T cell preparation compositions and methods

Abstract

Generating 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 preparation methods and therapeutic T cell compositions, which can be used to treat subjects with cancer and other conditions, diseases and disorders, individual antigen-specific T cell therapy.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 583,229, filed on November 8, 2017, U.S. Provisional Application No. 62 / 588,590, filed on November 20, 2017, U.S. Provisional Application No. 62 / 618,445, filed on January 17, 2018, and U.S. Provisional Application No. 62 / 737,625, filed on September 27, 2018, which are incorporated herein by reference in their entireties. Background Art

[0003] Tumor vaccines are usually composed of tumor antigens and immunostimulatory molecules (such as adjuvants, cytokines or TLR ligands), which work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. This type of vaccine contains a mixture of shared tissue-restricted tumor antigens or shared antigens and patient-specific antigens in the form of whole tumor cell preparations. Shared tissue-restricted tumor antigens are ideal immunogenic proteins that are selectively expressed in tumors of many individuals and are usually 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. Since whole tumor cells are isolated from autologous patients, the cells can include patient-specific tumor antigens as well as shared tumor antigens. Finally, there is a third type of tumor antigen - a new antigen rarely used in vaccines, which consists of proteins with tumor-specific mutations that cause changes in amino acid sequences (which can be patient-specific or shared). Such mutant proteins: (a) are unique to tumor cells as mutations and their corresponding proteins are only present in the tumor; (b) avoid central tolerance and are therefore more likely to be immunogenic; and (c) provide excellent targets for immune recognition, including recognition by humoral and cellular immunity.

[0004] Adoptive immunotherapy or adoptive cell therapy (ACT) is the transfer of genetically modified T lymphocytes to subjects to treat diseases. Adoptive immunotherapy has not yet realized its potential to treat numerous diseases, including cancer, infectious diseases, autoimmune diseases, inflammatory diseases and immunodeficiency. However, most (if not all) adoptive immunotherapy requires T cell activation and expansion steps to produce clinically effective therapeutic doses of T cells. Due to the inherent complexity of living cell culture and differences between patients, the current technology for producing therapeutic doses of T cells (including engineered T cells) is still limited by cumbersome T cell preparation processes. Existing T cell preparation processes are not easy to scale up, are not repeatable, unreliable or inefficient, and often produce inferior T cell products, which may be prone to exhaustion and loss of effector immune cell function. So far, engineered T cell adoptive immunotherapy has only achieved limited success and routinely shows variable clinical activity. Therefore, this type of therapy is not suitable for widespread clinical use. Therefore, it is still necessary to develop compositions and methods for expanding and inducing antigen-specific T cells with favorable phenotypes and functions. Summary of the invention

[0005] In some aspects, a pharmaceutical composition is provided herein, comprising: a population of immune cells from a biological sample and a pharmaceutically acceptable excipient, the biological sample comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence; wherein the amount of immune cells expressing CD14 and / or CD25 in the population is proportionally different from the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some aspects, a pharmaceutical composition is provided herein, comprising: a population of immune cells from a biological sample and a pharmaceutically acceptable excipient, the biological sample comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence; wherein the amount of immune cells expressing CD14 and / or CD25 in the population is different from the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some aspects, a pharmaceutical composition is provided herein, comprising: a population of immune cells from a biological sample and a pharmaceutically acceptable excipient, the biological sample comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the percentage of immune cells expressing CD14 and / or CD25 in the population is different from the percentage of immune cells expressing CD14 and / or CD25 in the biological sample. In some aspects, a pharmaceutical composition is provided herein, comprising: a population of immune cells from a biological sample and a pharmaceutically acceptable excipient, the biological sample comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the concentration of immune cells expressing CD14 and / or CD25 in the population is different from the concentration of immune cells expressing CD14 and / or CD25 in the biological sample.

[0006] In some aspects, a composition is provided herein, which includes an immune cell colony from a biological sample, wherein the amount of immune cells expressing CD14 and CD25 in the colony is proportionally less than the amount of immune cells expressing CD14 and CD25 in the biological sample. In some aspects, a composition is provided herein, which includes an immune cell colony from a biological sample, wherein the amount of immune cells expressing CD14 and CD25 in the colony is less than the amount of immune cells expressing CD14 and CD25 in the biological sample. In some aspects, a composition is provided herein, which includes an immune cell colony from a biological sample, wherein the percentage of immune cells expressing CD14 and CD25 in the colony is less than the percentage of immune cells expressing CD14 and CD25 in the biological sample. In some aspects, a composition is provided herein, which includes an immune cell colony from a biological sample, wherein the concentration of immune cells expressing CD14 and CD25 in the colony is less than the concentration of immune cells expressing CD14 and CD25 in the biological sample.

[0007] In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, wherein the immune cell population comprises T cells from a biological sample, wherein the T cells comprise at least one antigen-specific T cell, which antigen-specific T cell is a T cell stimulated by an antigen presenting cell (APC) and comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the APC is a FLT3L-stimulated APC.

[0008] In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, wherein the immune cell population comprises T cells from a biological sample, wherein the T cells comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence; wherein the at least one antigen-specific T cell is a total T cell, a total CD4 + T cells, total CD8 + 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, total CD4 T cells, or total immune cells in the biological sample; and wherein the biological sample comprises one or more antigen-specific T cells, wherein the one or more antigen-specific T cells in the biological sample are 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, total CD4 T cells, or total immune cells in the biological sample; + T cells, total CD8+ Total CD4 T cells or total immune cells + T cells, total CD8 +T cells, total T cells or total immune cells at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01% or 0.05%. In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, the immune cell population comprising T cells expanded or induced from a biological sample, wherein the T cells expanded or induced from the biological sample comprise at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence; wherein the amount, concentration or percentage of the at least one antigen-specific T cell is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1. , 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900 or 1000 times. In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, the immune cell population comprising T cells from a biological sample, wherein the T cells comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence; wherein the at least one antigen-specific T cell comprises an expanded or induced antigen-specific T cell, which is expanded or induced from the biological sample by at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, , 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900 or 1000 times. In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, the immune cell population comprising CD4 +T cells, of which the CD4 + T cells contain at least one antigen-specific CD4 + T cells, the antigen-specific CD4 + The T cell comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the at least one antigen-specific CD4 + T cells are total T cells, total CD4 + T cells, total CD8 + T cells or total immune cells; and wherein the biological sample comprises one or more antigen-specific CD4 T cells or total immune cells; and wherein the biological sample comprises one or more antigen-specific CD4 T cells or total immune cells; and wherein the biological sample comprises one or more antigen-specific CD4 T cells or total immune cells; and + T cells, wherein the one or more antigen-specific CD4 + T cells are the total T cells, total CD4 + T cells, total CD8 + Total CD4 T cells or total immune cells + T cells, total CD8 + T cells, total T cells, or total immune cells. In some embodiments, the pharmaceutical composition comprises an immune cell population and a pharmaceutically acceptable excipient, wherein the immune cell population comprises CD4 T cells from a biological sample. + T cells, of which the CD4 + T cells contain at least one antigen-specific CD4 + T cells, the antigen-specific CD4 + The T cell comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the at least one antigen-specific CD4 + T cells are total CD8 +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 T cells; and wherein the biological sample comprises one or more antigen-specific CD4 + T cells, wherein the one or more antigen-specific CD4 + T cells are the total CD4 + At most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01% or 0.05% of T cells. In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, the immune cell population comprising CD4 T cells expanded or induced from a biological sample. + T cells, wherein the CD4 + T cells contain at least one antigen-specific CD4 + T cells, the antigen-specific CD4 + The T cells comprise a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the amount, concentration or percentage of the at least one antigen-specific T cell is the antigen-specific CD4 + T cells, total CD4 + 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 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 9, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900 or 1000 times. In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, the immune cell population comprising CD4 + T cells, of which the CD4 + T cells contain at least one antigen-specific CD4 +T cells, the antigen-specific CD4 + The T cell comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the at least one antigen-specific CD4 + T cells contain expanded or induced antigen-specific CD4 + T cells, which are expanded or induced from the biological sample by at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 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, 9, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900 or 1000 times. In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, the immune cell population comprising CD4 from a biological sample depleted of cells expressing CD25 or cells expressing CD25 and CD14. + T cells, of which the CD4 + T cells contain at least one antigen-specific CD4 + T cells, the antigen-specific CD4 + The T cell comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the at least one antigen-specific CD4 + T cells contain expanded or induced antigen-specific CD4 + 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500-fold. In some aspects, the present invention provides a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, wherein the immune cell population comprises CD8 + T cells, among which the CD8 +T cells contain at least one antigen-specific CD8 + T cells, the antigen-specific CD8 + The T cell comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the at least one antigen-specific CD8 + T cells are total T cells, total CD4 + T cells, total CD8 + T cells or total immune cells; and wherein the biological sample comprises one or more antigen-specific CD8 T cells or total immune cells; and wherein the biological sample comprises one or more antigen-specific CD8 T cells or total immune cells; and + T cells, wherein the one or more antigen-specific CD8 + T cells are the total T cells, total CD4 + T cells, total CD8 + Total CD4 T cells or total immune cells + T cells, total CD8 + T cells, total T cells, or total immune cells. In some embodiments, the pharmaceutical composition comprises an immune cell population and a pharmaceutically acceptable excipient, wherein the immune cell population comprises CD8 T cells from a biological sample. + T cells, among which the CD8 + T cells contain at least one antigen-specific CD8 + T cells, the antigen-specific CD8 + The T cell comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the at least one antigen-specific CD8 + T cells are total CD8 + 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 T cells; and wherein the biological sample comprises one or more antigen-specific CD4 +T cells, wherein the one or more antigen-specific CD8 + T cells are the total CD8 + At most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01% or 0.05% of T cells. In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, the immune cell population comprising CD8 T cells expanded or induced from a biological sample. + T cells, wherein the CD8 + T cells contain at least one antigen-specific CD8 + T cells, the antigen-specific CD8 + The T cells comprise a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the amount, concentration or percentage of the at least one antigen-specific T cell is an antigen-specific CD8 + T cells, total CD8 + 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 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 9, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900 or 1000 times. In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, the immune cell population comprising CD8 + T cells, among which the CD8 + T cells contain at least one antigen-specific CD8 + T cells, the antigen-specific CD8 + The T cell comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the at least one antigen-specific CD8 + T cells contain expanded or induced antigen-specific CD4 +T cells, which are expanded or induced from the biological sample by at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900 or 1000 times. In some aspects, provided herein is a pharmaceutical composition comprising an immune cell population and a pharmaceutically acceptable excipient, the immune cell population comprising CD8 + T cells, among which the CD8 + T cells contain at least one antigen-specific CD8 + T cells, the antigen-specific CD8 + The T cell comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the at least one antigen-specific CD8 + T cells contain expanded or induced antigen-specific CD8 + T cells, which are expanded or induced from the biological sample by at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19,20,25,30,35,40,45,50,55,60,75,80,85,90,95,100,125,150,175,200 , 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900 times.

[0009] In some embodiments, the at least one antigen-specific T cell comprises at least one APC stimulated T cell. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the colony is proportionally less 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 colony is proportionally greater than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the percentage of immune cells expressing CD14 and / or CD25 in the colony is less than the percentage of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the percentage of immune cells expressing CD14 and / or CD25 in the colony is greater than the percentage of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the concentration of immune cells expressing CD14 and / or CD25 in the colony is less than the concentration of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the concentration of immune cells expressing CD14 and / or CD25 in the population is greater than the concentration of immune cells expressing CD14 and / or CD25 in the biological sample.

[0010] In some embodiments, the biological sample is from a subject. In some embodiments, the subject is a human. In some embodiments, the subject suffers from a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is selected from ovarian cancer, lung cancer, and melanoma.

[0011] In some embodiments, the at least one antigen-specific T cell comprises at least one CD4 + In some embodiments, the at least one antigen-specific T cell comprises at least one CD8 + 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 naive T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory CD4 + In some embodiments, the at least one antigen-specific T cell comprises an expanded memory CD4 + In some embodiments, the at least one antigen-specific T cell comprises a naive CD4 +In some embodiments, the at least one antigen-specific T cell comprises an induced naive CD4 + In some embodiments, the at least one antigen-specific T cell comprises a memory CD8 + In some embodiments, the at least one antigen-specific T cell comprises an expanded memory CD8 + In some embodiments, the at least one antigen-specific T cell comprises a naive CD8 + In some embodiments, the at least one antigen-specific T cell comprises an induced naive CD8 + T cells.

[0012] In some embodiments, the at least one antigen-specific T cell is stimulated in a 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. In some embodiments, the IDO inhibitor is epacadostat, navoximod, 1-methyltryptophan, or a combination thereof.

[0013] In some embodiments, the at least one antigenic peptide sequence comprises a mutation selected from the group consisting of: (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. In some embodiments, the at least one antigenic peptide sequence binds to the subject's HLA protein with a greater affinity than the corresponding wild-type peptide. In some embodiments, the at least one antigenic peptide sequence binds to the subject's HLA protein with a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 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, the TCR has a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 Binding peptide-HLA complex. In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in the non-cancerous cell of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or expressed gene of a cancer cell of the subject.

[0014] In some embodiments, one or more of the at least one antigenic peptide sequence is 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 naturally occurring amino acids in length. In some embodiments, one or more of the at least one antigenic peptide sequence binds to a protein encoded by a class I HLA allele and is 8-12 naturally occurring amino acids in length. In some embodiments, one or more of the at least one antigenic peptide sequence binds to a protein encoded by a class II HLA allele and is 16-25 naturally occurring amino acids in length.

[0015] 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 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 antigen is a new antigen, a tumor-associated antigen, an overexpressed antigen, a viral antigen, a minor histocompatibility antigen or a combination thereof.

[0016] In some embodiments, the APC is one or more APC preparations. In some embodiments, the APC includes an APC loaded with one or more antigenic peptides, wherein the one or more antigenic peptides include one or more of the at least one antigenic peptide sequence. In some embodiments, the APC is an autologous APC, an allogeneic APC, or an artificial APC. In some embodiments, the APC includes a dendritic cell (DC). In some embodiments, the APC is derived from a CD14 + In some embodiments, the APC is a CD14-enriched APC. In some embodiments, the APC is a CD141-enriched APC. In some embodiments, the CD14 + Monocytes are enriched from a biological sample from a subject comprising PBMCs. In some embodiments, the CD14 +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, IFN-α, R848, LPS, ss-rna40, poly I:C or a combination thereof.

[0017] In some embodiments, the CD14 + The monocytes are from a second biological sample comprising PBMCs. In some embodiments, the second biological sample is from the same subject.

[0018] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0019] In some embodiments, the at least one antigen-specific T cell comprises a plurality of antigen-specific T cells. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition 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%, 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 at least one antigen-specific CD8 + The percentage of T cells is total CD4 + T cells, total CD8 + In some embodiments, at least one antigen-specific CD4 T cells, total T cells, or total immune cells in the composition 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%, 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%. + The percentage of T cells is total CD4 + T cells, total CD8 +In some embodiments, the percentage of at least one antigen-specific T cell in the biological sample 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%, 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+ / - cells, total T cells, or total immune cells. + T cells, total CD8 + T cells, total T cells, or total immune cells. In some embodiments, the at least one antigen-specific CD8 + The percentage of T cells is total CD4 + T cells, total CD8 + T cells, total T cells, or total immune cells. In some embodiments, the at least one antigen-specific CD4 + The percentage of T cells is total CD4 + T cells, total CD8 + 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 T cells, total T cells or total immune cells.

[0020] In some embodiments, the pharmaceutical composition comprises: a population of immune cells from a biological sample, the biological sample comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the amount of immune cells expressing CD19 and / or CD16 in the population is different from the amount of immune cells expressing CD19 and / or CD16 in the biological sample. In some embodiments, the pharmaceutical composition comprises: a population of immune cells from a biological sample, the biological sample comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the amount of immune cells expressing CD19 and / or CD16 in the population is less than the amount of immune cells expressing CD19 and / or CD16 in the biological sample.

[0021] In some aspects, provided herein is a method of treatment comprising administering a composition described herein to a subject having a disease or disorder.

[0022] In some aspects, provided herein are methods of using the compositions described herein to prepare a medicament for use in therapy.

[0023] 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 antigen peptide sequence, the method comprising incubating APCs with a population of immune cells from a biological sample depleted of cells expressing CD25 or CD14 and CD25, wherein the at least one antigen-specific T cell comprises an amount of expanded or induced antigen-specific T cells that is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 3. .5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 times: incubating the APCs with a population of immune cells from a biological sample depleted of cells expressing CD14. In one aspect, the invention provides a method for preparing at least one antigen-specific CD4 T cell receptor (TCR) having specificity for at least one antigenic peptide sequence. +The method comprises incubating APCs with a population of immune cells from a biological sample depleted of cells expressing CD25 or CD14 and CD25, wherein at least one antigen-specific CD4 + T cells contain a certain amount of expanded or induced antigen-specific CD4 + The amount of antigen-specific CD4 T cells is expanded or induced using a method comprising the steps of + The amount of T cells is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 , 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 times: incubating the APCs with a population of immune cells from a biological sample depleted of cells expressing CD14. In one aspect, the present invention provides a method for preparing at least one antigen-specific CD8 T cell receptor (TCR) having specificity for at least one antigen peptide sequence. + The method comprises incubating APCs with a population of immune cells from a biological sample depleted of cells expressing CD25 or CD14 and CD25, wherein the at least one antigen-specific CD8 + T cells contain a certain amount of expanded or induced antigen-specific CD8 + The amount of antigen-specific CD8 T cells is expanded or induced using a method comprising the steps of + The amount of T cells is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 , 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 times: incubating the APCs with a population of immune cells from a biological sample depleted of cells expressing CD14.

[0024] In some embodiments, the biological sample is further depleted of cells expressing CD19. In some embodiments, the biological sample is further depleted of cells expressing CD19. In some embodiments, the APC is an APC stimulated by FLT3L. In some embodiments, the incubation of the immune cell population is carried out 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. In some embodiments, the IDO inhibitor is epacadostat, navoximod, 1-methyltryptophan, or a combination thereof.

[0025] In one 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 antigen 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; thereafter incubating at least one T cell in the biological sample with an APC, wherein the at least one antigen-specific T cell comprises an amount of expanded or induced antigen-specific T cells that is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.5, 3.6, 3.7, 3.8, 4.9, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 7.21, 7.22, 7.23, 7.24, 7.25 , 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 times: the method does not include 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 it includes subsequently incubating at least one T cell in the biological sample with APCs. In one aspect, the present invention provides a method for preparing a method comprising at least one antigen-specific CD4 + Methods of pharmaceutical compositions of T cells, the antigen-specific CD4 + The method comprises: incubating a population of immune cells from a biological sample with a FMS-like tyrosine kinase 3 receptor ligand (FLT3L) for a first period of time; then incubating at least one CD4 T cell in the biological sample with a T cell receptor (TCR) specific for at least one antigenic peptide sequence; +T cells are incubated with APCs, wherein the at least one antigen-specific CD4 + T cells contain a certain amount of expanded or induced antigen-specific CD4 + T cells, which are antigen-specific CD4 T cells that are expanded or induced using the following methods + The amount of T cells is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 times: the method does not include incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with the immune cell population from the biological sample for a first period of time; and it includes thereafter incubating at least one CD4 + T cells are incubated with APCs. In one aspect, the present invention provides a method for preparing a method comprising at least one antigen-specific CD8 + Method of pharmaceutical composition of T cells, the antigen specific CD8 + The method comprises: incubating a population of immune cells from a biological sample with a FMS-like tyrosine kinase 3 receptor ligand (FLT3L) for a first period of time; then incubating at least one CD8 + T cells are incubated with APCs, wherein the at least one antigen-specific CD8 + T cells contain a certain amount of expanded or induced antigen-specific CD8 + T cells, which are expanded or induced using the following methods: +The amount of T cells is at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 times: the method does not include incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with the immune cell population from the biological sample for a first period of time; and it includes thereafter incubating at least one CD8 + T cells were incubated with APCs.

[0026] 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 an APC with a population of immune cells from a biological sample depleted of cells expressing CD14 and / or CD25.

[0027] In one aspect, the present invention provides 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.

[0028] In one aspect, the present invention provides a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell, wherein the antigen-specific T cell comprises a T cell receptor (TCR) specific for at least one antigen peptide sequence, the method comprising: incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with an immune cell population from a biological sample for a first period of time; and then incubating at least one T cell from the biological sample with an APC.

[0029] In one aspect, the present invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen 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, the time period being less than 28 days from incubation of the immune cell population with the 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.

[0030] In one aspect, the present invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, the method comprising incubating a population of immune cells from a biological sample with three or fewer APC preparations for three or fewer separate time periods, wherein at least one antigen-specific memory T cell is expanded, or at least one antigen-specific naive T cell is induced.

[0031] In some embodiments, the population of immune cells is from a biological sample depleted of cells expressing CD14 and / or CD25.

[0032] In some embodiments, the APC is a FLT3L stimulated APC. In some embodiments, at least one of the APC preparations comprises a FLT3L stimulated APC. In some embodiments, at least two of the APC preparations comprise a FLT3L stimulated APC. In some embodiments, at least three of the APC preparations comprise a FLT3L stimulated APC. In some embodiments, each of the APC preparations comprises a FLT3L stimulated APC.

[0033] In some embodiments, the APC comprises one or more APC preparations. In some embodiments, the APC preparation comprises 3 or fewer APC preparations. In some embodiments, the APC preparation is sequentially incubated with the immune cells in one or more separate time periods. In some embodiments, the biological sample is from a subject. In some embodiments, the subject is a human. In some embodiments, the subject suffers from a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is selected from ovarian cancer, lung cancer, and melanoma.

[0034] In some embodiments, the at least one antigen-specific T cell comprises at least one CD4 + In some embodiments, the at least one antigen-specific T cell comprises at least one CD8 +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 at least one memory T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one naive T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one memory CD4 + In some embodiments, the at least one antigen-specific T cell comprises at least one naive CD4 + In some embodiments, the at least one antigen-specific T cell comprises at least one memory CD8 + In some embodiments, the at least one antigen-specific T cell comprises at least one naive CD8 + T cells.

[0035] In some embodiments, the at least one antigenic peptide sequence comprises a mutation selected from the group consisting of: (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. In some embodiments, the at least one antigenic peptide sequence comprises a point mutation and binds to the subject's HLA protein with a greater affinity than the corresponding wild-type peptide. In some embodiments, the at least one antigenic peptide sequence binds to the subject's HLA protein with a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 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, the TCR has a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50Binding peptide-HLA complex. In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in the non-cancerous cell of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or expressed gene of a cancer cell of the subject. In some embodiments, one or more of the at least one antigenic peptide sequence is 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 naturally occurring amino acids in length. In some embodiments, one or more of the at least one antigenic peptide sequence binds to a protein encoded by a class I HLA allele, and its length is 8-12 naturally occurring amino acids. In some embodiments, one or more of the at least one antigenic peptide sequence binds to a protein encoded by a class II HLA allele, and its length is 16-25 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 comprise 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 antigen is a neoantigen, a tumor-associated antigen, a viral antigen, a minor histocompatibility antigen, or a combination thereof.

