-21 addition improves the priming of specific T cells in NEO-STIM.RNA cultures.

[0212] [Figure 95] Figure 95 shows data demonstrating that IL-21 addition increases the Tcm population in Applicants' NEO-STIM.RNA cultures. DETAILED DESCRIPTION OF THE INVENTION

[0213] Detailed Description T cell therapeutics are expected to be relatively safe and well-tolerated adoptive T cell products. However, based on an evaluation of the risks associated with the products, there are three general classes of potential toxicities associated with T cell therapeutics: (a) treatment-related toxicity due to lymphodepletion, cell infusion, or cytokine release syndrome; (b) off-tumor, off-target toxicity due to expansion of autoreactive clones or cross-reactivity of neoantigen-specific T cells; and (c) off-tumor, on-target toxicity due to presentation of neoantigens in non-tumor tissues. Novel immunotherapeutic agents and their uses based on the discovery of neoantigens arising from mutational events unique to an individual's tumor are described herein. Accordingly, the disclosure described herein provides methods and protocols for generating antigen-specific immune cells, e.g., T cells, for use in the treatment of disease.

[0214] A composition of neoantigen-reactive T cells for cancer immunotherapy is presented herein. Adoptive T cell therapy is a promising new approach for cancer treatment, but requires several improvements. In general, T cells must be appropriately cytotoxic to cancer cells, spare non-cancerous cells in the body, not lose immunogenicity in the tumor environment, and provide long-term protection. Furthermore, the use of virally transduced cells presents its own challenges. Therefore, striking the right balance to achieve a therapeutically effective composition that specifically targets cancer cells while sparing healthy cells, halting disease progression, causing remission or at least substantial tumor regression, and preventing cancer recurrence requires several improvements in nearly every step of this complex process.

[0215] To aid in the understanding of this disclosure, several terms and phrases are defined below.

[0216] Antigens are foreign substances to the body that induce immune responses. "Neoantigens" refer to a class of tumor antigens that arise from tumor-specific changes in proteins. Neoantigens include, but are not limited to, tumor antigens that arise from, for example, substitutions in protein sequences, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, and expression of endogenous retroviral polypeptides.

[0217] A "neoepitope" refers to an epitope that is not present in a reference non-diseased cell, e.g., a non-cancerous cell or a germline cell, but is found in a diseased cell, e.g., a cancer cell. This includes situations where the corresponding epitope is found in a normal non-diseased cell or a germline cell, but one or more mutations have altered the sequence of the epitope in the diseased cell, e.g., a cancer cell, resulting in a neoepitope.

[0218] "Mutation" refers to a change or difference in a nucleic acid sequence (e.g., a nucleotide substitution, addition, or deletion) compared to a reference nucleic acid. "Somatic mutations" can occur in any cell of the body except germ cells (sperm and eggs) and are not passed on to offspring. Such changes may (but do not necessarily) cause cancer or other diseases. In some embodiments, the mutation is a nonsynonymous mutation. "Non-synonymous mutation" refers to a mutation (e.g., a nucleotide substitution) that results in an amino acid change, such as an amino acid substitution, in the translation product. "Frameshift" occurs when a mutation disrupts the normal phase of a gene's codon periodicity (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. It is possible for different mutations in a gene to achieve the same altered reading frame.

[0219] "Antigen processing" or "processing" refers to the degradation of a polypeptide or antigen into processing products that are fragments of the polypeptide or antigen (e.g., degradation of a polypeptide into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation by a cell, e.g., an antigen-presenting cell, to a specific T cell.

[0220] "Antigen-presenting cells" (APCs) refer to cells that present peptide fragments of protein antigens in association with MHC molecules on their cell surface. This term includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).

[0221] The term "affinity" refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide and a class I or II HLA, or a peptide-HLA complex and a T cell receptor (TCR)). D K refers to the dissociation constant between two members of a binding pair and has units of molar concentration. AK refers to the affinity constant between two members of a binding pair and is the reciprocal of the dissociation constant. Affinity can be determined experimentally, for example, by surface plasmon resonance (SPR) using a commercially available Biacore SPR unit. off K refers to the off-rate constant of the two members of a binding pair (e.g., the off-rate constant of an HLA-binding peptide and class I or II HLA, or a peptide-HLA complex and a TCR). on refers to the on-rate constant of the two members of a binding pair (eg, the on-rate constant of an HLA-binding peptide and class I or II HLA, or a peptide-HLA complex and a TCR).

[0222] Throughout this disclosure, "combined data" results are referred to as "IC 50 Affinity can be expressed in units of inhibitory concentration (IC 50 ), or the concentration at which 50% of the first member of the binding pair (e.g., a peptide) is displaced. Similarly, ln(IC 50 ) is IC 50 For example, IC 50 is the concentration of test peptide in a binding assay at which 50% inhibition of binding of the labeled reference peptide is observed. Given the conditions under which the assay is performed (e.g., limiting HLA protein concentration and / or labeled reference peptide concentration), these values ​​are DThe value can be approximated. Assays for determining binding are well known in the art and are described in detail in, for example, PCT Publications WO94 / 20127 and WO94 / 03205, as well as other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to the binding of a reference standard peptide. Binding can be measured using live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1996)), and immunoassays using purified MHC antibodies. (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high-flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and assays using class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

[0223] The term "derived" when used to describe an epitope is synonymous with "prepared." Derived epitopes can be isolated from natural sources or synthesized according to standard protocols in the art. Synthetic epitopes can include artificial amino acid residues, "amino acid mimetics," such as D-isomers of naturally occurring L-amino acid residues, such as cyclohexylalanine, or unnatural amino acid residues. Derived or prepared epitopes can be analogs of native epitopes. The term "derived from" refers to origin or source and can include naturally occurring, recombinant, unpurified, purified, or differentiated molecules or cells. For example, expanded or induced antigen-specific T cells can be derived from T cells. For example, expanded or induced antigen-specific T cells can be derived from antigen-specific T cells in a biological sample. For example, mature APCs (e.g., professional APCs) can be derived from immature APCs (e.g., immature APCs). For example, APCs can be derived from monocytes (e.g., CD14 + For example, dendritic cells can be derived from monocytes (e.g., CD14 + For example, APCs can be derived from bone marrow cells.

[0224] An "epitope" is a collective set of molecular features (e.g., the charge and primary, secondary, and tertiary structure of a peptide) that together form a site recognized by another molecule (e.g., an immunoglobulin, a T cell receptor, an HLA molecule, or a chimeric antigen receptor). For example, an epitope can be a set of amino acid residues involved in recognition by a particular immunoglobulin; a major histocompatibility complex (MHC) receptor; or, in the context of T cells, residues recognized by a T cell receptor protein and / or a chimeric antigen receptor. Epitopes can be prepared by isolation from natural sources or synthesized according to standard protocols in the art. Synthetic epitopes can include artificial amino acid residues, amino acid mimetics (such as D-isomers of naturally occurring L-amino acid residues or non-naturally occurring amino acid residues). Throughout this disclosure, epitopes may, in some cases, be referred to as peptides or peptide epitopes. In certain embodiments, the peptides of the present disclosure are limited in length. A length-limited embodiment occurs when a protein or peptide containing an epitope described herein contains a region (i.e., a contiguous series of amino acid residues) that is 100% identical to a native sequence. For example, to avoid defining an epitope by reading the entire native molecule, there is a limit to the length of any region that is 100% identical to a native peptide sequence. Thus, for a peptide containing an epitope described herein and a region that is 100% identical to a native peptide sequence, the region that is 100% identical to the native sequence generally has a length of less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues.In certain embodiments, an "epitope" as described herein is comprised by a peptide having a region having fewer than 51 amino acid residues with 100% identity to a native peptide sequence, in any increment up to 5 amino acid residues; for example, having 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue.

[0225] A "T cell epitope" refers to a peptide sequence that is bound by an MHC molecule in the form of a peptide-MHC (pMHC) complex that can be recognized and bound by the TCR of a T cell (e.g., a cytotoxic T lymphocyte or a T helper cell).

[0226] "T cells" are CD4 + T cells and CD8 +The term T cell includes T cells. The term T cell also includes both T helper type 1 T cells and T helper type 2 T cells. T cells can be generated by the methods described in this application for clinical applications. T cells or adoptive T cells, as referred to herein, for clinical applications, are cells isolated from a biological source, manipulated and cultured ex vivo, and prepared into drug candidates for specific treatment of cancer, such as melanoma. If the drug candidate cells pass specific qualitative and quantitative criteria for suitability for clinical application, the drug candidate can be designated a drug product. In some cases, the drug product is selected from a large number of drug candidates. In the context of this application, a drug product is a T cell, more specifically, a population of T cells, or more specifically, a population of T cells with heterogeneous characteristics and subtypes. For example, the drug product disclosed herein can have a population of T cells including CD8+ T cells and CD4+ T cells, where at least a certain percentage of the cells exhibit antigen specificity, and a certain percentage of each exhibits, among other things, a memory phenotype.

[0227] "Immune cell" refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0228] An "immunogenic" peptide or "immunogenic" epitope or "immunogenic" peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated or humoral response, e.g., a cytotoxic T lymphocyte (CTL) response, a helper T lymphocyte (HTL) response, and / or a B lymphocyte response. The immunogenic peptides described herein are capable of binding to an HLA molecule and subsequently inducing a cell-mediated or humoral response (e.g., a CTL (cytotoxic) response or an HTL response) against the peptide.

[0229] A "protective immune response" or "therapeutic immune response" refers to a CTL and / or HTL response against an antigen derived from a pathogenic antigen (e.g., a tumor antigen) that in some way prevents or at least partially arrests disease symptoms, side effects, or progression. The immune response can also include an antibody response, prompted by stimulation of helper T cells.

[0230] A "T cell receptor" ("TCR") refers to a molecule found on the surface of T lymphocytes (T cells) that recognizes antigens bound to major histocompatibility complex (MHC) molecules, whether natural or partially or wholly synthetically produced. The ability of a T cell to recognize antigens associated with various diseases (e.g., cancer) or infectious organisms is conferred by its TCR, which is composed of both alpha (α) and beta (β) chains or gamma (γ) and delta (δ) chains. The proteins that make up these chains are encoded by DNA, using a unique mechanism to generate the vast diversity of TCRs. This multisubunit immune recognition receptor associates with the CD3 complex and binds peptides presented by MHC class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of the TCR to peptides on the surface of APCs is a central event in T cell activation.

[0231] As used herein, "chimeric antigen receptor" or "CAR" refers to an antigen-binding protein comprising an immunoglobulin antigen-binding domain (e.g., an immunoglobulin variable domain) and a T cell receptor (TCR) constant domain. As used herein, the "constant domain" of a TCR polypeptide comprises the membrane-proximal TCR constant domain, the TCR transmembrane domain, and / or the TCR cytoplasmic domain, or fragments thereof. For example, in some embodiments, a CAR is a monomer comprising a polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCR β constant domain. In some embodiments, a CAR is a dimer comprising a first polypeptide comprising an immunoglobulin heavy or light chain variable domain linked to a TCR α or TCR β constant domain, and a second polypeptide comprising an immunoglobulin heavy or light chain variable domain (e.g., a kappa or lambda variable domain) linked to a TCR β or TCR α constant domain.

[0232] "Major histocompatibility complex" or "MHC" is a cluster of genes that plays a role in controlling cellular interactions that result in physiological immune responses. The term "major histocompatibility complex" and abbreviation "MHC" refer to a complex of genes that can include any class of MHC molecule, such as MHC class I and MHC class II molecules, and that occurs in all vertebrates. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. Thus, "human leukocyte antigen" or "HLA" refers to human major histocompatibility complex (MHC) proteins (see, e.g., Stites, et al., Immunology, 8 TH (Ed., Lange Publishing, Los Altos, Calif. (1994)). For a detailed description of the MHC and HLA complexes, see Paul, Fundamental Immunology, 3 rd Ed., Raven Press, New York (1993).

[0233] The major histocompatibility complex in the genome contains genetic regions whose gene products, expressed on the cell surface, are important for regulating immunological processes by binding and presenting endogenous and / or foreign antigens. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or diseased cells in immune responses. MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the surface of cells and can display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. MHC-bound peptides result from proteolytic cleavage of protein antigens and can represent potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes). MHC transports peptides to the cell surface, where they can be presented to specific cells such as cytotoxic T lymphocytes, T helper cells, or B cells. MHC regions can be divided into three subgroups: class I, class II, and class III. MHC class I proteins can contain an α chain and β2-microglobulin (not part of the MHC encoded by chromosome 15), which can present antigen fragments to cytotoxic T cells. MHC class II proteins can contain α and β chains and can present antigen fragments to T helper cells. MHC class III regions can encode other immune components, such as complement components and cytokines. MHCs can be both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).

[0234] A "receptor" refers to a biological molecule or group of molecules that can bind to a ligand. A receptor can function to transmit information in a cell, cell formation, or organism. A receptor includes at least one receptor unit, and for example, each receptor unit can be composed of a protein molecule. A receptor has a structure complementary to that of a ligand and can form a complex with the ligand as a binding partner. Information is transmitted, in particular, by a conformational change of the receptor after complexing with the ligand on the surface of a cell. In some embodiments, a receptor is understood to refer, in particular, to MHC class I and II proteins that can form a receptor / ligand complex with a ligand, in particular, a peptide or peptide fragment of a suitable length. A "ligand" refers to a molecule that has a structure complementary to that of a receptor and can form a complex with the receptor. In some embodiments, a ligand is understood to refer to a peptide or peptide fragment whose amino acid sequence has a suitable length and a suitable binding motif such that the peptide or peptide fragment can form a complex with an MHC protein, such as an MHC class I or MHC class II protein. In some embodiments, "receptor / ligand complex" is also understood to mean a "receptor / peptide complex" or "receptor / peptide fragment complex" comprising a peptide- or peptide fragment-presenting MHC molecule, such as an MHC class I or MHC class II molecule.

[0235] "Native" or "wild-type" sequence refers to a sequence found in nature. The term "naturally occurring" as used herein refers to the fact that something can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0236] The terms "peptide" and "peptide epitope" are used interchangeably herein with "oligopeptide" to designate a series of residues typically connected one after the other by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues. A "synthetic peptide" refers to a peptide obtained from a non-natural source, e.g., man-made. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. A "synthetic peptide" includes a "fusion protein."

[0237] The term "motif" refers to a pattern of residues in a peptide of a defined length that is recognized by a particular HLA molecule, e.g., an amino acid sequence less than about 15 amino acid residues in length or less than about 13 amino acid residues in length, e.g., about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13) for class I HLA motifs and about 6 to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for class II HLA motifs. Motifs typically differ for each HLA protein encoded by a given human HLA allele. These motifs differ in their patterns of primary and secondary anchor residues. In some embodiments, MHC class I motifs identify peptides that are 7, 8, 9, 10, 11, 12, or 13 amino acid residues in length. In some embodiments, the MHC class II motif identifies peptides that are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acid residues in length. A "cross-reactive binding" peptide refers to a peptide that binds to more than one member of a class of binding pair members (e.g., a peptide that is bound by both class I and class II HLA molecules).

[0238] The term "residue" refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or encoded by a nucleic acid (DNA or RNA). The nomenclature used to represent peptides or proteins follows conventional conventions. The amino group is presented to the left (amino or N-terminus) of each amino acid residue, and the carboxyl group is presented to the right (carboxy or C-terminus). Amino acid residue positions, when referenced in a peptide epitope, are numbered from amino to carboxyl, with position 1 being the residue located at the amino terminal end of the epitope or the peptide or protein of which the epitope may be a part. In formulas representing selected specific embodiments of the present invention, the amino and carboxyl terminal groups are not specifically shown but are of the type they assume at physiological pH values ​​unless otherwise specified. In amino acid structural formulas, each residue is generally represented by a standard three-letter or one-letter designation. The L-form of an amino acid residue is represented by a single capital letter or a three-letter symbol with the first letter capitalized, and the D-form for amino acid residues having the D-form is represented by a single lowercase letter or a three-letter symbol with the first letter capitalized. However, when the three-letter symbol or full name is used without capital letters, it can refer to an L-amino acid residue. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or "G." The amino acid sequences of the peptides depicted herein are generally named using standard one-letter symbols (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine).

[0239] A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, a phenylalanine substituted for a tyrosine is a conservative substitution. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate peptide function are well known in the art.

[0240] "Pharmaceutically acceptable" generally refers to a composition or component of a composition that is non-toxic, inert, and / or physiologically compatible. "Pharmaceutical excipients" or "excipients" include materials such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, etc. A "pharmaceutical excipient" is an excipient that is pharmaceutically acceptable.

[0241] In the present disclosure, the term "vaccine" relates to a pharmaceutical preparation (pharmaceutical composition) or product that, after administration, induces an immune response, e.g., a cellular or humoral immune response, that recognizes and attacks pathogens or diseased cells, such as cancer cells. Vaccines can be used for the prevention or treatment of diseases. The terms "personalized cancer vaccine" or "individualized cancer vaccine" or "personalized cancer vaccine" relate to a specific cancer patient and mean that the cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.

[0242] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length, including DNA and RNA, such as mRNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, polynucleotides and nucleic acids can be in vitro transcribed mRNA. In some embodiments, the polynucleotide administered using the methods of the present invention is mRNA.

[0243] The terms "isolated" or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany the material when found in its native state. Thus, an isolated peptide described herein does not contain some or all of the materials normally associated with the peptide in its in situ environment. For example, an "isolated" epitope may be one that does not contain the entire sequence of the protein from which it is derived. For example, a naturally occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide separated from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or peptide may be part of a composition, and such a vector or composition may still be "isolated" in that it is not part of its natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of DNA molecules described herein, and further include such molecules produced synthetically. In some embodiments, an isolated polypeptide, antibody, polynucleotide, vector, cell, or composition is substantially pure. The term "substantially pure," as used herein, refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0244] The term "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotide or amino acid residues that are the same when compared and aligned for maximum correspondence (introducing gaps, if necessary) without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences are well known in the art. Such include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaning that they share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequence that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the amino acid sequence of a peptide or the coding region of a nucleotide sequence.

[0245] The term "subject" refers to any animal (e.g., mammal), including but not limited to, a human, non-human primate, dog, cat, rodent, etc., who is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0246] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of a therapeutic agent effective to "treat" a disease or disorder in a subject or mammal. A therapeutically effective amount of a drug has a therapeutic effect and thus can prevent the onset of a disease or disorder; slow the onset of a disease or disorder; slow the progression of a disease or disorder; relieve to some extent one or more symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

[0247] The terms "treating" or "treatment" or "treat" or "alleviating" or "alleviating" refer to both (1) therapeutic measures that cure, slow, reduce the symptoms of, and / or halt the progression of, a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.

