Compositions and methods for producing T cells

Abstract

To provide T-cell production method, and a therapeutic T-cell composition that can be used as an individualized antigen-specific T-cell therapy for treating a subject having cancer and other pathosis, disease and disorder.SOLUTION: A pharmaceutical composition includes: (a) a population of immunocytes derived from a biological sample including at least one antigen-presenting cell (APC)-stimulated T-cell including a T-cell receptor (TCR) specific to at least one antigen peptide sequence, where (i) the amount of immunocytes expressing CD14 and CD25 in a population is relatively smaller than the amount of immunocytes expressing CD14 and CD25 in a biological sample, or (ii) APC is an FMS-like tyrosine kinase 3 receptor ligand (FLT3 L)-stimulated APC; and (b) pharmaceutically acceptable filler.SELECTED DRAWING: Figure 43

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 583,229, filed November 8, 2017; U.S. Provisional Application No. 62 / 588,590, filed November 20, 2017; U.S. Provisional Application No. 62 / 618,445, filed January 17, 2018; and U.S. Provisional Application No. 62 / 737,625, filed September 27, 2018, which applications are incorporated herein by reference in their entireties. [Background technology]

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

[0003] Adoptive immunotherapy, or adoptive cell therapy (ACT), is the transplantation of genetically modified T lymphocytes into a subject to treat a disease. Adoptive immunotherapy has yet to realize its potential for treating a wide variety of diseases, including cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiencies. However, most, if not all, adoptive immunotherapy strategies require T cell activation and expansion steps to generate clinically effective therapeutic doses of T cells. Due to the inherent complexity of live cell culture and interpatient variability, current technologies for generating therapeutic doses of T cells, including genetically engineered T cells, remain limited by the cumbersome T cell manufacturing process. Existing T cell manufacturing processes are not easily scalable, repeatable, reliable, or efficient, and often result in low-quality T cell products that can be prone to exhaustion and loss of effector immune cell function. To date, adoptive immunotherapy with genetically engineered T cells has met with limited success and routinely exhibits variable clinical activity. Therefore, such treatments are not suitable for widespread clinical use.Therefore, there remains a need for the development of compositions and methods for expanding and inducing antigen-specific T cells with suitable phenotype and function. Summary of the Invention

[0004] In some aspects, pharmaceutical compositions are provided that include a population of immune cells derived from a biological sample that includes at least one antigen-specific T cell that includes a T cell receptor (TCR) specific for at least one antigenic peptide sequence, and a pharmaceutically acceptable excipient, wherein the amount of immune cells expressing CD14 and / or CD25 in the population is different relative to the amount of immune cells expressing CD14 and / or CD25 in the biological sample. and a pharmaceutically acceptable excipient, wherein the amount of immune cells expressing CD14 and / or CD25 in the population differs from the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some aspects, pharmaceutical compositions are provided that comprise a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, and a pharmaceutically acceptable excipient, wherein the percentage of immune cells expressing CD14 and / or CD25 in the population differs from the percentage of immune cells expressing CD14 and / or CD25 in the biological sample. In some aspects, pharmaceutical compositions are provided that include a population of immune cells derived from a biological sample that includes at least one antigen-specific T cell that comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence, and a pharmaceutically acceptable excipient, wherein the concentration of immune cells expressing CD14 and / or CD25 in the population differs from the concentration of immune cells expressing CD14 and / or CD25 in the biological sample.

[0005]

[0005] In some aspects, compositions are provided that include a population of immune cells derived from a biological sample, wherein the amount of immune cells expressing CD14 and CD25 in the population is relatively low compared to the amount of immune cells expressing CD14 and CD25 in the biological sample. In some aspects, compositions are provided that include a population of immune cells derived from a biological sample, wherein the amount of immune cells expressing CD14 and CD25 in the population is relatively low compared to the amount of immune cells expressing CD14 and CD25 in the biological sample. In some aspects, compositions are provided that include a population of immune cells derived from a biological sample, wherein the percentage of immune cells expressing CD14 and CD25 in the population is less than the percentage of immune cells expressing CD14 and CD25 in the biological sample. In some aspects, compositions are provided that include a population of immune cells derived from a biological sample, wherein the concentration of immune cells expressing CD14 and CD25 in the population is less than the concentration of immune cells expressing CD14 and CD25 in the biological sample.