[0036] In some embodiments, the method includes depleting cells expressing CD14 and / or CD25 from a biological sample. In some embodiments, depleting cells expressing CD14 and / or CD25 includes binding a CD14 and / or CD25 binding agent to an APC or an APC of the APC preparation. In some embodiments, the CD14 and / or CD25 binding agent is biotinylated. In some embodiments, depleting cells expressing CD14 and / or CD25 further includes binding an anti-biotin reagent on a solid support to the CD14 and / or CD25 binding agent. In some embodiments, the CD14 and / or CD25 binding agent is attached to a solid support.

[0037] In some embodiments, the APC or APC preparation of APCs comprises an APC loaded with one or more antigenic peptides, wherein the antigenic peptide comprises one or more of the at least one antigenic peptide sequence. In some embodiments, the APC or APC preparation of APCs is an autologous APC or an allogeneic APC. In some embodiments, the APC or APC preparation of APCs comprises a dendritic cell (DC). In some embodiments, the APC or APC preparation of APCs is derived from a CD14 + In some embodiments, the APC preparation or the APC of the APC preparation is enriched from a biological sample. In some embodiments, the APC or the APC of the APC preparation is 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, IFN-α, R848, LPS, ss-rna40, poly I:C or a combination thereof.

[0038] In some embodiments, the APCs or APCs of the APC preparation are from a second biological sample. In some embodiments, the second biological sample is from the same subject. In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0039] In some embodiments, the at least one antigen-specific T cell comprises a plurality of antigen-specific T cells. In some embodiments, the at least one antigen-specific T cell is a percentage of 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 T cells, total T cells, or total immune cells. In some embodiments, at least one antigen-specific CD8 + The percentage of T cells is total CD4 + T cells, total CD8 +In some embodiments, at least one antigen-specific CD4 T cells, total T cells, or total immune cells is present in 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%. + The percentage of T cells is total CD4 + T cells, total CD8 + In some embodiments, the percentage of at least one antigen-specific T cell in a biological sample 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%, 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+ / - cells, total T cells, or total immune cells. + T cells, total CD8 + T cells, total T cells, or total immune cells. In some embodiments, the at least one antigen-specific CD8 + The percentage of T cells is total CD4 + T cells, total CD8 + T cells, total T cells, or total immune cells. In some embodiments, the at least one antigen-specific CD4 + The percentage of T cells is total CD4 + T cells, total CD8 +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 T cells, total T cells or total immune cells.

[0040] In some embodiments, the method further comprises administering one or more of the at least one antigen-specific T cell to a subject.

[0041] In some embodiments, the total time period of the individual time periods is less than 28 days. In some embodiments, incubation includes incubating the first APC product of the APC product with the T cells for more than 7 days. In some embodiments, the method includes incubating the APC in one or more APC products with a first medium comprising at least one cytokine or growth factor for a first time period. In some embodiments, the at least one cytokine or growth factor includes GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IFN-α, R848, LPS, ss-rna40, poly I: C or any combination thereof. In some embodiments, the method includes incubating the one or more APC products with at least one peptide for a second time period. In some embodiments, the method includes incubating the APC in one or more APC products with a second medium comprising one or more cytokines or growth factors for a third time period to obtain mature APCs. In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IFN-α, R848, LPS, ss-rna40, poly I:C or a combination thereof. In some embodiments, the method further includes removing the one or more cytokines or growth factors of the second medium after the third time period and before starting the fourth time period.

[0042] In some embodiments, the methods are performed ex vivo.

[0043] In some embodiments, the biological sample is freshly obtained from a subject or is a frozen sample.

[0044] In some embodiments, the method comprises obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC.

[0045] In some embodiments, the method comprises depleting cells expressing CD14 and / or CD25 from a biological sample, thereby obtaining a CD14 and / or CD25 cell-depleted sample.

[0046] In some embodiments, the method comprises incubating the CD14 and / or CD25 cell-depleted sample with FLT3L for a first period of time.

[0047] In some embodiments, the method comprises incubating at least one peptide with the CD14 and / or CD25 cell-depleted sample for a second period of time, thereby obtaining a first mature APC peptide-loaded sample.

[0048] In some embodiments, the method comprises incubating the first mature APC peptide-loaded sample with at least one PBMC for a third period of time, thereby obtaining a first stimulated PBMC sample.

[0049] In some embodiments, the method comprises incubating the PBMCs of the first stimulated PBMC sample with the FLT3L-stimulated APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample.

[0050] In some embodiments, the method comprises incubating PBMCs of the first stimulated PBMC sample with FLT3L and a second APC peptide-loaded sample of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample.

[0051] In some embodiments, the method comprises incubating PBMCs from the first stimulated PBMC sample with FLT3L-stimulated APCs from the FLT3L and mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample.

[0052] In some embodiments, the method comprises incubating the PBMCs of the second stimulated PBMC sample with the FLT3L-stimulated APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample.

[0053] In some embodiments, the method comprises incubating PBMCs of the second stimulated PBMC sample with FLT3L and a third APC peptide-loaded sample of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample.

[0054] In some embodiments, the method comprises incubating the PBMCs of the second stimulated PBMC sample with the FLT3L-stimulated APCs of the FLT3L and mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample.

[0055] In some embodiments, the method comprises administering to a subject in need thereof at least one T cell of a first stimulated PBMC sample. In some embodiments, the method comprises administering to a subject in need thereof at least one T cell of a second stimulated PBMC sample. In some embodiments, the method comprises administering to a subject in need thereof at least one T cell of a third stimulated PBMC sample.

[0056] In some embodiments, the incubation of the PBMCs of the first stimulated PBMC sample is carried out in the presence of IL-7, IL-15, or a combination thereof. In some embodiments, the incubation of the PBMCs of the first stimulated PBMC sample is carried out in the presence of an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof. In some embodiments, the incubation of the PBMCs of the second stimulated PBMC sample is carried out in the presence of IL-7, IL-15, or a combination thereof. In some embodiments, the incubation of the PBMCs of the second stimulated PBMC sample is carried out in the presence of an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof.

[0057] In one aspect, the present invention provides a method comprising: obtaining a biological sample from a subject, wherein the biological sample comprises at least one antigen presenting cell (APC); enriching cells expressing CD14 from the biological sample, thereby obtaining CD14 + Cell-enriched sample; the CD14 + The cell-enriched sample is incubated with at least one cytokine or growth factor for a first period of time; at least one peptide is combined with CD14 + The method comprises incubating the APC-enriched sample with the cell-enriched sample for a second time period to obtain an APC peptide-loaded sample; incubating the APC peptide-loaded sample with one or more cytokines or growth factors for a third time period to obtain a mature APC sample; incubating the APCs of the mature APC sample with a CD14 and / or CD25-depleted sample containing PBMCs for a fourth time period; incubating the PBMCs with the APCs of the mature APC sample for a fifth time period; incubating the PBMCs with the APCs of the mature APC sample for a sixth time period; and administering at least one T cell of the PBMCs to a subject in need thereof.

[0058] In one aspect, provided herein is a method comprising: obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; 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; incubating the CD14 and / or CD25 and / or CD19 cell-depleted sample with FLT3L for a first period of time; 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 an APC peptide. A loaded sample; incubating the APC peptide-loaded sample with at least one PBMC for a third time period to obtain a first stimulated PBMC sample; incubating the PBMCs of the first stimulated PBMC sample with APCs of a mature APC sample for a fourth time period to obtain a second stimulated PBMC sample; optionally, incubating the PBMCs of the second stimulated PBMC sample with APCs of a mature APC sample for a fifth time period to obtain a third stimulated PBMC sample; administering at least one T cell from the first, second or third stimulated PBMC sample to a subject in need.

[0059] In one aspect, the present invention provides a method comprising obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; 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; incubating the CD14 and / or CD25 and / or CD19 cell-depleted sample with FLT3L for a first period of time; 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 an APC peptide-loaded sample; and incubating the CD14 and / or CD25 and / or CD19 cell-depleted sample with FLT3L for a second period of time. The APC peptide-loaded sample is incubated with at least one PBMC for a third time period to obtain a first stimulated PBMC sample; optionally, the PBMCs of the first stimulated PBMC sample are incubated with FLT3L-stimulated APCs of the mature APC sample for a fourth time period to obtain a second stimulated PBMC sample; optionally, the PBMCs of the second stimulated PBMC sample are incubated with FLT3L-stimulated APCs of the mature APC sample for a fifth time period to obtain a third stimulated PBMC sample; at least one T cell from the first, second or third stimulated PBMC sample is administered to a subject in need.

[0060] In one aspect, provided herein is a method comprising: obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; 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; incubating the CD14 and / or CD25 and / or CD19 cell-depleted sample with FLT3L for a first period of time; 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 APC peptide-loaded sample; incubating the first APC peptide-depleted sample with FLT3L for a second period of time; The loaded sample is incubated with at least one PBMC for a third time period to obtain a first stimulated PBMC sample; optionally, the PBMCs of the first stimulated PBMC sample are incubated with a second APC peptide-loaded sample of FLT3L and mature APC sample for a fourth time period to obtain a second stimulated PBMC sample; optionally, the PBMCs of the second stimulated PBMC sample are incubated with a third APC peptide-loaded sample of FLT3L and mature APC sample for a fifth time period to obtain a third stimulated PBMC sample; administering at least one T cell from the first, second or third stimulated PBMC sample to a subject in need thereof.

[0061] In one aspect, the present invention provides a method comprising: obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; 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; incubating the CD14 and / or CD25 and / or CD19 cell-depleted sample with FLT3L for a first period of time; 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 APC peptide-loaded sample; incubating the first APC The peptide-loaded sample is incubated with at least one PBMC for a third time period to obtain a first stimulated PBMC sample; optionally, the PBMCs of the first stimulated PBMC sample are incubated with FLT3L-stimulated APCs of the FLT3L and mature APC sample for a fourth time period to obtain a second stimulated PBMC sample; optionally, the PBMCs of the second stimulated PBMC sample are incubated with FLT3L-stimulated APCs of the FLT3L and mature APC sample for a fifth time period to obtain a third stimulated PBMC sample; at least one T cell from the first, second or third stimulated PBMC sample is administered to a subject in need.

[0062] In one aspect, a method is provided herein, comprising determining the expression of one or more cell markers of at least one immune cell in a stimulated immune cell sample; and determining the binding of at least one immune cell in the stimulated immune cell sample to a peptide-MHC complex; wherein determining the expression and determining the binding are performed simultaneously. In some embodiments, the stimulated immune cell sample is an immune cell population stimulated with an APC comprising a peptide-MHC complex. In some embodiments, the immune cell population is from a biological sample.

[0063] In one aspect, the present invention provides a method comprising incubating a population of immune cells from a biological sample with an APC comprising a peptide-MHC complex to obtain a stimulated immune cell sample; determining the expression of one or more cell markers of at least one immune cell in the stimulated immune cell sample; and determining the binding of at least one immune cell in the stimulated immune cell sample to the peptide-MHC complex; wherein determining the expression and determining the binding are performed simultaneously.

[0064] In some embodiments, the one or more cell markers include 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 include cytokines. In some embodiments, the one or more cell markers include degranulation markers. In some embodiments, the one or more cell markers include cell surface markers. In some embodiments, the one or more cell markers include proteins. In some embodiments, determining the binding of at least one immune cell of the stimulated immune cell sample to a peptide-MHC complex includes determining the binding of at least one immune cell of the stimulated immune cell sample to an MHC tetramer containing the peptide and the peptide-MHC complex. In some embodiments, the MHC is class I MHC or class II MHC. In some embodiments, the peptide-MHC complex comprises one or more markers. In some embodiments, the immune cell colony from a biological sample comprises two or more samples, each sample comprising an immune cell colony from one or more biological samples. In some embodiments, the two or more samples are labeled with two or more sample markers. In some embodiments, determining expression and determining combination include fluorescence activated cell sorting (FACS). In some embodiments, determining expression and determining combination include single cell analysis. In some embodiments, determining expression and determining combination include determining the percentage of immune cells that both express one or more cell markers and are combined with peptide-MHC complexes. In some embodiments, the marker includes a fluorophore. In some embodiments, the immune cell colony includes an immune cell colony representing an immune cell colony of a composition described herein.

[0065] Where aspects or embodiments of the invention are described in terms of Markush groups or other alternative groupings, the invention includes not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also includes the main group lacking one or more of the group members. The invention also contemplates the explicit exclusion of one or more of any group members in the claimed invention.

[0066] Incorporation by reference

[0067] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety for all purposes, to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. For example, all publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the kits, compositions and methodology described in the publications, which can be used in combination with the methods, kits and compositions described herein. The documents discussed herein are provided only for their disclosures prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors described herein have no right to disclose prior to this due to prior invention or for any other reason. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1A An example schematic diagram depicting a protocol for the preparation of antigen-specific T cells.

[0069] Figure 1B An example schematic diagram depicting a protocol for the preparation of antigen-specific T cells.

[0070] Figure 2 Depicted are example results showing antigen-specific CD8 induced by long or short peptides + Fraction of memory T cells. "Bulk" means that the sample containing T cells used for induction is complete peripheral blood mononuclear cells (PBMC). "Treg" means that the sample containing T cells used for induction is PBMC depleted of cells expressing CD25.

[0071] Figure 3 Depicted is an example flow cytometry analysis showing antigen-specific CD8 + The fraction of naive T cells.

[0072] Figure 4 Depicted are example results showing antigen-specific CD8 + T cell responses to HIV short peptides, previously identified short neoantigens (PINs), or peptide pools of long PINs. "Complete PBMCs" means that the sample containing the T cells used for induction is complete PBMCs. "CD25 PBMCs" means that the sample containing the T cells used for induction is depleted of CD25+ cells.

[0073] Figure 5A Depicted are antigen-specific CD8 + Example flow cytometric analysis of naive T cell responses to a single previously identified neoantigen (PIN).

[0074] Figure 5B Depicted are antigen-specific CD8 +Example flow cytometric analysis of naive T cell responses to a single previously identified neoantigen (PIN).

[0075] Figure 6 Depicted are example results showing antigen-specific CD8 + T cell responses to the indicated peptides.

[0076] Figure 7 Figure 3 Antigen-specific CD8 + Example flow cytometry plots of T cell responses to the indicated mutant epitopes.

[0077] Fig. 8A Depicted is an exemplary bar graph showing antigen-specific memory CD8 + The result of T cell responses to viral antigens. After up to three rounds of stimulation, all CD8 + Approximately 50% of the T cells were specific for the indicated viral epitopes (CMV pp65, EBVYVL, EBV BMLF1 and Mart-1).

[0078] Figure 8B Depicts example results of a recall assay involving antigen-specific memory CD8 + T cell responses to peptide-loaded antigen-presenting cells followed by incubation with APCs loaded with and without viral antigens. The CD8 + T cell fraction.

[0079] Fig. 9 Depicted are example results of a cytotoxicity assay used to assess whether induced T cell cultures can kill tumor lines expressing an antigen. The fractions of live and dead caspase 3-positive tumor cells relative to total tumor cells are shown. Caspase 3-positive live tumor cells indicate that the cells are undergoing early cell death.

[0080] Fig.10 Depicted is an example flow cytometric analysis involving antigen-specific CD4 + T cell responses to peptide-loaded antigen-presenting cells were then incubated with APCs loaded with and without PINs. IFNγ-releasing CD4 + The percentage of T cells.

[0081] Fig.11 Delineation of antigen-specific CD4+ IFNγ release after restimulation with mutant or wild-type peptides + Example results for percentage of T cells.

[0082] Fig.12 Depicted is an example flow cytometry analysis showing antigen-specific CD8 + Naive T cell responses to short HTV5 peptides. Short-term and long-term induction are shown.

[0083] Fig.13 Depicted is an exemplary flow cytometry analysis showing antigen-specific CD8 + The fraction of naive T cells responding to the short ME1 peptide. Short-term and long-term induction are shown.

[0084] Fig.14 Depicted is an example flow cytometry analysis showing antigen-specific CD8 + Naive T cell responses to short HTV3 peptides. Short-term and long-term induction are shown.

[0085] Fig.15 Depicted is an example flow cytometry analysis showing antigen-specific CD8 + Naive T cell responses to long CSNK1A1 peptide. Short-term and long-term induction are shown.

[0086] Fig.16 Depicted is an example flow cytometry analysis showing antigen-specific CD8 + Naive T cell responses to long CSNK1A1 peptide. Short-term and long-term induction are shown.

[0087] Fig.17 Depicted is an example flow cytometry analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short GAS7 peptides. Short-term and long-term induction are shown.

[0088] Fig.18 Depicted is an example flow cytometry analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short ACTN4 peptides. Short-term and long-term induction are shown.

[0089] Fig.19A Depicted is an example flow cytometry analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short ACTN4 peptides. Short-term induction is shown.

[0090] Fig.19B Depicted is an example flow cytometry analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short HIV3 peptides. Long-term induction is shown.

[0091] Fig. 20 Using whole PBMC samples from human donors, antigen-specific CD8 + Example flow cytometric analysis of naive T cell responses to short HIV5 peptides. Short-term and long-term induction are shown.

[0092] Fig.21 Depicted is an example flow cytometry analysis showing antigen-specific CD8 + Naive T cell responses to short HIV3 peptides. Short-term induction is shown.

[0093] Fig. 22 Depicted is an example flow cytometry analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short PRDX5 peptides. Very short-term and long-term induction are shown.

[0094] Fig.23 Depicted is an example flow cytometry analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short HIV5 peptides. Short-term and long-term induction are shown.

[0095] Fig.24 A schematic diagram depicting an example of a method for producing a therapeutic T cell composition comprising expansion of memory T cells and induction of naive T cells.

[0096] Fig.25 Examples of methods of testing functionality, phenotype and / or function of T cells and / or T cell responses are depicted.

[0097] Fig.26 Depicted are examples of recall assays used to test functionality, phenotype and / or function of T cells and / or T cell responses.

[0098] Fig.27ADepicted is an example flow cytometry analysis showing the ability to deconvolute multiplexed samples from labeled samples acquired individually or as a mixture in a recall assay. Uniquely labeled samples are resolved with no or minimal cross-contamination with other barcodes.

[0099] Fig.27B Depicted is an example flow cytometry analysis showing detection of antigen-specific CD8 in a recall assay by multimeric staining of a mixture of nine uniquely labeled samples. + T cells.

[0100] Fig.28A Depicted is an example flow cytometric analysis of a recall assay using six uniquely barcoded samples, in which recall was performed with unloaded DCs and DCs loaded with neoantigens.

[0101] Fig.28B Figure 3 shows the functional number of CD4 T cells in the recall response assay incubated with DCs loaded with the indicated concentrations of peptides. + Example bar graph of percentage of T cells. Analysis of de novo CD4 T cells alone or mixed with irrelevant samples without barcoding. + Samples from both induced cultures of T cell responses. Barcoding did not alter detectable functionality. The number of functions elicited from the cells and the magnitude of the response did not change significantly with barcoding of the samples.

[0102] Fig.29A Depicted is an exemplary bar graph showing antigen-specific memory CD8 + Results of T cell responses to viral antigens. CD8 + Memory responses can be initiated from CD8 + T cells increased from 0.23% to >60%.

[0103] Fig.29B Depicts example results of a recall assay involving antigen-specific memory CD8 + T cell responses to viral antigens followed by recall with DCs loaded and unloaded with viral antigens. The figure depicts the CD8 T cells at two time points releasing the indicated cytokines. + T cell fraction.

[0104] Fig. 30A Describes the de novo induction of CD4 +Example results of hit identification with response detection and functional characterization. In the example shown, induction was performed in four replicate cultures against 10 HIV-derived antigenic determinants that were naive targets from HIV-negative healthy donors. Antigen-specific responses were detected in 4 / 4 biological replicates, with varying magnitudes of responses.

[0105] Fig. 30B Describes the de novo induction of CD4 + Example results of pooled deconvolution for detection and functional characterization of responses. Multiple responses were detected in each test replicate, and in each case the same two epitopes (HIV#5 and HIV#7) produced the highest magnitude responses.

[0106] Fig. 30C Describes the de novo induction of CD4 + Example results of responses tested and functionally characterized to determine sensitivity. In the pooled deconvolution analysis, similar magnitudes were observed for each response. Responses to HIV#5, HIV#6, and HIV#4 showed EC 50 They are 0.45 μM, 0.43 μM and 9.1 μM respectively.

[0107] Fig.31 An example schematic diagram depicting a protocol for the preparation of antigen-specific T cells.

[0108] Fig.32 An example schematic diagram of a T cell induction protocol is depicted.

[0109] Fig.33 An example schematic diagram depicting a dendritic cell generation protocol.

[0110] Fig.34 Depicted is an exemplary pMHC multimer diagram showing pMHC from a patient-specific epitope SRSF1. E>K 、ARAP1 Y>H and PKDREJ G>R melanoma patients and targeting patient-specific epitopes (AASDHneoORF and seven model neoantigens: ACTN4 K>N 、CSNK1A1 S>L 、DHX40neoORF、GLI3 P>L QARSR >W 、FAM178B P>L and RPS26 P>L CD8 induced in leukocyte separation material of melanoma patients + T cell responses. The first graph in the first and second rows indicates memory responses, and the remaining graphs indicate de novo responses.

[0111] Fig.35 Delineation of SRSF1 E>K and ARAP1 Y>H Example data of pMHC multimer plots before and after peptide stimulation (left), pie chart depicts functionality of neoantigen-specific T cells after rechallenge with DC loaded with neoantigen; pMHC multimer + CD8 + or CD4 + Gating of T cells. CD8 T cells induced in melanoma patients + Memory, CD8 + De novo and CD4 + The multifunctional profile of a de novo response is shown by a combination of 1, 2 or 3 functions (eg, one or more functions is the production of one or more factors selected from IFNγ, TNFα, CD107a and 4-1BB).

[0112] Fig.36 Delineating the specificity of memory and de novo responses induced against mutant and wild-type peptides in melanoma patients. E>K and ARAP1 Y>H Specific T cell responses DCs were challenged 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 total CD8 + T cells were IFN-γ+ and / or TNFα+ and / or CD107a+ (Y axis); both responses showed significant differences from 0 μM concentration, while there was no reactivity to wild-type neoantigen peptide. Statistical analysis: FDR adjusted p value, P value: *≤0.05, ***≤0.001, ****≤0.0001.

[0113] Fig.37A Depicted according to CD8 + CD107a + The cytotoxicity profile of induced memory responses in melanoma patients was quantified by the frequency of T cells. It also depicts the target cell killing of these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The induced CD8 + Cytotoxic capacity of T cell responses. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The construct contained either mutant or wild-type sequences, with a mutation in the center. CD8 + Upregulation of CD107a on T cells and upregulation of active caspase 3 on tumor cells. Target ratio: 3.3:1 (SRSF1 E>K ).

[0114] Fig.37B Depicted according to CD8 + CD107a + Another example of the cytotoxicity profile of induced memory responses in melanoma patients is shown, quantified by the frequency of T cells. It also depicts the target cell killing of these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The induced CD8 + Cytotoxic capacity of T cell responses. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The construct contained either mutant or wild-type sequences, with a mutation in the center. CD8 + Upregulation of CD107a on T cells and upregulation of active caspase 3 on tumor cells. Red circles highlight the pMHC+ fraction. Effector:target ratio: 5:1 (SRSF1 E>K ). Statistical analysis: Unpaired T test, **≤0.01, ****≤0.0001.