[0248] The term "depleted" when used to describe a cell sample (e.g., a peripheral blood mononuclear cell (PBMC) sample) refers to a cell sample in which a subpopulation of cells has been removed or depleted. For example, an immune cell sample in which CD25-expressing cells have been depleted refers to an immune cell sample in which CD25-expressing cells have been removed or depleted. For example, one or more binding agents can be used to remove or deplete one or more cells or cell types from the sample. For example, CD14 + Cells can be depleted or removed from the PBMC sample, such as by using an antibody that binds to CD14.

[0249] "Stimulation" refers to a response induced by the binding of a stimulatory molecule to its cognate ligand, thereby mediating a signal transduction event. For example, T cell stimulation can refer to the binding of a T cell's TCR to a peptide-MHC complex. For example, T cell stimulation can refer to the step in Protocol 1 or Protocol 2 in which PBMCs are cultured with peptide-loaded APCs.

[0250] The term "enriched" refers to a composition or fraction in which a target species has been partially purified such that the concentration of the target species is substantially greater than the naturally occurring level of that species in the finished product without enrichment. The term "induced cells" refers to cells that have been treated with an inducer compound, cell, or population of cells that affects the protein expression, gene expression, differentiation state, shape, morphology, viability, etc. of the cell.

[0251] A "reference" can be used to correlate and / or compare the results obtained in the disclosed method with a diseased sample. Typically, a "reference" can be based on one or more normal samples, particularly disease-free samples, obtained from either an individual, such as an individual of the same species, or one or more different individuals (e.g., healthy individuals). A "reference" can be empirically determined by testing a sufficiently large number of normal samples.

[0252] As used herein, a tumor is a cancerous tumor unless otherwise specified, and the terms cancer and tumor are used interchangeably throughout this document. Although tumors are cancers of solid tissue, some of the compositions and methods described herein are generally applicable to leukemia, a cancer of the blood.

[0253] Overview of T-cell therapy The generation of antigen-specific T cells by controlled ex vivo induction or expansion of T cells (e.g., autologous T cells) can provide highly specific and beneficial T cell therapies (e.g., adoptive T cell therapies). The present disclosure provides T cell manufacturing methods and therapeutic T cell compositions that can be used to treat subjects with cancer and other conditions, diseases, and disorders. The goal is to expand and induce antigen-specific T cells with favorable phenotypes and functions. The present disclosure provides compositions and methods for producing T cells that can be used for antigen-specific T cell therapy (e.g., personalized or individualized T cell therapy). The T cell compositions provided herein can be personalized antigen-specific T cell therapies. Figure 1 graphically depicts an overview of the process involved in T cell therapy: on the one hand, it includes the identification of cancer and cancer-specific antigens in a subject with cancer, resulting in the production of neoantigenic peptides; and on the other hand, it includes the preparation of activated antigen-specific cells for immunotherapy and the administration of the cell products.

[0254] Neoantigens for T cell-based therapy Traditional antigen-targeted immunotherapy has focused on antigens, including tumor-associated antigens (TAAs), cancer-testis antigens (gene products ectopically expressed in tumors, typically germline-restricted), or antigens derived from genes that exhibit tissue-specific expression. However, tumors also display mutated gene protein products called neoantigens. The number and type of mutations can be easily defined using next-generation sequencing approaches and include single amino acid missense mutations, fusion proteins, and novel open reading frames (neoORFs) that vary in length from one to up to 100 or more amino acids. Neoantigens are antigens that contain non-silent mutations in epitopes, meaning the same antigen is not expressed in non-cancer cells within the same human body. Mutation-based antigens are particularly useful because they bypass central tolerance (a process that occurs during normal thymic development that eliminates autoreactive T cells) and demonstrate exquisite tumor specificity. Each non-synonymous (i.e., protein-coding) mutation has the potential to generate neoantigens that can be recognized by the patient's T cells. T cells that recognize such neoantigens can function to both directly kill tumor cells and catalyze a broader immune response against tumors. The methods described herein aim to induce and expand such neoantigen-reactive T cells in a patient-specific manner and to utilize such cells for adoptive cell therapy.

[0255] In some embodiments, a neoantigen as used herein comprises a point mutation.

[0256] In some embodiments, a neoantigen as used herein comprises a frameshift mutation.

[0257] In some embodiments, a neo-antigen as used herein comprises a crossover mutation.

[0258] In some embodiments, a neoantigen as used herein comprises an insertion mutation resulting from the insertion of one or more nucleotides.

[0259] In some embodiments, neoantigens as used herein include deletion mutations resulting from the deletion of one or more nucleotides.

[0260] In some embodiments, the neoantigen can result from an insertion-deletion (in-del) mutation.

[0261] In some embodiments, the antigen or neo-antigenic peptide binds to an HLA protein (e.g., HLA class I or HLA class II). In specific embodiments, the antigen or neo-antigenic peptide binds to an HLA protein with greater affinity than the corresponding wild-type peptide. In specific embodiments, the antigen or neo-antigenic peptide has an IC of at least 5000 nM or less, at least 500 nM or less, at least 100 nM or less, at least 50 nM or less. 50 or K D It has.

[0262] In some embodiments, the antigen or neo-antigenic peptide can be about 8 to about 50 amino acid residues in length, or about 8 to about 30, about 8 to about 20, about 8 to about 18, about 8 to about 15, or about 8 to about 12 amino acid residues in length. In some embodiments, the antigen or neo-antigenic peptide can be about 8 to about 500 amino acid residues in length, or about 8 to about 450, about 8 to about 400, about 8 to about 350, about 8 to about 300, about 8 to about 250, about 8 to about 200, about 8 to about 150, about 8 to about 100, about 8 to about 50, or about 8 to about 30 amino acid residues in length.

[0263] In some embodiments, the antigen or neo-antigenic peptide may be at least 8, 9, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues in length. In some embodiments, the neo-antigenic peptide may be at least 8, 9, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acid residues in length. In some embodiments, the antigen or neo-antigenic peptide may be at most 8, 9, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or fewer amino acid residues in length. In some embodiments, the antigen or neo-antigenic peptide may be at most 8, 9, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or fewer amino acid residues in length.

[0264] In some embodiments, the antigen or neo-antigenic peptide has an overall length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

[0265] In some embodiments, the antigen or neo-antigen peptide has an overall length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids. In some embodiments, the peptide length is between 8 and 15 amino acids in length. In some embodiments, the peptide is 8-12 amino acids in length for targeting CD8+ T cells, 8-11 amino acids in length for targeting CD8+ T cells, and 25 amino acids in length for targeting CD4+ T cells.

[0266] In some embodiments, the neo-antigenic peptide can have a pI value of about 0.5 to about 12, about 2 to about 10, or about 4 to about 8. In some embodiments, the neo-antigenic peptide can have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or more. In some embodiments, the neo-antigenic peptide can have a pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or less.

[0267] In some embodiments, the antigen or neo-antigenic peptide can have an HLA binding affinity of about 1 pM to about 1 mM, about 100 pM to about 500 μM, about 500 pM to about 10 μM, about 1 nM to about 1 μM, or about 10 nM to about 1 μM. In some embodiments, the antigen or neo-antigenic peptide can have an HLA binding affinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 μM or more. In some embodiments, the antigen or neo-antigenic peptide can have an HLA binding affinity of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 μM.

[0268] In some embodiments, the antigen or neo-antigenic peptides described herein can include a carrier such as those known in the art, for example, thyroglobulin, albumin such as human serum albumin, tetanus toxoid, polyamino acid residues such as poly-L-lysine, poly-L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and others.

[0269] In some embodiments, the antigens or neo-antigenic peptides described herein are acylated by terminal NH2 acylation, e.g., alkanoyl (C1-C 20 ) or thioglycolyl acetylation, terminal carboxylamidation, e.g., with ammonia, methylamine, etc. In some embodiments, these modifications can provide sites for linking to a support or other molecules.

[0270] In some embodiments, the antigen or neo-antigen peptides described herein can contain modifications, including but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surface active materials, e.g., lipids, etc., or can be chemically modified, e.g., acetylated, etc. Additionally, the bonds in the peptides can be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0271] In some embodiments, the antigenic or neo-antigenic peptides described herein can contain substitutions that alter the physical properties (e.g., stability or solubility) of the resulting peptide. For example, antigenic or neo-antigenic peptides can be modified by substitution of cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical nature, cysteine ​​has a tendency to form disulfide bridges, sufficiently altering the peptide structurally to reduce binding capacity. Substitution of α-aminobutyric acid for C not only alleviates this problem, but in certain instances, actually improves binding and cross-linking capacity. Substitution of cysteine ​​with α-aminobutyric acid can occur at any residue in the antigenic or neo-antigenic peptide, for example, at either anchor or non-anchor positions of the epitope or analog within the peptide, or at other positions in the peptide.

[0272] In some embodiments, the antigenic peptides or neo-antigenic peptides described herein contain amino acid mimetics or unnatural amino acid residues, such as D- or L-naphtylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1, 2, 3, or 4-pyreneylalanine; D- or L-3-thienylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine. The peptides may include lysine, D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine, D-ρ-fluorophenylalanine, D- or L-ρ-biphenyl-phenylalanine, D- or L-ρ-methoxybiphenylphenylalanine, D- or L-2-indole(allyl)alanine, and D- or L-alkylalanine, where the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, isopentyl, or a non-acidic amino acid residue. Aromatic rings of unnatural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides containing various amino acid mimetics or unnatural amino acid residues are particularly useful because they tend to exhibit increased stability in vivo. Such peptides may also possess improved shelf life or manufacturing characteristics.

[0273] In some embodiments, the peptide is contacted with an immune cell to activate the cell and make it antigen-responsive.

[0274] In some embodiments, the peptide is contacted with the immune cells ex vivo.

[0275] In some embodiments, the peptide is contacted with an immune cell in a biological system, for example, a human.

[0276] In some embodiments, the immune cell is an antigen-presenting cell.

[0277] In some embodiments, the immune cell is a T cell.

[0278] The present disclosure relates to methods for producing T cells that are specific for an immunogenic antigen.

[0279] The present disclosure also relates to a composition comprising antigen-specific T cells stimulated by APC. In some embodiments, one or more antigen peptides are loaded onto APC, and then the peptide-loaded APC is used to stimulate T cells to produce antigen-specific T cells. In some embodiments, the antigen is a neoantigen. In some embodiments, the APC used for peptide loading is a dendritic cell.

[0280] In some embodiments, the peptide sequence comprises a mutation that is not present in the non-cancer cells of the subject. In some embodiments, the peptide is encoded by a gene or expressed gene in the cancer cells of the subject. In some embodiments, the peptide sequence has a length of at least 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 40; 50; 60; 70; 80; 90; 100; 150; 200; 250; 300; 350; 400; 450; 500; 600; 700; 800; 900; 1,000; 1,500; 2,000; 2,500; 3,000; 4,000; 5,000; 7,500; or 10,000 or more naturally occurring amino acids.

[0281] In some embodiments, the peptide sequence binds to proteins encoded by class I HLA alleles and has a length of 8 to 12 naturally occurring amino acids. In some embodiments, the peptide sequence binds to proteins encoded by class II HLA alleles and has a length of 16 to 25 naturally occurring amino acids. In some embodiments, the peptide sequence comprises a plurality of antigenic peptide sequences. In some embodiments, the plurality of antigenic peptide sequences comprises at least 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, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 antigenic peptide sequences. In some embodiments, the antigenic peptide sequences comprise unmodified peptide bonds between the amino acids in the sequence. In some embodiments, multiple antigenic peptide sequences are linked together by a linker. In some embodiments, the linker sequence comprises G and S amino acids, such as GSS, GSSS, or GGGS. In some embodiments, the linker may be a modified linker having a cleavable sequence. Exemplary cleavable sequences include self-cleaving sequences such as T2A and P2A. In some embodiments, the antigenic peptide sequence may comprise a modification, for example, an MITD sequence or an SP1 signaling domain.

[0282] In some embodiments, APCs are transfected or transduced with a nucleic acid encoding a peptide sequence comprising one or more antigenic peptide sequences. The APCs express the antigenic peptide sequences and present the antigen in association with MHC to T cells, thereby activating the T cells. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA.

[0283] In some embodiments, the antigens described herein are neoantigens. Candidate immunogenic neoantigen sequences can be identified by any suitable method known in the art. The methods of the present disclosure can be useful, for example, for producing a treatment specific to a disease of a subject or for producing a vaccine against a disease. Candidate immunogenic neoantigens can be previously identified neoantigens. In some embodiments, candidate immunogenic neoantigens may not have been previously identified. Candidate immunogenic neoantigens for use in the methods and compositions described herein can be specific to a subject. In some embodiments, candidate neoantigens for use in the methods and compositions described herein can be specific to multiple subjects.

[0284] In both animals and humans, mutated epitopes can be potentially effective in inducing immune responses or activating T cells. In one embodiment, potentially immunogenic epitopes of an infectious pathogen, such as a virus, can be determined in a subject. In one embodiment, potentially immunogenic mutated epitopes can be determined in a subject with a disease, such as cancer. In some embodiments, potentially immunogenic antigens or neoantigens for use in the methods described herein can be differentiation antigens expressed in tumors and cells of the tissue type in which they were generated. In some embodiments, potentially immunogenic antigens or neoantigens for use in the methods described herein can be cancer / germline antigens that are not expressed in other differentiated tissues. In some embodiments, potentially immunogenic antigens or neoantigens for use in the methods described herein can be mutated antigens. For example, candidate immunogenic antigen or neoantigen peptides for use in the methods described herein can include antigens or neoantigens of fusion proteins generated by missense point mutations or tumor-specific translocations of gene segments. In some embodiments, potentially immunogenic antigens or neoantigens for use in the methods described herein can be overexpressed antigens. In some embodiments, the potentially immunogenic antigen or neoantigen can be found in a tumor. For example, a potentially immunogenic antigen or neoantigen for use in the methods described herein can include a protein whose expression is tightly regulated in cells of differentiated normal tissue.

[0285] Potentially immunogenic mutated epitopes can be determined by genome or exome sequencing of tumor tissues and healthy tissues from cancer patients using next-generation sequencing technology. For example, genes selected based on their mutation frequency and ability to act as antigens or neoantigens can be sequenced using next-generation sequencing technology. In one embodiment, the sequencing data can be analyzed to identify potentially immunogenic mutated peptides that can bind to the target HLA molecule. In one embodiment, the data can be analyzed using a computer. In another embodiment, the sequence data can be analyzed for the presence of antigens or neoantigen peptides. In one embodiment, potentially immunogenic antigens or neoantigen peptides can be determined by their affinity for MHC molecules.

[0286] Potentially immunogenic antigens or neo-antigenic peptides can be determined by direct protein sequencing. For example, protein sequencing of enzymatic protein digests using multidimensional mass spectrometry techniques (e.g., tandem mass spectrometry (MS / MS)) can be used to identify potentially immunogenic antigens or neo-antigenic peptides for use in the methods described herein.

[0287] High-throughput methods for de novo sequencing of unknown proteins can be used to identify potentially immunogenic antigens or neo-antigenic peptides. For example, high-throughput methods for de novo sequencing of unknown proteins, such as meta-shotgun protein sequencing, can be used to analyze the proteome of a subject's tumor to identify potentially immunogenic expressed neo-antigens.

[0288] Potentially immunogenic antigens or neo-antigen peptides can also be identified using MHC multimers to identify antigen-specific T cell responses. For example, high-throughput analysis of antigen-specific T cell responses in patient samples can be performed using MHC tetramer-based screening techniques. Tetramer-based screening techniques can be used for the initial identification of potentially immunogenic tumor-specific antigens or as a secondary screening protocol to assess which potentially immunogenic antigens a patient may have already been exposed to, thereby facilitating the selection of potentially immunogenic antigens for use in the methods described herein.