[0006]

[0006] In some aspects, a pharmaceutical composition is provided comprising: a population of immune cells comprising T cells derived from a biological sample, wherein the T cells are antigen-presenting cell (APC)-stimulated T cells and comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, and the APC is an FLT3L-stimulated APC; and a pharmaceutically acceptable excipient.

[0007] In some embodiments, the method includes: a population of immune cells comprising T cells derived from a biological sample, the population of immune cells comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence; and a pharmaceutically acceptable excipient; the at least one antigen-specific T cell is selected from the group consisting of total T cells, total CD4 + T cells, total CD8 + T cells, or 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 immune cells in the biological sample; the biological sample comprises one or more antigen-specific T cells; 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 immune cells in the biological sample; + T cells, total CD8 + In some embodiments, pharmaceutical compositions are provided that contain at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, or 0.05% of T cells, total T cells, or total immune cells. In some embodiments, the pharmaceutical compositions include a population of immune cells comprising T cells expanded or derived from a biological sample, wherein the T cells expanded or derived from the biological sample comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence; and a pharmaceutically acceptable excipient; and the amount, concentration, or percentage of the at least one antigen-specific T cell is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, or 0.05% of the total T cells, total T cells, or total immune cells. 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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 1 , 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 the original amount. In some embodiments, a method for producing a medicament for the treatment of ... Pharmaceutical compositions are provided that include antigen-specific T cells expanded or induced 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 fold. + A population of immune cells, including T cells, + T cells express at least one antigen-specific CD4 T cell receptor (TCR) specific for at least one antigenic peptide sequence. + a population of immune cells, including T cells; a pharmaceutically acceptable excipient; and at least one antigen-specific CD4 + T cells are total T cells, total CD4 + T cells, total CD8 +T cells, or 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 immune cells; and the biological sample is one or more antigen-specific CD4 + T cells; and one or more antigen-specific CD4 + T cells are the total CD4 + T cells, total CD8 + Pharmaceutical compositions are provided that contain up to about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, or 0.05% of T cells, total T cells, or total immune cells. In some embodiments, the pharmaceutical composition comprises a CD4 T cell derived from a biological sample. + A population of immune cells, including T cells, + T cells express at least one antigen-specific CD4 T cell receptor (TCR) specific for at least one antigenic peptide sequence. + a population of immune cells, including T cells; and a pharmaceutically acceptable excipient, wherein at least one antigen-specific CD4 + T cells are all CD4 + 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 T cells; and the biological sample is one or more antigen-specific CD4 + T cells; and one or more antigen-specific CD4 + T cells are the total CD4 + In some embodiments, the CD4 T cells expanded or derived from the biological sample are at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, or 0.05% of the T cells.+ A population of immune cells, including T cells, expanded or derived from a biological sample. + T cells express at least one antigen-specific CD4 T cell receptor (TCR) specific for at least one antigenic peptide sequence. + a population of immune cells, including T cells, and a pharmaceutically acceptable excipient; and the amount, concentration, or percentage of at least one antigen-specific T cell is determined by measuring the antigen-specific CD4 T cell in the biological sample. + T cells, total CD4 + at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 In some embodiments, pharmaceutical compositions are provided that are 0.8, 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 higher than the CD4 concentration of the biological sample. + A population of immune cells, including T cells, + T cells express at least one antigen-specific CD4 T cell receptor (TCR) specific for at least one antigenic peptide sequence. + a population of immune cells, including T cells; a pharmaceutically acceptable excipient; and at least one antigen-specific CD4 +T cells are isolated from a biological sample and are 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-fold expanded or induced antigen-specific CD4 + In some embodiments, pharmaceutical compositions comprising CD4 T cells derived from a biological sample that have been depleted of cells expressing CD25 or cells expressing