[0115] Fig.37C Depicted according to CD8 + CD107a + The cytotoxicity profile of de novo responses induced in melanoma patients was quantified by the frequency of T cells. It also depicts the target cell killing of these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The induced CD8 + Cytotoxic capacity of T cell responses. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The construct contained either mutant or wild-type sequences, with a mutation in the center. CD8 + Upregulation of CD107a on T cells and active caspase 3 on tumor cells. Red circles highlight the pMHC+ fraction. Effector:target ratio: 0.66:1 (ARAP1Y>H). Statistical analysis: Unpaired T test, **≤0.01, ****≤0.0001.

[0116] Fig.38A Delineation of neoantigen-specific CD4 in melanoma patients + Identification of T cell responses. Responses were identified based on the production of IFN-γ and TNFα (Y axis) when DCs were re-challenged with mutant neoantigenic peptides (0.8 μM). MKRN1 S>L ,CREBBP S>L and TPCN1K>E were identified as positive responses.

[0117] Fig.38B Depicted are the mutant and wild-type peptides shown. Fig.38A CD4 depicted in + Specificity of T cell responses. In validation studies, Fig.38A The CD4 T cell responses shown in the Figure were challenged with mutant and 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 the total CD4+ IFNγ+ and / or TNFα+ in the samples were measured (Y axis). Two of the CD4+ T cell responses (MKRN1 S>L and CREEBP S>L ) showed significant differences to 10 μM concentration and were unresponsive to wild-type neoantigenic peptides, but TPCN1 K>E The responses were reactive to both mutant and wild-type neoantigenic peptides.Statistical analysis: FDR-adjusted p-value, P value < 0.05;

[0118] Fig.38C Depict the multifunctional spectrum of these CD4+T cell responses, as shown in a combination of 1, 2, 3 or 4 functions (e.g., one or more functions are one or more selected from the production of factors of IFNγ, TNFα, CD107a and 4-1BB). The multifunctionality of the CD4+T cell responses identified was assessed by attacking again with DC (0.8 μm) loaded with mutant neoantigen peptides. The percentages in the pie chart represent the percentages of functional CD4+T cells (1, 2 and / or 3 functions). The representative data depicted are generated by CD4+T cell responses after stimulation induced in the patient.

[0119] Fig.39 The functionality of the memory response induced in two healthy donors (e.g., HD66 and HD63) with or without the addition of Epacadostat is depicted, as indicated by a combination of 1, 2, or 3 functions (e.g., one or more functions is the production of one or more factors selected from IFNγ, TNFα, and CD107α).

[0120] Fig.40 De novo CD8 T cells induced in six replicate inductions with or without the addition of Epacadostat are depicted. + Percent T cell response ("hit rate", averaged across four healthy donors).

[0121] Fig.41A Depicted are the absolute numbers of antigen-specific cells from donor HD55 following induction using the T cell preparation protocol provided herein with or without the addition of PD-1 blocking antibodies.

[0122] Fig.41B Depicted are the absolute numbers of antigen-specific cells from donor HD67 after induction using the T cell preparation protocol provided herein with or without the addition of PD-1 blocking antibodies.

[0123] Fig.42A De novo CD8 + pMHC of T cell responses + CD8 + T cell fraction.

[0124] Fig.42B De novo CD8 + CD8 T cell responses + The percentage of T cells.

[0125] Fig.43 An example of the method described herein is depicted. Patient-specific neoantigens are predicted using a bioinformatics engine, and synthetic long peptides covering the predicted neoantigens are used as immunogens in a stimulation protocol to assess immunogenicity. The stimulation protocol includes feeding these neoantigen encoding peptides to patient-derived APCs, which are then co-cultured with patient-derived T cells to elicit antigen-specific T cells. DETAILED DESCRIPTION

[0126] Described herein are novel immunotherapeutic agents and their uses based on the discovery of new antigens arising from mutational events unique to individual tumors. Accordingly, the disclosure described herein provides methods and protocols for creating antigen-specific immune cells, such as T cells, for treating disease.

[0127] definition

[0128] The terms used herein are for the purpose of describing a particular situation only and are not intended to be limiting. Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" as used herein are intended to include the plural forms as well. In addition, to the extent that the terms "includes", "comprising", "having", or variations thereof are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising".

[0129] It should be understood that terms such as "comprising" may have the meaning assigned to it in U.S. patent law; for example, the term may mean "including", "containing", etc.; and terms such as "consisting essentially of" and "consisting essentially of" have the meaning assigned to them in U.S. patent law, for example, it allows for elements not expressly recited but excludes elements found in the prior art or that affect the basic or novel features of the invention. Nothing herein is a commitment.

[0130] The term "and / or" as used in phrases such as "A and / or B" is intended herein to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0131] The term "about" or "approximately" may mean within an acceptable error range for a particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the relevant art, "about" may mean within 1 or more than 1 standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within the order of magnitude of a value, within 5 times, and more preferably within 2 times. Where specific values ​​are described in the present application and claims, unless otherwise indicated, the term "about" shall be presumed to mean within an acceptable error range for that specific value.

[0132] To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0133] "New antigens" refer to a class of tumor antigens produced by tumor-specific changes in proteins. New antigens include, but are not limited to, tumor antigens produced by, for example, substitutions of protein sequences, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, and expression of endogenous retroviral polypeptides.

[0134] A "neo-epitope" refers to an epitope that is not present in a reference such as a 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 the case where the corresponding epitope is found in a normal non-cancerous cell or a germline cell, but due to one or more mutations in the diseased cell, e.g., a cancer cell, the sequence of the epitope is altered to generate a neo-epitope.

[0135] A "reference" can be used to correlate and / or compare the results obtained in the methods of the present disclosure with disease specimens. Typically, a "reference" can be obtained based on one or more normal specimens, particularly specimens not affected by disease, obtained from an individual or one or more different individuals (e.g., healthy individuals), such as individuals of the same species. A "reference" can be determined empirically by testing a sufficiently large number of normal specimens.

[0136] "Mutation" refers to a change or difference (e.g., nucleotide substitution, addition or deletion) in a nucleic acid sequence compared to a reference nucleic acid. "Somatic mutation" can occur in any cell of the body except germ cells (sperm and eggs) and will not be passed on to children. These changes can (but not always) lead to cancer or other diseases. In some embodiments, the mutation is a non-synonymous mutation. "Non-synonymous mutation" refers to a mutation (e.g., nucleotide substitution) that causes an amino acid change in the translation product, such as an amino acid substitution. When a mutation disrupts the normal phase (also referred to as a "reading frame") of the periodicity of a gene codon, a "frameshift" occurs, resulting in the translation of a non-native protein sequence. Different mutations in a gene can achieve the same altered reading frame.

[0137] 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 class I or class II HLA, or a peptide-HLA complex and a T cell receptor (TCR)). D Refers to the dissociation constant between two members of a binding pair and has units of molar concentration. K A K is 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 dissociation rate constant of two members of a binding pair (e.g., the dissociation rate constant of an HLA-binding peptide from class I or II HLA or a peptide-HLA complex from a TCR). on Refers to the association rate constant of two members of a binding pair (e.g., the association rate constant of an HLA-binding peptide with class I or II HLA or a peptide-HLA complex with a TCR).

[0138] Throughout this disclosure, “combined data” results may be referred to as “IC 50 ”. Affinity can also be expressed as inhibitory concentration 50 (IC 50 ), or the concentration at which 50% of the first member of the binding pair (e.g., peptide) is displaced. Similarly, ln(IC 50 ) refers to IC 50 The natural logarithm of . For example, IC 50 It can be the concentration of the test peptide at which 50% inhibition of the binding of the labeled reference peptide is observed in the binding assay. Taking into account the conditions under which the assay is run (e.g., limiting HLA protein concentration and / or labeled reference peptide concentration), these values ​​can be close to K DValues. Assays for determining binding are well known in the art and are described in detail, for example, in PCT Publications WO 94 / 20127 and WO 94 / 03205, and 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 binding of a reference standard peptide. Binding can also be determined using other assay systems, including those using living 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. Immunol. 148:194 (1992); 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 (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-throughput soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurements of 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)).

[0139] When used to discuss epitopes, the term "derived" is a synonym for "prepared." Derived epitopes can be isolated from natural sources or can be synthesized according to standard protocols in the art. Synthetic epitopes can contain artificial amino acid residues "amino acid mimetics," such as the D isomers of naturally occurring L amino acid residues or non-natural amino acid residues such as cyclohexylalanine. Derived or prepared epitopes can be analogs of natural epitopes. The term "derived from" refers to the 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.

[0140] An "epitope" is a collection of features of a molecule (e.g., charge of a peptide and primary, secondary, and tertiary peptide structures) 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 group of amino acid residues that participate in recognition by a specific immunoglobulin; a major histocompatibility complex (MHC) receptor; or in the case of a T cell, those residues recognized by a T cell receptor protein and / or a chimeric antigen receptor. Epitopes can be prepared by isolation from natural sources, or they can be 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 the disclosure, epitopes may be referred to as peptides or peptide epitopes in some cases. In certain embodiments, there are restrictions on the length of the peptides disclosed herein. Length-restricted embodiments occur when a protein or peptide comprising an epitope described herein comprises a region having 100% identity with a natural sequence (i.e., a continuous series of amino acid residues). In order to avoid that the definition of an epitope is read, for example, over the entire native molecule, the length of any region with 100% identity to the native peptide sequence is limited. Thus, for peptides comprising an epitope as described herein and a region with 100% identity to a native peptide sequence, the region with 100% identity to the native sequence typically has the following length: 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 contained in a peptide having a region of less than 51 amino acid residues, in any increment down to 5 amino acid residues, which region is 100% identical to the native peptide sequence; for example, 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.

[0141] "T cell epitope" refers to a peptide sequence that is bound by an MHC molecule in the form of a peptide-MHC (pMHC) complex. The peptide-MHC complex can be recognized and bound by the TCR of a T cell (eg, a cytotoxic T lymphocyte or a T helper cell).

[0142] "T cells" include CD4 +T cells and CD8 + T cell. The term T cell also includes T helper type 1 T cells and T helper type 2 T cells.

[0143] "Immune cells" refer to cells that play a role in an immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0144] An "immunogenic" peptide or "immunogenic" epitope or "immunogenic" peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated response or a 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 then inducing a cell-mediated response or a humoral response (e.g., a CTL (cytotoxic) response or an HTL response) to the peptide.

[0145] A "protective immune response" or "therapeutic immune response" refers to a CTL and / or HTL response to an antigen derived from a pathogenic antigen (e.g., a tumor antigen) that in some way prevents or at least partially prevents disease symptoms, side effects, or progression. The immune response may also include an antibody response promoted by stimulating helper T cells.

[0146] "T cell receptor" ("TCR") refers to a natural or partially or completely synthetically produced molecule found on the surface of T lymphocytes (T cells) that recognize antigens bound to major histocompatibility complex (MHC) molecules. The ability of a T cell to recognize antigens associated with a variety of diseases (e.g., cancer) or infectious organisms is conferred by its TCR, which is composed of an alpha (α) chain and a beta (β) chain or a gamma (γ) and delta (δ) chain. The proteins that make up these chains are encoded by DNA, which uses a unique mechanism to generate the huge diversity of TCRs. This multi-subunit immune recognition receptor is associated with the CD3 complex and binds to peptides presented by MHC class I and class II proteins on the surface of antigen presenting cells (APCs). The binding of the TCR to the peptide on the APC is a central event in T cell activation.

[0147] As used herein, "chimeric antigen receptor" or "CAR" refers to an antigen binding protein, which includes an immunoglobulin antigen binding domain (e.g., immunoglobulin variable domain) and a T cell receptor (TCR) constant domain. As used herein, the "constant domain" of a TCR polypeptide includes a membrane-proximal TCR constant domain, a TCR transmembrane domain, and / or a TCR cytoplasmic domain or a fragment thereof. For example, in some embodiments, CAR is a monomer comprising a polypeptide comprising an immunoglobulin heavy chain variable domain connected to a TCRβ constant domain. In some embodiments, CAR is a dimer comprising: a first polypeptide comprising an immunoglobulin heavy chain or light chain variable domain connected to a TCRα or TCRβ constant domain, and a second polypeptide comprising an immunoglobulin heavy chain or light chain variable domain (e.g., κ or λ variable domain) connected to a TCRβ or TCRα constant domain.

[0148] "Major histocompatibility complex" or "MHC" is a gene cluster that plays a role in controlling cell interactions that lead to physiological immune responses. The term "major histocompatibility complex" and the abbreviation "MHC" can include any class of MHC molecules, such as MHC class I and MHC class II molecules, and refers to a complex of genes present in all vertebrates. In humans, the MHC complex is also called the human leukocyte antigen (HLA) complex. Therefore, "human leukocyte antigen" or "HLA" refers to human major histocompatibility complex (MHC) proteins (see, e.g., Stites et al., Immunology, 8th edition, Lange Publishing, Los Altos, Calif. (1994)). For a detailed description of MHC and HLA complexes, see Paul, Fundamental Immunology, 3rd edition, Raven Press, New York (1993).

[0149] The major histocompatibility complex in the genome contains genetic regions whose gene products expressed on the cell surface are essential for binding and presenting endogenous and / or exogenous antigens, and are therefore used to regulate immune processes. MHC proteins or molecules are essential for signal transduction 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 can be expressed on the cell surface and display self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. MHC-bound peptides can be produced by proteolytic cleavage of protein antigens and represent potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes). MHC can transport peptides to the cell surface and present them there to specific cells, such as cytotoxic T lymphocytes, T helper cells, or B cells. The MHC region can be divided into three subgroups: class I, class II, and class III. MHC class I proteins can contain α chains and β2-microglobulin (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T cells. MHC class II proteins can contain alpha and beta chains, which can present antigen fragments to T helper cells. MHC class III regions can encode other immune components, such as complement components and cytokines. MHC can be either polygenic (there are several MHC class I and MHC class II genes) or polymorphic (each gene has multiple alleles).

[0150] "Antigen processing" or "processing" refers to the degradation of a polypeptide or antigen into processing products, which are fragments of the polypeptide or antigen (e.g., degradation of a polypeptide into peptides) and the association (e.g., via binding) of one or more of these fragments with MHC molecules for presentation by a cell (e.g., an antigen presenting cell) to a specific T cell.

[0151] "Antigen presenting cell" (APC) refers to a cell that presents peptide fragments of a protein antigen associated with an MHC molecule on its cell surface. The 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).

[0152] "Receptor" refers to a biological molecule or a grouping of molecules that can bind to a ligand. Receptors can be used to transmit information in cells, cell formations or organisms. Receptors include at least one receptor unit, for example, wherein each receptor unit can be composed of a protein molecule. Receptors have a structure that is complementary to the structure of the ligand, and can be used as a binding partner to complex with the ligand. Information is transmitted specifically by the conformational changes of the receptor after the ligand is complexed on the cell surface. In some embodiments, receptors should be understood to refer especially to proteins of MHC class I and class II that can form receptor / ligand complexes with ligands (particularly peptides or peptide fragments of suitable length). "Ligand" refers to a molecule that has a structure that is complementary to the structure of a receptor and can form a complex with the receptor. In some embodiments, a ligand should be understood to mean a peptide or peptide fragment having a suitable length and a suitable binding motif in its amino acid sequence, so 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" should also be understood to mean a "receptor / peptide complex" or a "receptor / peptide fragment complex", which includes an MHC molecule, such as an MHC class I or class II molecule, presenting the peptide or peptide fragment.

[0153] A "native" or "wild-type" sequence refers to a sequence found in nature. As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a source in nature and has not been intentionally modified by man in the laboratory is naturally occurring.

[0154] The terms "peptide" and "peptide epitope" are used interchangeably with "oligopeptide" in this specification and refer to a series of residues generally linked to each other by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues. "Synthetic peptide" refers to a peptide obtained from a non-natural source, for example, artificial. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. "Synthetic peptide" includes "fusion proteins".

[0155] The term "motif" refers to a pattern of residues in an amino acid sequence of a defined length, e.g., a peptide of less than about 15 amino acid residues in length or less than about 13 amino acid residues in length, e.g., for a class I HLA motif, having about 8 to about 13 (e.g., 8, 9, 10, 11, 12, or 13) amino acid residues, and for a class II HLA motif, having about 6 to about 25 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acid residues, which are recognized by a particular HLA molecule. The motif is typically different for each HLA protein encoded by a given human HLA allele. The patterns of primary and secondary anchor residues of these motifs are different. In some embodiments, an MHC class I motif recognizes a peptide of 7, 8, 9, 10, 11, 12, or 13 amino acid residues in length. In some embodiments, the MHC class II motif recognizes a peptide that is 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 binding pair member class (e.g., a peptide that is bound by both a class I HLA molecule and a class II HLA molecule).

[0156] The term "residue" refers to an amino acid residue or amino acid simulation residue that is incorporated into a peptide or protein or encoded by a nucleic acid (DNA or RNA) through an amide bond or an amide bond mimetic. The nomenclature used to describe a peptide or protein follows conventional practice. The amino group is presented on the left side (amino terminal or N-terminal) of each amino acid residue and the carboxyl group is presented on the right side (carboxyl terminal or C-terminal). When the amino acid residue position is mentioned in a peptide epitope, the amino acid residue is numbered in the amino to carboxyl direction, wherein the first position is the residue at the amino terminal of the peptide or protein of which the epitope or epitope may be a part. In the general formula representing the specific embodiment selected by the present invention, unless otherwise indicated, the amino terminal and carboxyl terminal groups (although not specifically shown) are the forms they present at physiological pH values. In the amino acid structural formula, each residue is usually represented by a standard three-letter or single-letter nomenclature. The L-form of an amino acid residue is represented by a capital single letter or a three-letter symbol with a capital initial, while the D-form of those amino acid residues with a D-form is represented by a lowercase single letter or a lowercase three-letter symbol. However, when the three-letter symbols or full names without capital letters are used, they may also refer to L amino acid residues. Glycine has no asymmetric carbon atom and is referred to as "Gly" or "G" for short. The amino acid sequences of the peptides described herein are generally represented using standard single-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; Y, tyrosine).

[0157] A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including 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), non-polar 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, replacing tyrosine with phenylalanine is a conservative substitution. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate peptide function are well known in the art.

[0158] "Pharmaceutically acceptable" refers to compositions or components of compositions that are generally non-toxic, inert and / or physiologically compatible. "Pharmaceutically acceptable excipients" or "excipients" include materials such as adjuvants, carriers, pH adjusters and buffers, tonicity adjusters, wetting agents, preservatives, etc. "Pharmaceutically acceptable excipients" are pharmaceutically acceptable excipients.

[0159] According to the present disclosure, the term "vaccine" refers to a pharmaceutical preparation (pharmaceutical composition) or product that induces an immune response (e.g., a cellular or humoral immune response) after administration, which recognizes and attacks pathogens or diseased cells, such as cancer cells. Vaccines can be used to prevent or treat diseases. The terms "individualized cancer vaccine" or "personalized cancer vaccine", "personal cancer vaccine" refer to specific cancer patients and mean that the cancer vaccine is adapted to the needs or special circumstances of individual cancer patients.

[0160] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to nucleotide polymers of any length, including DNA and RNA (e.g., 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 polynucleotides administered using the methods of the present invention are mRNA.

[0161] The term "isolated" or "biologically pure" refers to a material that is substantially or essentially free of components that normally accompany the material when it is found in its natural state. Thus, the isolated peptides described herein do not contain some or all of the substances that are normally associated with the peptide in its native environment. For example, an "isolated" epitope can be an epitope that does not include the full sequence of the protein from which the epitope is derived. For example, a naturally occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide isolated from some or all of the coexisting substances in the natural system is isolated. Such a polynucleotide can be part of a vector, and / or such a polynucleotide or peptide can be part of a composition, and still be "isolated" because such a vector or composition is not part of its natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of DNA molecules described herein, and also include such molecules produced synthetically. In some embodiments, an isolated polypeptide, antibody, polynucleotide, vector, cell or composition is substantially pure. As used herein, the term "substantially pure" refers to a material that is at least 50% pure (ie, free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0162] In the context of two or more nucleic acids or polypeptides, the term "identical" or percentage "identity" refers to when compared and aligned (if necessary, introducing gaps) for maximum correspondence, two or more sequences or subsequences are identical or have a specific percentage of identical nucleotides or amino acid residues, without considering any conservative amino acid substitutions as part of 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 obtain amino acid or nucleotide sequence alignments are well known in the art. These include but are not limited to BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and variations thereof. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaning that when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and in some embodiments have at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. In some embodiments, identity exists in 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, or any integer value of residues therebetween. In some embodiments, identity exists in 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 full length of the compared sequences, such as the amino acid sequence of a peptide or the coding region of a nucleotide sequence.

[0163] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that is to be the recipient of a particular treatment. Generally, when referring to a human subject, the terms "subject" and "patient" are used interchangeably herein.

[0164] The term "effective amount" or "therapeutically effective amount" or "therapeutic effect" refers to an amount of a therapeutic agent that is effective for "treating" a disease or condition in a subject or mammal. A therapeutically effective amount of a drug has a therapeutic effect, thereby preventing the development of a disease or condition; slowing the development of a disease or condition; slowing the progression of a disease or condition; alleviating to some extent one or more symptoms associated with the disease or condition; reducing morbidity and mortality; improving quality of life; or a combination of these effects.

[0165] The terms "treat" or "alleviate" refer to: (1) therapeutic measures that cure, slow down, alleviate 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 development of a target pathological condition or disorder. Thus, subjects in need of treatment include subjects already suffering from the disorder; subjects susceptible to the disorder; and subjects in whom the disorder is to be prevented.

[0166] When used to describe a cell sample (e.g., a peripheral blood mononuclear cell (PBMC) sample), the term "depleted" refers to a cell sample in which a subpopulation of cells has been removed or depleted. For example, an immune cell sample depleted of cells expressing CD25 refers to an immune cell sample in which cells expressing CD25 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 a sample. For example, CD14 can be depleted or removed from a PBMC sample, for example, by using an antibody that binds to CD14. + cell.

[0167] "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, stimulation of a T cell may refer to the binding of the TCR of a T cell to a peptide-MHC complex. For example, stimulation of a T cell may refer to the step in Scheme 1 or Scheme 2, where PBMCs are cultured with APCs loaded with peptides.

[0168] The term "enriched" refers to a composition or fraction in which a target species has been partially purified so that the concentration of the target species is substantially greater than the level of the species naturally present in the unenriched final product. The term "induced cells" refers to cells that have been treated with an inducing compound, cell, or cell population that affects the protein expression, gene expression, differentiation state, shape, morphology, viability, etc. of the cell.

[0169] Overview of T cell therapy and its preparation

[0170] The production of antigen-specific T cells by controlled in vitro 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 preparation methods and therapeutic T cell compositions, which can be used to treat subjects with cancer and other conditions, diseases, and disorders. The purpose is to expand and induce antigen-specific T cells with good phenotype and function. The present disclosure provides compositions and methods for preparing T cells that can be used for antigen-specific T cell therapies (e.g., personal or personalized T cell therapies). The T cell compositions provided herein can be personal antigen-specific T cell therapies.