[0289] In some embodiments, specific neoantigens are targeted for immunotherapy. In some embodiments, the neoantigenic peptides are synthetic. The neoantigenic peptides used herein are designed so that each peptide is specific to an HLA antigen and can bind to the HLA antigen with high binding affinity and specificity. In some embodiments, the peptides used herein are designed based on a high-performance HLA binding prediction model created by the inventors, as described, for example, in the following patent applications / publications: WO2011143656, WO2017184590, and U.S. Provisional Patent Applications Nos. 62 / 783,914 and 62 / 826,827, all of which are incorporated herein by reference. NetMHCIIpan, even though it is the current prediction standard, cannot be considered foolproof. Of the three class II loci (DR, DP, and DQ), data may exist only for certain common alleles of HLA-DR. Briefly, the newly created predictive model is useful for identifying immunogenic antigen peptides and can be used for the development of drugs such as personalized medicines and the isolation and characterization of antigen-specific T cells. The machine learning HLA-peptide presentation predictive model includes a plurality of predictor variables identified based at least on training data, where the training data includes: sequence information of peptide sequences presented by HLA proteins expressed in cells and identified by mass spectrometry; training peptide sequence information including amino acid position information, the training peptide sequence information associated with the HLA proteins expressed in cells; and a function representing the relationship between the amino acid position information received as input and the presentation probability created as output based on the amino acid position information and the predictive variables. CD4+ T cell responses can have antitumor activity. In existing prediction methods, a high rate of CD4+ T cell responses can be demonstrated without using class II prediction (e.g., 60% of SLP epitopes in the NeoVax study (49% in NT-001) and 48% of mRNA epitopes in the BioNTech study). It may not be clear whether these epitopes are typically presented natively (by tumors or by phagocytic DCs).Therefore, improving the identification of naturally presented class II epitopes is desirable to translate high CD4+ T cell response rates into therapeutic efficacy. The roles of gene expression, enzymatic cleavage, and pathway / localization bias may not have been robustly quantified. While most existing MS data can be presumed to be derived from autophagy, it may be unclear whether autophagy (class II presentation by tumor cells) or phagocytosis (class II presentation of tumor epitopes by APCs) is the more relevant pathway. Different data generation approaches for learning the rules of class II presentation may exist, including field standards and proposed approaches. Field standards may include affinity measurements, which offer low throughput and require radioactive reagents and may be the basis for NetMHCIIpan predictors, which miss the role of processing. Emerging approaches include mass spectrometry; data from cell lines / tissues / tumors can help determine processing rules for autophagy (much of this data has already been published), and single-allelic MS can enable the determination of allele-specific binding rules (multi-allelic MS data is presumed to be overly complex for efficient learning). The newly created prediction method includes a step of training a machine-learned HLA-peptide presentation prediction model, wherein the training step includes a step of inputting, using a computer processor, amino acid positional information sequences of HLA-peptides isolated from one or more HLA-peptide complexes derived from cells expressing HLA class II alleles into the HLA-peptide presentation prediction model, wherein the machine-learned HLA-peptide presentation prediction model includes a plurality of predictor variables identified based at least on training data, wherein the training data includes: sequence information of peptide sequences presented by HLA proteins expressed in cells and identified by mass spectrometry; training peptide sequence information including amino acid positional information of training peptides, the training peptide sequence information being associated with the HLA proteins expressed in cells; and a function representing the relationship between the amino acid positional information received as input and the presentation likelihood created as output, based on the amino acid positional information and the predictor variables.In some embodiments, the proposed model has a positive predictive value of at least 0.25 at a recall of 0.1% to 10%. In some embodiments, the proposed model has a positive predictive value of at least 0.4 at a recall of 0.1% to 10%. In some embodiments, the proposed model has a positive predictive value of at least 0.6 at a recall of 0.1% to 10%. In some embodiments, the mass spectrometry is single-allele mass spectrometry. In some embodiments, the peptides are presented by HLA proteins expressed in cells by autophagy. In some embodiments, the peptides are presented by HLA proteins expressed in cells by phagocytosis. In some embodiments, the quality of the training data is increased by using multiple quality metrics. In some embodiments, the multiple quality metrics include common contaminant peptide removal, high scored peak intensity, high score, and high mass accuracy. In some embodiments, the scored peak intensity is at least 50%. In some embodiments, the scored peak intensity is at least 70%. In some embodiments, the peptides presented by HLA proteins expressed in cells are peptides presented by a single immunoprecipitated HLA protein expressed in cells. In some embodiments, the plurality of predictor variables includes a peptide-HLA affinity predictor variable. In some embodiments, the plurality of predictor variables includes a source protein expression level predictor variable. In some embodiments, the plurality of predictor variables includes a peptide cleavability predictor variable. In some embodiments, the peptides presented by HLA proteins include peptides identified by searching a peptide database using a reversed-database search strategy. In some embodiments, the HLA protein is an HLA-DR protein, and an HLA-DP or HLA-DQ protein. In some embodiments, the HLA protein is an HLA-DR protein selected from the group consisting of an HLA-DR protein, and an HLA-DP or HLA-DQ protein.In some embodiments, the HLA proteins are HLA-DPB1*01:01 / HLA-DPA1*01:03, HLA-DPB1*02:01 / HLA-DPA1*01:03, HLA-DPB1*03:01 / HLA-DPA1*01:03, HLA-DPB1*04:01 / HLA-DPA1*01:03, HLA-DPB1*04:02 / HLA-DPA1*01:03, HLA-DPB1*06:01 / HLA-DPA1*01:03, HLA-DQB1*02:01 / HLA -DQA1*05:01, HLA-DQB1*02:02 / HLA-DQA1*02:01, HLA-DQB1*06:02 / HLA-DQA1*01:02, HLA-DQB1*06:04 / HLA-DQA1*01:02, HLA-DRB 1*01:01, HLA-DRB1*01:02, HLA-DRB1*03:01, HLA-DRB1*03:02, HLA-DRB1*04:01, HLA-DRB1*04:02, HLA-DRB1*04:03, HLA-DRB1*04 :04, HLA-DRB1*04:05, HLA-DRB1*04:07, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*08:02, HLA-DRB1*08:03, HLA-DRB1*08:04, HLA-DRB1*09:01, HLA-DRB1*10:01, HLA-DRB1*11:01, HLA-DRB1*11:02, HLA-DRB1*11:04, HLA-DRB1*12:01, HLA-DRB1*12:02, HLA- In some embodiments, the peptides presented by the HLA proteins include peptides identified by comparing the MS / MS spectrum of the HLA-peptide to the MS / MS spectrum of one or more HLA-peptides in a peptide database.

[0290] In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a readthrough mutation, and a gene fusion mutation.

[0291] In some embodiments, the peptides presented by the HLA proteins have a length of 15 to 40 amino acids. In some embodiments, the peptides presented by the HLA proteins include peptides identified by (a) isolating one or more HLA complexes from a cell line expressing a single HLA class II allele; (b) isolating one or more HLA-peptides from the one or more isolated HLA complexes; (c) obtaining MS / MS spectra for the one or more isolated HLA-peptides; and (d) obtaining peptide sequences corresponding to the MS / MS spectra of the one or more isolated HLA-peptides from a peptide database, wherein the one or more sequences obtained from step (d) identify the sequences of the one or more isolated HLA-peptides.

[0292] Various antigenic peptides can be used to induce or expand T cells. Various antigenic peptides can be used to activate antigen-presenting cells (APCs), and then, by contacting T cells with the antigen-loaded APCs, the APCs can activate T cells.

[0293] In some embodiments, the peptide comprises a mutation selected from (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a readthrough mutation, (E) a gene fusion mutation, and combinations thereof. In some embodiments, the peptide comprises a point mutation and binds to an HLA protein of interest with greater affinity than the corresponding wild-type peptide.

[0294] In some embodiments, the peptide has an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM.50 In some embodiments, the peptide binds to an HLA protein of interest with an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K D In some embodiments, each peptide binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, the TCR of the induced or expanded antigen-specific T cells has an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K D In some embodiments, the TCR binds to a peptide-HLA complex with an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K D and bind to a peptide-HLA complex. In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in non-cancer cells of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or expressed gene in cancer cells of the subject.

[0295] In some embodiments, the peptide has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 or more naturally occurring amino acids. In some embodiments, the peptide binds to a protein encoded by a class I HLA allele and has a length of 8 to 12 naturally occurring amino acids. In some embodiments, the peptide binds to a protein encoded by a class II HLA allele and has a length of 16 to 25 naturally occurring amino acids. In some embodiments, the peptide comprises a plurality of peptides. In some embodiments, the plurality of peptides comprises at least 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, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 or more antigenic peptides.

[0296] In some aspects, the present disclosure provides peptides or polynucleotides encoding peptides identified using the methods briefly described herein above (e.g., peptides with tumor-specific mutations, viral peptides, or peptides associated with non-cancerous diseases).

[0297] In some embodiments, optical methods are used to select or identify immunogenic antigens. In some embodiments, barcoded probes are used to select or identify immunogenic antigens. In some embodiments, barcoded probes comprising a target-specific region and a barcoded region are used to select or identify immunogenic antigens. In some embodiments, the target-specific region comprises a nucleic acid sequence that hybridizes to or has at least about 90%, 95%, or 100% sequence complementarity to the nucleic acid sequence of the target polynucleotide.

[0298] Preparation of activated antigen-specific T cells Provided herein is a method for stimulating T cells. For example, the method provided herein can be used to stimulate antigen-specific T cells. The method provided herein can be used to induce or activate T cells. For example, the method provided herein can be used to expand activated T cells. For example, the method provided herein can be used to induce naive T cells. For example, the method provided herein can be used to stimulate antigen-specific CD8 + T cells can be expanded, for example, antigen-specific CD4 T cells can be expanded using the methods provided herein. + T cells can be expanded, for example, to produce antigen-specific CD8 T cells with a memory phenotype using the methods provided herein. + T cells can be expanded. For example, the therapeutic composition can include antigen-specific CD8+ T cells. For example, the therapeutic composition can include antigen-specific memory T cells.

[0299] T cells can be activated ex vivo with a composition comprising a neo-antigenic peptide or a polynucleotide encoding the neo-antigenic peptide.

[0300] T cells can be activated ex vivo with a composition comprising antigen-loaded antigen-presenting cells.

[0301] In some embodiments, the APCs and / or T cells are derived from a biological sample obtained from a subject.

[0302] In some embodiments, the APCs and / or T cells are derived from a biological sample that is a peripheral blood mononuclear cell (PBMC).

[0303] In some embodiments, the subject is administered FLT3L prior to obtaining the biological sample for preparing APCs and / or T cells.

[0304] In some embodiments, the APCs and / or T cells are derived from a biological sample that is a leukapheresis sample.

[0305] In some embodiments, antigen-presenting cells are first loaded with neo-antigenic peptides ex vivo and used to prepare neo-antigen-activated T cells. In some embodiments, the compositions provided herein include T cells stimulated by APCs, such as APCs pre-loaded with antigenic peptides. The compositions can include a population of immune cells, including T cells, derived from a sample (e.g., a biological sample), where the T cells include T cells stimulated by APCs. In some embodiments, mRNA encoding one or more neo-antigenic peptides is introduced into the APCs for expression of the neo-antigenic peptides. Such APCs are used to stimulate or activate T cells.

[0306] In some embodiments, the biological sample comprises a percentage of at least one antigen-specific T cell in the composition that is at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%. In some embodiments, the biological sample comprises less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% of antigen-activated T cells of the total cell count in a biological sample derived from peripheral blood or leukapheresis. In some embodiments, the biological sample comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% antigen-activated T cells of the total cell count in a biological sample derived from peripheral blood or leukapheresis. In some embodiments, the biological sample comprises antigen-naive T cells. In some embodiments, the biological sample comprises greater than about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% antigen naive cells of the total cell count in a biological sample derived from peripheral blood or leukapheresis. In some embodiments, the at least one antigen-specific CD8 + The percentage of T cells is less than about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% in a biological sample derived from peripheral blood or leukapheresis. In some embodiments, the composition contains at least one antigen-specific CD4 +The percentage of T cells is at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% in a biological sample derived from peripheral blood or leukapheresis. In some embodiments, the percentage of at least one antigen-specific T cell in a biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of total immune cells. In some embodiments, the percentage of at least one antigen-specific CD8 T cell in a biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of total immune cells. + The percentage of T cells is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total immune cells. In some embodiments, the percentage of T cells is at most about 0.00001%, 0.00005%, 0.0001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total immune cells. + The percentage of T cells is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of total immune cells. In some embodiments, the percentage of antigen-specific T cells in the biological sample is at most about 0.5%. In some embodiments, the percentage of neoantigen-specific CD8 T cells in the biological sample is at most about 0.5%. + In some embodiments, the percentage of T cells in the biological sample is at most about 0.5%. + The percentage of T cells is at most about 0.5% in a biological sample.

[0307] Preparation of neo-antigen-loaded APCs In some embodiments, the composition comprises a population of immune cells incubated with one or more cytokines, growth factors, or ligands, such as ligands that bind to cell surface receptors on APCs or T cells. Non-limiting examples of such cytokines, growth factors, and ligands include, but are not limited to, GM-CSF, IL-4, IL-7, FLT3L, TNF-α, IL-1β, IL-15, PGE1, IL-6, IFN-α, IFN-γ, R848, LPS, ss-RNA40, and poly I:C. In some embodiments, the composition comprises a population of immune cells incubated with one or more APCs or an APC preparation. For example, the composition can comprise APCs stimulated with one or more cytokines, growth factors, and / or ligands, or a population of immune cells incubated with an APC preparation stimulated with cytokines, growth factors, and / or ligands. For example, the composition can comprise APCs stimulated with one or more cytokines, growth factors, and / or ligands, or a population of immune cells incubated with an APC preparation stimulated with cytokines, growth factors, and / or ligands. For example, the composition can include a population of immune cells incubated with APCs stimulated with one or more growth factors or a growth factor-stimulated APC preparation. For example, the composition can include a population of immune cells incubated with APCs stimulated with one or more ligands or a ligand-stimulated APC preparation.

[0308] In some embodiments, the APCs are autologous APCs, allogeneic APCs, or artificial APCs.

[0309] Immune cells are characterized by cell surface molecules.In some embodiments, immune cells are preferably selected based on cell surface markers, for example, by using antibodies that can bind to cell surface receptors from biological samples.In some embodiments, some cells are negatively selected to enrich one or more cell types that do not express the cell surface molecules that are negatively selected.

[0310] In some embodiments, antigen-presenting cells (APCs) are prepared from a biological sample by selecting from APCs or precursor cells, which can be cultured in the presence of neo-antigenic peptides to generate neo-antigen-loaded APCs, which are used to activate T cells. Some relevant cell surface markers for selecting and / or enriching sets of cells are described below.

[0311] CD1 (cluster of differentiation 1) is a family of glycoproteins expressed on the surface of various human antigen-presenting cells. It associates with class I MHC molecules and is involved in the presentation of lipid antigens to T cells.

[0312] CD11b or integrin alpha M (ITGAM) is a heterodimeric integrin alpha-M beta-2 (α), also known as macrophage-1 antigen (Mac-1) or complement receptor 3 (CR3). M ITGAM is one protein subunit that forms the αβ2 molecule. ITGAM is also known as CR3A and cluster of differentiation molecule 11b (CD11b). M The second chain of β2 is the common integrin β2 subunit known as CD18, and thus integrin α M β2 belongs to the β2 subfamily (or leukocyte) integrins. M Integrin β2 is expressed on the surface of many leukocytes involved in the innate immune system, including monocytes, granulocytes, macrophages, and natural killer cells. It mediates inflammation by regulating leukocyte adhesion and migration, and has been implicated in several immune processes, such as phagocytosis, cell-mediated cytotoxicity, chemotaxis, and cell activation. It participates in the complement system through its ability to bind inactivated complement component 3b (iC3b). Integrin α M The ITGAM (alpha) subunit of β2 is directly involved in initiating cell adhesion and spreading, but cannot mediate cell migration without the presence of the β2 (CD18) subunit.

[0313] CD11c, also known as integrin, alpha X (complement component 3 receptor subunit 4) (ITGAX), is the gene encoding CD11c. CD11c is an integrin alpha X chain protein. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. This protein combines with a beta 2 chain (ITGB2) to form a leukocyte-specific integrin called inactivated C3b (iC3b) receptor 4 (CR4). The alpha X beta 2 complex appears to overlap with the properties of alpha M beta 2 integrin in the adhesion of neutrophils and monocytes to stimulated endothelial cells and in the phagocytosis of complement-coated particles. CD11c is a type I transmembrane protein found at high levels on the surface of most human dendritic cells, but also on the surface of monocytes, macrophages, neutrophils, and some B cells; it induces cell activation and helps trigger the neutrophil respiratory burst; it is expressed in hairy cell leukemia, acute nonlymphocytic leukemia, and some B-cell chronic lymphocytic leukemias.

[0314] CD14 is a surface antigen preferentially expressed on monocytes / macrophages. It mediates the innate immune response to bacterial lipopolysaccharides in cooperation with other proteins. Alternative splicing results in multiple transcript variants encoding the same protein. CD14 exists in two forms: one membrane-anchored by a glycosylphosphatidylinositol tail (mCD14) and the other soluble form (sCD14). Soluble CD14 appears after shedding of mCD14 (48 kDa) or is directly secreted from intracellular vesicles (56 kDa). CD14 acts as a coreceptor (together with the Toll-like receptors TLR4 and MD-2) for the detection of bacterial lipopolysaccharide (LPS). CD14 can bind LPS only in the presence of lipopolysaccharide-binding protein (LBP). Although LPS is considered its primary ligand, CD14 also recognizes other pathogen-associated molecular patterns such as lipoteichoic acid.

[0315] CD25 is expressed by conventional T cells after stimulation and is expressed by CD4 + CD25 hi Only T cells have been shown to be "suppressors."

[0316] In some embodiments, the APCs comprise dendritic cells (DCs). In some embodiments, the APCs comprise CD14 + In some embodiments, APCs are derived from monocytes. In some embodiments, APCs can be obtained from skin, spleen, bone marrow, thymus, lymph nodes, peripheral blood, or umbilical cord blood. In some embodiments, CD14 + Monocytes are derived from a biological sample from a subject, including PBMCs. For example, CD14 + Monocytes may be isolated, enriched, or purified from a biological sample from a subject, including PBMCs. + The monocytes are stimulated with one or more cytokines or growth factors. In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, R848, LPS, ss-rna40, poly I:C, or a combination thereof. In some embodiments, CD14 + The monocytes are derived from a second biological sample containing PBMCs.

[0317] In some embodiments, the isolated population of APCs can be enriched or substantially enriched. In some embodiments, the isolated population of APCs is at least 30%, at least 50%, at least 75%, or at least 90% homogeneous. In some embodiments, the isolated population of APCs is at least 60%, at least 75%, or at least 90% homogeneous. APCs, such as APCs, can include, for example, APCs derived from monocytic dendritic cell precursors in culture, as well as endogenously derived APCs present in tissues such as peripheral blood, umbilical cord blood, skin, spleen, bone marrow, thymus, and lymph nodes.

[0318] APCs and cell populations substantially enriched in APCs can be isolated by methods also provided by the present invention, which generally involve obtaining a population of cells containing APC precursors and differentiating the APC precursors into immature or mature APCs, and can also involve isolating APCs from the population of differentiated immature or mature APCs.

[0319] APC precursor cells can be obtained by methods known in the art. APC precursors can be isolated, for example, by density gradient separation, fluorescence-activated cell sorting (FACS), immunological cell separation techniques such as panning, complement lysis, rosetting, magnetic cell separation techniques, nylon wool separation, and combinations of such methods. Methods for immunoselecting APCs include using antibodies against cell surface markers associated with APC precursors, such as anti-CD34 and / or anti-CD14 antibodies coupled to a substrate.

[0320] It is also possible to obtain a population enriched in APC precursors.Methods for obtaining such enriched precursor populations are known in the art.For example, enriched populations of APC precursors can be isolated from tissue sources by selectively removing cells that adhere to a substrate.For example, using tissue sources such as bone marrow or peripheral blood, adhesive monocytes can be removed from cell preparations using commercially available plastic substrates (for example, beads or magnetic beads) to obtain a population enriched in non-adherent APC precursors.

[0321] Monocytic APC precursors can also be obtained from tissue sources by using an APC precursor adhesion substrate. For example, peripheral blood leukocytes isolated by leukapheresis are contacted with a monocytic APC precursor adhesion substrate having a high surface area-to-volume ratio, and adherent monocytic APC precursors are isolated. In additional embodiments, the coupled substrate can be a granular or fibrous substrate having a high surface-to-volume ratio, such as microbeads, microcarrier beads, pellets, granules, powders, capillaries, microvillous membranes, or the like. Furthermore, the granular or fibrous substrate can be glass, polystyrene, plastic, glass-coated polystyrene microbeads, or the like.

[0322] APC precursors can also be cultured in vitro for differentiation and / or expansion. Methods for differentiation / expansion of APC precursors are known in the art. Generally, expansion can be achieved by culturing the precursors in the presence of at least one cytokine that induces APC (e.g., dendritic cell) differentiation / proliferation. Typically, such cytokines are granulocyte colony-stimulating factor (G-CSF) or granulocyte / macrophage colony-stimulating factor (GM-CSF). In addition, other agents can be used to inhibit proliferation and / or maturation of non-APC cell types in culture, thereby further enriching the population of APC precursors. Typically, such agents include cytokines such as IL-13, IL-4, or IL-15, among others.

[0323] The isolated population of APC precursors is cultured and differentiated to obtain immature or mature APCs. Suitable tissue culture media include, but are not limited to, AIM-V®, RPMI 1640, DMEM, X-VIVO, and others. The tissue culture medium is typically supplemented with amino acids, vitamins, divalent cations, and cytokines to promote differentiation of the precursors toward an APC phenotype. Typically, the differentiation-promoting cytokines are GM-CSF and / or IL-4.