CD25 and CD14 are provided. + A population of immune cells, including T cells, + T cells express at least one antigen-specific CD4 T cell receptor (TCR) specific for at least one antigenic peptide sequence. + a population of immune cells, including T cells, and a pharmaceutically acceptable excipient; + The T cells are expanded or induced to express antigen-specific CD4 T cells 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, or 500 fold compared to the biological sample. + Pharmaceutical compositions comprising T cells are presented. In some embodiments, the T cells are derived from a biological sample. + A population of immune cells, including T cells, + T cells express at least one antigen-specific CD8 T cell receptor (TCR) specific for at least one antigenic peptide sequence. +a population of immune cells, including T cells; a pharmaceutically acceptable excipient; and at least one antigen-specific CD8 + T cells are total T cells, total CD4 + T cells, total CD8 + T cells, or 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 immune cells; and the biological sample is one or more antigen-specific CD8 + T cells; one or more antigen-specific CD8 + T cells are the total CD4 + T cells, total CD8 + Pharmaceutical compositions are provided that contain up to about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, or 0.05% of T cells, total T cells, or total immune cells. In some embodiments, the pharmaceutical composition comprises CD8 T cells derived from a biological sample. + A population of immune cells, including T cells, + T cells express at least one antigen-specific CD8 T cell receptor (TCR) specific for at least one antigenic peptide sequence. + a population of immune cells, including T cells; and a pharmaceutically acceptable excipient, wherein at least one antigen-specific CD8 + T cells are all 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%, 95%, 100%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 1 %, 85%, 90%, or 95%; and the biological sample is one or more antigen-specific CD4 + T cells; one or more antigen-specific CD8 + T cells are the total CD8+ At most, approximately 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, and 0% of T cells In some embodiments, the CD8 expanded or derived from the biological sample is 0.005%, 0.01%, or 0.05%. + A population of immune cells, including T cells, expanded or derived from a biological sample. + T cells express at least one antigen-specific CD8 T cell receptor (TCR) specific for at least one antigenic peptide sequence. + a population of immune cells, including T cells, and a pharmaceutically acceptable excipient; and determining the amount, concentration, or percentage of at least one antigen-specific T cell in the biological sample by measuring the antigen-specific CD8 + T cells, total CD8 + 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, In some embodiments, pharmaceutical compositions are provided that are 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 higher than the CD8 antibody concentration from the biological sample. + A population of immune cells, including T cells, + T cells express at least one antigen-specific CD8 T cell receptor (TCR) specific for at least one antigenic peptide sequence. + a population of immune cells, including T cells; a pharmaceutically acceptable excipient; and at least one antigen-specific CD8 +T cells are isolated from a biological sample and are 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-fold expanded or induced antigen-specific CD4 + Pharmaceutical compositions comprising CD8 T cells derived from a biological sample are provided. + A population of immune cells, including T cells, + T cells express at least one antigen-specific CD8 T cell receptor (TCR) specific for at least one antigenic peptide sequence. + a population of immune cells, including T cells; a pharmaceutically acceptable excipient; and at least one antigen-specific CD8 + T cells are isolated from a biological sample and are 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-fold expanded or induced antigen-specific CD8 + A pharmaceutical composition comprising the T cells is presented.

[0008] 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 population is relatively 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 population is relatively 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 population 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 population 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 population 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.

[0009] In some embodiments, the biological sample is from a subject. In some embodiments, the subject is human. In some embodiments, the subject has a disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, lung cancer, and melanoma.

[0010] 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, at least one antigen-specific T cell comprises an expanded memory CD4 + In some embodiments, the at least one antigen-specific T cell is a naive CD4 + In some embodiments, the at least one antigen-specific T cell comprises an induced naive CD4 T cell. + In some embodiments, 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 cell. + Contains T cells.