[0171] Provided herein are methods for stimulating T cells. For example, the methods provided herein can be used to stimulate antigen-specific T cells. The methods provided herein can be used to expand or induce antigen-specific T cells. For example, the methods provided herein can be used to expand antigen-specific memory T cells. For example, the methods provided herein can be used to induce antigen-specific naive T cells. For example, the methods provided herein can be used to expand antigen-specific CD8 + Memory T cells. For example, the methods provided herein can be used to induce antigen-specific CD8 + For example, the methods provided herein can be used to expand antigen-specific CD4 + Memory T cells. For example, the methods provided herein can be used to induce antigen-specific CD4 + Naive T cells. Also provided herein are therapeutic compositions comprising antigen-specific T cells. For example, the therapeutic composition may comprise antigen-specific memory T cells. For example, the therapeutic composition may comprise antigen-specific naive T cells. Also provided herein are methods of using the therapeutic compositions described herein or methods of using them for treatment.

[0172] T cell composition

[0173] Provided herein are compositions (e.g., pharmaceutical compositions) comprising an immune cell colony. The composition may include at least one antigen-specific T cell comprising a T cell receptor (TCR). The composition may include at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence.

[0174] In some embodiments, provided herein are compositions comprising T cells stimulated by APC such as APCs preloaded with antigenic peptides. The composition may include an immune cell colony, which includes T cells from a sample (e.g., a biological sample), wherein the T cells include T cells stimulated by APCs. In some embodiments, the composition includes an immune cell colony incubated with a ligand such as a cell surface receptor binding with one or more cytokines, growth factors, or ligands such as APCs or T cells. Non-limiting examples of such cytokines, growth factors, and ligands include, but are not limited to, GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IFN-α, R848, LPS, ss-rna40, and poly-I: C. In some embodiments, the composition includes an immune cell colony incubated with one or more APCs or APC products. For example, the composition may include an immune cell colony incubated with an APC or APC product stimulated with one or more cytokines, growth factors, and / or ligands. For example, the composition may comprise a population of immune cells that have been incubated with one or more cytokine-stimulated APCs or cytokine-stimulated APC preparations. For example, the composition may comprise a population of immune cells that have been incubated with one or more growth factor-stimulated APCs or growth factor-stimulated APC preparations. For example, the composition may comprise a population of immune cells that have been incubated with one or more ligand-stimulated APCs or ligand-stimulated APC preparations.

[0175] In some embodiments, the APC is an autologous APC, an allogeneic APC, or an artificial APC. In some embodiments, the APC comprises a dendritic cell (DC). In some embodiments, the APC is derived from a CD14 + 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 + The monocytes are derived from a biological sample from a subject comprising PBMCs. For example, CD14 can be isolated, enriched or purified from a biological sample from a subject comprising PBMCs. + In some embodiments, CD14 cells 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, 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 comprising PBMCs.

[0176] In some embodiments, the isolated CD14 + APC populations can be enriched or substantially enriched. In some embodiments, the isolated CD14 + The APC population is at least 30%, at least 50%, at least 75%, or at least 90% homogeneous. + The APC population is at least 60%, at least 75%, or at least 90% homogeneous. APCs, such as CD14 + APCs may include, for example, APCs derived from mononuclear dendritic 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.

[0177] CD14 can be isolated by the method provided by the present invention. + APC and CD14 + The method generally comprises obtaining a cell population comprising APC precursors, differentiating the APC precursors into immature or mature APCs, and may further comprise isolating CD14 from the differentiated immature or mature APC population. + APC.

[0178] APC precursor cells can be obtained by methods known in the art. APC precursors can be separated by, for example, density gradient separation, fluorescence activated cell sorting (FACS), immune cell separation techniques such as panning, complement lysis, rosettes, magnetic cell separation techniques, nylon wool separation, and combinations of such methods. Methods for immunoselection of APCs include, for example, using antibodies against cell surface markers associated with APC precursors, such as anti-CD34 and / or anti-CD14 antibodies coupled to a substrate.

[0179] Enriched APC precursor populations can also be obtained. Methods for obtaining such enriched precursor populations are known in the art. For example, enriched APC precursor populations can be isolated from tissue sources by selectively removing cells adhering to a substrate. Using tissue sources such as bone marrow or peripheral blood, commercially processed plastic substrates (e.g., beads or magnetic beads) can be used to remove adherent monocytes from cell products to obtain non-adhesive APC precursor enriched populations.

[0180] Mononuclear cell APC precursors can also be obtained from tissue sources by using APC precursor adhesion substrates. For example, peripheral blood leukocytes separated by, for example, leukocyte separation are contacted with a mononuclear APC precursor adhesion substrate with a high surface area: volume ratio, and the adhered mononuclear APC precursors are separated. In other embodiments, the coupled substrate can be a granular or fibrous substrate with a high surface: volume ratio, such as microbeads, microcarrier beads, pellets, particles, powders, capillaries, microporous membranes, etc. In addition, the granular or fibrous substrate can be glass, polystyrene, plastics, glass-coated polystyrene microbeads, etc.

[0181] 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. Typically, expansion can be achieved by culturing precursors in the presence of at least one cytokine that induces APC (e.g., dendritic cells) differentiation / proliferation. Typically, these 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 the proliferation and / or maturation of non-APC cell types in the culture, thereby further enriching the APC precursor population. Typically, such agents include cytokines, for example, IL-13, IL-4, or IL-15, etc.

[0182] The isolated APC precursor population is cultured and differentiated to obtain immature or mature APCs. Suitable tissue culture media include, for example, but are not limited to RPMI 1640, DMEM, X-VIVO Etc. Tissue culture medium is usually supplemented with amino acids, vitamins, divalent cations and cytokines to promote the differentiation of precursors to APC phenotype. Typically, the cytokines that promote differentiation are GM-CSF and / or IL-4.

[0183] In addition, cultures of APC precursors can include plasma to promote CD14 + APC development. Typical plasma concentrations are about 5%. In addition, for example, in the case of isolating APC precursors by adhesion to a substrate, plasma can be included in the culture medium during the adhesion step to promote the early CD14 + Phenotype. Typical plasma concentrations during adhesion are approximately 1% or higher.

[0184] The mononuclear cell APC precursors can be cultured for any suitable time. In certain embodiments, a suitable culture time for differentiating the precursors into immature APCs can be from about 1 day to about 10 days, such as from about 4 days to about 7 days. The differentiation of the precursors into immature APCs can be performed by methods known to those skilled in the art, such as by the presence or absence of cell surface markers (e.g., CD11c + 、CD83低 、CD86 - / 低 , HLA-DR + ) to monitor the differentiation of immature APCs from precursors. Immature APCs can also be cultured in appropriate tissue culture media to keep the immature APCs in a state of further differentiation or antigen uptake, processing and presentation. For example, immature APCs can be maintained in the presence of GM-CSF and IL-4.

[0185] After differentiation from APC precursors, CD14 + To obtain isolated CD14 + APC populations. Typically, CD14 is isolated from enriched or substantially enriched APC prior to maturation. + In the case of APC, for immature CD14 + For APC, the isolated population will be enriched or substantially enriched. Typically, CD14 + Isolation of APCs involves isolating CD14 + The cell population of cells is contacted with a CD14-specific probe. In an exemplary embodiment, cells expressing CD14 are detected by FACS using a CD14-specific probe directly conjugated to a fluorescent molecule (e.g., FITC or PE), or using an unlabeled antibody specific for CD14 and a labeled secondary antibody specific for the first antibody. CD14 can also be sorted by FACS. + Cells and CD14 低 and CD14 - Cell separation. CD14 can be determined by, for example, CD14 staining on PBMC-derived monocytes. 高 Positive gating. Typically, a CD14-specific binding agent is, for example, an anti-CD14 antibody (eg, a monoclonal or antigen-binding fragment thereof). Many anti-CD14 antibodies suitable for use in the present invention are known to the skilled artisan, and many are commercially available.

[0186] In another embodiment, a CD14-specific probe is coupled to a substrate and CD14 is isolated by affinity selection. + cells. This will include CD14 + The cell population is exposed to the coupled substrate and CD14 + The cells adhered specifically to the substrate. The non-adherent CD14 - cells, and then eluted the adherent cells to obtain cells substantially enriched for CD14 +The cell population of APC separation. 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 separation of cell populations using substrate-coupled surface marker-specific antibodies are well known.

[0187] During culture, immature APCs (CD14 - The isolated population of immature APCs or the total immature APCs before separation) can be optionally exposed to a predetermined antigen. Suitable predetermined antigens may include any antigen to which T cell regulation 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 may 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 surfaces, autoantigens, and any other antigens. Any antigen may also be presented as a peptide or recombinantly produced protein or part thereof. After contact with the antigen, the cells may be cultured for any suitable time to allow antigen uptake and processing to expand the antigen-specific APC population, etc.

[0188] For example, in one embodiment, immature APCs can be cultured after antigen uptake to promote the maturation of immature APCs into mature APCs that present antigens in the context of MHC molecules. Methods for APC maturation are known. For example, such maturation can be performed by culturing in the presence of known maturation factors such as cytokines (e.g., TNF-α, IL-1β, or CD40 ligands), bacterial products (e.g., LPS or BCG), etc. The maturation of immature APCs to mature APCs can be monitored by methods known in the art, for example, by measuring the presence or absence of cell surface markers (e.g., upregulation of CD83, CD86, and MHC molecules) or using, for example, oligonucleotide arrays to detect the expression of mature APC-specific mRNA or protein.

[0189] Optionally, immature APCs can be cultured in a suitable 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 the maturation of immature APCs.

[0190] On the other hand, isolated CD14 +The APC population is enriched for mature APCs. The isolated CD14 + Immature APC populations can be induced to mature and isolated CD14 + Mature APC population. Optionally, CD14 + and CD14 - A mixed population of immature APCs (differentiated from APC precursors) is induced to mature, the maturation stage is monitored as described above, and at the appropriate stage of mature APC enrichment, CD14 + Cells enriched or substantially enriched for CD14 + Isolated populations of mature APCs.

[0191] According to another aspect of the present invention, for example, APC can be preserved by cryopreservation before or after being exposed to prostate cancer antigen. Available cryopreservatives include but are not limited to dimethyl sulfoxide (DMSO), glycerol, polyvinyl pyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, isoerythritol, D-ribitol, D-mannitol, D-sorbitol, inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, monoacetin and inorganic salts. Controlled slow cooling rate may be critical. Different cryoprotectants and different cell types usually have different optimal cooling rates. The heat of the melting stage of water into ice should usually be minimum. The cooling process can be carried out by using, for example, a programmable freezing device or a methanol bath program. Programmable freezing equipment allows to determine the optimal cooling rate and helps standard reproducible cooling. The freezer of programmable control rate, such as Cryomed or Planar, allows the freezing scheme to be adjusted to the required cooling rate curve.

[0192] After thorough freezing, the APCs can be quickly transferred to a long-term cryogenic storage container. In a typical embodiment, the sample can be cryogenically stored in liquid nitrogen (-196°C) or its vapor (-165°C). Considerations and procedures for the handling, cryopreservation and long-term storage of hematopoietic stem cells, particularly hematopoietic stem cells from bone marrow or peripheral blood, are largely applicable to the APCs of the present invention.

[0193] The frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37-41° C.) and cooled immediately after thawing. In order to prevent the cells from clumping when thawing, it may be necessary to treat the cells. In order to prevent clumping, various procedures can be used, including but not limited to adding DNA enzymes, low molecular weight dextran and citrate, hydroxyethyl starch, etc. before and / or after freezing. If the cryoprotectant is toxic to the human body, it should be removed before the thawed APC is used therapeutically. One method of removing the cryoprotectant is to dilute it to a very small concentration. Once the frozen APC has been thawed and recovered, they can be used to activate T cells as described herein for non-frozen APC.

[0194] In some embodiments, the composition comprises an immune cell population that has been depleted of one or more types of immune cells. For example, the composition may comprise an immune cell population that has been 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 an immune cell population from a biological sample, the biological sample comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) with specificity for at least one antigenic peptide sequence, wherein the amount of immune cells expressing CD14 and / or CD25 in the population is proportionally different from the amount of immune cells expressing CD14 and / or CD25 in the biological sample. For example, the composition may comprise an immune cell population from a biological sample, the biological sample comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) with specificity for at least one antigenic peptide sequence, wherein the amount of immune cells expressing CD14 in the population is proportionally different from the amount of immune cells expressing CD14 in the biological sample. For example, a composition may include a population of immune cells from a biological sample, the biological sample including at least one antigen-specific T cell, the antigen-specific T cell including a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the amount of immune cells expressing CD25 in the population is proportionally different from the amount of immune cells expressing CD25 in the biological sample. For example, a composition may include a population of immune cells from a biological sample, the biological sample including at least one antigen-specific T cell, the antigen-specific T cell including a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the amount of immune cells expressing CD14 and CD25 in the population is proportionally different from the amount of immune cells expressing CD14 and CD25 in the biological sample. For example, a composition may include a population of immune cells from a biological sample, wherein the amount of immune cells expressing CD14 and CD25 in the population is proportionally less than the amount of immune cells expressing CD14 and CD25 in the biological sample.

[0195] In some embodiments, the composition comprises an immune cell colony, which comprises T cells from a sample (e.g., a biological sample), wherein the T cells comprise T cells stimulated by APC, wherein the APC is an APC stimulated by FLT3L. For example, the composition may comprise an immune cell colony, which comprises T cells from a sample (e.g., a biological sample), wherein the T cells include T cells stimulated by APC and antigen-specific T cells comprising T cell receptors (TCR) with specificity for at least one antigenic peptide sequence, wherein the APC is an APC stimulated by FLT3L. In some embodiments, the composition comprises an immune cell colony, which comprises T cells from a biological sample, wherein the T cells comprise at least one antigen-specific T cell, which is a T cell stimulated by APC and comprises a T cell receptor (TCR) with specificity for at least one antigenic peptide sequence, wherein the APC is an APC stimulated by FLT3L, and wherein the amount of antigen-specific T cells in the colony is proportionally greater than the amount of antigen-specific T cells in the biological sample. In some embodiments, the T cell comprises a plurality of antigen-specific T cells, the antigen-specific T cells comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence. In some embodiments, the T cell comprises a plurality of antigen-specific T cells, the antigen-specific T cells comprising a plurality of T cell receptors (TCR) having specificity for at least one antigenic peptide sequence. In some embodiments, the T cell comprises a plurality of antigen-specific T cells, the antigen-specific T cells comprising a plurality of T cell receptors (TCR) having specificity for a plurality of antigenic peptide sequences. For example, the plurality of antigen-specific T cells in the composition can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000 , 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1x10 6 , 2x10 6、3x10 6 、4x10 6 、5x10 6 、6x10 6 、7x10 6 、8x10 6 、9x10 6 、1x10 7 、2x10 7 、3x10 7 、4x10 7 、5x10 7 、6x10 7 、7x10 7 、8x10 7 、9x10 7 、1x10 8 、2x10 8 、3x10 8 、4x10 8 、5x10 8 、6x10 8 、7x10 8 、8x10 8 、9x10 8 、1x10 9 、2x10 9 、3x10 9 、4x10 9 、5x10 9 、6x10 9 、7x10 9 、8x10 9 、9x10 9 、1x10 10 、2x10 10 、3x10 10 、4x10 10 、5x10 10 、6x10 10 、7x10 10 、8x10 10 、9x10 10 、1x10 11 、2x10 11 、3x10 11 、4x10 11 、5x10 11 、6x10 11 、7x10 11 、8x10 11 、9x10 11 、1x10 12 、2x10 12 、3x10 12 、4x1012 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 or 9x10 12 For example, the plurality of T cell receptors (TCRs) specific for at least one antigenic peptide sequence may include 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 1000 different TCRs specific for at least one antigenic peptide sequence. For example, the plurality of T cell receptors (TCRs) specific for a plurality of antigenic peptide sequences may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 1000 different TCRs specific for the plurality of antigenic peptide sequences. For example, the plurality of antigenic peptide sequences may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 1000 different antigenic peptide sequences.

[0196] In some embodiments, the composition or pharmaceutical composition comprises an immune cell colony from a biological sample. In some embodiments, the immune cell comprises a plurality of antigen-specific T cells. In some embodiments, each antigen-specific T cell comprises a T cell receptor (TCR) specific for at least one antigen peptide sequence. In some embodiments, the composition or pharmaceutical composition comprises an immune cell colony, wherein the amount of immune cells expressing CD14 and / or CD25 in the colony is less than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0197] In some embodiments, the pharmaceutical composition provided herein comprises: a population of immune cells from a biological sample and a pharmaceutically acceptable excipient, the 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 immune cells expressing CD14 and / or CD25 in the population is different in proportion to the number of immune cells expressing CD14 and / or CD25 in the biological sample.

[0198] In some embodiments, the compositions provided herein comprise a population of immune cells from a biological sample, wherein the amount of immune cells in the population that express CD14 and CD25 is proportionally less than the amount of immune cells that express CD14 and CD25 in the biological sample.

[0199] In some embodiments, the pharmaceutical composition provided herein comprises: an immune cell colony and a pharmaceutically acceptable excipient, the immune cell colony comprising T cells from a biological sample, wherein the T cell comprises at least one antigen-specific T cell, the antigen-specific T cell is a T cell stimulated by an APC and comprises a T cell receptor (TCR) specific for at least one antigen peptide sequence, wherein the APC is an APC stimulated by FLT3L. In some embodiments, the at least one antigen-specific T cell comprises at least one APC stimulated T cell. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the colony is proportionally less 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 colony is proportionally greater than the amount of immune cells expressing CD14 and / or CD25 in the biological sample.

[0200] In some embodiments, the pharmaceutical composition comprises CD4 + T cells, including CD4 + The percentage of T cells in the antigen-specific T cells is at least about 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In some embodiments, the pharmaceutical composition comprises naive CD8 + T cells, of which naive CD8 + The percentage of T cells to antigen-specific T cells is at least about 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the pharmaceutical composition comprises memory CD8 + T cells, including memory CD8 + The percentage of T cells to antigen-specific T cells is at least about 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0201] In some embodiments, a pharmaceutical composition comprises an immune cell population comprising T cells from a biological sample and a pharmaceutically acceptable excipient, wherein the T cells comprise APC-stimulated T cells and antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the APC is a FLT3L-stimulated APC.

[0202] In some embodiments, a pharmaceutical composition comprises a population of immune cells comprising T cells from a biological sample and a pharmaceutically acceptable excipient, wherein the T cells comprise a plurality of neo-antigen-specific T cells, wherein the neo-antigen-specific T cells comprise a T cell receptor (TCR) specific for at least one antigenic peptide sequence; wherein the percentage of antigen-specific T cells among the T cells is at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%.

[0203] In some embodiments, the neoantigen-specific T cells comprise APC-stimulated T cells. In some embodiments, the percentage of immune cells expressing CD14 and / or CD25 in the population is less than the percentage of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the biological sample is from a subject. In some embodiments, the subject is a human. In some embodiments, the subject has a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the neoantigen-specific T cells comprise CD4 + and / or CD8 + T cells. In some embodiments, the neoantigen-specific T cells comprise CD4-enriched T cells and / or CD8-enriched T cells. For example, CD4-enriched T cells can be isolated, enriched or purified from a biological sample from a subject comprising PBMCs. + T cells or CD8 + In some embodiments, the neoantigen-specific T cells are naive CD4 + and / or naive CD8 +T cells. In some embodiments, naive T cells are characterized by surface expression of L-selectin (CD62L). In some embodiments, naive T cells are characterized by the absence of one or more activation markers CD25, CD44 or CD69. In some embodiments, naive T cells are characterized by the absence of memory CD45RO isoforms. In some embodiments, naive T cells are characterized by the expression of a functional IL-7 receptor, which is composed of subunits IL-7 receptor-α, CD127 and a common γ chain, CD132. In some embodiments, the at least one neoantigenic peptide sequence comprises a mutation selected from the following: (A) point mutations, and the cancer neoantigenic peptide has an IC of less than 500nM 50 and binding to the subject's HLA protein with greater affinity than the corresponding wild-type peptide, (B) splice site mutations, (C) frameshift mutations, (D) read-through mutations, (E) gene fusion mutations, and combinations thereof. In some embodiments, each of the at least one new 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 new antigenic peptide sequence comprises a mutation that is not present in the subject's non-cancerous cell. In some embodiments, each of the at least one new antigenic peptide sequence is encoded by an expressed gene of a cancer cell of the subject.

[0204] In some embodiments, one or more of the at least one new antigenic peptide sequence has a length of 8-50 naturally occurring amino acids. In some embodiments, the at least one new antigenic peptide sequence comprises a plurality of new antigenic peptide sequences. In some embodiments, the plurality of new antigenic peptide sequences comprises 2-50, 3-50, 4-50, 5-50, 6-50, 7-50, 8-50, 9-50 or 10-50 new antigenic peptide sequences.

[0205] In some embodiments, the APC is one or more APC preparations. In some embodiments, the APC includes an APC loaded with one or more neoantigenic peptides, wherein the one or more neoantigenic peptides comprise one or more of the at least one neoantigenic peptide sequence. In some embodiments, the APC is an autologous APC or an allogeneic APC.

[0206] In some embodiments, the APC comprises a dendritic cell (DC). In some embodiments, the APC is derived from a CD14 + In some embodiments, CD14 + Monocytes are enriched from a biological sample from a subject comprising PBMCs. For example, CD14 can be isolated, enriched or purified from a biological sample from a subject comprising PBMCs. + .

[0207] In some embodiments, CD14 is 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, CD14 + The monocytes are from a second biological sample comprising PBMCs. In some embodiments, the second biological sample is from the same subject.

[0208] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the percentage of the at least one antigen-specific T cell in the composition 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%, 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 at least one antigen-specific CD8 + The percentage of T cells is total CD4 + T cells, total CD8 + In some embodiments, at least one antigen-specific CD4 T cells, total T cells, or total immune cells in the composition 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%, 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%. +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%, 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. In some embodiments, the percentage of at least one antigen-specific T cell in the biological sample is a percentage of total CD4 + T cells, total CD8 + T cells, total T cells, or total immune cells. In some embodiments, the at least one antigen-specific CD8 + The percentage of T cells is total CD4 + T cells, total CD8 + T cells, total T cells, or total immune cells. In some embodiments, the at least one antigen-specific CD4 + The percentage of T cells is total CD4 + T cells, total CD8 + 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 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5%. + The percentage of T cells is at most about 0.5%. In some embodiments, the antigen-specific CD4 + The percentage of T cells was about 0.5% at most.

[0209] In some embodiments, the percentage of antigen-specific T cells in the pharmaceutical composition is 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 T cells, total T cells, or total immune cells. In some embodiments, the pharmaceutical composition contains 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 antigen-specific CD8 + The percentage of total CD4 T cells + T cells, total CD8 + T cells, total T cells, or total immune cells. In some embodiments, the pharmaceutical composition contains 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 antigen-specific naive CD8 + The percentage of total CD4 T cells + 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 T cells, total T cells, or total immune cells. In some embodiments, the pharmaceutical composition contains 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 antigen-specific memory CD8 + The percentage of total CD4 T cells + T cells, total CD8 +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 T cells, total T cells or total immune cells. In some embodiments, the antigen-specific CD4 + The percentage of total CD4 T cells + T cells, total CD8 + or 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 T cells, total T cells or total immune cells.