[0324] Furthermore, cultures of APC precursors during expansion, differentiation, and maturation to the APC phenotype can contain plasma to promote APC development. A typical plasma concentration is about 5%. In addition, for example, when APC precursors are isolated by adherence to a substrate, CD14 + Plasma may be included in the culture medium during the adherence step to enhance the phenotype, with typical plasma concentrations during adherence being about 1% or more.

[0325] The monocytic APC precursors can be cultured for any suitable period of time. In certain embodiments, the culture time suitable for differentiation of the precursors into immature APCs can be about 1 to about 10 days, for example, about 4 to about 7 days. Differentiation of the precursors into immature APCs can be achieved by detecting cell surface markers (e.g., CD11c + , CD83 low , CD86 - / low , HLA-DR + Immature APCs can be monitored by methods known to those skilled in the art, such as by the presence or absence of IL-4. Immature APCs can also be cultured in an appropriate tissue culture medium to maintain them in a state for further differentiation or antigen uptake, processing, and presentation. For example, immature APCs can be maintained in the presence of GM-CSF and IL-4.

[0326] In some embodiments, APC precursors can be isolated prior to differentiation. In some embodiments, the isolated population can be enriched or substantially enriched for APC precursors. In some embodiments, APC precursors are isolated using a CD14-specific probe. In an exemplary embodiment, CD14-expressing cells are detected by FACS using a CD14-specific probe directly conjugated to a fluorescent molecule (e.g., FITC or PE), or by an unlabeled antibody specific to CD14 and a labeled second antibody specific to this first antibody. CD14 + Cells were purified by FACS sorting to identify CD14 low and CD14 - It can also be isolated from cells.high Positive gating can be determined, for example, by reference to CD14 staining in PBMC-derived monocytes. Typically, the CD14-specific binding agent is, for example, an anti-CD14 antibody (e.g., a monoclonal antibody or an antigen-binding fragment thereof). Many anti-CD14 antibodies suitable for use in the present invention are well known to those skilled in the art, and many of them can be purchased commercially. Differentiation into immature APCs (CD14 negative) can be performed after isolation.

[0327] In another embodiment, a CD14-specific probe is coupled to the substrate and CD14 + The cells are isolated by affinity selection. + A population of cells containing CD14 cells is exposed to the coupled substrate. + The cells are allowed to adhere specifically. Then, non-adherent CD14 - The cells are washed off the substrate, and then the adherent cells are eluted to obtain an isolated cell population that is substantially enriched in APC precursors. The CD14-specific probe can be, for example, an anti-CD14 antibody. The substrate can be, for example, a commercially available tissue culture plate or beads (e.g., glass or magnetic beads). Methods for affinity isolation of cell populations using antibodies coupled to substrates specific for surface markers are generally known.

[0328] During culture, the immature APCs can be exposed to a predetermined antigen as needed. Suitable predetermined antigens can include any antigen for which T cell modulation is desired. In one embodiment, immature APCs are cultured in the presence of prostate-specific membrane antigen (PSMA) for cancer immunotherapy and / or tumor growth inhibition. Other antigens can include, for example, bacterial cells, viruses, partially purified or purified bacterial or viral antigens, tumor cells, tumor-specific or tumor-associated antigens (e.g., tumor cell lysates, tumor cell membrane preparations, antigens isolated from tumors, fusion proteins, liposomes, etc.), recombinant cells expressing antigens on their surface, autoantigens, and any other antigens. Any of the antigens can also be presented as peptides or recombinantly produced proteins or portions thereof. After contact with the antigen, the cells can be cultured for any suitable period of time to allow antigen uptake and processing, thereby expanding the population of antigen-specific APCs, or otherwise.

[0329] For example, in one embodiment, immature APCs can be cultured after antigen uptake to promote maturation of immature APCs into mature APCs that present antigen in the context of MHC molecules. Methods for APC maturation are known. Such maturation can be achieved, for example, by culturing in the presence of known maturation factors, such as cytokines (e.g., TNF-α, IL-1β, or CD40 ligand), bacterial products (e.g., LPS or BCG), and others. Maturation of immature APCs into mature APCs can be monitored by methods known in the art, such as, for example, measuring the presence or absence of cell surface markers (e.g., upregulation of CD83, CD86, and MHC molecules), or by testing for the expression of mature APC-specific mRNA or protein using, for example, an oligonucleotide array.

[0330] If desired, immature APCs can be cultured in an appropriate tissue culture medium to expand the cell population and / or maintain the immature APCs in a state for further differentiation or antigen uptake. For example, immature APCs can be maintained and / or expanded in the presence of GM-CSF and IL-4. Immature APCs can also be cultured in the presence of anti-inflammatory molecules, such as anti-inflammatory cytokines (e.g., IL-10 and TGF-β), to inhibit immature APC maturation.

[0331] In another embodiment, the isolated population of APCs is enriched for mature APCs. The differentiated population of immature APCs can be cultured in the presence of the above-mentioned maturation factors (e.g., bacterial products and / or pro-inflammatory cytokines), thereby inducing maturation, to obtain an isolated population of mature APCs. Immature APCs can be isolated by removing CD14+ cells.

[0332] In yet another embodiment of the present invention, APCs can be preserved, for example, by cryopreservation, either before or after exposure to an appropriate antigen. Cryopreservatives that can be used include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate, and inorganic salts. A controlled, slow cooling rate can be crucial. Different cryoprotectants and different cell types typically have different optimal cooling rates. The heat of the fusion phase, in which water turns to ice, should typically be minimized. For example, the cooling procedure can be performed using a programmable freezing device or a methanol bath procedure. A programmable freezing device allows for the determination of the optimal cooling rate and facilitates standard, reproducible cooling. Programmable rate-controlled freezers, such as the Cryomed or Planar, allow for adjustment of the freezing regimen to a desired cooling rate profile.

[0333] After complete freezing, the APCs can be rapidly transferred to a long-term cryogenic storage container. In a typical embodiment, the samples can be cryogenically stored in liquid nitrogen (-196°C) or its vapor (-165°C). Considerations and procedures for the manipulation, cryopreservation, and long-term storage of hematopoietic stem cells, particularly those derived from bone marrow or peripheral blood, are largely applicable to the APCs of the present invention.

[0334] Frozen cells are preferably thawed rapidly (e.g., in a water bath maintained at 37-41°C) and cooled immediately after thawing. It may be desirable to treat the cells to prevent cell clumping after thawing. Various procedures can be used to prevent clumping, including, but not limited to, the pre- and / or post-freezing addition of DNAse, low-molecular-weight dextran and citrate, hydroxyethyl starch, and others. If cryoprotectants are toxic in humans, they should be removed prior to therapeutic use of thawed APCs. One way to remove the cryoprotectant is by dilution to an insignificantly low concentration. Once thawed and recovered, frozen APCs can be used for T cell activation as described herein for unfrozen APCs.

[0335] In one aspect, a composition for T cell activation comprises a population of immune cells depleted of one or more types of immune cells. For example, the composition can comprise a population of immune cells depleted of one or more types of immune cells expressing one or more proteins, such as one or more cell surface receptors. In some embodiments, the composition comprises a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the amount of CD14- and / or CD25-expressing immune cells in the population is different relative to the amount of CD14- and / or CD25-expressing immune cells in the biological sample. For example, the composition can comprise a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the amount of CD14-expressing immune cells in the population is different relative to the amount of CD14-expressing immune cells in the biological sample. For example, the composition can include a population of immune cells derived from a biological sample, the population including at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, wherein the amount of CD25-expressing immune cells in the population is relatively different from the amount of CD25-expressing immune cells in the biological sample. For example, the composition can include a population of immune cells derived from a biological sample, the population including at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, wherein the amount of CD14- and CD25-expressing immune cells in the population is relatively different from the amount of CD14- and CD25-expressing immune cells in the biological sample. For example, the composition can include a population of immune cells derived from a biological sample, the amount of CD14- and CD25-expressing immune cells in the population is relatively lower than the amount of CD14- and CD25-expressing immune cells in the biological sample.

[0336] 1. A method for preparing a cell composition for cancer immunotherapy, comprising: I. preparing antigen-loaded antigen-presenting cells (APCs), the method comprising: (a) obtaining peripheral blood mononuclear cells (PBMCs) from a subject pretreated with fms-like tyrosine kinase 3 ligand (FLT3L); (b) administering to the subject (i) a plurality of cancer neo-antigenic peptides, or one or more polynucleotides encoding a plurality of cancer neo-antigenic peptides, each of which binds to a protein encoded by an HLA allele expressed in the subject, (ii) a stimulant for activating the cells; (iii) an agent for promoting cell growth and maintenance ex vivo to obtain a cell population; and (iv) CD11b. low or contacting PBMCs ex vivo with an agent for reducing or depleting CD11b+ cells from the cell population to obtain CD11b-depleted antigen-loaded APCs; II. CD11b low or contacting ex vivo isolated T cells with CD11b-depleted antigen-loaded APCs; and III. preparing antigen-primed T cells for a cell composition for cancer immunotherapy.

[0337] Provided herein is an improved method for preparing tumor antigen-specific T cells ex vivo, the method comprising: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells, wherein the population of immune cells is derived from a biological sample from a human subject; and (b) inducing the first population of APCs and T cells of step (a) for a first period of time with (i) an FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) cancer cells from (A) a human subject having cancer. (B) incubating the stimulated T cells of step (b) in the presence of a polypeptide comprising at least one tumor antigen epitope sequence expressed by (B) or a polynucleotide encoding the polypeptide, thereby forming a population of cells comprising stimulated T cells; and (c) expanding the stimulated T cells of step (b), thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) the at least one tumor antigen epitope sequence of step (b)(ii) and (ii) an MHC protein expressed by the cancer cells or APCs of the human subject of (b)(ii). Administering the expanded population of cells of (c) to a human subject, wherein the expanded population of cells of step (c) is greater than or equal to 1 x 10 8 ~1×10 11Provided herein are methods for preparing activated antigen-specific T cells, including CD8+ T cells and CD4+ T cells, from naive T cell compartments; the method includes depleting CD14+ cells from PBMCs prior to antigen stimulation and expansion; or depleting CD14+ cells and CD25+ cells, CD14+, CD25+, and CD11b+ cells. Provided herein are methods for preparing activated antigen-specific T cells, including CD8+ T cells and CD4+ T cells, from naive T cell compartments; the method includes depleting CD25+ cells from PBMCs prior to antigen stimulation and expansion; or depleting CD14+ cells and CD25+ cells, CD14+, CD25+, and CD11b+ cells. In some embodiments, provided herein are methods for preparing activated antigen-specific T cells, including CD8+ T cells and CD4+ T cells, from the naive T cell compartment; the process includes depleting CD11b+ cells from PBMCs prior to antigen stimulation and expansion; or depleting CD14+ and CD25+ cells, or CD14+, CD25+ and CD11b+ cells.

[0338] In some embodiments, the subject is pretreated with FLT3L for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week prior to PBMC isolation or leukapheresis. In some embodiments, the subject is pretreated with FLT3L for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks prior to PBMC isolation or leukapheresis.

[0339] In some embodiments, the cell population is enriched for CD11c+ cells. In some embodiments, the antigen-loaded APCs comprise dendritic cells (DCs). In some embodiments, the antigen-loaded APCs comprise plasmacytoid dendritic cells (pDCs). In some embodiments, the antigen-loaded APCs comprise CD1c+ DCs. In some embodiments, the antigen-loaded APCs comprise CD141+ DCs. In some embodiments, the cell population comprises macrophages. In some embodiments, the method further comprises reducing or depleting CD19+ cells from the cell population to activate or enrich neo-antigen-activated T cells. In some embodiments, the method further comprises reducing or depleting both CD11b+ and CD19+ cells from the cell population to activate or enrich neo-antigen-activated T cells.

[0340] In some embodiments, the method further comprises depleting or depleting CD14+ cells from the cell population to prepare and enrich for antigen-activated T cells. In some embodiments, the method further comprises depleting or depleting CD25+ cells from the cell population to prepare and enrich for antigen-activated T cells. In some embodiments, the method further comprises depleting or depleting one or more of CD19+, CD14+, CD25+, or CD11b+ cells from the cell population to activate or enrich for neo-antigen-activated T cells.

[0341] In some embodiments, the stimulant for activating the cells comprises FL3TL.

[0342] In some embodiments, agents that promote cell growth and maintenance ex vivo include growth factors, cytokines, amino acids, supplements, or combinations thereof.

[0343] In some embodiments, the antigen-loaded APCs are capable of stimulating T cells for 2, 3, 4, 5, 6, or 7 days.

[0344] In some embodiments, each of the multiple cancer neo-antigenic peptides is between 8 and 30 amino acids in length.

[0345] In some embodiments, each of the plurality of neo-antigenic peptides comprises a neo-antigenic epitope. In some embodiments, the plurality of cancer neo-antigenic peptides comprises 2, 3, 4, 5, 6, 7, or 8 neo-antigenic peptides; each of the plurality of neo-antigenic peptides has the neo-antigenic peptide characteristics described in the previous section.

[0346] In some embodiments, the neo-antigenic peptide used in preparing antigen-loaded APCs is a long peptide containing at least 20 amino acids, or at least 30 amino acids, or at least 40 amino acids, or at least 50 amino acids, or any number of amino acids therebetween. In some embodiments, the neo-antigenic peptide used in preparing antigen-loaded APCs contains adjacent amino acids on either side of mutations that facilitate endogenous processing of the neo-antigenic peptide for increased presentation to T cells.

[0347] Longer immunogenic peptides can be designed in several ways. In some embodiments, when HLA-binding peptides are predicted or known, the longer immunogenic peptides can consist of (1) individual binding peptides with extensions of 2-5 amino acids toward the N- and C-termini of each corresponding gene product; or (2) concatenation of the respective extension sequences with part or all of the binding peptide. In other embodiments, if sequencing reveals long (>10 residue) epitope sequences, e.g., neoepitopes, present in the tumor (e.g., due to frameshift, readthrough, or intron inclusion resulting in a novel peptide sequence), the longer neo-antigenic peptide can consist of the entire novel tumor-specific stretch of amino acids, either as a single longer peptide or several overlapping longer peptides. In some embodiments, the use of longer peptides is presumed to allow endogenous processing by patient cells, resulting in more effective antigen presentation and induction of T cell responses. In some embodiments, two or more peptides can be used, where these peptides overlap and tile across the long neo-antigenic peptide.

[0348] In some embodiments, each of the plurality of neo-antigenic peptides comprises the same neo-antigenic epitope. In some embodiments, the plurality of neo-antigenic peptides comprises two or more neo-antigenic epitopes.

[0349] In some embodiments, the one or more polynucleotides encoding the multiple cancer neo-antigenic peptides are DNA.

[0350] In some embodiments, one or more polynucleotides encoding multiple cancer neo-antigenic peptides are inserted into one or more mammalian expression vectors.

[0351] In some embodiments, the one or more polynucleotides encoding the multiple cancer neo-antigenic peptides are messenger RNA.

[0352] In some embodiments, the present invention provides RNA, oligoribonucleotide and polyribonucleotide molecules comprising modified nucleosides.

[0353] In some embodiments, the present invention provides gene therapy vectors comprising RNA, oligoribonucleotides and polyribonucleotides.

[0354] In some embodiments, the present invention provides methods of gene therapy and gene transcription silencing, including those described above.

[0355] In some embodiments, the polynucleotide encodes a single neo-antigenic peptide.

[0356] In some embodiments, one polynucleotide encodes two or more neo-antigenic peptides.

[0357] In some embodiments, the polynucleotide is a messenger RNA, hi some embodiments, each messenger RNA comprises coding sequences for two or more neo-antigenic peptides in tandem.

[0358] In some embodiments, each messenger RNA contains tandem coding sequences for two, three, four, five, six, seven, eight, nine, or ten or more neo-antigenic peptides. Typically, the mRNA contains a 5'-UTR, a protein-coding region, and a 3'-UTR. The mRNA has a limited half-life in cells and in vitro. In some embodiments, the mRNA is self-amplifying. In the context of the present invention, the mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methodologies are known to those skilled in the art. For example, various commercially available in vitro transcription methods are available. In vitro transcription kits exist.

[0359] The stability and translation efficiency of RNA can be modified.For example, RNA can be stabilized and its translation can be increased by one or more modifications that have the effect of stabilizing RNA and / or increasing its translation efficiency.Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference.In order to increase the expression of the RNA used according to the present invention, RNA can be modified in the coding region, i.e., the sequence that codes for the peptide or protein to be expressed, so that the GC content can be increased to increase mRNA stability and codon optimization can be performed, thereby enhancing translation in cells, without changing the sequence of the peptide or protein to be expressed.

[0360] In some embodiments, the mRNA can contain multiple neo-antigenic epitopes. In some embodiments, a long polyribonucleotide sequence capable of encoding a neo-ORF can be used, such as a mutated GATA3 sequence encoding a neo-ORF. In some embodiments, the mRNA of most or even the entire coding region of a gene containing a sequence encoding a neo-antigenic peptide is delivered to immune cells for endogenous processing and presentation of the antigen.

[0361] In some embodiments, the coding sequence for each neo-antigenic peptide is between 24 and 120 nucleotides in length.

[0362] In some embodiments, the mRNA is 50 to 10,000 nucleotides in length. In some embodiments, the mRNA is 100 to 10,000 nucleotides in length. In some embodiments, the mRNA is 200 to 10,000 nucleotides in length. In some embodiments, the mRNA is 50 to 5,000 nucleotides in length. In some embodiments, the mRNA is 100 to 5,000 nucleotides in length. In some embodiments, the mRNA is 100 to 1,000 nucleotides in length. In some embodiments, the mRNA is 300 to 800 nucleotides in length. In some embodiments, the mRNA is 400 to 700 nucleotides in length. In some embodiments, the mRNA is 450 to 600 nucleotides in length. In some embodiments, the mRNA is at least 200 nucleotides in length. In some embodiments, the mRNA is more than 250 nucleotides, more than 300 nucleotides, more than 350 nucleotides, more than 400 nucleotides, more than 450 nucleotides, more than 500 nucleotides, more than 550 nucleotides, more than 600 nucleotides, more than 650 nucleotides, more than 700 nucleotides, more than 750 nucleotides, more than 800 nucleotides, more than 850 nucleotides, more than 900 nucleotides, more than 950 nucleotides, more than 1000 nucleotides, more than 2000 nucleotides, more than 3000 nucleotides, more than 4000 nucleotides or more than 5000 nucleotides long.

[0363] In some embodiments, mRNA encoding one or more neo-antigenic peptides is modified, and the modification relates to the 5'-UTR. In some embodiments, the modification relates to providing RNA with a 5'-cap or 5'-cap analog in the 5'-UTR. The term "5'-cap" refers to the cap structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide attached to the mRNA via an unusual 5'-to-5' triphosphate linkage. In some embodiments, this guanosine is methylated at position 7. The term "conventional 5'-cap" refers to the naturally occurring RNA 5'-cap, the 7-methylguanosine cap (mG). In the context of the present invention, the term "5'-cap" includes 5'-cap analogs that have been modified to resemble the RNA cap structure and retain the ability to stabilize RNA and / or enhance RNA translation when attached to RNA in vivo and / or in cells. In some embodiments, the mRNA is co-transcriptionally capped.

[0364] In some embodiments, the mRNA encoding one or more neo-antigenic peptides comprises a 3'-UTR that includes a poly-A tail. In some embodiments, the poly-A tail is 100-200 bp in length. In some embodiments, the poly-A tail is longer than 20 nucleotides. In some embodiments, the poly-A tail is longer than 50 nucleotides. In some embodiments, the poly-A tail is longer than 60 nucleotides. In some embodiments, the poly-A tail is longer than 70 nucleotides. In some embodiments, the poly-A tail is longer than 80 nucleotides. In some embodiments, the poly-A tail is longer than 90 nucleotides. In some embodiments, the poly-A tail is longer than 100 nucleotides. In some embodiments, the poly-A tail is longer than 110 nucleotides. In some embodiments, the poly-A tail is longer than 120 nucleotides. In some embodiments, the poly-A tail is longer than 130 nucleotides. In some embodiments, the poly-A tail is longer than 140 nucleotides. In some embodiments, the poly-A tail is longer than 150 nucleotides. In some embodiments, the poly-A tail is longer than 160 nucleotides. In some embodiments, the poly-A tail is longer than 170 nucleotides. In some embodiments, the poly-A tail is longer than 180 nucleotides. In some embodiments, the poly-A tail is longer than 190 nucleotides. In some embodiments, the poly-A tail is longer than 200 nucleotides. In some embodiments, the poly-A tail is longer than 210 nucleotides. In some embodiments, the poly-A tail is longer than 220 nucleotides. In some embodiments, the poly-A tail is longer than 230 nucleotides. In some embodiments, the poly-A tail is longer than 100 nucleotides. In some embodiments, the poly-A tail is longer than 240 nucleotides. In some embodiments, the poly-A tail is longer than 100 nucleotides. In some embodiments, the poly-A tail is about 250 nucleotides.

[0365] In some embodiments, the poly-A tail comprises 100-250 adenosine units. In some embodiments, the poly-A tail comprises 120-130 adenine units. In some embodiments, the poly-A tail comprises 120 adenine units. In some embodiments, the poly-A tail comprises 121 adenine units. In some embodiments, the poly-A tail comprises 122 adenine units. In some embodiments, the poly-A tail comprises 123 adenine units. In some embodiments, the poly-A tail comprises 124 adenine units. In some embodiments, the poly-A tail comprises 125 adenine units. In some embodiments, the poly-A tail is 129 bases long.

[0366] In some embodiments, the coding sequences for two consecutive neo-antigenic peptides are separated by a spacer or linker.

[0367] In some embodiments, the spacer or linker comprises up to 5000 nucleotide residues. An exemplary spacer sequence is GGCGGCAGCGGCGGCGGCGGCAGCGGCGGC. Another exemplary spacer sequence is GGCGGCAGCCTGGGCGGCGGCGGCAGCGGC. Another exemplary spacer sequence is GGCGTCGGCACC. Another exemplary spacer sequence is CAGCTGGGCCTG. Another exemplary spacer is a sequence encoding lysine, such as AAA or AAG. Another exemplary spacer sequence is CAACTGGGATTG.

[0368] In some embodiments, the mRNA comprises one or more additional structures to enhance antigen epitope processing and presentation by APCs.

[0369] In some embodiments, the linker or spacer region can contain a cleavage site. The cleavage site ensures that the protein product containing the string of epitope sequences is cleaved into separate epitope sequences for presentation. In order to avoid accidental cleavage of epitopes within the sequence, the preferred cleavage site is located adjacent to a specific epitope. In some embodiments, the design of the epitope and cleavage region in the mRNA encoding the string of epitopes is non-random.

[0370] In certain embodiments, mRNA encoding the neo-antigenic peptides of the present invention is administered to a subject in need thereof. In some embodiments, the mRNA to be administered comprises at least one modified nucleoside-phosphate.

[0371] In some embodiments, T cells are activated with neo-antigenic peptides by artificial antigen-presenting cells. In some embodiments, T cells are activated with neo-antigenic peptides using an artificial scaffold loaded with neo-antigenic peptides coupled to MHC antigens to which the neo-antigenic peptides can bind with high affinity.

[0372] In some embodiments, the additional structure comprises a structure encoding a specific domain from a protein selected from the group consisting of MITD, SP1, and the tenth fibronectin domain: 10FnIII.

[0373] In some embodiments, cells derived from peripheral blood or leukapheresis are contacted one or more times with multiple cancer neo-antigenic peptides or one or more polynucleotides encoding multiple cancer neo-antigenic peptides to prepare antigen-loaded APCs.

[0374] In some embodiments, the method comprises incubating the APCs, or one or more of the APC preparations, with a first culture medium comprising at least one cytokine or growth factor for a first period of time.

[0375] In some embodiments, the method includes incubating one or more of the APC preparations with at least one peptide for a second period of time.

[0376] In some embodiments, the enriched cells further comprise CD1c+ cells.

[0377] In some embodiments, the cell population is enriched for CD11c+ and CD141+ cells.

[0378] In some embodiments, the cell population comprising antigen-loaded APCs comprises greater than or equal to 1%, 2%, 3%, 4%, 5%, 6, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD11c+ cells.

[0379] In some embodiments, the cell population comprising antigen-loaded APCs comprises less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, 4% or fewer CD11b+ expressing cells.

[0380] In some embodiments, the cell population comprising antigen-loaded APCs comprises greater than 1%, 2%, 3%, 4%, 5%, 6, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% neo-antigenic peptide-expressing cells that are CD11c+.

[0381] In some embodiments, the cell population comprising antigen-loaded APCs comprises greater than 1%, 2%, 3%, 4%, 5%, 6, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% neo-antigenic peptide-expressing cells that are CD11c+CD1c+ or CD141+ cells.

[0382] In some embodiments, the neo-antigen-loaded APCs comprise mature APCs.

[0383] In some embodiments, the method includes obtaining a biological sample from the subject, the biological sample comprising at least one APC and at least one PBMC or at least one T cell.

[0384] In some embodiments, the method comprises depleting cells expressing CD14 and / or CD25 and / or CD19 from the biological sample, thereby obtaining a CD14 and / or CD25 and / or CD19 cell-depleted sample.

[0385] In some embodiments, the method comprises incubating a CD14 and / or CD25 and / or CD19 cell-depleted sample with FLT3L for a first period of time.

[0386] In some embodiments, the method includes incubating at least one peptide with the CD14 and / or CD25 and / or CD19 cell-depleted sample for a second period of time, thereby obtaining a first mature APC peptide-loaded sample.

[0387] Preparation of neoantigen-activated T cells using neoantigen-loaded APCs In some embodiments, neoantigen-loaded APCs (APCs) prepared by the above-described method are incubated with T cells to obtain antigen-activated T cells. The method can include generating at least one antigen-specific T cell, wherein the antigen is a neoantigen. In some embodiments, generating at least one antigen-specific T cell includes generating a plurality of antigen-specific T cells.

[0388] In some embodiments, the T cells are obtained from a biological sample from a subject.

[0389] In some embodiments, the T cells are obtained from a biological sample from the same subject from which the APCs are derived. In some embodiments, the T cells are obtained from a biological sample from a subject different from the subject from which the APCs are derived.

[0390] In some embodiments, the APCs and / or T cells are derived from a biological sample that is a peripheral blood mononuclear cell (PBMC). In some embodiments, the APCs and / or T cells are derived from a biological sample that is a leukapheresis sample.

[0391] In some embodiments, the APC comprises a dendritic cell (DC).

[0392] In some embodiments, the APCs are derived from CD14+ monocytes, or are CD14-enriched APCs, or are CD141-enriched APCs.

[0393] In some embodiments, CD14+ monocytes are enriched from a subject-derived biological sample that contains peripheral blood mononuclear cells (PBMCs).

[0394] In some embodiments, the APCs are PBMCs. In some embodiments, the PBMCs are freshly isolated PBMCs. In some embodiments, the PBMCs are frozen PBMCs. In some embodiments, the PBMCs are autologous PBMCs isolated from a subject or patient.

[0395] In some embodiments, PBMCs are loaded with antigens, which can be peptides or polypeptides, or polynucleotides such as mRNA encoding peptides and polypeptides. PBMCs (monocytes, DCs, phagocytes) can take up antigens by phagocytosis, process them, and present them on their surface for T cell activation. The peptides or polypeptides loaded onto PBMCs can be supplemented with adjuvants to increase immunogenicity. In some embodiments, PBMCs are loaded with nucleic acid antigens. The nucleic acid antigens can be in the form of mRNA containing sequences encoding one or more antigens. In some embodiments, mRNA antigen loading does not require adjuvant supplementation, for example, because RNA can act as a self-adjuvant. In some embodiments, APCs are loaded with 20-40 antigens. In some embodiments, APCs express 20-40 antigens. In some embodiments, the antigens are neoantigens. In some embodiments, at least a majority of the antigens are neoantigens. In some embodiments, APCs (or PBMCs) are loaded with or express nucleic acid sequences encoding short peptides (8-12 amino acids each) for CD8+ T cell stimulation. In some embodiments, APCs (or PBMCs) are loaded with or express nucleic acid sequences encoding short peptides (16-25 amino acids each) for CD4+ T cell stimulation. In some embodiments, APCs (e.g., PBMCs) can be loaded with both short and long antigenic peptide sequences; or APCs express both short and long antigenic peptide sequences. In some embodiments, APCs are loaded with up to or about 40 short antigenic peptide sequences and up to or about 20 long antigenic peptide sequences. In some embodiments, APCs are transduced or transfected with nucleic acids comprising up to or about 40 short antigenic peptide sequences and up to or about 20 long antigenic peptide sequences.

[0396] In some embodiments, PBMCs are directly isolated or thawed from a frozen sample and incubated with one or more antigens, such as neoantigens, a composition comprising neoantigens, or one or more nucleic acids or polynucleotides encoding one or more antigens. In some embodiments, the PBMC sample is not further cultured for differentiation or further maturation of one or more cellular components within the PBMCs (e.g., maturation of antigen-presenting cells or differentiation of monocytes into dendritic cells) before exposing the PBMCs to one or more antigens or nucleic acids encoding one or more antigens. In some embodiments, one or more cell types are depleted or removed from a freshly isolated or freshly thawed PBMC population before exposing or incubating the cells with one or more antigens or nucleic acids encoding one or more antigens. In some embodiments, CD14+ cells are depleted from PBMCs. In some embodiments, CD25+ cells are depleted from PBMCs. In some embodiments, CD11b+ cells are depleted from PBMCs. In some embodiments, CD14+ and CD25+ cells are depleted from PBMCs before incubation with one or more antigens or one or more nucleic acids encoding one or more antigens. In some embodiments, CD11b+ and / or CD14+ and / or CD25+ cells are depleted from PBMCs. In some embodiments, the methods provided herein include preparing tumor antigen-specific T cells by depleting CD14+ cells and / or CD25+ cells from a PBMC sample from a human subject containing a percentage of immature dendritic cells (DCs) approximately equal to the percentage of immature DCs in the peripheral blood of the human subject. In some embodiments, the methods provided herein include preparing tumor antigen-specific T cells by depleting CD14+ cells and / or CD25+ cells from a PBMC sample from a human subject containing a percentage of mature DCs approximately equal to the percentage of mature DCs in the peripheral blood of the human subject.In some embodiments, the methods provided herein comprise preparing tumor antigen-specific T cells by depleting CD14+ cells and / or CD25+ cells from a PBMC sample from a human subject that contains a ratio of immature to mature DCs that is approximately the same as the ratio of immature to mature DCs in the peripheral blood of the human subject. In some embodiments, the methods provided herein comprise preparing tumor antigen-specific T cells by depleting CD14+ cells and / or CD25+ cells from a PBMC sample from a human subject that has not been subjected to a step of maturing the immature DCs into mature DCs.

[0397] In some embodiments, the CD14+ monocytes are stimulated with one or more cytokines or growth factors.

[0398] In some embodiments, the one or more cytokines or growth factors comprise GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, R848, LPS, ss-rna40, poly I:C, or a combination thereof.

[0399] In some embodiments, the CD14+ monocytes are derived from a second biological sample comprising PBMCs.

[0400] In some embodiments, the second biological sample is from the same subject.

[0401] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0402] In some embodiments, at least one antigen-specific T cell is stimulated in medium comprising IL-7, IL-15, an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof.

[0403] In some embodiments, the IDO inhibitor is epacadostat, navoximod, 1-methyltryptophan, or a combination thereof.

[0404] In some embodiments, the subject is administered FLT3L prior to obtaining the biological sample for preparing APCs and / or T cells.

[0405] In some embodiments, the T cells are obtained from a biological sample from a subject described in the previous section of this disclosure.

[0406] In some embodiments, the biological sample is a freshly obtained or frozen sample from a subject.

[0407] In some embodiments, the incubating step is performed in the presence of at least one cytokine or growth factor comprising GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, IL-15, R848, LPS, ss-rna40, poly I:C, or any combination thereof.

[0408] In some embodiments, the method includes stimulating T cells with IL-7, IL-15, or a combination thereof. In some embodiments, the method includes stimulating T cells with IL-7, IL-15, or a combination thereof in the presence of an IDO inhibitor, a PD-1 antibody, or IL-12. In some embodiments, the stimulated T cells are expanded ex vivo under suitable T cell growth conditions in the presence of one or more tumor antigen epitope sequences, or APCs loaded with one or more tumor antigen epitope sequences, or APCs loaded with (e.g., expressing) a nucleic acid sequence (e.g., mRNA sequence) encoding one or more tumor antigen epitope sequences, and one or more cytokines or growth factors including GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, R848, LPS, ss-RNA40, poly I:C, or a combination thereof, and FLT3L. In some embodiments, the method further comprises administering antigen-specific T cells to the subject.

[0409] In some embodiments, the method comprises incubating APCs prepared as described in the previous section with T cells in the presence of a medium comprising at least one cytokine or growth factor to generate neo-antigen-activated T cells.

[0410] In some embodiments, the incubating step comprises incubating a first of the APC preparations with the T cells for more than 7 days, hi some embodiments, the incubated T cells are stimulated T cells expanded in vitro in the presence of the APC preparation, cytokines and growth factors for more than 7 days.

[0411] In some embodiments, the incubating step comprises incubating a first of the APC preparations with the T cells for more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.

[0412] In some embodiments, the first of the one or more periods is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 days.

[0413] In some embodiments, the total duration of the separate periods is less than 28 days, in some embodiments, the total duration of the separate periods is 20 to 27 days, in some embodiments, the total duration of the separate periods is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 days.

[0414] In some embodiments, the method comprises incubating a first APC preparation of the APC preparation with the T cells for more than 7 days. In some embodiments, the method comprises incubating a first APC preparation of the APC preparation with the T cells for more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the method comprises incubating a first APC preparation of the APC preparation with the T cells for 7-20, 8-20, 9-20, 10-20, 11-20, or 12-20 days. In some embodiments, the method comprises incubating a first APC preparation of the APC preparation with the T cells for about 10-15 days.

[0415] In some embodiments, the method includes incubating a second APC preparation of the APC preparation with the T cells for 5 to 9 days. In some embodiments, the method includes incubating a second APC preparation of the APC preparation with the T cells for 5, 6, 7, 8, or 9 days. In some embodiments, the method further includes removing one or more cytokines or growth factors of the second culture medium after the third period and before the start of a fourth period.

[0416] In some embodiments, the method comprises incubating a third one of the APC preparations with the T cells for 5 to 9 days, hi some embodiments, the method comprises incubating a third one of the APC preparations with the T cells for 5, 6, 7, 8, or 9 days.

[0417] In some embodiments, the method comprises incubating a first one of the APC preparations with the T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days; incubating a second one of the APC preparations with the T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days; and incubating a third one of the APC preparations with the T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days.

[0418] In some embodiments, the method is performed ex vivo. In some embodiments, the T cells are cultured in a medium containing a cytokine. In some embodiments, an example cytokine includes IL-7. In some embodiments, an example cytokine includes IL-15. In some embodiments, an example cytokine includes IL-7 and IL-15. In some embodiments, the T cells are cultured in a medium containing IL-7 and / or IL-15. In some embodiments, the cytokine in the T cell culture or medium has a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, IL-7 in the T cell culture or medium has a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL or 20 ng / mL. In some embodiments, IL-15 in the T cell culture or medium has a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the T cells are cultured in medium further containing FLT3L.In some embodiments, FLT3L in the T cell culture or medium has a final concentration of at least 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, or 200 ng / mL. In some embodiments, T cells are incubated, induced, or stimulated in medium containing FLT3L for a first period of time. In some embodiments, T cells are incubated, induced, or stimulated in medium containing additionally added FLT3L for a second period of time. In some embodiments, T cells are incubated, induced, or stimulated in medium containing additionally added FLT3L for a third period of time. In some embodiments, the T cells are incubated, induced, or stimulated for a fourth, fifth, or sixth period in medium containing additional added FLT3L, with freshly added FLT3L in each period.

[0419] In some embodiments, the T cells are cultured in the presence of a neoantigen, e.g., a neoantigen presented by an APC, and the medium is supplemented with high potassium [K] + In some embodiments, the T cells are incubated with the APC or T cells for at least a period of time in a medium containing a high [K] + In some embodiments, the medium is cultured in the presence of [K]. + The content is varied over at least a period of incubation with APCs or T cells. In some embodiments, the content in the medium is kept constant throughout the period of T cell ex vivo culture. In some embodiments, the [K] content in the T cell culture medium is varied over at least a period of incubation with APCs or T cells. In some embodiments, the content in the medium is kept constant throughout the period of T cell ex vivo culture. + In some embodiments, the [K] in the T cell culture medium is ≧5 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧6 mM.+ In some embodiments, the [K] content in the T cell culture medium is ≧7 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧8 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧9 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧10 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧11 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧12 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧13 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧14 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧15 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧16 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧17 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧18 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧19 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧20 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧22 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧25 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧30 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧35 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧40 mM. + The content is about 40 mM.

[0420] In some embodiments, the [K] in the T cell culture medium + The neoantigen may be present in an amount of about 40 mM for at least a period of time during incubation of the neoantigen with the T cells. In some embodiments, the neoantigen may be presented by neoantigen-loaded APCs. In some embodiments, the T cells may be exposed to [K] + In some embodiments, T effector function, CD8+ cytotoxicity, cytokine production, and memory phenotype are examined in the presence of a high [K] + T cells grown in the presence of a high [K] express an effector T cell phenotype. + In some embodiments, T cells grown in the presence of a high [K] express memory cell markers. + T cells grown in the presence of do not express T cell exhaustion markers.