[0210] Preparation method

[0211] Provided herein are methods for preparing antigen-specific T cells. Provided herein are methods for preparing T cell compositions, such as therapeutic T cell compositions. For example, the methods may include expanding or inducing antigen-specific T cells. Preparing (e.g., inducing or expanding) T cells may also refer to preparing T cells, and broadly encompasses isolating, stimulating, culturing, inducing and / or expanding any type of T cell (e.g., CD4 + T cells and CD8 + In a first 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.

[0212] In a second aspect, the present invention provides 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.

[0213] In a third aspect, the present invention provides 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 an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with an immune cell population from a biological sample for a first period of time; thereafter incubating at least one T cell from the biological sample with an APC.

[0214] In a fourth aspect, the present invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, the method comprising incubating an immune cell population from a biological sample with one or more APC preparations for one or more separate time periods, the time period being less than 28 days from incubation of the immune cell population with the 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.

[0215] In a fifth aspect, the present invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating a population of immune cells from a biological sample with three or fewer APC preparations for three or fewer time periods, wherein at least one antigen-specific memory T cell is expanded, or at least one antigen-specific naive T cell is induced.

[0216] 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 one or more APC preparations for one or more separate time periods, thereby stimulating the T cells to become antigen-specific T cells, wherein the percentage of antigen-specific T cells is 1:1 of the total CD4 + T cells, total CD8 +or 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 T cells, total T cells or total immune cells. In some embodiments, the method for preparing antigen-specific T cells comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence comprises incubating an immune cell population from a biological sample with 3 or fewer APC preparations for 3 or fewer separate time periods, thereby stimulating the T cells to become antigen-specific T cells. In some embodiments, the method for preparing antigen-specific T cells comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence comprises incubating an immune cell population from a biological sample with 2 or fewer APC preparations for 2 or fewer separate time periods, thereby stimulating the T cells to become antigen-specific T cells.

[0217] In some embodiments, the method comprises: (a) obtaining a biological sample from a subject, the biological sample comprising at least one antigen presenting cell (APC); (b) enriching cells expressing CD14 from the biological sample, thereby obtaining CD14 + Cell-enriched sample; (c) the CD14 + The cell-enriched sample is incubated with at least one cytokine or growth factor for a first period of time; (d) at least one peptide is combined with the CD14 of (c) + (e) incubating the APC peptide-loaded sample with one or more cytokines or growth factors for a third time period to obtain a mature APC sample; (f) incubating the APCs of the mature APC sample with a CD14 and / or CD25-depleted sample containing PBMCs for a fourth time period; (g) incubating the PBMCs with the APCs of the mature APC sample for a fifth time period; (h) incubating the PBMCs with the APCs of the mature APC sample for a sixth time period; and (i) administering at least one T cell of the PBMCs to a subject in need.

[0218] In some embodiments, the method comprises: (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 cell-depleted sample; (c) incubating the CD14 and / or CD25 cell-depleted sample with FLT3L for a first period of time; (d) incubating at least one peptide with the CD14 and / or CD25 cell-depleted sample of (c) for a second period of time, thereby obtaining an APC peptide-loaded sample; (e) incubating the APC peptide-loaded sample with at least one PBMC for a third time period, 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 time period, thereby obtaining a second stimulated T cell sample; (g) incubating the PBMCs of the second stimulated PBMC sample with the APCs of the mature APC sample for a fifth time period, thereby obtaining a third stimulated PBMC sample; (h) administering at least one T cell from the third stimulated PBMC sample to a subject in need thereof.

[0219] 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 the APC with a population of immune cells from a biological sample depleted of cells expressing CD14 and / or CD25.

[0220] In some embodiments, the present invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, the method comprising incubating an immune cell population from a biological sample with one or more APC preparations for one or more separate time periods, the time period being less than 28 days from the incubation of the immune cell population with the 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, the present invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, the method comprising incubating an immune cell population from a biological sample with 3 or less APC preparations for 3 or less separate time periods, wherein at least one antigen-specific memory T cell is expanded, or at least one antigen-specific naive T cell is induced.

[0221] In some embodiments, the method for preparing an antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence includes contacting an immune cell (e.g., PBMC) colony with an APC. In some embodiments, the method for preparing an antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence includes incubating an immune cell (e.g., PBMC) colony with an APC for a period of time. In some embodiments, the immune cell colony is from a biological sample. In some embodiments, the immune cell colony is from a sample (e.g., a biological sample) that has been depleted of cells expressing CD14. In some embodiments, the immune cell colony is from a sample (e.g., a biological sample) that has been depleted of cells expressing CD25. In some embodiments, the immune cell colony is from a sample (e.g., a biological sample) that has been depleted of cells expressing CD14 and cells expressing CD25.

[0222] In some embodiments, the method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigen peptide sequence comprises incubating an APC stimulated by a FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with an immune cell population from a biological sample. In some embodiments, provided herein is a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigen peptide sequence, the method comprising: incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with an immune cell population from a biological sample for a first period of time; thereafter incubating at least one T cell of the biological sample with an APC.

[0223] In some embodiments, the method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence comprises contacting an immune cell population from a sample (e.g., a biological sample) with an FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, the method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence comprises contacting an immune cell population from a sample (e.g., a biological sample) with an APC stimulated by an FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, the method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence comprises incubating an immune cell population from a sample (e.g., a biological sample) with an APC stimulated by an 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 an 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); then contacting the T cells of the biological sample with an APC. 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 time periods. In some embodiments, the 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 an immune cell population 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 time periods. In some embodiments, the one or more separate time periods are less than 28 days, calculated from the incubation of the immune cell population with the first APC preparation in the one or more APC preparations.

[0224] In some embodiments, the method for preparing antigen-specific T cells comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence comprises incubating an immune cell colony with APC for a period of time, wherein the immune cell colony is from a biological sample comprising PBMC. In some embodiments, the method for preparing antigen-specific T cells comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence comprises incubating an immune cell colony with APC for a period of time, wherein the immune cell colony is from a biological sample depleted of cells expressing CD14 and / or CD25.

[0225] 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 APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) for a period of time.

[0226] In some embodiments, a method for 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 an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample; and then contacting the T cells from the biological sample with APCs.

[0227] 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 an immune cell population from a biological sample with one or more APC preparations for one or more separate time periods, thereby inducing or expanding antigen-specific T cells, wherein the one or more separate time periods are less than 28 days from the time the immune cell population is incubated with the first APC preparation in the one or more APC preparations. In some embodiments, the immune cell population from a biological sample is incubated with one or more APC preparations for one or more separate time periods 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, an IDO inhibitor can increase antigen-specific CD8 + In some embodiments, IDO inhibitors can maintain memory CD8 + Functional spectrum of T cell responses. PD-1 antibodies can increase antigen-specific memory CD8 +Absolute number of T cell responses. PD-1 antibodies can increase the proliferation rate of cells treated with this antibody. Addition of IL-12 can lead to an increase in antigen-specific cells and / or CD8 + The frequency of T cells increases.

[0228] 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 one or more APC preparations for one or more separate time periods, thereby expanding or inducing antigen-specific T cells, wherein the antigen-specific T cells, antigen-specific CD4 + T cells or antigen-specific CD8 + The percentage of T cells is total T cells, total CD4 + T cells, total CD8 + or 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 T cells, total immune cells, or total cells.

[0229] 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 fewer APC preparations for three or fewer separate time periods, thereby stimulating the T cells to become antigen-specific T cells.

[0230] In some embodiments, the immune cell colony is from a biological sample that is depleted of cells expressing CD14 and / or CD25. In some embodiments, the APC is an APC stimulated by FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, the APC comprises one or more APC products. In some embodiments, the APC products comprise 3 or less APC products. In some embodiments, the APC products are sequentially incubated with immune cells in one or more separate time periods.

[0231] In some embodiments, the biological sample is from a subject. In some embodiments, the subject is a human. For example, the subject can be a patient or a donor. In some embodiments, the subject has a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the antigen-specific T cells comprise 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-enriched T cells can be isolated, enriched or purified from a biological sample from a subject comprising PBMCs. + T cells and / or CD8 + In some embodiments, the 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.

[0232] In some embodiments, the at least one antigenic peptide sequence comprises a mutation selected from the group consisting of: (A) a point mutation, and the cancer antigenic peptide has an IC of less than 500 nM. 50 and binding to the HLA protein of the subject with greater affinity than the corresponding wild-type peptide, (B) splice site mutation, (C) frameshift mutation, (D) read-through mutation, (E) gene fusion mutation, and combinations thereof. In some embodiments, each of the at least one antigenic peptide sequence binds to the protein encoded by the 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 the non-cancerous cell of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by an expression gene of a cancer cell of the subject. In some embodiments, one or more of the at least one antigenic peptide sequence has a length of 8-50 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 comprise 2-50, 3-50, 4-50, 5-50, 6-50, 7-50, 8-50, 9-50 or 10-50 antigenic peptide sequences.

[0233] In some embodiments, the APC includes an APC loaded with one or more antigenic peptides, wherein the one or more antigenic peptides include 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 includes a dendritic cell (DC).

[0234] In some embodiments, the method comprises depleting cells expressing CD14 and / or CD25 from a biological sample. In some embodiments, depleting CD14 + The cell comprises contacting a CD14 binding agent with an APC. In some embodiments, the APC is derived from a CD14 + In some embodiments, APCs are enriched from a biological sample. For example, APCs can be isolated, enriched or purified from a biological sample from a subject that contains PBMCs.

[0235] In some embodiments, APC is 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, GM-CSF, TNF-α, IL-1β, PGE1, IL-6, IL-7 or a combination thereof.

[0236] In some embodiments, the APCs are from a second biological sample. In some embodiments, the second biological sample is from the same subject.

[0237] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMC). 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 total T cells or total immune cells. In some embodiments, the percentage of antigen-specific T cells in the method 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 the total T cells or total immune cells. In some embodiments, the percentage of antigen-specific 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. In some embodiments, the method comprises antigen-specific naive 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. In some embodiments, the method comprises 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. In some embodiments, the method includes antigen-specific CD4 + 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 method includes antigen-specific CD4 + 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 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 + 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 naive CD8 + The percentage of T cells is up to 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 T cells in the biological sample is up to 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 up to 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 T cells in a biological sample is up to 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 up to about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0238] In some embodiments, the method comprises stimulating T cells with IL-7, IL-15, or a combination thereof. In some embodiments, the method comprises 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 method further comprises administering antigen-specific T cells to a subject.

[0239] In some embodiments, the first of the one or more time periods is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 days.

[0240] In some embodiments, the total time period of the individual time periods is less than 28 days. In some embodiments, the total time period of the individual time periods is 20-27 days. In some embodiments, the total time period of the individual time 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.

[0241] In some embodiments, the method comprises incubating the first APC preparation of the APC preparation with the T cells for more than 7 days. In some embodiments, the method comprises incubating the 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 the 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 the first APC preparation of the APC preparation with the T cells for about 10-15 days.

[0242] In some embodiments, the method comprises incubating the second APC preparation of the APC preparation with the T cells for 5-9 days. In some embodiments, the method comprises incubating the second APC preparation of the APC preparation with the T cells for 5, 6, 7, 8 or 9 days.

[0243] In some embodiments, the method comprises incubating a third APC preparation of the APC preparation with T cells for 5-9 days. In some embodiments, the method comprises incubating a third APC preparation of the APC preparation with T cells for 5, 6, 7, 8, or 9 days.

[0244] In some embodiments, the method comprises incubating a first preparation of APCs with 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 preparation of APCs with 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 preparation of APCs with 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.

[0245] In some embodiments, the biological sample is a freshly obtained or frozen sample from a subject.

[0246] In some embodiments, the method comprises 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, or 17 or 18 days. In some embodiments, the first period of time is no 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 no 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-α, R848, LPS, ss-rna40, poly I:C, or any combination thereof.

[0247] In some embodiments, the method comprises incubating one or more APC preparations with at least one peptide for a second period of time. In some embodiments, the second period of time does not exceed 1 hour.

[0248] In some embodiments, the method includes incubating one or more APC products with a second medium comprising one or more cytokines or growth factors for a third time period to obtain mature APCs. In some embodiments, the one or more cytokines or growth factors include GM-CSF (granulocyte macrophage colony stimulating factor), IL-4, FLT3L, 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 time period is no 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 third time period 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 time period is no more than 2, 3, 4 or 5 days. In some embodiments, the third period of time is at least 1, 2, 3, or 4 days.

[0249] In some embodiments, the method further comprises removing the one or more cytokines or growth factors from the second medium after the third time period and before the start of the fourth time period.

[0250] In some embodiments, the methods are performed ex vivo.

[0251] In some embodiments, the method of preparing T cells comprises obtaining a biological sample comprising APCs from a subject. In some embodiments, the method comprises enriching CD14 from the biological sample. + cells, thereby acquiring CD14 + In some embodiments, the method comprises adding CD14 + The enriched sample is incubated with a first medium comprising at least one cytokine or growth factor for a first period of time. In some embodiments, the method comprises combining at least one peptide with CD14 +In some embodiments, the method comprises incubating the APC peptide-loaded sample with the second medium containing one or more cytokines or growth factors for a third time period to obtain a mature APC sample. In some embodiments, the method comprises contacting the APC of the mature APC sample with peripheral blood mononuclear cells (PBMC) and the third medium containing at least one cytokine or growth factor for a fourth time period. In some embodiments, the method comprises incubating the PBMC with the APC of the mature APC sample for a fifth time period. In some embodiments, the method comprises incubating the PBMC with the APC of the mature APC sample for a sixth time period. In some embodiments, the method comprises administering the T cells of the PBMC to a subject in need thereof.

[0252] In some other embodiments, the method of preparing T cells comprises obtaining a biological sample comprising APCs from a subject. In some embodiments, the method comprises enriching CD14 from the biological sample. + cells, thereby acquiring CD14 + In some embodiments, the method comprises adding CD14 + The enriched sample is incubated with a first medium comprising at least one cytokine or growth factor for at least, at most, or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0253] In some embodiments, the method comprises combining at least one peptide with CD14 +The enriched samples are incubated together for at least or up to or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days to obtain an APC peptide-loaded sample. In some embodiments, the method comprises incubating the APC peptide-loaded sample with a medium comprising one or more cytokines or growth factors for at least or up to or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days to obtain a mature APC sample. In some embodiments, the method comprises contacting the APCs of mature APCs with PBMCs and a medium comprising at least one cytokine or growth factor for at least or up to or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, a method comprises incubating PBMCs with APCs of a mature APC sample for at least or up to or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the method comprises incubating PBMCs with APCs of a mature APC sample for at least or up to or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the method comprises administering T cells of PBMCs to a subject in need thereof.

[0254] In some embodiments, the method comprises: (a) obtaining a biological sample from a subject, the biological sample comprising at least one antigen presenting cell (APC); (b) enriching cells expressing CD14 from the biological sample, thereby obtaining CD14 + Cell-enriched samples; (c) CD14 + The cell-enriched sample is incubated with at least one cytokine or growth factor for a first period of time; (d) at least one peptide is combined with the CD14 of (c) +(e) incubating the APC peptide-loaded sample with one or more cytokines or growth factors for a third time period to obtain a mature APC sample; (f) incubating the APCs of the mature APC sample with a CD14 and / or CD25-depleted sample containing PBMCs for a fourth time period; (g) incubating the PBMCs with the APCs of the mature APC sample for a fifth time period; (h) incubating the PBMCs with the APCs of the mature APC sample for a sixth time period; and (i) administering at least one T cell of the PBMCs to a subject in need. In some embodiments, the first period of time is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the second time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the third time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the fourth time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the fifth time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0255] In some embodiments, the method comprises: (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 and / or CD19 from the biological sample, thereby obtaining a CD14 and / or CD25 and / or CD19 cell-depleted sample; (c) incubating the CD14 and / or CD25 and / or CD19 cell-depleted sample with FLT3L for a first period of time; (d) incubating at least one peptide with the CD14 and / or CD25 and / or CD19 cell-depleted sample of (c) for a second period of time, thereby obtaining APC. (e) incubating the APC peptide-loaded sample with at least one PBMC for a third time period, thereby obtaining a first stimulated PBMC sample; (f) incubating the PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth time period, thereby obtaining a second stimulated PBMC sample; (g) optionally, incubating the PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth time period, thereby obtaining a third stimulated PBMC sample; (h) administering at least one T cell from the first, second or third stimulated PBMC sample to a subject in need thereof. In some embodiments, the first period of time is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the second time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the third time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.In some embodiments, the fourth time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the fifth time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0256] In some other embodiments, the method of preparing T cells includes: (a) obtaining a biological sample comprising APC and T cells from a subject; (b) incubating the biological sample with a first medium comprising at least one cytokine or growth factor for a first time period; (c) incubating at least one peptide with the biological sample of (c) for a second time period to obtain a sample loaded with APC peptides; (d) incubating the sample loaded with APC peptides with a second medium comprising one or more cytokines or growth factors for a third time period to obtain a mature APC sample; (e) after the third time period, incubating the mature APC sample with human serum for a fourth time period; (f) incubating the biological sample with one or more cytokines for a fifth time period; (g) administering the T cells of the biological sample to a subject in need. In some embodiments, at least one cytokine or growth factor comprises FLT3L. In some embodiments, the first time period is at least 5 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 15 hours, at least 20 hours, at least 22 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days. In some embodiments, the second time period is at least 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours or 3 hours. In some embodiments, the third time period is at least 10 hours, at least 12 hours, at least 15 hours, at least 20 hours, at least 22 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days or at least 5 days. In some embodiments, the fourth time period is about 2, 3 or 4 days. In some embodiments, the fifth time period is at least 4, 5, 6, 7, 8, 9, 10, 11 or 12 days. In some embodiments, the first period of time is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the second time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.In some embodiments, the third time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the fourth time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the fifth time period is at least or at most or about 30, 40, or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0257] The induced or expanded T cells may include various types of T cells. In some embodiments, the antigen-specific T cells include at least one CD4 + In some embodiments, the antigen-specific T cells comprise at least one CD8 + T cells. In some embodiments, the antigen-specific T cells comprise at least one CD4-enriched T cell. In some embodiments, the antigen-specific T cells comprise at least one CD8-enriched T cell. In some embodiments, the antigen-specific T cells comprise at least one memory T cell. In some embodiments, the antigen-specific T cells comprise at least one naive T cell. In some embodiments, the antigen-specific T cells comprise at least one memory CD4 + In some embodiments, the antigen-specific T cells comprise at least one naive CD4 + In some embodiments, the antigen-specific T cells comprise at least one memory CD8 + In some embodiments, the antigen-specific T cells comprise at least one naive CD8 + T cells.

[0258] Various antigenic peptides can be used to induce or expand T cells. In some embodiments, the peptide comprises a mutation selected from the following: (A) point mutation, (B) splice site mutation, (C) frameshift mutation, (D) read-through mutation, (E) gene fusion mutation, and combinations thereof. In some embodiments, the peptide comprises a point mutation and binds to the subject's HLA protein with greater affinity than the corresponding wild-type peptide. In some embodiments, the peptide binds to the subject's HLA protein with an IC of less than 500nM, 250nM, 150nM, 100nM, 50nM, 25nM, or 10nM. 50 In some embodiments, the peptide binds to the HLA protein of the subject at 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 the 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 DBinding peptide-HLA complex. In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in the non-cancerous cell of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or an expressed gene of a cancer cell of the subject. In some embodiments, the peptide is 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 length. In some embodiments, the peptide binds to a protein encoded by a class I HLA allele and has a length of 8-12 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 16-25 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.

[0259] In various embodiments, APC is used to stimulate / induce T cells. In some embodiments, APC or APC products APC is loaded with one or more antigenic peptides. In some embodiments, APC or APC products APC is autologous APC or allogeneic APC. In some embodiments, APC or APC products APC includes dendritic cells (DC). In some embodiments, the method includes consuming cells expressing CD14 and / or CD25 from a biological sample. In some embodiments, the method includes consuming cells expressing CD19 from a biological sample. In some embodiments, consuming cells expressing CD14 and / or CD25 includes combining CD14 and / or CD25 binding agents with APC products APC or APC. In some embodiments, CD14 and / or CD25 binding agents are biotinylated. In some embodiments, consuming cells expressing CD14 and / or CD25 includes combining an anti-biotin reagent on a solid support with a CD14 and / or CD25 binding agent. In some embodiments, CD14 and / or CD25 binding agents are connected to a solid support. In some embodiments, depleting cells expressing CD19 comprises binding a CD19 binding agent to the APC or the APC of the APC preparation. In some embodiments, the CD19 binding agent is biotinylated. In some embodiments, depleting cells expressing CD19 comprises binding an anti-biotin agent on a solid support to the CD19 binding agent. In some embodiments, the CD19 binding agent is attached to a solid support. In some embodiments, the APC or the APC of the APC preparation is derived from CD14 + Monocytes. In some embodiments, the APC or the APC of the APC preparation is a CD141-enriched APC or a CD141-enriched dendritic cell.

[0260] In some embodiments, the APC or the APC in the APC preparation is enriched from a biological sample. In some embodiments, the APC or the APC in the APC preparation is 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, IFN-α, R848, LPS, ss-rna40, poly I: C or a combination thereof. In some embodiments, the APC or the APC in the APC preparation is from a second biological sample. In some embodiments, the second biological sample is from the same subject. In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMC). In some embodiments, the biological sample is freshly obtained from the subject or is a frozen sample.

[0261] In some embodiments, the percentage of at least one antigen-specific T cell is 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 T cells, total T cells, or total immune cells. In some embodiments, at least one antigen-specific CD8 + The percentage of T cells is total CD4 + T cells, total CD8 + In some embodiments, at least one antigen-specific CD4 T cells, total T cells, or total immune cells is present in 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%. + The percentage of T cells is total CD4 + T cells, total CD8 + or 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 T cells, total T cells or total immune cells.

[0262] In some embodiments, the percentage of at least one antigen-specific T cell in the biological sample is a percentage of total CD4 + T cells, total CD8 +T cells, total T cells, or total immune cells. In some embodiments, at least one antigen-specific CD8 T cell 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%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%. + The percentage of T cells is total CD4 + T cells, total CD8 + At most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of T cells, total T cells, or total immune cells in the biological sample. In some embodiments, at least one antigen-specific CD4 + The percentage of T cells is total CD4 + T cells, total CD8 + At most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1% of T cells, total T cells or total immune cells.