[0421] In some embodiments, the stimulated T cells are a population of immune cells comprising activated T cells stimulated by APCs comprising neo-antigenic peptide-MHC complexes. In some embodiments, the method can include incubating a population of immune cells from a biological sample with APCs comprising peptide-MHC complexes, thereby obtaining a stimulated immune cell sample; determining expression of one or more cell markers of at least one immune cell of the stimulated immune cell sample; and determining binding of at least one immune cell of the stimulated immune cell sample to the peptide-MHC complexes, wherein determining expression of certain cell surface markers or other determinant markers, such as intracellular factors or released agents, such as cytokines, and determining binding to the neo-antigen-MHC complexes are performed simultaneously. In some embodiments, the one or more cell markers comprise TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, Granzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3, or any combination thereof. In some embodiments, the one or more cell markers comprise a cytokine. In some embodiments, the one or more cell markers comprise a degranulation marker. In some embodiments, the one or more cell markers comprise a cell surface marker. In some embodiments, the one or more cell markers comprise a protein. In some embodiments, determining binding of at least one immune cell of the stimulated immune cell sample to a peptide-MHC complex comprises determining binding of at least one immune cell of the stimulated immune cell sample to an MHC tetramer comprising the peptide and MHC of the peptide-MHC complex. In some embodiments, the MHC is a class I MHC or a class II MHC. In some embodiments, the peptide-MHC complex comprises one or more labels.

[0422] In some embodiments, T cell activation is verified by detecting cytokine release by activated T cells. In some embodiments, the cytokine is one or more of TNF-α, IFN-γ, or IL-2. In some embodiments, T cell activation is verified by its specific antigen binding and cytokine release. In some embodiments, T cell activation is verified by its ability to kill tumor cells in vitro. A sample of activated T cells can be used to verify the activation status of the T cells. In some embodiments, a sample from T cells is removed from a T cell culture and the cell composition and activation status are determined by flow cytometry.

[0423] In some embodiments, the percentage of at least one antigen-specific T cell in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total T cells or total immune cells. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 5%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 7%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 10%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 12%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 15%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 20%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 25%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 30%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 40%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 50%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 60%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 70%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 80%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 90%.

[0424] In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 5%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 7%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 10%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 12%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 15%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 20%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 25%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 30%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 40%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 50%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 60%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 70% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0425] In some embodiments, the percentage of at least one antigen-specific CD4+ T cells in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells.

[0426] In some embodiments, the percentage of at least one antigen-specific T cells in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0427] In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0428] In some embodiments, the percentage of at least one antigen-specific CD4+ T cells in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0429] In some embodiments, the antigen is a neoantigen, a tumor-associated antigen, an overexpressed antigen, a viral antigen, a minor histocompatibility antigen, or a combination thereof.

[0430] In some embodiments, the number of at least one antigen-specific CD8+ T cells in the composition is at least about 1x10^6, 2x10^6, 5x10^6, 1x10^7, 2x10^7, 5x10^7, 1x10^8, 2x10^8 or 5x10^8 antigen-specific CD8+ T cells.

[0431] In some embodiments, the number of at least one antigen-specific CD4+ T cells in the composition is at least about 1x10^6, 2x10^6, 5x10^6, 1x10^7, 2x10^7, 5x10^7, 1x10^8, 2x10^8 or 5x10^8 antigen-specific CD4+ T cells.

[0432] Pharmaceutical Composition Provided herein is a composition (e.g., a pharmaceutical composition) comprising a population of immune cells. The composition can comprise at least one antigen-specific T cell comprising a T cell receptor (TCR). The composition can comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence.

[0433] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, including excipients and auxiliary agents that facilitate the processing of active agents into preparations that can be used as pharmaceuticals.Suitable formulations can depend on the selected route of administration.Any well-known techniques, carriers and excipients can be used as appropriate and as understood in the art.

[0434] In some cases, the pharmaceutical composition is formulated as a cell-based therapy, e.g., a T cell therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapy, a nucleic acid-based therapy, an antibody-based therapy, and / or a cell-based therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic or a nucleic acid-based therapeutic that encodes a polypeptide. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic or a nucleic acid-based therapeutic that encodes a polypeptide, wherein the peptide-based therapeutic or nucleic acid-based therapeutic is contained in a cell, and the cell is a T cell. In some embodiments, the pharmaceutical composition comprises an antibody-based therapeutic. The composition can comprise T cells specific for two or more immunogenic antigens or neo-antigen peptides.

[0435] In one aspect, provided herein is a pharmaceutical composition comprising: (a) a population of immune cells comprising T cells derived from a biological sample, wherein the T cells are APC-stimulated T cells and comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, and the APC is a FLT3L-stimulated APC; and (b) a pharmaceutically acceptable excipient.

[0436] In one aspect, provided herein is a pharmaceutical composition comprising: (a) a population of immune cells derived from a biological sample, the population comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence; and (b) a pharmaceutically acceptable excipient, wherein the amount of immune cells expressing CD14 and / or CD25 in the population is relatively different from the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the at least one antigen-specific T cell comprises T cells stimulated by at least one APC. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the population is relatively lower than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the population is relatively higher than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the at least one antigen-specific T cell comprises at least one CD4+ T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD8+ T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD4-enriched T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD8-enriched T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory CD4+ T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory CD8+ T cell. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total T cells or total immune cells.In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0437] In addition to the active ingredient, a pharmaceutical composition may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.

[0438] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0439] Acceptable carriers are physiologically tolerable to the patient to which they are administered and retain the therapeutic properties of the compound with which / in which it is administered. Acceptable carriers and their formulations are generally described in, for example, Remington's Pharmaceutical Sciences (18 thed. A. Gennaro, Mack Publishing Co., Easton, PA 1990). An example of a carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in the transport or transfer of a compound of interest from an administration site in one organ or body part to another, or involved in an in vitro assay system. An acceptable carrier is compatible with the other ingredients of the formulation and is not harmful to the subject to which it is administered. Furthermore, an acceptable carrier should not alter the specific activity of the neoantigen.

[0440] In one aspect, provided herein is a pharmaceutically acceptable or physiologically acceptable composition that includes a solvent (aqueous or non-aqueous), a solution, an emulsion, a dispersion medium, a coating, an isotonic agent, and an absorption enhancer or retardant, which is compatible with pharmaceutical administration. Thus, a pharmaceutical composition or pharmaceutical preparation refers to a composition suitable for pharmaceutical use in a subject. The composition can be formulated to be compatible with a specific administration route (i.e., systemic or local). Thus, the composition includes a carrier, diluent, or excipient that is suitable for administration by various routes.

[0441] In some embodiments, the composition may further contain an acceptable additive to improve the stability of immune cells in the composition. The acceptable additive may not alter the specific activity of the immune cells. Examples of acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. The acceptable additive may be combined with an acceptable carrier and / or excipient, such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 to increase peptide stability and reduce gelation of the solution. Surfactants may be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0442] Pharmaceutical compositions can be administered, for example, by injection. Compositions for injection include aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride may be included in the composition. The resulting solution can be packaged for use as is or lyophilized; the lyophilized preparation can then be combined with a sterile solution prior to administration. For intravenous injection or injection at the affected site, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability.Those skilled in the art can easily prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed.Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount into a suitable solvent with one or a combination of the ingredients listed above as required, followed by filtration sterilization.Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from the ingredients listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method can be vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients from the solution previously sterile-filtered.

[0443] The composition can be conventionally administered intravenously, for example, by injection of a unit dose.For injection, the active ingredient can be in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has suitable pH, isotonicity and stability.For example, suitable solutions can be prepared using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as required.In addition, the composition can be administered by aerosolization.

[0444] When a composition is contemplated for use in medicine or any of the methods provided herein, it is contemplated that the composition may be substantially free of pyrogens, so that the composition does not provoke an inflammatory or dangerous allergic reaction when administered to a human patient. Testing a composition for pyrogens and preparing a composition that is substantially free of pyrogens is well understood by one of ordinary skill in the art and can be accomplished using commercially available kits.

[0445] Acceptable carriers can contain compounds that act as stabilizers, increase or delay absorption, or increase or delay clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, or dextran; low-molecular-weight proteins; compositions that reduce peptide clearance or hydrolysis; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents, including liposomal carriers, can also be used to stabilize, increase, or decrease the absorption of pharmaceutical compositions. To protect against digestion, the compound can be complexed with the composition to make it resistant to acidic and enzymatic hydrolysis, or the compound can be complexed in a suitable resistant carrier such as liposomes. Means of protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135; and U.S. Pat. No. 5,391,377).

[0446] The composition can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be administered depends on the subject to be treated, the ability of the subject's immune system to utilize the active ingredient, and the desired degree of binding capacity. The exact amount of active ingredient required to be administered depends on the judgment of the physician and is unique to each individual. Suitable regimens for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more time intervals by subsequent injections or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations is contemplated.

[0447] In some embodiments, the present invention is directed to immunogenic compositions, e.g., pharmaceutical compositions, capable of generating a neoantigen-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic composition comprises a neoantigen therapeutic agent described herein (e.g., a peptide, a polynucleotide, a TCR, a CAR, a cell containing a TCR or a CAR, a dendritic cell containing a polypeptide, a dendritic cell containing a polynucleotide, an antibody, etc.) that corresponds to a tumor-specific antigen or neoantigen.

[0448] In some embodiments, the pharmaceutical compositions described herein are capable of generating a specific cytotoxic T cell response, a specific helper T cell response, or a B cell response.

[0449] In some embodiments, antigenic polypeptides or polynucleotides can be provided as antigen-presenting cells (e.g., dendritic cells) containing such polypeptides or polynucleotides. In other embodiments, such antigen-presenting cells are used to stimulate T cells for use in a patient. In some embodiments, the antigen-presenting cells are dendritic cells. In related embodiments, the dendritic cells are autologous dendritic cells pulsed with neo-antigenic peptides or nucleic acids. The neo-antigenic peptides can be any suitable peptide that generates an appropriate T cell response. In some embodiments, the T cells are CTLs. In some embodiments, the T cells are HTLs. Thus, one embodiment of the present disclosure is an immunogenic composition containing at least one antigen-presenting cell (e.g., dendritic cell) pulsed or loaded with one or more neo-antigenic polypeptides or polynucleotides described herein. In some embodiments, such APCs are autologous (e.g., autologous dendritic cells). Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient can be loaded with neo-antigenic peptides or polynucleotides ex vivo. In related embodiments, such APCs or PBMCs are injected back into the patient. The polynucleotide can be any suitable polynucleotide that can transduce dendritic cells, thereby resulting in presentation of neo-antigenic peptides and induction of immunity. In some embodiments, such antigen-presenting cells (APCs) (e.g., dendritic cells) or peripheral blood mononuclear cells (PBMCs) are used to stimulate T cells (e.g., autologous T cells). In related embodiments, the T cells are CTLs. In other related embodiments, the T cells are HTLs. In some embodiments, the T cells are CD8 + In some embodiments, the T cells are CD4 + These T cells are then injected into the patient.

[0450] In some embodiments, CTLs are injected into a patient. In some embodiments, HTLs are injected into a patient. In some embodiments, both CTLs and HTLs are injected into a patient. Administration of either therapeutic agent can be simultaneous or sequential, in any order.

[0451] In some embodiments, pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment can be formulated for parenteral, topical, nasal, oral, or local administration. In some embodiments, pharmaceutical compositions described herein are administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the compositions can be administered intratumorally. The compositions can be administered at the site of surgical resection to induce a local immune response against the tumor. In some embodiments, compositions for parenteral administration are described herein, comprising a solution of neo-antigenic peptides, wherein the immunogenic composition is dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques or sterile filtered. The resulting aqueous solutions can be packaged for use as is or can be lyophilized, with the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0452] The ability of adjuvants to increase the immune response to antigens is typically manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms.For example, an increase in humoral immunity can be manifested by a significant increase in the titer of antibodies raised against antigens, and an increase in T cell activity can be manifested in increased cell proliferation, cellular cytotoxicity, or cytokine secretion.Adjuvants can also modify immune responses, for example, by changing a predominantly humoral or T helper 2 response into a predominantly cellular or T helper 1 response.

[0453] Suitable adjuvants are known in the art (see WO2015 / 095811) and include poly(I:C), poly-ICLC, STING agonists, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA Examples of adjuvants include, but are not limited to, 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel® vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Pam3CSK4, saponin, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA), which is derived from mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Several immunological adjuvants specific for dendritic cells and their preparations have been described (e.g., MF59) (Dupuis M, et al., Cell Immunol. 1998; 186(1):18-27; Allison AC; Dev Biol Stand. 1998; 92:3-11) (Mosca et al. Frontiers in Bioscience, 2007; 12:4050-4060) (Gamvrellis et al. Immunol & Cell Biol. 2004; 82: 506-516). Cytokines can also be used.Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating dendritic cell maturation into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1β, IL-4, IL-6, and CD40L) (U.S. Pat. No. 5,849,589, incorporated herein by reference in its entirety), and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418).

[0454] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in therapeutic settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cell-mediated immunogenic pharmaceutical compositions, and polysaccharide conjugates in both prophylactic and therapeutic immunogenic pharmaceutical compositions. Importantly, this enhances the CD4 +Even without T cell help, CpG oligonucleotides enhance dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and strong cytotoxic T-lymphocyte (CTL) production. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of adjuvants such as alum or Freund's incomplete adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which are particularly useful for inducing strong responses when the antigen is relatively weak. This can also accelerate immune responses, and in some experiments has allowed the antigen dose to be reduced while still achieving antibody responses comparable to those of full-dose immunogenic pharmaceutical compositions without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484). US Patent No. 6,406,705 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. A commercially available CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) by Mologen (Berlin, DE), which is a component of the pharmaceutical composition described herein. Other TLR binding molecules, such as RNA that binds to TLR7, TLR8, and / or TLR9, can also be used.

[0455] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly(I and / or polyC) (e.g., polyI:CI2U), non-CpG bacterial DNA or RNA, ssRNA40 for TLR8, and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which can act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of ordinary skill in the art without undue experimentation. Additional adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0456] In some embodiments, immunogenic compositions according to the present disclosure can include two or more different adjuvants. Additionally, the present invention encompasses pharmaceutical compositions comprising any adjuvant agent, including any of the above or combinations thereof. In some embodiments, immunogenic compositions include neo-antigen therapeutics (e.g., peptides, polynucleotides, TCRs, CARs, cells containing a TCR or CAR, dendritic cells containing a polypeptide, dendritic cells containing a polynucleotide, antibodies, etc.), and adjuvants can be administered separately and in any suitable order.

[0457] Lipidation can be classified into several different types, including N-myristoylation, palmitoylation, GPI-anchor attachment, prenylation, and several additional types of modifications. N-myristoylation is the covalent attachment of myristic acid, a C14 saturated acid, to a glycine residue. Palmitoylation is the thioester linkage of a long-chain fatty acid (C16) to a cysteine ​​residue. GPI-anchor attachment is the attachment of glycosylphosphatidylinositol (GPI) via an amide bond. Prenylation is the thioether linkage of an isoprenoid lipid (e.g., farnesyl (C-15), geranylgeranyl (C-20)) to a cysteine ​​residue. Additional types of modifications include the attachment of S-diacylglycerol via the sulfur atom of cysteine, O-octanoyl conjugation via serine or threonine residues, S-archaeol conjugation to cysteine ​​residues, and cholesterol attachment.

[0458] Fatty acids for producing lipidated peptides can include C2-C30 saturated, monounsaturated, or polyunsaturated fatty acid acyl groups. Exemplary fatty acids include palmitoyl, myristoyl, stearoyl, and decanoyl groups. In some instances, lipid moieties with adjuvant properties are attached to a polypeptide of interest to induce or enhance immunogenicity in the absence of exogenous adjuvants. Lipidated peptides or lipopeptides can be referred to as self-adjuvanting lipopeptides. Any of the fatty acids described above and elsewhere herein can induce or enhance the immunogenicity of a polypeptide of interest. Fatty acids capable of inducing or enhancing immunogenicity can include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, and decanoyl groups.

[0459] A polypeptide, such as a naked peptide or a lipidated peptide, can be incorporated into a liposome. In some cases, a lipidated peptide can be incorporated into a liposome. For example, the lipid portion of the lipidated peptide can spontaneously integrate into the lipid bilayer of the liposome. Thus, the lipopeptide can be presented on the "surface" of the liposome. Exemplary liposomes suitable for incorporation into the formulation include, but are not limited to, multilamellar vesicles (MLVs), oligolamellar vesicles (OLVs), unilamellar vesicles (UVs), small unilamellar vesicles (SUVs), medium-sized unilamellar vesicles (MUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), multivesicular vesicles (MVVs), single or oligolamellar vesicles made by the reverse phase evaporation method (REVs), multilamellar vesicles made by the reverse phase evaporation method (MLV-REVs), stable plurilamellar vesicles (SPLVs), freeze-thawed MLVs (FATMLVs), vesicles prepared by the extrusion method (VETs), vesicles prepared by French press (FPVs), vesicles prepared by fusion (FUVs), dehydrated-rehydrated vesicles (DRVs), and bubblesomes (BSVs).

[0460] Depending on the preparation method, liposomes can be unilamellar or multilamellar, and can vary in size, ranging from about 0.02 μm to more than about 10 μm in diameter. Liposomes can adsorb to many types of cells and release incorporated agents (e.g., peptides described herein). In some cases, liposomes fuse with target cells, which then release the contents of the liposomes into the target cells. Liposomes can be endocytosed by phagocytic cells. Endocytosis can be followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agent.

[0461] The liposome provided herein can comprise a carrier lipid.In some embodiments, the carrier lipid is a phospholipid.The carrier lipid that can form liposomes includes but is not limited to dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Other suitable phospholipids further include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic acid (DPPA); dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), etc., or combinations thereof. In some embodiments, the liposomes further comprise a sterol (e.g., cholesterol) that modulates liposome formation. The carrier lipid can be any known non-phosphate polar lipid.

[0462] The pharmaceutical compositions may also be encapsulated in liposomes using well known technology.Biodegradable microspheres may also be employed as carriers for the pharmaceutical compositions of the present invention.

[0463] Pharmaceutical compositions can be administered in liposome or microsphere (or microparticle).The method for preparing liposome and microsphere for administration to patient is well known to those skilled in the art.Basically, material is dissolved in aqueous solution, and suitable phospholipid and lipid are added, if necessary, together with surfactant, and material is dialyzed or sonicated as necessary.

[0464] Microspheres formed from polymers or proteins are well known to those skilled in the art and can be tailored to pass through the gastrointestinal tract directly into the bloodstream. Alternatively, compounds can be loaded and the microspheres or composites of microspheres can be implanted for slow release over periods ranging from days to months.

[0465] Cell-based immunogenic pharmaceutical compositions can also be administered to subjects.For example, antigen-presenting cell (APC)-based immunogenic pharmaceutical compositions can be formulated using any of well-known techniques, carriers and excipients, as appropriate and understood in the art.APCs include monocytes, monocyte-derived cells, macrophages and dendritic cells.Optionally, APC-based immunogenic pharmaceutical compositions can be dendritic cell-based immunogenic pharmaceutical compositions.

[0466] Dendritic cell-based immunogenic pharmaceutical compositions can be prepared by any method known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared by ex vivo or in vivo methods. Ex vivo methods can include the use of autologous DCs pulsed ex vivo with a polypeptide described herein to activate or load the DCs prior to administration to a patient. In vivo methods can include targeting specific DC receptors using an antibody coupled to a polypeptide described herein. DC-based immunogenic pharmaceutical compositions can further include a DC activator, such as a TLR3, TLR-7-8, or CD40 agonist. DC-based immunogenic pharmaceutical compositions can further include an adjuvant and a pharmaceutically acceptable carrier.

[0467] Adjuvants can be used to enhance the immune response (humoral and / or cellular) induced in patients receiving the immunogenic pharmaceutical composition. In some cases, adjuvants can induce a Th1-type response. In other cases, adjuvants can induce a Th2-type response. In contrast to Th2-type responses, which can be characterized by the production of cytokines such as IL-4, IL-5, and IL-10, Th1-type responses can be characterized by the production of cytokines such as IFN-γ.

[0468] In some embodiments, lipid-based adjuvants such as MPLA and MDP can be used together with the immunogenic pharmaceutical compositions disclosed herein. Monophosphoryl lipid A (MPLA) is, for example, an adjuvant that causes increased presentation of liposomal antigens to specific T lymphocytes. In addition, muramyl dipeptide (MDP) can also be used as a suitable adjuvant in conjunction with the immunogenic pharmaceutical formulations described herein.

[0469] Adjuvants can also include stimulatory molecules such as cytokines. Non-limiting examples of cytokines include CCL20, α-interferon (IFNα), β-interferon (IFNβ), γ-interferon (IFNγ), platelet-derived growth factor (PDGF), TNFα, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1-, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant form of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Ap o-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, and TAP2.

[0470] Additional adjuvants include MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, and IL-18. Cell growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0471] In some embodiments, the adjuvant can be a toll-like receptor modulator. Examples of toll-like receptor modulators include TLR9 agonists, but are not limited to small molecule toll-like receptor modulators such as imiquimod. Optionally, the adjuvant is selected from bacterial toxoids, polyoxypropylene-polyoxyethylene block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or combinations thereof. Optionally, the adjuvant is an oil-in-water emulsion. The oil-in-water emulsion can include at least one oil and at least one surfactant, wherein the oil(s) and surfactant(s) are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion can have a diameter of less than 5 μm, and can even have a submicron diameter; such small sizes can be achieved by a microfluidic device to provide a stable emulsion. Droplets having a size of less than 220 nm can be subjected to filtration sterilization.

[0472] In some examples, immunogenic pharmaceutical compositions can include carriers and excipients (including, but not limited to, buffers, carbohydrates, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents, and / or preservatives), water, oils including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., saline solution, aqueous dextrose and glycerol solution, flavors, colorants, detackifiers and other acceptable additives, adjuvants, or binders, and other pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifiers, wetting agents, etc., as required. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. In another example, the pharmaceutical preparation is substantially free of preservatives. In another example, the pharmaceutical preparation may contain at least one preservative. The pharmaceutical compositions described herein can be administered using any suitable carrier known to those skilled in the art, although it will be recognized that the type of carrier will vary depending on the mode of administration.

[0473] The immunogenic pharmaceutical composition may include a preservative, such as thiomersal or 2-phenoxyethanol. In some examples, the immunogenic pharmaceutical composition is substantially free of mercurial materials (e.g., <10 μg / mL), e.g., thiomersal-free. Alpha-tocopherol succinate can be used as an alternative to mercurial compounds.

[0474] Physiological salts such as sodium salts may be included in the immunogenic pharmaceutical composition to control tonicity. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride, among others.

[0475] The immunogenic pharmaceutical composition can have an osmolality within the range of between 200 mOsm / kg and 400 mOsm / kg, between 240 and 360 mOsm / kg, or between 290 and 310 mOsm / kg.

[0476] The immunogenic pharmaceutical composition can include one or more buffers, such as Tris buffer, borate buffer, succinate buffer, histidine buffer (especially with an aluminum hydroxide adjuvant), or citrate buffer, in some cases in the 5-20 or 10-50 mM range.

[0477] The pH of the immunogenic pharmaceutical composition can be between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8.

[0478] The immunogenic pharmaceutical composition may be sterile. The immunogenic pharmaceutical composition may be pyrogen-free, for example, containing <1 EU (endotoxin unit, a standard measure) per dose, or <0.1 EU per dose. The composition may be gluten-free.

[0479] The immunogenic pharmaceutical composition may include a detergent, such as a polyoxyethylene sorbitan ester surfactant (known as "Tween® class") or an octoxynol (such as octoxynol-9 (Triton® X-100) or t-octylphenoxypolyethoxyethanol). The detergent may be present in trace amounts. The immunogenic pharmaceutical composition may include less than 1 mg / mL each of octoxynol-10 and polysorbate 80. Other residual components present in trace amounts may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0480] The immunogenic pharmaceutical composition can be formulated as a sterile solution or suspension in a suitable medium known in the art.The pharmaceutical composition can be sterilized by conventional well-known sterilization techniques or can be sterile filtered.The resulting aqueous solution can be packaged for use as is or can be lyophilized, and the lyophilized preparation is combined with a sterile solution before administration.

[0481] For example, pharmaceutical compositions comprising active agents, such as immune cells, described herein in combination with one or more adjuvants can be formulated to contain a particular molar ratio. For example, a molar ratio of about 99:1 to about 1:99 of active agents, such as immune cells, described herein in combination with one or more adjuvants can be used. In some examples, the molar ratio of active agents, such as immune cells, described herein in combination with one or more adjuvants can be selected from the following ranges: about 80:20 to about 20:80; about 75:25 to about 25:75; about 70:30 to about 30:70; about 66:33 to about 33:66; about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90. The molar ratio of active agents, such as immune cells, described herein in combination with one or more adjuvants can be about 1:9, and in some cases, about 1:1. Active agents, such as immune cells described herein, combined with one or more adjuvants can be formulated together in the same dosage unit, e.g., one vial, suppository, tablet, capsule, aerosol spray; or each agent, form and / or compound can be formulated in separate units, e.g., two vials, suppositories, tablets, two capsules, a tablet and a vial, aerosol spray, etc.

[0482] In some examples, the immunogenic pharmaceutical composition can be administered with an additional agent. The choice of additional agent can depend, at least in part, on the condition being treated. The additional agent can include, for example, a checkpoint inhibitor agent such as an anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 agent (e.g., an anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 antibody); or any agent that has a therapeutic effect against pathogen infection (e.g., a viral infection), including, for example, an NSAID, e.g., ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin, a drug used to treat an inflammatory condition. For example, the checkpoint inhibitor can be a PD-1 / PD-L1 antagonist selected from the group consisting of nivolumab (ONO-4538 / BMS-936558, MDX1 106, OPDIVO), pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011), and MPDL328OA (ROCHE). As another example, the formulation can additionally contain one or more supplements, such as vitamins C, E, or other antioxidants.

[0483] Pharmaceutical compositions comprising an active agent, such as an immune cell described herein, in combination with one or more adjuvants can be formulated in a conventional manner using one or more physiologically acceptable carriers, including, for example, excipients, diluents, and / or adjuvants that facilitate processing of the active agent into a preparation that can be administered. The appropriate formulation can depend, at least in part, on the chosen route of administration. The agent(s) described herein can be delivered to a patient using a number of administration routes or mechanisms, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular application, as well as by inhalation.

[0484] The active agent can be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and can be presented in unit dose form in ampoules, prefilled syringes, small volume injections, or in multi-dose containers with added preservatives. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous medium, for example, a solution in aqueous polyethylene glycol, etc.

[0485] In some embodiments, the pharmaceutical composition comprises a preservative or stabilizer. In some embodiments, the preservative or stabilizer is selected from a cytokine, a growth factor, an adjuvant, or a chemical. In some embodiments, the composition comprises at least one agent that helps preserve cell viability through at least one freeze-thaw cycle. In some embodiments, the composition comprises at least one agent that helps preserve cell viability through at least two or more freeze-thaw cycles.

[0486] For injectable formulations, the vehicle can be selected from vehicles known in the art as suitable, including aqueous solutions or oil suspensions, or emulsions in sesame, corn, cottonseed, or peanut oil, as well as elixirs, mannitol, dextrose, or sterile aqueous solutions and similar pharmaceutical vehicles. The formulation can also contain biocompatible, biodegradable polymer compositions, such as poly(lactic-co-glycolic) acid. These materials can be fabricated into drug-loaded micro- or nanospheres that are further coated or derivatized to provide superior sustained-release performance. Vehicles suitable for periocular or intraocular injection include, for example, suspensions of therapeutic agents in injection-grade water, liposomes, and vehicles suitable for lipophilic substances. Other vehicles for periocular or intraocular injection are known in the art.

[0487] In some cases, pharmaceutical compositions are routinely formulated as pharmaceutical compositions adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the compositions can contain solubilizers and local anesthetics such as lidocaine to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the content of active agent. When the composition is to be administered by injection, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0488] Manufacturing method: Methods for producing antigen-specific T cells are provided herein. Methods for preparing T cell compositions, such as therapeutic T cell compositions, are provided herein. For example, the methods can include expanding or inducing antigen-specific T cells. Preparing (e.g., inducing or expanding) T cells can also refer to producing T cells, and can include any type of T cell (e.g., CD4 + T cells and CD8 +The present invention broadly encompasses procedures for isolating, stimulating, culturing, inducing, and / or expanding antigen-specific T cells (e.g., T cells). In one aspect, provided herein is a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating APCs with a population of immune cells from a biological sample depleted of cells expressing CD14 and / or CD25. In some embodiments, the method comprises preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating APCs with a population of immune cells from a biological sample depleted of cells expressing CD11b and / or CD19. In some embodiments, the method comprises incubating APCs with a population of immune cells from a biological sample depleted of cells expressing any CD11b and / or CD19 and / or CD14 and / or CD25, or any combination thereof.

[0489] In a second aspect, provided herein is a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample.

[0490] In a third aspect, provided herein is a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample for a first period of time, and thereafter incubating at least one T cell of the biological sample with an APC.

[0491] In a fourth aspect, provided herein is a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods of less than 28 days following incubation of the population of immune cells with a first APC preparation of the one or more APC preparations, wherein at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is induced.

[0492] In a fifth aspect, provided herein is a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating a population of immune cells from a biological sample with three or less APC preparations for three or less separate periods of time, wherein at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is induced.

[0493] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate periods of time, thereby stimulating the T cells into antigen-specific T cells, wherein the percentage of antigen-specific T cells is greater than or equal to the total CD4 + T cells, total CD8 +at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the T cells, total T cells or total immune cells. In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with three or less APC preparations for three or less separate periods of time, thereby stimulating the T cells to become antigen-specific T cells. In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with two or less APC preparations for two or less separate periods of time, thereby stimulating the T cells to become antigen-specific T cells.

[0494] In some embodiments, provided herein are methods comprising incubating a population of immune cells derived from a biological sample with one or more APC preparations for one or more separate periods of time, thereby stimulating T cells into antigen-specific T cells, wherein the APC preparation is a PBMC cell population from which cells expressing one or more cell surface markers have been depleted prior to antigen loading of the APC population. In some embodiments, CD14+ cells are depleted prior to antigen loading of the APC population. In some embodiments, CD25+ cells are depleted prior to antigen loading of the APC population. In some embodiments, CD11b+ cells are depleted prior to antigen loading of the APC population. In some embodiments, CD19+ cells are depleted prior to antigen loading of the APC population. In some embodiments, CD3+ cells are depleted prior to antigen loading of the APC population. In some embodiments, CD25+ cells and CD14+ cells are depleted prior to antigen loading of the APC population. In some embodiments, CD11b+ and CD25+ cells are depleted before antigen loading of an APC population. In some embodiments, CD11b+ and CD14+ cells are depleted before antigen loading of an APC population. In some embodiments, CD11b+, CD14+ and CD25+ cells are depleted before antigen loading of an APC population. In some embodiments, CD11b+ and CD19+ cells are depleted before antigen loading of an APC population. In some embodiments, CD11b+, CD19+ and CD25+ cells are depleted before antigen loading of an APC population. In some embodiments, CD11b+, CD14+, CD19+ and CD25+ cells are depleted before antigen loading of an APC population. In some embodiments, the method comprises adding a PBMC-derived population of APC-enriched cells depleted of CD3+ cells to any of the above-mentioned depleted APC populations. In some embodiments, the PBMC-derived population of APC-enriched cells is depleted of CD3+, and such population is depleted of any one or more of CD11b+, CD14+, CD19+ or CD25+ cells.

[0495] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the method comprises adding to the PBMC sample a composition comprising one or more antigenic peptides or nucleic acids encoding same, thereby loading APCs within the PBMCs with antigen for antigen presentation to T cells in the PBMCs.

[0496] In some embodiments, the method includes (a) obtaining a biological sample from a subject, the biological sample comprising at least one antigen-presenting cell (APC); and (b) enriching cells expressing CD11c from the biological sample, thereby obtaining CD11c. + (c) obtaining a sample enriched for CD11c cells; + (d) incubating the cell-enriched sample with at least one cytokine or growth factor for a first period of time; and (e) incubating the cell-enriched sample with at least one peptide for a second period of time. + (e) incubating the APC peptide-loaded sample with one or more cytokines or growth factors for a third period of time, thereby obtaining an APC peptide-loaded sample; (f) incubating APCs from the mature APC sample with a CD11b- and / or CD14- and / or CD25-depleted sample comprising PBMCs for a fourth period of time; (g) incubating the PBMCs with the APCs from the mature APC sample for a fifth period of time; (h) incubating the PBMCs with the APCs from the mature APC sample for a sixth period of time; and (i) administering at least one T cell from the PBMCs to a subject in need thereof.

[0497] In some embodiments, the method includes (a) obtaining a biological sample from a subject, the biological sample comprising at least one antigen-presenting cell (APC); and (b) enriching cells that express CD14 from the biological sample, thereby enriching CD14. + (c) obtaining a sample enriched for CD14 cells; +(d) incubating the cell-enriched sample with at least one cytokine or growth factor for a first period of time; and (e) incubating the cell-enriched sample with at least one peptide for a second period of time. + (e) incubating the APC peptide-loaded sample with one or more cytokines or growth factors for a third period of time, thereby obtaining an APC peptide-loaded sample; (f) incubating APCs from the mature APC sample with a CD14- and / or CD25-depleted sample comprising PBMCs for a fourth period of time; (g) incubating the PBMCs with APCs from the mature APC sample for a fifth period of time; (h) incubating the PBMCs with APCs from the mature APC sample for a sixth period of time; and (i) administering at least one T cell from the PBMCs to a subject in need thereof.

[0498] In some embodiments, the method includes (a) obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD11b and / or CD19 from the biological sample, thereby obtaining a CD11b- and / or CD19-cell-depleted sample; (c) incubating the CD11b- and / or CD19-cell-depleted sample with FLT3L for a first period of time; (d) incubating at least one peptide with the CD11b- and / or CD19-cell-depleted sample of (c) for a second period of time, thereby obtaining an APC-peptide-loaded sample; and (e) (f) incubating the PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; (g) incubating the PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and (h) administering at least one T cell of the third stimulated PBMC sample to a subject in need thereof.

[0499] In some embodiments, the method includes (a) obtaining a biological sample from a subject comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD11b and / or CD19 and / or CD14 and / or CD25 from the biological sample, thereby obtaining a CD11b and / or CD19 cell-depleted sample; (c) incubating the CD11b and / or CD19 and / or CD14 and / or CD25 cell-depleted sample with FLT3L for a first period of time; and (d) incubating at least one peptide with the CD11b and / or CD19 and / or CD14 and / or CD25 cell-depleted sample of (c) for a second period of time. (e) incubating the APC peptide-loaded sample with at least one PBMC for a third period of time, thereby obtaining a first stimulated PBMC sample; (f) incubating the PBMCs of the first stimulated PBMC sample with the APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; (g) incubating the PBMCs of the second stimulated PBMC sample with the APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and (h) administering at least one T cell of the third stimulated PBMC sample to a subject in need thereof.

[0500] In some embodiments, the method includes (a) obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD14 and / or CD25 from the biological sample, thereby obtaining a CD14- and / or CD25-depleted sample; (c) incubating the CD14- and / or CD25-depleted sample with FLT3L for a first period of time; (d) incubating at least one peptide with the CD14- and / or CD25-depleted sample of (c) for a second period of time, thereby obtaining an APC-peptide-loaded sample; and (e) incubating the CD14- and / or CD25-depleted sample with FLT3L for a second period of time. (f) incubating the PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; (g) incubating the PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and (h) administering at least one T cell of the third stimulated PBMC sample to a subject in need thereof.

[0501] In some embodiments, a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating APCs with a population of immune cells from a biological sample that has been depleted of cells expressing CD14 and / or CD25.

[0502] In some embodiments, provided herein are methods for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate periods of less than 28 days following incubation of the population of immune cells with a first APC preparation of the one or more APC preparations, wherein at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is induced. In some embodiments, provided herein are methods for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating a population of immune cells from a biological sample with three or less APC preparations for three or less separate periods of time, wherein at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is induced.

[0503] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells (e.g., PBMCs) with APCs. In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating the population of immune cells (e.g., PBMCs) with APCs for a period of time. In some embodiments, the population of immune cells is derived from a biological sample. In some embodiments, the population of immune cells is derived from a sample (e.g., a biological sample) that has been depleted of CD14-expressing cells. In some embodiments, the population of immune cells is derived from a sample (e.g., a biological sample) that has been depleted of CD25-expressing cells. In some embodiments, the population of immune cells is derived from a sample (e.g., a biological sample) that has been depleted of CD14-expressing cells and CD25-expressing cells.

[0504] In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample. In some embodiments, provided herein are methods for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample for a first period of time, and then incubating at least one T cell of the biological sample with APCs.

[0505] In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells from a sample (e.g., a biological sample) with FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells from a sample (e.g., a biological sample) with APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a sample (e.g., a biological sample) with APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample (e.g., for a period of time) and then contacting the T cells of the biological sample with APCs. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells from a sample (e.g., a biological sample) with one or more APC preparations. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a sample (e.g., a biological sample) with one or more APC preparations for one or more separate periods of time.In some embodiments, a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a sample (e.g., a biological sample) with one or more APC preparations for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate periods. In some embodiments, the one or more separate periods are less than 28 days calculated from incubation of the population of immune cells with a first APC preparation of the one or more APC preparations.