[0263] 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 total time period of the individual time periods is less than 28 days. In some embodiments, incubation comprises incubating the APC preparation of the APC preparation with the T cells for more than 7 days. In some embodiments, incubation comprises incubating the first, second, third or fourth APC preparation of the APC preparation with the T cells for more than 7 days. In some embodiments, the method comprises incubating the APCs in one or more APC preparations with a first medium comprising at least one cytokine or growth factor for a first time period. In some embodiments, the first time period is at least or at most or about 30, 40 or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. In some embodiments, the at least one cytokine or growth factor includes GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-rna40, poly I: C or any combination thereof. In some embodiments, the method includes incubating one or more APC preparations with at least one peptide for a second time period. In some embodiments, the second time period is at least or up to or about 30, 40 or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. In some embodiments, the method comprises incubating the APCs in the one or more APCs with a second medium comprising one or more cytokines or growth factors for a third time period, thereby obtaining mature APCs. In some embodiments, the third time period is at least or up to or about 30, 40 or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. 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, IFN-α, R848, LPS, ss-rna40, poly I: C or a combination thereof.In some embodiments, the method further includes removing the one or more cytokines or growth factors of the second medium after the third time period and before starting the fourth time period. In some embodiments, the fourth time period is at least or at most or about 30, 40 or 50 minutes; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. In some embodiments, the antigen is a new antigen, a tumor-associated antigen, a viral antigen, a minor histocompatibility antigen or a combination thereof. In some embodiments, the method is performed in vitro. In some embodiments, the at least one antigen-specific T cell comprises a plurality of antigen-specific T cells.

[0264] Antigen presenting cell (APC) and preparation method thereof

[0265] In some embodiments, the method includes inducing or stimulating or expanding T cells with antigen presenting cells (APC). Before contacting T cells, antigen peptides can be preloaded into APC. In some embodiments, the method of expanding or inducing antigen-specific T cells includes stimulating an immune cell colony containing T cells with APC. In some embodiments, the immune cell colony is from a biological sample that has exhausted cells expressing CD14 and / or CD25. In some embodiments, APC is an APC stimulated by FLT3L. In some embodiments, the APC includes one or more APC products. In some embodiments, at least one of the one or more APC products includes an APC stimulated by FLT3L. In some embodiments, the one or more APC products include 3 or less APC products. In some embodiments, one or more APC products are sequentially incubated with immune cells in one or more separate time periods.

[0266] Antigen presenting cells (APCs) present peptide fragments of protein antigens in combination with MHC molecules on their cell surface. The presented peptides are associated with MHC molecules as peptide-MHC complexes (pMHC) on the APC cell surface. The processing and presentation of peptide-MHC complexes may involve a series of sequential stages, including: protease-mediated protein digestion; peptide transport into the endoplasmic reticulum (ER) mediated by transporters associated with antigen processing (TAP); formation of peptide-MHC I molecules with newly synthesized MHC molecules; and transport of peptide-MHC molecules to the cell surface.

[0267] Some APCs can activate antigen-specific T cells. For example, T cells comprising a T cell receptor (TCR) that interacts with pMHC can be activated, stimulated, induced or expanded when forming TCR-pMHC. In some embodiments, the MHC (e.g., class I MHC or class II MHC) of an antigen presenting cell can be loaded with a peptide and presented by introducing a polypeptide nucleic acid (e.g., RNA) encoding an antigenic peptide or comprising a peptide sequence to be presented into the APC.

[0268] From a biological perspective, in order for a somatic mutation to generate an immune response, several conditions need to be met: the allele containing the mutation should be expressed in the cell, the mutation should be within a protein-coding region and non-synonymous, the translated protein should be cleaved by the proteasome or other cellular protein degradation pathways, and the epitope containing the mutation should be presented by the MHC complex, the presented epitope should be recognized by the TCR, and finally, the TCR-pMHC complex should initiate a signaling cascade that activates the T cell.

[0269] Monocytes can circulate in the blood and then enter tissues where they can differentiate into macrophages and dendritic cells. Typical monocytes are often characterized by high levels of expression of the CD14 cell surface receptor. Monocytes and B cells may be competent APCs, although their antigen presenting capacity appears to be limited to the reactivation of previously primed T cells. These cell types may not be able to directly activate functionally naive or unprimed T cell populations. Professional antigen presenting cells internalize antigens very efficiently, either by phagocytosis or by receptor-mediated endocytosis, and subsequently display antigen fragments bound to MHC molecules on their membranes. T cells recognize and interact with the antigen-MHC molecule complexes on the antigen presenting cell membrane. Additional co-stimulatory signals are subsequently generated by the antigen presenting cells, leading to the activation of T cells. The expression of co-stimulatory molecules is a typical feature of professional antigen presenting cells.

[0270] Professional antigen presenting cells can internalize antigens very effectively by phagocytosis or by receptor-mediated endocytosis, and then display antigen fragments bound to MHC molecules on their membranes. T cells can recognize the antigen-MHC molecule complex on the APC membrane and interact with it. APC can then produce other co-stimulatory signals, thereby leading to the activation of T cells. The expression of co-stimulatory molecules can be the defining feature of professional antigen presenting cells. The example of professional APC can include but is not limited to dendritic cells (DC), macrophages and B cells. Professional APC may express high levels of MHC class II, ICAM-1 and B7-2.

[0271] One of the main types of professional antigen presenting cells is dendritic cells, which have the widest range of antigen presentation. Other main types of professional antigen presenting cells include macrophages, B cells and some activated epithelial cells. Dendritic cells are white blood cell colonies that present antigens (e.g., antigens captured in peripheral tissues) to T cells via MHC Class II and Class I antigen presentation pathways. Dendritic cells can activate immature and previously triggered T cells (e.g., memory T cells). Dendritic cells (DCs) can be white blood cell colonies that present antigens captured in peripheral tissues to T cells via MHC Class I and Class II antigen presentation pathways. Dendritic cells can be effective inducers of immune responses, and the activation of these cells can be a key step in inducing anti-tumor immunity. Dendritic cells are effective inducers of immune responses, and the activation of these cells is a key step in inducing anti-tumor immunity.

[0272] Dendritic cells can be classified as "immature" and "mature" cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate differentiation stages. Immature dendritic cells can be characterized as antigen presenting cells with high antigen uptake and processing capacity, which is associated with high expression of Fcγ receptors and mannose receptors. The mature phenotype can generally be characterized by lower expression of these markers and high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and co-stimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB). Mature dendritic cells can be CD11b + 、CD11c + , HLA-DR + 、CD80 + 、CD86 + 、CD54 + 、CD3 - 、CD19 - 、CD14 - 、CD141 + (BDCA-3) and / or CD1a +Dendritic cell maturation can be referred to as a state of dendritic cell activation, in which such antigen-presenting dendritic cells cause T cell initiation, while the presentation of immature dendritic cells causes tolerance. Dendritic cell maturation can be caused by biomolecules with microbial characteristics detected by the following substances: innate receptors (e.g., bacterial DNA, viral RNA, endotoxins, etc.), proinflammatory cytokines (e.g., TNF, interleukins, and interferons), CD40L connection to CD40 on the surface of dendritic cells, and substances released from cells undergoing cell death. Other non-limiting examples of cytokines that can induce dendritic cell maturation include IL-4, GM-CSF, TNF-α, IL-1β, PGE1, and IL-6. For example, dendritic cells can be obtained by culturing bone marrow cells in vitro with cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor α (TNF-α). For example, dendritic cells can be derived from CD14 isolated from PBMC. + Monocytes. Cytokines or growth factors that can be used to derive monocytes into dendritic cells include, but are not limited to, GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-RNA40, and poly I:C.

[0273] Normally, non-professional antigen-presenting cells do not constitutively express MHC class II proteins. MHC class II proteins are usually expressed only when non-professional antigen-presenting cells are stimulated by certain cytokines such as IFN-γ.

[0274] The source of antigen presenting cells (APC) can generally be tissue derived, including APC or APC precursors capable of expressing and presenting antigenic peptides in vitro. In some embodiments, when loaded with target RNA and / or treated with necessary cytokines or factors, APC can proliferate and become professional APC.

[0275] In one aspect, APC precursor cells can proliferate and mature into dendritic cells (DC) in vitro. Although many tissue sources can be used, typical tissue sources can include spleen, thymus, tissue biopsy, tumor, infused lymph, lymph node, bone marrow, apheresis or leukocyte separation products and / or peripheral blood. In certain embodiments, apheresis blood products, bone marrow and peripheral blood can be sources. Fetal tissue, fetal or umbilical cord blood, which is also rich in growth factors, can also be used as a blood source for obtaining APC and / or precursor APC. Examples of precursor cells include, but are not limited to, embryonic stem cells, CD34 + Cells, monocyte progenitor cells, monocytes, and pre-B cells. For example, APCs can be derived from monocytes or CD34 + Cell precursors.

[0276] In one aspect, the source of APC and / or precursor APC can be apheresis or leukocyte separation product. Cells can be collected using apheresis procedures known in the art (e.g., Bishop et al., Blood, vol.83, No.2, pp.610-616 (1994)). Apheresis products can typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma portion and the cells can be placed in a suitable buffer or culture medium for subsequent processing steps. In another embodiment of the present invention, cells can be washed with phosphate buffered saline (PBS). In alternative embodiments, the washing solution may lack calcium, and may lack magnesium, or may lack many (even if not all) divalent cations. The washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free 2+ , Mg-free 2+ Alternatively, the aliquots can be stripped of unwanted components and the cells resuspended directly in culture medium.

[0277] APCs can be prepared from a variety of sources, including humans and non-human primates, other mammals, and vertebrates. In certain embodiments, APCs can be prepared from human or non-human vertebrate blood. APCs can also be isolated from an abundant leukocyte population. Leukocyte populations can be prepared by methods known to those skilled in the art. Such methods typically include collecting heparinized blood, apheresis or leukocyte removal, preparation of buffy coat, rosetting, centrifugation, density gradient centrifugation (e.g., using Ficoll, silica gel, and sucrose), differential lysis of non-leukocytes, and filtration. Leukocyte populations can also be prepared by collecting blood from a subject, defibrinating to remove platelets, and dissolving red blood cells. The leukocyte population can optionally be enriched for mononuclear dendritic cell precursors.

[0278] Depending on the desired use of the leukocyte enriched population, blood cell populations can be obtained from a variety of subjects. The subject can be a healthy subject. Alternatively, blood cells can be obtained from a subject in need of immunostimulation (e.g., a cancer patient or other patient to whom immunostimulation is beneficial). Similarly, blood cells can be obtained from a subject in need of immunosuppression, such as a patient with an autoimmune disease (e.g., rheumatoid arthritis, diabetes, lupus, multiple sclerosis, etc.). White blood cell populations can also be obtained from healthy individuals with HLA matching.

[0279] When blood is used as a source of APC, blood leukocytes can be obtained using conventional methods that maintain their vitality. According to one aspect of the present invention, blood can be diluted into a medium that may or may not contain heparin or other suitable anticoagulants. The volume of blood to medium is about 1 to 1. Cells can be concentrated by centrifuging blood in a medium at about 1,000rpm (150g) at 4°C. Platelets and erythrocytes can be eliminated by resuspending the cells in any solution known in the art that dissolves erythrocytes, such as ammonium chloride. For example, the mixture can be a medium and ammonium chloride with a volume ratio of about 1:1. Cells can be concentrated by centrifugation and washed in the desired solution until a white blood cell population substantially free of platelets and erythrocytes is obtained. Any isotonic solution commonly used in tissue culture can be used as a medium for separating leukocytes from platelets and erythrocytes. Examples of such isotonic solutions can be phosphate buffered saline, Hanks balanced salt solution, and complete growth medium. APC and / or APC precursor cells can also be purified by elutriation.

[0280] In one embodiment, the isolation of APCs and / or precursor APCs can be performed by mixing ficoll-treated whole blood or apheresis-treated peripheral blood with one or more unrelated or non-antibody-coupled paramagnetic particles (about 1 vial of beads or 4×10 9 beads (usually about 5×10 8 About 2×10 10 The cells are pre-incubated at a temperature of 22 to 37° C. for about 30 minutes to 2 hours, and then the cells that have attached to or engulfed the paramagnetic particles are magnetically removed. This separation can be performed using standard methods available in the art. For example, any magnetic separation method can be used, including various commercially available methods (e.g., Magnetic Particle Concentrator(DYNAL )). Confirmation of separation can be monitored by a variety of methods known to those of ordinary skill in the art, including flow cytometric analysis of cells before and after said separation.

[0281] APCs can be cultured in a suitable culture medium in an appropriate culture container or vessel to form a primary culture. In certain embodiments, the culture medium can be supplemented with one or more cytokines. A suitable culture container or vessel can be any container with a tissue culture compatible surface. Examples include various bags, flasks, roller bottles, culture dishes, and multi-well plates for tissue culture. Surfaces treated with substances such as collagen or poly-L-lysine, or surfaces with antibodies specific to specific cell types to promote cell adhesion can also be used, provided that they allow differential attachment of cells as described below. The surface can also be chemically treated, for example by ionization. The surface can be treated with about 105 Up to 10 7 cells / cm 2 In one aspect, cells can be seeded at an initial cell density of 10 6 cells / cm 2 Seeding cells.

[0282] In one embodiment, primary cultures from a selected tissue source are maintained under humidity, CO 2 Incubate at about 37°C under standard tissue culture conditions of 40°C and pH until the cell population is sufficiently adhered to the substrate to allow non-adherent cells to be separated. In contrast to monocytes, some immature APCs in the blood do not initially adhere to plastic, particularly immature DCs, so precursors can be separated after overnight culture. Monocytes and fibroblasts may comprise most adherent cells, typically adhering to the substrate within about 30 minutes to about 24 hours. In some aspects, non-adherent cells may be separated from adherent cells between about 1 and 16 hours. Non-adherent cells may be separated in about 1 to 2 hours. Any method that does not remove a large number of adherent cells may be used to separate adherent cells from non-adherent cells. In some aspects, cells may be removed by simple shaking or pipetting. In some aspects, pipetting may be most preferred.

[0283] The adherent cells (e.g., monocytes) constituting precursor APCs separated by the method according to the present invention can be cultured under humidity, CO 2 The cells are incubated at about 37° C. under standard tissue culture conditions of pH and pH until the cell population reaches the immature APC stage. In certain aspects, the adherent cells can be incubated together for a period of 4 hours to 7 days according to the present disclosure. However, one of ordinary skill in the art will readily appreciate that the incubation time and conditions can vary. Depending on the source of the precursor cells, the immature APCs can be CD14 - or CD14 + Immature APCs may also express intermediate levels of CD1a, CD40, CD86, CD54, and MHC class II (marker expression levels on sample cells can be compared to expression levels on MHC class II-negative cells and cells known to express high levels of MHC class II by flow cytometric analysis). Immature APCs generally do not express CCR7.

[0284] In certain aspects of the disclosure, it is not necessary to isolate T cells from APCs. For example, in one embodiment, PBMCs comprising APCs and T cells can be exposed to an antigen as described herein, and the resulting antigen-specific T cells further expanded as described herein.

[0285] In certain aspects of the invention, it is not necessary that the APCs or T cells described herein be derived from autologous sources. Thus, the APCs and T cells may be obtained from matched or unmatched donors, or from cell lines, T cell lines, or other cells grown in vitro. Methods for matching haplotypes are known in the art. In addition, the APCs and T cells or their supernatants may be obtained from xenogeneic sources, for example, mouse, rat, non-human primate, and pig cells may be used.

[0286] Suitable preparations of APC include, for example, dendritic cells and monocytes. In other embodiments, APC can be activated non-nominal APC, such as B cells, cells, or epithelial or endothelial cells. APC can be immature or mature. APC and T cells are usually co-cultured for about 6 to about 48 hours, although longer and shorter times are within the scope of the present invention. Co-culture can usually be carried out for a sufficient time to activate T cells, but is shorter than the time required for differentiation and / or maturation of a large number of immature APC or APC precursors.

[0287] In certain embodiments, mononuclear dendritic cell precursors can be separated, for example, by contacting the enriched leukocytes or monocytes with the mononuclear dendritic cell precursor adhesion substrate. In short, when the enriched leukocyte or monocyte colony contacts the substrate, the mononuclear dendritic cell precursors or monocytes in the cell colony can adhere to the substrate. Other leukocytes can show a reduced binding affinity with the substrate, so that the mononuclear dendritic cell precursors are preferentially enriched on the substrate surface. Suitable substrates include granular substrates, such as glass particles, plastic particles, glass-coated plastic particles, glass-coated polystyrene particles, microcapillaries and microvilli. The surface of the substrate can be optionally treated to enhance the adhesion of mononuclear dendritic cell precursors to the substrate. The surface of the substrate can be coated with, for example, protein, cytokine, plasma and / or monocyte binding protein. After the cell colony rich in leukocytes or monocytes is contacted with the substrate adhering to the mononuclear dendritic cell precursor, the mononuclear dendritic cell precursor adheres to the substrate, thereby forming a complex comprising the mononuclear dendritic cell precursor on the substrate. Mononuclear dendritic cell precursor binding can be monitored, for example, by antibody detection using anti-cell surface marker antibodies (e.g., anti-CD14 antibodies), by FACS forward and side scatter analysis, etc. In some embodiments, the leukocyte population can be in contact with the substrate for about 5 to about 300 minutes, more typically about 30 to about 120 minutes. The mononuclear dendritic cell precursor complex can be optionally washed with a suitable wash buffer to remove non-specifically bound leukocytes. Suitable wash buffers include tissue culture medium, phosphate buffered saline, Dulbecco's phosphate buffered saline, etc. The culture medium can be supplemented with amino acids, vitamins and / or hormones to promote the vitality and / or proliferation of mononuclear dendritic cell precursors. The washing effect can be monitored by forward and side scatter analysis of the wash buffer by FACS, by staining the eluted cells with cell surface markers, and the like. Typically, the complex can be washed several times to remove non-specifically bound leukocytes. Adherent mononuclear dendritic cell precursors can be eluted from the substrate. For example, the precursors can be eluted from the substrate by treatment with phosphate buffered saline containing 0.4% EDTA or other non-toxic chelating agents.Monocytic dendritic cell precursors can generally be eluted from the substrate without the use of trypsin or other proteases.

[0288] In other embodiments, dendritic cells can be isolated according to other methods known to the skilled artisan (e.g., O'Doherty et al., J. Exp. Med. 178: 1067-76 (1993); Young and Steinman, J. Exp. Med. 171: 1315-32 (1990); Freudenthal and Steinman, Proc. Natl. Acad. Sci. USA 87: 7698-702 (1990); Macatonia et al., Immunol. 67: 285-89 (1989); Markowicz and Engleman, J. Clin. Invest. 85: 955-61 (1990); U.S. Pat. Nos. 5,994,126 and 5,851,756). Methods for immunoselecting dendritic cells include, for example, using antibodies against cell surface markers associated with dendritic cell precursors, such as matrix-coupled anti-CD34 and / or anti-CD14 antibodies (e.g., Bernhard et al., Cancer Res. 55:1099-104 (1995); Caux et al., Nature 360:258-61 (1992)) or those associated with fully differentiated dendritic cells, such as CD11c, CD54, CD83, CD80, and CD86.

[0289] In other embodiments, the APC can be a non-nominal APC under inflammatory or other activation conditions. For example, the non-nominal APC can include epithelial cells, T cells, B cells stimulated by interferon-γ, and / or monocytes activated by factors or conditions that induce APC activity. Such non-nominal APCs can be prepared according to methods known in the art.

[0290] Depending on the type of APC, the APC can be cultured, expanded, differentiated and / or matured as desired. The APC can be cultured in any suitable culture container, such as a culture plate, a flask, a culture bag, and a bioreactor.

[0291] In certain embodiments, APCs can be cultured in a suitable culture medium or growth medium to maintain and / or expand the number of APCs in the preparation. The culture medium can be selected according to the type of APC separated. For example, mature APCs, such as mature dendritic cells, can be cultured in a growth medium suitable for their maintenance and expansion. The culture medium can be supplemented with amino acids, vitamins, antibiotics, divalent cations, etc. In addition, cytokines, growth factors, and / or hormones can be included in the growth medium. For example, in order to maintain and / or expand mature dendritic cells, cytokines such as granulocyte / macrophage colony stimulating factor (GM-CSF) and / or interleukin 4 (IL-4) can be added. In other embodiments, immature APCs can be cultured and / or expanded. Immature dendritic cells can retain the ability to take in target mRNA and process new antigens. In some embodiments, immature dendritic cells can be cultured in a culture medium suitable for their maintenance and cultivation. The culture medium can be supplemented with amino acids, vitamins, antibiotics, divalent cations, etc. In addition, cytokines, growth factors, and / or hormones can be included in the growth medium.

[0292] Other immature APCs can be similarly cultured or expanded. The preparation of immature APCs can be matured to form mature APCs. The maturation of APCs can occur during or after exposure to antigenic peptides. In certain embodiments, the preparation of immature dendritic cells can be mature. Suitable maturation factors include, for example, cytokine TNF-α, bacterial products (e.g., BCG), etc. In another aspect, separated APC precursors can be used to prepare the preparation of immature APCs. APC precursors can be cultured, differentiated and / or matured. In certain embodiments, mononuclear dendritic cell precursors can be cultured in the presence of a suitable culture medium supplemented with amino acids, vitamins, cytokines and / or divalent cations to promote the differentiation of mononuclear dendritic cell precursors to immature dendritic cells. In some embodiments, APC precursors are separated from PBMCs. PBMCs can be obtained from donors, such as human donors, and can be used fresh or frozen for future use. In some embodiments, APCs are prepared by one or more APC preparations. In some embodiments, APCs include APCs loaded with one or more antigenic peptides, and the one or more antigenic peptides include one or more of at least one antigenic peptide sequence. In some embodiments, the APC is an autologous APC, an allogeneic APC, or an artificial APC.

[0293] In some embodiments, the APC precursor is a monocyte. In some embodiments, the monocyte is a CD14 + In some embodiments, the monocytes are isolated by anti-CD14 antibodies. In some embodiments, the isolated monocytes are isolated at 10 5 Up to 10 7Cells / well are seeded in 2 mL of culture medium. In some embodiments, the isolated mononuclear cells are plated at about 3×10 6 Cells / wells are seeded in 2mL culture medium. In some embodiments, the isolated monocytes are cultured in a culture medium containing cytokines or growth factors. In some embodiments, the isolated monocytes are cultured in a culture medium containing GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-rna40, poly I: C or a combination thereof. In some embodiments, the isolated monocytes are cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days or at least 6 days before maturation. In some embodiments, monocytes are derived in vitro as dendritic cells in culture medium. In some embodiments, derived dendritic cells are further matured in vitro and loaded with antigenic peptides. In some embodiments, derived dendritic cells are cultured in a culture medium containing one or more antigenic peptides. In some embodiments, the antigenic peptide is a new antigenic peptide. Examples of new antigenic peptides include but are not limited to HIV short peptides, HIV long peptides, previously identified new antigens (PIN) short peptides and PIN long peptides. In some embodiments, the derived dendritic cells are cultured in a medium containing one or more neoantigenic peptides for at least 30 minutes, at least 50 minutes, at least 1 hour, or at least 2 hours. In some embodiments, after incubation with the antigenic peptides, the derived dendritic cells are further incubated with one or more cytokines. In some embodiments, the one or more cytokines include GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-rna40, poly I:C, or a combination thereof.