[0506] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells with APCs for a period of time, wherein the population of immune cells is derived from a biological sample comprising PBMCs. In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells with APCs for a period of time, wherein the population of immune cells is derived from a biological sample that has been depleted of CD14- and / or CD25-expressing cells.

[0507] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) for a period of time.

[0508] In some embodiments, a method of preparing a pharmaceutical composition comprising antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample, and then contacting the T cells of the biological sample with APCs.

[0509] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate periods of time, thereby inducing or expanding antigen-specific T cells, wherein the one or more separate periods of time are less than 28 days calculated from the incubation of the population of immune cells with a first of the one or more APC preparations. In some embodiments, incubating the population of immune cells from the biological sample with the one or more APC preparations for one or more separate periods of time is performed in a medium containing IL-7, IL-15, or a combination thereof. In some embodiments, the medium further comprises an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof. The IDO inhibitor can be epacadostat, navoximod, 1-methyltryptophan, or a combination thereof. In some embodiments, the IDO inhibitor inhibits antigen-specific CD8 + In some embodiments, the IDO inhibitor may increase the number of memory CD8 cells. + The functional profile of T cell responses can be maintained. PD-1 antibodies can increase the absolute number of antigen-specific memory CD8+ T cell responses. PD-1 antibodies can increase the proliferation rate of cells treated with such antibodies. The addition of IL-12 can increase the expansion of antigen-specific cells and / or CD8 + This may result in an increase in the frequency of T cells.

[0510] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate periods of time, thereby expanding or inducing the antigen-specific T cells, the antigen-specific T cells, the antigen-specific CD4 + T cells or antigen-specific CD8 +The percentage of T cells is shown as total T cells, total CD4 + T cells, total CD8 + at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the T cells, total immune cells or total cells.

[0511] In some embodiments, a method of preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with three or less APC preparations for three or less separate periods of time, thereby stimulating the T cells to become antigen-specific T cells.

[0512] In some embodiments, the population of immune cells is derived from a biological sample that has been depleted of CD14 and / or CD25-expressing cells. In some embodiments, the APCs are APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, the APCs comprise one or more APC preparations. In some embodiments, the APC preparation comprises three or fewer APC preparations. In some embodiments, the APC preparations are sequentially incubated with immune cells within one or more separate periods.

[0513] In some embodiments, the biological sample is from a subject. In some embodiments, the subject is a human. For example, the subject may be a patient or a donor. In some embodiments, the subject has a disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the antigen-specific T cells are CD4 + and / or CD8 +In some embodiments, the antigen-specific T cells include CD4-enriched T cells and / or CD8-enriched T cells. For example, CD4 + T cells and / or CD8 + T cells can be isolated, enriched, or purified from a biological sample from a subject, including PBMCs. In some embodiments, antigen-specific T cells are naive CD4 + and / or naive CD8 + In some embodiments, the antigen-specific T cells are memory CD4 + and / or Memory CD8 + T cells.

[0514] In some embodiments, the at least one antigenic peptide sequence comprises: (A) a cancer antigen peptide having an IC of less than 500 nM; 50 The at least one antigenic peptide sequence comprises a mutation selected from (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a read-through mutation, (E) a gene fusion mutation, and combinations thereof, that binds to an HLA protein of interest at a specific site and with higher affinity than the corresponding wild-type peptide. In some embodiments, each of the at least one antigenic peptide sequence binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in non-cancer cells of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by an expressed gene in cancer cells of the subject. In some embodiments, one or more of the at least one antigenic peptide sequence has a length of 8 to 50 naturally occurring amino acids. In some embodiments, the at least one antigenic peptide sequence comprises a plurality of antigenic peptide sequences. In some embodiments, the plurality of antigenic peptide sequences comprises 2 to 50, 3 to 50, 4 to 50, 5 to 5, 6 to 50, 7 to 50, 8 to 50, 9 to 50, or 10 to 50 antigenic peptide sequences.

[0515] In some embodiments, the APC comprises an APC loaded with one or more antigenic peptides comprising one or more of at least one antigenic peptide sequence. In some embodiments, the APC is an autologous APC or an allogeneic APC. In some embodiments, the APC comprises a dendritic cell (DC).

[0516] In some embodiments, the method comprises depleting CD14 and / or CD25 expressing cells from the biological sample. + The step of depleting the cells comprises contacting the APCs with a CD14 binding agent. In some embodiments, the APCs + Derived from monocytes. In some embodiments, APCs are enriched from biological samples. For example, APCs can be isolated, enriched or purified from subject-derived biological samples, including PBMCs.

[0517] In some embodiments, the APCs are stimulated with one or more cytokines or growth factors. In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, or a combination thereof. In some embodiments, the one or more cytokines or growth factors include IL-4, IFN-γ, LPS, GM-CSF, TNF-α, IL-1β, PGE1, IL-6, IL-7, or a combination thereof.

[0518] In some embodiments, the APCs are from a second biological sample. In some embodiments, the second biological sample is from the same subject.

[0519] In some embodiments, the percentage of antigen-specific T cells in the method is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. In some embodiments, the percentage of antigen-specific T cells in the methods is about 0.1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to 65%, or about 65% to about 70% of total T cells or total immune cells. + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. In some embodiments, the antigen-specific memory CD8 + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. +The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. In some embodiments, the percentage of antigen-specific T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the percentage of antigen-specific CD8 T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the antigen-specific naive CD8 T cells in the biological sample are at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the percentage of antigen-specific memory CD8 T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the antigen-specific CD4 T cells in the biological sample are at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0520] In some embodiments, the biological sample is a freshly obtained or frozen sample from a subject.

[0521] In some embodiments, the method includes incubating one or more APC preparations with a first medium comprising at least one cytokine or growth factor for a first period of time. In some embodiments, the first period of time is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days. In some embodiments, the first period of time is not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days. In some embodiments, the first period of time is at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 days. In some embodiments, the first period of time is not more than 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the at least one cytokine or growth factor comprises GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-γ, LPS, IFN-α, R848, LPS, ss-rna40, poly I:C, or any combination thereof.

[0522] In some embodiments, the method includes incubating one or more APC preparations with at least one peptide for a second period of time, which in some embodiments is one hour or less.

[0523] In some embodiments, the method includes incubating one or more APC preparations with a second medium containing one or more cytokines or growth factors for a third period of time, thereby obtaining mature APCs. In some embodiments, the one or more cytokines or growth factors include GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-4, FLT3L, IFN-γ, LPS, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848 (resiquimod), LPS, ss-rna40, poly I:C, CpG, or a combination thereof. In some embodiments, the third period of time is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days or less. In some embodiments, the third period of time is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days. In some embodiments, the third period of time is no longer than 2, 3, 4, or 5 days. In some embodiments, the third period of time is at least 1, 2, 3, or 4 days.

[0524] In some embodiments, the method further comprises removing the one or more cytokines or growth factors of the second culture medium after the third period of time and before the start of the fourth period of time.

[0525] Antigen-loaded PBMCs for in vitro T cell induction In some embodiments, the methods provided herein include isolating PBMCs from a human blood sample and directly loading the PBMCs with antigen. PBMCs directly contacted with antigen can readily take up the antigen by phagocytosis and present the antigen to T cells that may be present in or added to the culture. In some embodiments, the methods provided herein include isolating PBMCs from a human blood sample and nucleofecting or electroporating polynucleotides, such as mRNA encoding one or more antigens, into the PBMCs. In some embodiments, antigen delivery to PBMCs instead of antigen-presenting cells that mature into DCs offers significant advantages in terms of time and manufacturing efficiency. PBMCs can be further depleted of one or more cell types. In some embodiments, PBMCs can be depleted of CD3+ cells during the initial antigen loading period, and the CD3+ cells can be returned to culture for the PBMCs to stimulate CD3+ T cells. In some embodiments, PBMCs can be depleted of CD25+ cells. In some embodiments, PBMCs can be depleted of CD14+ cells. In some embodiments, PBMCs can be depleted of CD19+ cells. In some embodiments, PBMCs can be depleted of both CD14-expressing cells and CD25-expressing cells. In some embodiments, CD11b+ cells are depleted from PBMC samples before antigen loading. In some embodiments, CD11b+ and CD25+ cells are depleted from PBMC samples before antigen loading.

[0526] In some embodiments, PBMCs isolated from human blood samples may be handled as minimally as possible prior to loading with antigen. Increased handling of PBMCs, such as freezing and thawing cells, multiple cell depletion steps, etc., may compromise cell health and viability.

[0527] In some embodiments, the PBMCs are allogeneic to the subject of the therapy. In some embodiments, the PBMCs are allogeneic to the subject of adoptive cell therapy using antigen-specific T cells.

[0528] In some embodiments, the PBMCs are HLA-matched to the subject of therapy. In some embodiments, the PBMCs are allogeneic and HLA subtype-matched to the subject, but the CD3+ T cells are autologous. The PBMCs are loaded with the respective antigens (e.g., derived from analysis with a peptide presentation analysis platform such as RECON) and co-cultured with the subject's PBMCs containing T cells to stimulate antigen-specific T cells.

[0529] In some embodiments, mRNA is used as an immunogen for uptake and antigen presentation. One advantage of using mRNA to load PBMCs over peptide antigens is that RNA is self-adjuvanting and does not require additional adjuvants. Another advantage of using mRNA is that peptides are endogenously processed and presented. In some embodiments, the mRNA contains shortmer constructs encoding 9-10 amino acid peptides that constitute the epitope. In some embodiments, the mRNA contains longmer constructs encoding approximately 25 amino acid peptides. In some embodiments, the mRNA contains concatenations of multiple epitopes. In some embodiments, concatemers may contain one or more epitopes from the same antigenic protein. In some embodiments, concatemers may contain one or more epitopes from several different antigenic proteins. Some embodiments are described in the Examples section. Antigen loading of PBMCs by antigen loading can involve various mechanisms of nucleic acid delivery and incorporation into PBMCs. In some embodiments, delivery or integration mechanisms include transfection, electroporation, nucleofection, chemical delivery, eg, lipid-encapsulated or liposome-mediated delivery.

[0530] The use of antigen-loaded PBMCs to stimulate T cells saves the maturation time required in methods of generating DCs from PBMC samples prior to T cell stimulation. In some embodiments, the use of antigen-loaded PBMCs, e.g., mRNA-loaded PBMCs, as APCs reduces the total production time by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the use of antigen-loaded PBMCs as APCs reduces the total production time by 3 days. In some embodiments, the use of antigen-loaded PBMCs as APCs reduces the total production time by 4 days. In some embodiments, the use of antigen-loaded PBMCs as APCs reduces the total production time by 5 days. In some embodiments, the use of antigen-loaded PBMCs as APCs reduces the total production time by 6 days. In some embodiments, the use of antigen-loaded PBMCs as APCs reduces the total production time by 7 days.

[0531] In some embodiments, the use of mRNA as an antigen may be preferred because it is easy to design and manufacture the nucleic acid and transfect PBMCs. In some embodiments, PBMCs loaded with mRNA can stimulate T cells and generate a higher number of antigen-specific T cells. In some embodiments, PBMCs loaded with mRNA can stimulate T cells and generate a higher yield of antigen-specific T cells. In some embodiments, PBMCs loaded with mRNA can stimulate T cells and generate antigen-specific T cells that express more input antigens, i.e., reactive to a variety of antigens. In some embodiments, PBMCs loaded with mRNA can stimulate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigen-reactive T cells in an expanded cell pool. In some embodiments, PBMCs loaded with mRNA can stimulate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigen-reactive T cells than conventional antigen-loaded APCs (e.g., peptide-loaded DCs). How to treat

[0532] Provided herein is a method for treating cancer in a subject, the method comprising the steps of: I. contacting cancer neoantigen-loaded antigen-presenting cells (APCs) with isolated T cells ex vivo, wherein the cancer neoantigen-loaded antigen-presenting cells (APCs) are CD11b-depleted; II. preparing ex vivo cancer neoantigen-primed T cells for a cellular composition for cancer immunotherapy; and III. administering the cellular composition for cancer immunotherapy to the subject, wherein at least one or more conditions or symptoms associated with cancer are reduced or alleviated by the administering step, thereby treating the subject, wherein the cancer neoantigen-loaded APCs and the cancer neoantigen-primed T cells each express a protein to which the neoantigen can specifically bind, encoded by an HLA allele expressed in the subject.

[0533] In some embodiments, the method further comprises administering one or more of the at least one antigen-specific T cells to a subject. In some embodiments, the therapeutic composition comprising the T cells is administered by injection. In some embodiments, the therapeutic composition comprising the T cells is administered by infusion. When administration is by injection, the active agent may be formulated in an aqueous solution, particularly a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution may contain a formulator agent, such as a suspending agent, a stabilizing agent, and / or a dispersing agent. In another embodiment, the pharmaceutical composition does not contain an adjuvant or any other substance added to enhance the immune response stimulated by the peptide. In some embodiments, the method further comprises administering one or more of the at least one antigen-specific T cells to a subject as a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition comprises a preservative or stabilizer. In some embodiments, the preservative or stabilizer is selected from a cytokine, a growth factor, an adjuvant, or a chemical. In some embodiments, the at least one antigen-specific T cell is administered to the subject within 28 days of collecting the PBMC sample from the subject.

[0534] In addition to the formulations described above, active agent can also be formulated as depot preparation.This long-acting formulation can be administered by implantation or transdermal delivery (for example, subcutaneous or intramuscular), intramuscular injection, or transdermal patch.Therefore, for example, agent can be formulated with suitable polymeric or hydrophobic material (for example, as emulsion in acceptabl...

Claims

1. An ex vivo method for preparing tumor antigen-specific T cells, comprising: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen presenting cells (APCs) and T cells, thereby forming a population of CD14- and / or CD25-depleted immune cells comprising a first population of APCs and T cells; (b) treating the first population of APCs and T cells of step (a) for a first period of time; (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) a first polynucleotide encoding a first polypeptide comprising two or more short epitope sequences, and a second polynucleotide encoding a second polypeptide comprising two or more long epitope sequences, wherein the short epitope sequence comprises 8 to 12 amino acids and the long epitope sequence comprises 16 to 25 amino acids, and wherein the two or more short epitope sequences and the two or more long epitope sequences are derived from a human subject with cancer. thereby forming a population of cells comprising stimulated T cells; (c) expanding the population of cells comprising the stimulated T cells, thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one epitope sequence and (ii) an MHC protein expressed by the cancer cells or APCs of the human subject of (b)(ii), wherein at least 30% of the expanded population of cells comprising tumor antigen-specific T cells are effector memory T cells, and wherein the human subject has ovarian cancer. A method comprising:

2. The ex vivo method described in claim 1, wherein the first polynucleotide encoding the first polypeptide is mRNA, and the second polynucleotide encoding the second polypeptide is mRNA.

3. The method described in claim 2, wherein the first mRNA encodes a polypeptide comprising up to 40 short tumor antigen epitope sequences expressed by cancer cells of the human subject having cancer, and the second mRNA encodes a polypeptide comprising up to 20 long tumor antigen epitope sequences expressed by cancer cells of the human subject having cancer.

4. 4. The method of any one of claims 1 to 3, wherein steps (b) and (c) are carried out for less than 28 days.

5. The method of claim 1, wherein the expanded population of cells comprising the tumor antigen-specific T cells in step (c) comprises a dose of 0.75 x 10^8 to 1.25 x 10^10 total cells.

6. The expanded population of cells comprising: 0.75 x 10^8 to 1 x 10^9 total cells, 0.75 x 10^8 to 0.75 x 10^9 total cells, 1 x 10^8 to 1.25 x 10^9 total cells, 1x10^8 to 1x10^9 total cells, 1 x 10^8 to 0.75 x 10^9 total cells, 1.25 x 10^8 to 1.25 x 10^9 total cells, 1.25 x 10^8 to 1 x 10^9 total cells, 1.25 x 10^8 to 0.75 x 10^9 total cells, 1.5 x 10^9 to 1 x 10^10 total cells, 1.5 x 10^9 to 0.75 x 10^10 total cells, 2 x 10^9 to 1.25 x 10^10 total cells, 2 x 10^9 to 1 x 10^10 total cells, 2 x 10^9 to 0.75 x 10^10 total cells, 2.5 x 10^9 to 1.25 x 10^10 total cells, 2.5 x 10^9 to 1 x 10^10 total cells, or 2.5 x 10^9 to 0.75 x 10^10 total cells 6. The ex vivo method of claim 5, comprising:

7. The ex vivo method of claim 1, wherein the percentage of IFNγ+ cells in the expanded population of cells comprising the tumor antigen-specific T cells is at least 15% of the tumor antigen-specific T cells.

8. The ex vivo method of claim 1, wherein the percentage of TNFα+ and IFNγ+ cells in the expanded population of cells comprising the tumor antigen-specific T cells is at least 2% of the tumor antigen-specific T cells.

9. The ex vivo method of claim 1, wherein the percentage of TNFα+ and CD107a+ cells in the expanded population of cells comprising the tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cells.

10. The ex vivo method of claim 1, wherein the percentage of IFNγ+ and CD107a+ cells in the expanded population of cells comprising the tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cells.

11. The ex vivo method of claim 1, wherein the percentage of TNFα+ and IFNγ+ and CD107a+ cells in the expanded population of cells comprising the tumor antigen-specific T cells is at least 0.1% of the tumor antigen-specific T cells.

12. 2. The ex vivo method of claim 1, wherein the percentage of CD4+ T cells in the expanded population of cells comprising the tumor antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 15%.

13. 2. The ex vivo method of claim 1, wherein the percentage of CD4+ T cells in said expanded population of cells, including said tumor antigen-specific T cells, that are effector memory T cells (CD62L- and CD45RA-), is at least 60%.

14. 2. The ex vivo method of claim 1, wherein step (b) comprises incubating the APCs and the first population of T cells of step (a) in the presence of IL-21 for a first period of time.

15. 2. The ex vivo method of claim 1, wherein step (c) comprises expanding said population of cells comprising stimulated T cells in the presence of IL-21.

16. A pharmaceutical composition for use in treating cancer in a human subject, comprising an expanded population of cells comprising tumor antigen-specific T cells prepared by the ex vivo method of claim 1.

17. A pharmaceutical composition for use in combination with an immune checkpoint inhibitor in the treatment of cancer in a human subject, the pharmaceutical composition comprising an expanded population of cells comprising tumor antigen-specific T cells prepared by the ex vivo method of claim 1.

18. The pharmaceutical composition of claim 17, wherein the expanded population of cells is administered to the human subject followed by administration of the immune checkpoint inhibitor.

19. 19. The pharmaceutical composition of claim 18, wherein the immune checkpoint inhibitor comprises an anti-PD1 antibody.

20. 20. The pharmaceutical composition of claim 19, wherein the immune checkpoint inhibitor further comprises an anti-CTLA4 antibody.

Images