[0294] In some embodiments, whole PBMCs are used to prepare APCs, which can be further used to stimulate T cells. In some embodiments, PBMCs are cultured in a medium containing FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, T regulatory cells (Tregs) of PBMCs are depleted. reg cells) and then cultured in a medium containing FLT3L. In some embodiments, depletion of CD14 + The cells are then cultured in a medium containing FLT3L. In some embodiments, the PBMCs are depleted of CD25 + The cells are then cultured in a medium containing FLT3L. In some embodiments, the PBMCs are depleted of CD25 + and CD14 + The cells are then cultured in a medium containing FLT3L. In some embodiments, the PBMCs are depleted of CD25 +、CD14 + In some embodiments, PBMCs are cultured in a medium containing FLT3L and then CD14+ cells are removed. + In some embodiments, PBMCs are cultured in medium containing FLT3L and then depleted of CD25 + In some embodiments, PBMCs are cultured in a medium containing FLT3L and then depleted of CD14 + Cells and CD25 + In some embodiments, the isolated CD14 + Monocytes are cultured in a medium containing FLT3L. In some embodiments, after culture in a medium containing FLT3L, PBMCs (complete, CD14 + Depleted, CD25 + Depleted, CD25 + / CD14 + Depletion or CD25 + / CD14 + / CD19 + depleted) or isolated CD14 + Monocytes are cultured in a medium containing one or more antigens. In some embodiments, PBMCs or isolated CD14 +Monocytes are cultured in a culture medium containing one or more mature cytokines. Examples of mature cytokines include, but are not limited to, GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-rna40, poly-I:C or a combination thereof. Mature cytokines can be added to cell culture or culture medium at various concentrations. In some embodiments, mature cytokines are added to cell culture or culture medium at a final concentration of at least 0.05ng / mL, 0.1ng / mL, 0.2ng / mL, 0.3ng / mL, 0.4ng / mL, 0.5ng / mL, 0.8ng / mL, 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 12ng / mL, 15ng / mL, 18ng / mL or 20ng / mL. In some embodiments, the mature cytokine is added to the cell culture or culture medium at a final concentration of at least 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.8 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL. In some embodiments, mature cytokines are added to cell culture or culture medium with a final concentration of at least 10U / mL, 20U / mL, 30U / mL, 40U / mL, 50U / mL, 80U / mL, 100U / mL, 200U / mL, 500U / mL, 800U / mL, 1000U / mL, 1500U / mL, 2000U / mL or 2500U / mL (as used herein, enzyme units are calculated according to the manufacturer's scheme). In some embodiments, the PBMC cultures (complete PBMC or PBMCs that exhaust certain cells) prepared for APC are further incubated or cytokine-treated to induce or stimulate T cells. In this case, APC preparation (e.g., maturation and peptide loading) and T cell induction or stimulation are carried out using the same cell culture. In other cases, APC products are cell colonies separated from PBMC colonies for T cell stimulation.

[0295] In some embodiments, the method comprises incubating one or more APCs or one or more APC preparations with a peptide, thereby producing an APC sample loaded with the peptide. For example, the method may comprise incubating one or more APCs or one or more APC preparations with one or more or more peptides at a concentration of 0.001-100 μM, thereby producing an APC sample loaded with the peptide. For example, the method can include incubating one or more APCs or one or more APC preparations with one or more peptides at a concentration of at least about 0.001 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM or 100 μM. For example, the method can include incubating one or more APCs or one or more APC preparations with one or more peptides at a concentration of up to about 0.001 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM or 100 μM. For example, the method can include incubating one or more APCs or one or more APC preparations with one or more peptides at a concentration of about 0.001 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM or 100 μM.For example, the method can include treating one or more APCs or one or more APC preparations with a concentration of at least about 0.001 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM, 57 μM, 58 μM, 59 μM, 60 μM, 61 μM, 62 μM, 63 μM, 64 μM, 65 μM, 66 μM, 67 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides are incubated together at 9μM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM or 100μM. For example, the method can include treating one or more APCs or one or more APC preparations with a concentration of up to about 0.001 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM, 57 μM, 58 μM, 59 μM, 60 μM, 61 μM, 62 μM, 63 μM, 64 μM, 65 μM, 66 μM, 67 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides are incubated together at 9μM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM or 100μM. For example, the method may include mixing one or more APCs or one or more APC preparations with a concentration of about 0.001 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more peptides are incubated together at 1 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM or 100 μM.

[0296] T cells

[0297] T cells belong to a group of white blood cells called lymphocytes and play a central role in cell-mediated immunity. T cells include CD4 + T cells (helper T cells) and CD8 + T cells (cytotoxic T cells). CD4 + T cells help other white blood cells in immune processes, including the maturation of B cells and the activation of cytotoxic T cells and macrophages. + T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen presenting cells (APCs). Once activated, T cells can rapidly divide and secrete cytokines that regulate active immune responses. CD8 + T cells can destroy virus-infected cells and tumor cells and may also participate in transplant rejection. + T cells can recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of almost every cell in the body. Most T cells have a T cell receptor (TCR). The ability of T cells to recognize antigens associated with a variety of cancers or infectious organisms is conferred by their TCR, which is composed of an alpha (α) chain and a beta (β) chain or a gamma (γ) and delta (δ) chain. The proteins that make up these chains are encoded by DNA, which uses a unique mechanism to generate the diversity of TCRs. This multi-subunit immune recognition receptor can associate with the CD3 complex and bind to peptides presented by MHC class I and class II proteins on the surface of antigen presenting cells (APCs). The first signal in T cell activation can be provided by the binding of the T cell receptor to a short peptide presented by MHC on another cell. This ensures that only T cells with TCRs specific for the peptide are activated. The companion cell is usually an antigen presenting cell, such as a professional antigen presenting cell, and in the case of a naive response, it is usually a dendritic cell, although B cells and macrophages may be important APCs. Binding of the TCR to an antigenic peptide on an APC may be a core event in T cell activation, which occurs at the immunological synapse, the point of contact between the T cell and the APC.

[0298] Each TCR contains variable complementary determining regions (CDRs), as well as framework regions (FRs) and constant regions. The amino acid sequences of the third complementary determining region (CDR3) loops of the α and β chain variable domains largely determine the sequence diversity of αβT cells caused by recombination between variable (Vβ), diversity (Dβ) and joining (Jβ) gene segments in the β chain locus, and between similar Vα and Jα gene segments in the α chain locus. The presence of multiple such gene segments in the TCRα and β chain loci allows a large number of different CDR3 sequences to be encoded. During TCR gene rearrangement, independent additions and deletions of nucleotides at the Vβ-Dβ, Dβ-Jβ and Vα-Jα junctions further increase CDR3 sequence diversity. In this regard, immune competence is reflected in the diversity of TCRs. γδTCRs differ from αβTCRs in that the receptors they encode interact closely with the innate immune system. TCRγδ is expressed early in development, has a specific anatomical distribution, has unique pathogen and small molecule specificity, and has a wide spectrum of innate and adaptive cell interactions. Early in ontogeny, as limited subsets of TCRγδ cells prenatally populate multiple tissues, biased patterns of TCRγV and J segment expression are established.

[0299] T cells can be prepared according to methods known in the art. T cells can be enriched T cell preparations, APC-depleted cell preparations, or substantially purified T cell preparations. T cells can be mixed T cell populations or purified T cell subsets. T cells can be enriched T cell preparations containing T cells with an increased number or percentage relative to the isolated T cell population.

[0300] T cells or subsets of T cells can be obtained from various lymphoid tissues. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen tissue, lymphoid tissue and tumor. As used herein, the term "peripheral blood lymphocyte" (PBL) and its grammatical equivalents may refer to lymphocytes circulating in blood (e.g., peripheral blood). Peripheral blood lymphocytes may refer to lymphocytes that are not localized to organs. Peripheral blood lymphocytes may include T cells, NK cells, B cells or any combination thereof.

[0301] The method may include isolating T cells from a subject. The method may include obtaining T cells isolated from a subject. T cells may be obtained from a T cell line. T cells may be obtained from an autologous source. T cells may be obtained from an allogeneic source. T cells may also be obtained from a xenogeneic source, such as a mouse, rat, non-human primate, and pig.

[0302] The T cells can be APC-depleted cell products. The T cells can be substantially free of APCs. For example, the T cells can comprise T cells from which more than 75% of the APCs have been separated. In some embodiments, peripheral blood mononuclear cells (PBMCs) can be obtained from blood, for example in heparinized vials. The PBMCs can be separated from the red blood cells by centrifugation, and the PBMCs can be recovered from the interface. The recovered PBMCs can optionally be washed (e.g., with PBS).

[0303] T cell purification can be achieved, for example, by positive or negative selection, including but not limited to the use of antibodies against CD2, CD3, CD4, CD5, CD8, CD14, CD16, CD19 and / or CD25. Specific T cell subsets, such as CD28, can be isolated by positive or negative selection techniques. + 、CD4 + 、CD8 + 、CD45RA + and / or CD45RO + T cells. For example, CD45RO can be isolated by positive or negative selection techniques. + 、CD14 - and / or CD25 - T cells. For example, CD45RA can be isolated by positive or negative selection techniques + 、CD14 - and / or CD25 - T cells. For example, CD3 + 、CD14 - and / or CD25 - T cells. For example, CD28 + 、CD14 - and / or CD25 - T cells. For example, CD4 + 、CD14 - and / or CD25 - T cells. For example, CD8 + 、CD14 - and / or CD25 - T cells. For example, CD14 - and / or CD25 - T cells. For example, CD19 - T cells. For example, CD16 - T cells. For example, CD3 / CD28 conjugated magnetic beads can be used to positively select CD3 +and CD28 + T cells. In one aspect of the invention, enrichment of a T cell population by negative selection can be achieved by combining a combination of antibodies against surface markers specific to negatively selected cells. For example, enrichment of a T cell population can be accomplished by negative selection using antibodies against CD19, CD16, CD14, CD25, or any combination thereof. For example, enrichment of a T cell population can be accomplished by negative selection using a combination of antibodies against CD19, CD16, CD25, and / or CD14.

[0304] For example, a T cell sample can include cells from the subject's circulating blood and can be obtained by apheresis or leukapheresis. The T cell sample can include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and / or platelets. Undesirable components of the T cell sample can be removed and the remaining T cells can be suspended in a culture medium. For example, the cells can be washed to remove the plasma portion. For example, the red blood cells can be lysed and passed through a PERCOLL TM T cells were isolated from peripheral blood lymphocytes by gradient centrifugation.

[0305] In various embodiments, compositions and methods comprising T cells are provided herein. In some embodiments, the T cells comprise a TCR having TCRα and TCRβ chains. In some embodiments, the T cells comprise a TCR having TCRγ and TCRδ chains. In some embodiments, the T cells comprise a T cell receptor (TCR) specific for at least one antigenic peptide sequence. In some embodiments, the antigen-specific T cells comprise at least one CD4 + In some embodiments, the antigen-specific T cells comprise at least one CD8 + T cells. In some embodiments, the antigen-specific T cells comprise at least one CD4-enriched T cell. In some embodiments, the antigen-specific T cells comprise at least one CD8-enriched T cell. In some embodiments, the antigen-specific T cells comprise memory T cells. In some embodiments, the antigen-specific T cells comprise naive T cells. In some embodiments, the antigen-specific T cells comprise memory CD4 + In some embodiments, the antigen-specific T cells comprise naive CD4 + In some embodiments, the antigen-specific T cells are memory CD8 + In some embodiments, the antigen-specific T cells comprise naive CD8 +T cells. In some embodiments, the antigenic peptide sequence comprises a mutation selected from the group consisting of: (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. In some embodiments, the antigenic peptide sequence binds to the subject's HLA protein with a greater affinity than the corresponding wild-type peptide. In some embodiments, the antigenic peptide sequence binds to the subject's HLA protein with a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, each peptide sequence binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, the TCR of a T cell of a composition described herein has a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 Binds to peptide-HLA complexes.

[0306] In some embodiments, T cells are cultured in a culture medium containing cytokines. Examples of cytokines include IL-7 and IL-15. In some embodiments, the final concentration of the cytokine in T cell culture or culture medium is 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 final concentration of IL-7 in the T cell culture or culture medium is 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 final concentration of IL-15 in the T cell culture or culture medium is 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 a culture medium further comprising FLT3L. In some embodiments, the final concentration of FLT3L in T cell culture or culture medium is at least 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 12ng / mL, 15ng / mL, 18ng / mL, 20ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL or 200ng / mL. In some embodiments, T cells are incubated, induced or stimulated in a culture medium containing FLT3L for a first period of time. In some embodiments, T cells are incubated, induced or stimulated in a culture medium containing additionally added FLT3L for a second period of time.In some embodiments, T cells are incubated, induced or stimulated in a medium containing additional FLT3L for a third period of time. In some embodiments, T cells are incubated, induced or stimulated in a medium containing additional FLT3L for a fourth, fifth or sixth period of time, with freshly added FLT3L in each period of time.

[0307] antigen

[0308] The present disclosure relates to methods for preparing T cells that are specific for immunogenic antigens. The present disclosure also relates to compositions comprising antigen-specific T cells stimulated with APCs. In some embodiments, one or more antigenic peptides are loaded onto APCs, wherein the T cells are then stimulated with the peptide-loaded APCs to produce antigen-specific T cells. In some embodiments, the antigen is a new antigen. In some embodiments, the APCs used for peptide loading are dendritic cells.

[0309] In some embodiments, the peptide sequence is included in a mutation that is not present in a non-cancerous cell of a subject. In some embodiments, the peptide is encoded by a gene of a cancer cell of a subject or a gene expressed. In some embodiments, the length of the peptide sequence is 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 sequence is in conjunction with the protein encoded by class I HLA allele, and has the length of 8-12 natural amino acids. In some embodiments, the peptide sequence is in conjunction with the protein encoded by class II HLA allele, and has the length of 16-25 naturally occurring amino acids. In some embodiments, the peptide sequence comprises a plurality of antigenic peptide sequences. In some embodiments, a 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.

[0310] 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 disclosed herein can be used, for example, to produce specific therapies for a subject's disease or to produce vaccines for a disease. Candidate immunogenic neoantigens may be previously identified neoantigens. In some embodiments, candidate immunogenic neoantigens may not have been previously identified. Candidate immunogenic neoantigens used in the methods and compositions described herein may be specific to a subject. In some embodiments, candidate neoantigens used in the methods and compositions described herein may be specific to multiple subjects.

[0311] In both animals and humans, mutated epitopes may be effective in inducing an immune response or activating T cells. In one embodiment, potential immunogenic epitopes of infectious agents such as viruses in subjects can be determined. In one embodiment, potential immunogenic mutated epitopes of subjects with diseases such as cancer can be determined. In some embodiments, potential immunogenic antigens or neoantigens for use in the methods described herein may be differentiation antigens expressed in cells of tumors and tissue types that produce them. In some embodiments, potential immunogenic antigens or neoantigens for use in the methods described herein may be cancer / germline antigens that are not expressed in another differentiated tissue. In some embodiments, potential immunogenic antigens or neoantigens for use in the methods described herein may be mutated antigens. For example, candidate immunogenic antigens or neoantigenic peptides for use in the methods described herein may contain missense point mutations or antigens or neoantigens of fusion proteins produced by tumor-specific translocation of gene segments. In some embodiments, potential immunogenic antigens or neoantigens for use in the methods described herein may be overexpressed antigens. In some embodiments, potential immunogenic antigens or neoantigens may be found in tumors. For example, potentially immunogenic antigens or neoantigens for use with the methods described herein may include proteins whose expression is tightly regulated in cells of differentiated normal tissues.

[0312] Next generation sequencing technology can be used to determine potential immunogenic mutation epitopes by sequencing the genome or exome of tumor tissue and healthy tissue of cancer patients. For example, next generation sequencing technology can be used to sequence genes selected based on their mutation frequency and ability to act as antigens or new antigens. In one embodiment, sequencing data can be analyzed to identify peptides with potential immunogenic mutations that can bind to the subject's HLA molecules. In one embodiment, a computer can be used to analyze the data. In another embodiment, the presence of antigens or new antigenic peptides in the sequence data can be analyzed. In one embodiment, it can be determined by the affinity of potential immunogenic antigens or new antigenic peptides to MHC molecules.

[0313] Potential immunogenic antigens or new 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 potential immunogenic antigens or new antigenic peptides for use in the methods described herein.

[0314] High-throughput methods for de novo sequencing of unknown proteins can be used to identify potentially immunogenic antigens or neoantigenic 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 neoantigens.

[0315] MHC polymers can also be used to identify potential immunogenic antigens or new antigenic peptides 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 preliminary identification of potential immunogenic tumor-specific antigens, or alternatively can be used as a secondary screening scheme to assess which potential immunogenic antigens a patient may have been exposed to, thereby facilitating the selection of potential immunogenic antigens for use in the methods described herein.

[0316] In some embodiments, immune cells can be analyzed or characterized. For example, immune cells of the compositions described herein can be analyzed or characterized. In some embodiments, the method may include determining the expression of one or more cell markers of at least one immune cell in the stimulated immune cell sample; and determining the binding of at least one immune cell in the stimulated immune cell sample to the peptide-MHC complex; wherein determining the expression and determining the binding are performed simultaneously. In some embodiments, the stimulated immune cell sample is an immune cell population stimulated with an APC comprising a peptide-MHC complex. In some embodiments, the immune cell population is from a biological sample. In some embodiments, the method may include incubating an immune cell population from a biological sample with an APC comprising a peptide-MHC complex, thereby obtaining a stimulated immune cell sample. Determine the expression of one or more cell markers of at least one immune cell in the stimulated immune cell sample; and determine the binding of at least one immune cell in the stimulated immune cell sample to the peptide-MHC complex; wherein determining the expression and determining the binding are performed simultaneously. In some embodiments, the one or more cell markers include TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, enzyme 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 include cytokines. In some embodiments, the one or more cell markers include degranulation markers. In some embodiments, the one or more cell markers include cell surface markers. In some embodiments, the one or more cell markers include proteins. In some embodiments, determining the binding of at least one immune cell of the stimulated immune cell sample to the peptide-MHC complex includes determining the binding of at least one immune cell of the stimulated immune cell sample to the MHC tetramer of the MHC containing the peptide and the peptide-MHC complex. In some embodiments, the MHC is class I MHC or class II MHC. In some embodiments, the peptide-MHC complex comprises one or more markers. In some embodiments, the immune cell colony from a biological sample comprises two or more samples, each sample comprising an immune cell colony from one or more biological samples. In some embodiments, two or more samples are labeled with two or more sample markers. In some embodiments, determining expression and determining binding include fluorescence activated cell sorting (FACS). In some embodiments, determining expression and determining binding include single cell analysis. In some embodiments, determining expression and determining binding include determining the percentage of immune cells that both express one or more cell markers and bind to peptide-MHC complexes. In some embodiments, the marker includes a fluorophore.In some embodiments, the immune cell colony includes an immune cell colony representing an immune cell colony of a composition described herein. In some embodiments, an immune cell colony expressing 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, CD3, CD28, CD4, CD8, or any combination thereof and / or not expressing CD14, CD19, CD16, CD25, or any combination thereof can be analyzed and characterized. For example, the method may include analyzing or characterizing a specific T cell subset, such as expressing 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, CD3, CD28, CD4, CD8 or any combination and / or not expressing CD14, CD19, CD16, CD25 or any combination thereof T cell subset. For example, the method may include analyzing or characterizing an immune cell colony that does not express CD14, CD25, CD19, CD16 or any combination thereof.

[0317] In some embodiments, the expression of one or more cell markers in the immune cell population can be determined. For example, the method can include determining the expression of 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, CD14, CD25, CD19, CD16, or any combination thereof. For example, the method may include incubating a population of immune cells from a biological sample with an APC comprising a peptide-MHC complex to obtain a stimulated immune cell sample; determining the expression of 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, CD14, CD25, CD19, CD16, or any combination thereof, of at least one immune cell in the stimulated immune cell sample; determining the binding of at least one immune cell in the stimulated immune cell sample to the peptide-MHC complex; wherein determining the expression and determining the binding are performed simultaneously.

[0318] Potential immunogenic antigens or new antigenic peptides for use in the methods described herein can be known antigen or new antigen sequences. For example, potential immunogenic antigens or new antigenic peptides for use in the methods described herein can be from a database of antigen or new antigen sequences.

[0319] In some aspects, the present disclosure provides peptides (eg, peptides having tumor-specific mutations, viral peptides, or peptides associated with non-cancerous diseases) identified using the methods described herein, or polynucleotides encoding the peptides.

[0320] In some embodiments, an optical method is used to select or identify an immunogenic antigen. In some embodiments, a barcoded probe is used to select or identify an immunogenic antigen. In some embodiments, a barcoded probe comprising a target-specific region and a barcoded region is used to select or identify an immunogenic antigen. In some embodiments, the target-specific region comprises a nucleic acid sequence that hybridizes with a nucleic acid sequence of a target polynucleotide or has at least about 90%, 95%, or 100% sequence complementarity.

[0321] In some embodiments, sequencing methods are used to identify immunogenic antigens. Any suitable sequencing method can be used according to the present invention, for example, next generation sequencing (NGS) technology. The third generation sequencing method may replace NGS technology in the future to accelerate the sequencing step of the method. For the purpose of clarification: the term "next generation sequencing" or "NGS" in the context of the present invention means all high-throughput sequencing technologies compared with the "conventional" sequencing method known as Sanger chemistry, which reads nucleic acid templates randomly along the entire genome by dividing the entire genome into small pieces. This type of NGS technology (also referred to as massively parallel sequencing technology) can provide a full genome, exon group, transcriptome (all transcribed sequences of a genome) or methylation group (all methylation sequences of a genome) in a very short time, such as within 1-2 weeks, such as within 1-7 days or less than 24 hours. Nucleic acid sequence information, and in principle allow single cell sequencing methods. Multiple NGS platforms commercially available or mentioned in the literature can be used in the context of the present invention, for example, those NGS platforms described in detail in WO 2012 / 159643.

[0322] In certain embodiments, the antigen or neoantigenic peptide or epitope thereof may include, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues and any range derivable therein. In specific embodiments, the immunogenic antigen or epitope thereof is equal to or less than 100 amino acids.

[0323] In some embodiments, the length of the antigen or neoantigenic peptide or epitope thereof of MHC class I is 13 residues or less, and generally consists of about 8 to about 11 residues, especially 9 or 10 residues. In some embodiments, the length of the immunogenic antigen or neoantigenic peptide or epitope thereof of MHC class II is 9-24 residues.

[0324] 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) a single binding peptide extending 2-5 amino acids to the N-terminus and C-terminus of each corresponding gene product; or (2) a concatenation of some or all binding peptides with the extended sequence of each binding peptide. In other embodiments, when sequencing reveals the presence of long (>10 residues) epitope sequences, such as new epitopes, in tumors (e.g., due to frameshifting, read-through, or intron inclusion resulting in new peptide sequences), the longer new antigenic peptides can consist of the entire new tumor-specific amino acid stretch as a single longer peptide or several overlapping longer peptides. In some embodiments, it is speculated that the use of longer peptides allows endogenous processing by patient cells and can result in more efficient antigen presentation and T cell response induction. In some embodiments, two or more peptides can be used, where the peptides overlap and are layered on the long new antigenic peptide.

[0325] In some embodiments, the antigen or neoantigenic peptide binds to an HLA protein (e.g., HLA class I or HLA class II). In specific embodiments, the antigen or neoantigenic peptide binds to an HLA protein with greater affinity than the corresponding wild-type peptide. In specific embodiments, the IC of the antigen or neoantigenic peptide is 50 or K D At least less than 5000 nM, at least less than 500 nM, at least less than 100 nM, at least less than 50 nM or less.

[0326] In some embodiments, the length of the antigen or neoantigenic peptide can be about 8 to about 50 amino acid residues, 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 some embodiments, the length of the antigen or neoantigenic peptide can be about 8 to about 500 amino acid residues, 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.

[0327] In some embodiments, the antigen or neoantigenic 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 neoantigenic peptide can 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 length of the antigenic or neoantigenic peptide can be up to 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 some embodiments, the antigen or neoantigenic peptide may be up to 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.

[0328] In some embodiments, the antigen or neo-antigenic peptide has a total 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.

[0329] In some embodiments, the antigen or neo-antigenic peptide has a total 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.

[0330] In some embodiments, the neoantigenic peptides may 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 neoantigenic peptides may have a pi value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the neoantigenic peptides may have a pi value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

[0331] In some embodiments, the antigen or neoantigenic peptide may 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 neoantigenic peptide may 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 neoantigenic peptide may 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.

[0332] In some embodiments, the antigens or neoantigenic peptides described herein may include carriers 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, etc.

[0333] In some embodiments, the antigens or neoantigenic peptides described herein may be cleaved by a terminal -NH 2 Acylation (e.g., by alkanoyl (C 1 -C 20 ) or thioacetylation), terminal carboxyl amidation (e.g., ammonia, methylamine, etc.). In some embodiments, these modifications can provide sites for connection to supports or other molecules.

[0334] In some embodiments, the antigens or neoantigenic peptides described herein may include modifications such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surfactants (e.g., lipids), or may be chemically modified, such as acetylation, etc. In addition, the bonds in the peptides may be bonds other than peptide bonds, such as covalent bonds, ester bonds or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0335] In some embodiments, the antigens or neoantigenic peptides described herein may contain substitutions to change the physical properties (e.g., stability or solubility) of the resulting peptide. For example, an antigen or neoantigenic peptide can be modified by replacing cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical properties, cysteine ​​has a tendency to form disulfide bonds and structurally changes the peptide sufficiently to reduce binding ability. Replacing C with α-aminobutyric acid not only alleviates this problem, but actually improves binding and cross-binding ability in some cases. The replacement of cysteine ​​with α-aminobutyric acid can occur at any residue of the antigen or neoantigenic peptide, such as at an anchored or non-anchored position of an epitope or analog within the peptide or at other positions of the peptide.

[0336] In some embodiments, the antigenic peptides or neoantigenic peptides described herein may comprise amino acid mimetics or non-natural amino acid residues, such as D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1, 2, 3 or 4-pyrenylalanine; D- or L-3-thienylalanine; D- or L-(2-pyridyl)-alanine; D- or L-(3-pyridyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenyl Glycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoro-methyl)-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-alkyl alanine, wherein the alkyl group may be a substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-butyl (sec-isotyl), isopentyl or non-acidic amino acid residue. The aromatic ring of the non-natural amino acid includes, for example, thiazolyl, thienyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl and pyridyl aromatic rings. Modified peptides with various amino acid mimetics or non-natural amino acid residues are particularly useful because they tend to show increased in vivo stability. Such peptides may also have improved shelf life or preparation properties.

[0337] Peptide stability can be determined in a variety of ways. For example, peptidases and various biological media such as human plasma and serum have been used to determine stability. See, for example, Verhoef et al., Eur. J. Drug Metab. Pharmacokinetics 11: 291 (1986). The half-life of the peptides described herein is conveniently determined using 25% human serum (v / v). The protocol is as follows: the combined human serum (AB type, non-heat-inactivated) is destroyed by centrifugation before use. The serum is then diluted to 25% with RPMI-1640 or another suitable tissue culture medium. At predetermined time intervals, a small amount of the reaction solution is removed and added to a 6% trichloroacetic acid (TCA) aqueous solution or ethanol. The turbid reaction sample is cooled (4° C.) for 15 minutes and then rotated to agglomerate the precipitated serum proteins. The presence of the peptide is then determined by reverse phase HPLC using stability-specific chromatographic conditions.

[0338] In some embodiments, the antigens or neo-antigenic peptides described herein may be in solution, lyophilized, or may be in crystalline form.

[0339] In some embodiments, the antigens or neoantigenic peptides described herein can be prepared by synthesis, either by recombinant DNA technology or chemical synthesis, or can be isolated from natural sources such as original tumors or pathogenic organisms. The epitope can be synthesized alone or directly or indirectly linked to the peptide. Although the antigens or neoantigenic peptides described herein are substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments, the peptide can be conjugated by synthesis to be linked to natural fragments or particles.

[0340] In some embodiments, peptides can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. (See, e.g., Stewart & Young, OligoPhase Peptide Synthesis, 2nd edition, Pierce Chemical Co., 1984). In addition, chemical connections can be used to connect individual peptides to produce larger peptides still within the scope of the invention.

[0341] Alternatively, recombinant DNA techniques can be used, wherein the nucleotide sequence encoding the peptide inserted into an expression vector is transformed or transfected into a suitable host cell and cultured under conditions suitable for expression. These methods are generally known in the art, as summarized in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). Therefore, a recombinant peptide comprising or consisting of one or more epitopes described herein can be used to present a suitable T cell epitope.

[0342] In one aspect, the disclosure described herein also provides a composition comprising one, at least two or more than two antigenic peptides or neoantigenic peptides. In some embodiments, the composition described herein contains at least two different peptides. In some embodiments, the at least two different peptides are derived from the same polypeptide. Different polypeptides refer to peptides of different lengths, different amino acid sequences, or both. The peptides are derived from any polypeptide known or found to contain tumor-specific mutations. In some embodiments, the isolated antigen or neoantigenic peptide is encoded by a gene having a point mutation (resulting in an amino acid substitution of a natural peptide).

[0343] Pharmaceutical composition

[0344] The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, including excipients and adjuvants, which assist in processing the active agent into a pharmaceutically acceptable product. Suitable formulations may depend on the selected route of administration. Any known techniques, carriers and excipients may be used as suitable techniques, carriers and excipients as understood in the art.

[0345] In some cases, the pharmaceutical composition is formulated as a cell-based therapeutic agent, such as a T cell therapeutic agent. 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 agent or a nucleic acid-based therapeutic agent, wherein the nucleic acid encodes a polypeptide. In some embodiments, the pharmaceutical composition comprises an antibody-based therapeutic agent. The composition may comprise T cells specific for two or more immunogenic antigens or neoantigenic peptides.

[0346] In addition to the active ingredient, the pharmaceutical composition may also contain a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other substances known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance will depend on the route of administration.

[0347] Acceptable carriers, excipients, or stabilizers are those that are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, 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; proteins such as serum albumin, gelatin, or immunoglobulins; 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 nonionic surfactants such as or polyethylene glycol (PEG).

[0348] An acceptable carrier is physiologically acceptable to the patient to be administered and maintains the therapeutic properties of the compound to be administered. Acceptable carriers and their preparations are generally described in, for example, Remington'pharmaceuticalSciences (18th edition, A.Gennaro, Mack Publishing Co., Easton, PA 1990). An example of a carrier is normal saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is involved in carrying or transporting the subject compound from the administration site of one organ or body part to another organ or body part, or in an in vitro assay system. An acceptable carrier is compatible with the other ingredients of the preparation and is harmless to the subject to be administered. An acceptable carrier should also not change the specific activity of the new antigen.

[0349] In one aspect, pharmaceutically acceptable or physiologically acceptable compositions are provided herein, comprising solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coating materials, isotonic agents and absorption enhancers or delay agents compatible with drug administration. Therefore, pharmaceutical compositions or pharmaceutical preparations refer to compositions suitable for drug use in subjects. Compositions can be formulated to be compatible with specific routes of administration (i.e., systemic or local). Therefore, compositions include carriers, diluents or excipients suitable for administration by various routes.

[0350] In some embodiments, the composition may further include an acceptable additive to improve the stability of immune cells in the composition. Acceptable additives may not change the specific activity of 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. Acceptable additives may be combined with acceptable carriers and / or excipients such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 used to increase the stability of the peptide and reduce the 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 in storage.

[0351] The pharmaceutical composition can be administered, for example, by injection. Compositions for injection include aqueous solutions (which are water-soluble) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, by using coatings such as lecithin, in the case of dispersions, by maintaining the desired particle size, and by using a surfactant to maintain fluidity. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic agents, such as sugars, polyols such as mannitol, sorbitol and sodium chloride, can be included in the composition. The resulting solution can be packaged for use as is, or lyophilized; the lyophilized product can then be merged with a sterile solution before administration. For intravenous injection, or injection at the affected part, the active ingredient will be in the form of an aqueous solution acceptable to the parenteral, which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art can use, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection to prepare suitable solutions. As required, preservatives, stabilizers, buffers, antioxidants and / or other additives may be included. A sterile injectable solution may be prepared by incorporating the required amount of active ingredient into a suitable solvent together with a combination of ingredients or ingredients listed above (if necessary), and then filtering and sterilizing. Typically, dispersions are prepared by incorporating active ingredient into a sterile vehicle containing an alkaline dispersion medium and other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods may be vacuum drying and freeze drying, which obtain powders of active ingredient plus any additional required ingredients from their previously filtered and sterilized solutions.

[0352] For example, the composition can be routinely administered intravenously, for example, by injecting a unit dose. For injection, the active ingredient can be in the form of a parenterally acceptable aqueous solution, which is substantially pyrogen-free and has a suitable pH, isotonicity and stability. Suitable solutions can be prepared using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed. In addition, the composition can be administered via atomization.

[0353] When a composition is contemplated for use in a pharmaceutical or any of the methods provided herein, it is contemplated that the composition may be substantially free of pyrogens, such that the composition does not induce an inflammatory response or an unsafe 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 known to those of ordinary skill in the art and can be accomplished using commercially available kits.

[0354] Acceptable carriers may contain compounds that stabilize, 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 clearance or hydrolysis of peptides; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents may also be used to stabilize or increase or decrease the absorption of pharmaceutical compositions (including liposome carriers). To prevent digestion, the compound may be complexed with a composition to make it resistant to acid and enzymatic hydrolysis, or the compound may be complexed in an appropriate resistant carrier such as a liposome. Means for protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13: 1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48: 119135; and U.S. Pat. No. 5,391,377).

[0355] The composition can be administered in a therapeutically effective amount in a manner compatible with the dosage formulation. 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 degree of desired binding ability. The exact amount of active ingredient to be administered depends on the practitioner's judgment and is specific to each individual. Suitable regimens for initial administration and booster injections are also different, but typically initial administration is followed by repeated administration at intervals of one or more hours by subsequent injections or other administrations. Alternatively, continuous intravenous infusions sufficient to maintain blood concentrations are contemplated.

[0356] In some embodiments, the present invention relates to an immunogenic composition, such as a pharmaceutical composition capable of inducing 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 corresponding to a tumor-specific antigen or neoantigen (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.).

[0357] In some embodiments, the pharmaceutical compositions described herein are capable of eliciting a specific cytotoxic T cell response, a specific helper T cell response, or a B cell response.

[0358] In some embodiments, the antigenic polypeptide or polynucleotide can be provided as an antigen presenting cell (e.g., dendritic cell) containing such polypeptide or polynucleotide. In other embodiments, such antigen presenting cells are used to stimulate T cells for patients. In some embodiments, the antigen presenting cell is a dendritic cell. In related embodiments, the dendritic cell is an autologous dendritic cell pulsed with a new antigenic peptide or nucleic acid. The new antigenic peptide can be any suitable peptide that produces an appropriate T cell response. In some embodiments, the T cell is a CTL. In some embodiments, the T cell is a HTL. Therefore, 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 new 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 patients can be loaded with new antigenic peptides or polynucleotides in vitro. In related embodiments, such APCs or PBMCs are injected back into the patient. The polynucleotide can be any suitable polynucleotide capable of transducing dendritic cells, resulting in presentation of new antigenic peptides and induction of immunity. In some embodiments, such antigen presenting cells (APC) (e.g., dendritic cells) or peripheral blood mononuclear cells (PBMC) 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 cell is a CD4 + In some embodiments, CTLs are injected into the patient. In some embodiments, HTLs are injected into the patient. In some embodiments, both CTLs and HTLs are injected into the patient. The administration of any therapeutic agent can be performed simultaneously or sequentially and in any order.

[0359] In some embodiments, the pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment may be formulated for parenteral, topical, nasal, oral, or external administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, such as intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the composition may be administered intratumorally. The composition may be administered at the site of surgical resection to induce a local immune response to the tumor. In some embodiments, compositions for parenteral administration are described herein, comprising a solution of a neoantigenic peptide, and the immunogenic composition is dissolved or suspended in an acceptable carrier such as an aqueous carrier. A variety of aqueous carriers may be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterilized by filtration. The resulting aqueous solution may be packaged for use as is, or lyophilized, and the lyophilized product may be combined with a sterile solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0360] The ability of an adjuvant to increase the immune response to an antigen is generally manifested as a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity can be manifested as a significant increase in antibody titers to the antigen, and an increase in T cell activity can be manifested as increased cell proliferation or cytotoxicity or cytokine secretion. An adjuvant can also alter the immune response, for example, by changing a response that is primarily humoral or type 2 T helper cells to a response that is primarily cellular or type 1 T helper cells.

[0361] Suitable adjuvants are known in the art (see WO2015 / 095811), and include, but are not limited to, poly(I:C), poly-ICLC, STING agonists, 1018ISS, 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 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, carrier systems, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulin derived from saponin (Aquila Biotech, Worcester, Mass., USA), mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Several immunoadjuvants specific for dendritic cells have been described (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 may also be used. Several cytokines have been directly implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into potent antigen-presenting cells of 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, which is incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0362] It has also been reported that CpG immunostimulatory oligonucleotides enhance the effect of adjuvants in therapeutic environments. Without being bound by theory, CpG oligonucleotides work by activating the innate (non-adaptive) immune system via Toll-like receptors (TLRs) (mainly TLR9). The TLR9 activation triggered by CpG enhances the antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live viruses or killed viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cell immunogenic pharmaceutical compositions, and polysaccharide conjugates in both preventive and therapeutic immunogenic pharmaceutical compositions. Importantly, it enhances the maturation and differentiation of dendritic cells, thereby leading to the activation of enhanced TH1 cells and the generation of strong cytotoxic T lymphocytes (CTL), even in the absence of CD4 T cells to help. Even in the presence of adjuvants that usually promote TH2 biases such as alum or incomplete Freund's adjuvant (IFA), the TH1 bias induced by TLR9 stimulation is also maintained. When formulated or co-administered with other adjuvants or in preparations such as microparticles, nanoparticles, lipid emulsions or similar preparations, CpG oligonucleo...

Claims

1. A pharmaceutical composition comprising: (a) an ex vivo expanded immune cell population from a biological sample, the biological sample comprising antigen presenting cells (APCs) and T cells, wherein the ex vivo expanded immune cell population comprises tumor antigen-specific T cells stimulated by APCs, the T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, in: The amount of immune cells expressing CD14 and the amount of immune cells expressing CD25 in the population are proportionally less than the amount of immune cells expressing CD14 and the amount of immune cells expressing CD25 in the biological sample; and (b) a pharmaceutically acceptable excipient; and in: (i) in the ex vivo expanded immune cell population comprising APC-stimulated tumor antigen-specific T cells, at least 0.1% of the CD8+ T cells are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells; and (ii) in the in vitro expanded immune cell population comprising APC-stimulated tumor antigen-specific T cells, at least 0.1% of the CD4+ T cells are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

2. The pharmaceutical composition according to claim 1, wherein the biological sample is peripheral blood mononuclear cells (PBMCs) from a human subject suffering from cancer, and wherein immune cells expressing CD14 and immune cells expressing CD25 in the biological sample are depleted from the biological sample, respectively.

3. The pharmaceutical composition according to claim 2, wherein the at least one antigenic peptide sequence has a K of less than 500 nM. D or IC 50 Binds to a protein encoded by an HLA allele expressed by a human subject.

4. The pharmaceutical composition of claim 2, wherein each of the at least one antigenic peptide sequence is encoded by a gene expressed by a cancer cell of a human subject and comprises a mutation that is not present in a non-cancerous cell of the human subject.

5. The pharmaceutical composition according to claim 1 or 2, wherein one or more of the at least one antigenic peptide sequence has a length of 8 to 50 naturally occurring amino acids.

6. The pharmaceutical composition according to claim 1 or 2, wherein the at least one antigenic peptide sequence comprises at least 2 antigenic peptide sequences.

7. The pharmaceutical composition according to claim 1 or 2, wherein at least one antigen-specific CD8 + The percentage of T cells is the total CD8 + At least 1% of T cells.

8. The pharmaceutical composition according to claim 7, wherein at least one antigen-specific CD8 + The percentage of T cells is the total CD8 + At least 5% of T cells.

9. The pharmaceutical composition according to claim 1 or 2, wherein at least one antigen-specific CD8 + The number of T cells is at least 1x10 6 Antigen-specific CD8 + T cells.

10. The pharmaceutical composition according to claim 1, wherein the APC-stimulated tumor antigen-specific T cells are incubated with APC, the at least one antigen peptide sequence and FLT3L.

11. Use of the pharmaceutical composition of claim 1 or 2 in the preparation of a medicament for cancer treatment.

12. An ex vivo method for preparing a population of antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising (a) providing an immune cell population from a biological sample depleted of cells expressing CD14 and cells expressing CD25, the immune cell population comprising antigen presenting cells (APCs) and T cells; (b) incubating FLT3L and at least one peptide comprising the at least one antigenic peptide sequence with the immune cell population from (a) for a first period of time, thereby obtaining a first stimulated T cell sample; as well as (c) expanding the first stimulated T cell sample to obtain an antigen-specific T cell population, in: (i) in the antigen-specific T cell population, at least 0.1% of the CD8+ T cells are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells; and (ii) in the antigen-specific T cell population, at least 0.1% of the CD4+ T cells are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

13. The method of claim 12, wherein the incubation in (b) comprises incubating the immune cell population from the biological sample with one or more APC preparations for one or more separate time periods, the first time period being less than 28 days from incubation of the immune cell population.

14. The method of claim 13, wherein the method further comprises incubating the population of immune cells from the biological sample with three or fewer APC preparations for three or fewer separate time periods.

15. The method of claim 14, wherein at least two of the one or more APC preparations comprise FLT3L-stimulated APCs.

16. The method of claim 15, wherein at least three of the one or more APC preparations comprise FLT3L-stimulated APCs.

17. The method of claim 12 or 13, wherein incubating comprises incubating the immune cell population comprising APCs and T cells for more than 7 days.

18. The method of claim 17, wherein the APC comprises an APC loaded with one or more antigenic peptides, the antigenic peptides comprising one or more of the at least one antigenic peptide sequence.

19. The method of claim 17, wherein the APC is autologous APC or allogeneic APC.

20. The method of claim 19, wherein the APC comprises a dendritic cell (DC).

21. The method according to claim 20, wherein the DC is CD141 + DC.

22. The method of claim 12 or 13, wherein the method further comprises depleting CD14-expressing cells and CD25-expressing cells from the biological sample, thereby obtaining the immune cell population from a biological sample depleted of CD14-expressing cells and CD25-expressing cells.

23. The method of claim 12 or 13, wherein the method further comprises depleting cells expressing CD19.

24. The method of claim 22, wherein depleting cells expressing CD14 and cells expressing CD25 comprises binding a CD14 or CD25 binding agent to cells of the biological sample.

25. The method of claim 12 or 13, wherein the method further comprises producing a medicament for treating cancer in a subject in need thereof, wherein the medicament comprises one or more of the at least one antigen-specific T cells.

26. The method of claim 12 or 13, wherein incubating further comprises incubating one or more APC preparations with the first stimulated T cell sample for more than 7 days.

27. The method of claim 26, wherein the method comprises incubating at least one of the one or more APC preparations with a first medium comprising at least one cytokine or growth factor for a first period of time.

28. The method of claim 27, wherein the method comprises incubating at least one of the one or more APC preparations with a second medium comprising one or more cytokines or growth factors for a second or third period of time, thereby obtaining mature APCs.

29. The method of claim 28, wherein the method further comprises removing the one or more cytokines or growth factors from the second medium after the third period of time.

30. The method of claim 26, wherein the APCs in the APC preparation are stimulated with one or more cytokines or growth factors.

31. The method of claim 30, wherein the one or more cytokines or growth factors comprise GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-rna40, poly I:C, or a combination thereof.

32. The method of claim 12 or 13, wherein the antigen is a neoantigen, a tumor-associated antigen, a viral antigen, a minor histocompatibility antigen, or a combination thereof.

33. The method of claim 12 or 13, wherein the method is performed ex vivo.

34. The method of claim 28, wherein the method comprises incubating the population of immune cells from the biological sample depleted of cells expressing CD14 and cells expressing CD25 with FLT3L for a first period of time.

35. The method of claim 12 or 13, wherein the method comprises incubating at least one peptide with a population of immune cells from a biological sample depleted of cells expressing CD14 and cells expressing CD25 for a second period of time, thereby obtaining a first mature APC peptide-loaded sample.

36. The method of claim 35, wherein the method comprises incubating the first mature APC peptide-loaded sample with at least one T cell for a third period of time, thereby obtaining a first stimulated T cell sample.

37. The method of claim 13, wherein the one or more separate time periods are at least 1 hour.

38. The method of claim 37, wherein the one or more individual time periods are from 4 to 40 hours.

39. The method of claim 14, wherein the immune cell population comprises at least one of the APCs or the one or more APC preparations.

40. The method of claim 12 or 13, wherein the method comprises incubating FLT3L and at least one peptide with a population of immune cells from the biological sample depleted of cells expressing CD14 and cells expressing CD25, wherein the FLT3L is incubated with the population of immune cells for a first period of time, and wherein the at least one peptide is incubated with the population of immune cells for a first peptide stimulation period of time, thereby obtaining a first stimulated T cell sample, wherein the population of immune cells comprises at least one T cell and at least one APC.

41. The method of claim 40, wherein the method further comprises incubating FLT3L and at least one peptide with at least one APC, wherein the FLT3L is incubated with the at least one APC for a second time period, and wherein the at least one peptide is incubated with the at least one APC for a second peptide stimulation time period, thereby obtaining a first mature APC peptide-loaded sample; and incubating the first mature APC peptide-loaded sample with the first stimulated T cell sample, thereby obtaining a second stimulated T cell sample.

42. The method of claim 41, wherein the method further comprises incubating FLT3L and at least one peptide with at least one APC, wherein the FLT3L is incubated with the at least one APC for a third time period, and wherein the at least one peptide is incubated with the at least one APC for a third peptide stimulation time period, thereby obtaining a second mature APC peptide-loaded sample; and incubating the second mature APC peptide-loaded sample with the second stimulated T cell sample, thereby obtaining a third stimulated T cell sample.

43. The method of claim 42, wherein the method comprises preparing a medicament using at least one T cell from the first or second or third stimulated T cell sample.

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