[0011] In some embodiments, at least one antigen-specific T cell is stimulated in a medium containing 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.

[0012] In some embodiments, the at least one antigenic peptide sequence comprises a mutation selected from (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a read-through mutation, (E) a gene fusion mutation, and combinations thereof. In some embodiments, the at least one antigenic peptide sequence binds to an HLA protein of interest with greater affinity than the corresponding wild-type peptide. In some embodiments, the at least one antigenic peptide sequence binds to an HLA protein of interest 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 binds to the peptide-HLA complex 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 comprises a mutation that is not present in non-cancer cells of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or expressed gene in cancer cells of the subject.

[0013]

[0013] In some embodiments, one or more of the at least one antigenic peptide sequence has a length of at least 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 40; 50; 60; 70; 80; 90; 100; 150; 200; 250; 300; 350; 400; 450; 500; 600; 700; 800; 900; 1,000; 1,500; 2,000; 2,500; 3,000; 4,000; 5,000; 7,500; or 10,000 naturally occurring amino acids. In some embodiments, one or more of the at least one antigenic peptide sequences binds to proteins encoded by class I HLA alleles and has a length of 8 to 12 naturally occurring amino acids. In some embodiments, one or more of the at least one antigenic peptide sequences binds to proteins encoded by class II HLA alleles and has a length of 16 to 25 naturally occurring amino acids.

[0014] 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 neoantigen, a tumor-associated antigen, an overexpressed antigen, a viral antigen, a minor histocompatibility antigen, or a combination thereof.

[0015] In some embodiments, the APC is one or more APC preparations. In some embodiments, the APC comprises APCs loaded with one or more antigenic peptides comprising one or more of at least one antigenic peptide sequence. In some embodiments, the APC is an autologous APC, an allogeneic APC, or an artificial APC. In some embodiments, the APC comprises a dendritic cell (DC). In some embodiments, the APC is a CD14 +In some embodiments, the APCs are derived from monocytes. In some embodiments, the APCs are CD14-enriched APCs. In some embodiments, the APCs are CD141-enriched APCs. In some embodiments, the CD14 + Monocytes are enriched from a biological sample derived from a subject, including PBMCs. + The monocytes are stimulated with one or more cytokines or growth factors, hi 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.

[0016] In some embodiments, CD14 + The monocytes are derived from a second biological sample comprising PBMCs. In some embodiments, the second biological sample is from the same subject.

[0017] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0017]

[0018] 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 at least one antigen-specific T 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% of total T cells or total immune cells. In some embodiments, at least one antigen-specific CD8 + The percentage of T cells 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%, of 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 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 percentage of at least one antigen-specific T cell in the biological sample is determined by measuring the percentage of total CD4 + T cells, total CD8 + In some embodiments, the 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%, or 0.5% of the total T cells or total immune cells. + The percentage of T cells is the total CD4 + T cells, total CD8 + In some embodiments, the at least one antigen-specific CD4 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%, or 0.5% of the total T cells or total immune cells. +The percentage of T cells is the 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.

[0018]

[0019] In some embodiments, a pharmaceutical composition comprises a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, and a pharmaceutically acceptable excipient; 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, a pharmaceutical composition comprises a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, and a pharmaceutically acceptable excipient; 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.

[0019]

[0020] In some aspects, methods of treatment are provided that include administering a composition described herein to a subject having a disease or disorder.

[0021] In some aspects, methods are provided for using the compositions described herein for the manufacture of a medicament for use in treatment.

[0020]

[0022] In one aspect, 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 is provided, the method comprising incubating APCs with a population of immune cells derived from a biological sample depleted of cells expressing CD25 or CD14 and CD25, wherein the at least one antigen-specific T cell is derived from a biological sample depleted of cells expressing CD14. 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, In one embodiment, the amount of antigen-specific T cells expanded or induced is 60, 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 one embodiment, the amount of antigen-specific CD4 ... + A method for preparing T cells is presented, the method comprising incubating APCs with a population of immune cells derived from a biological sample depleted of cells expressing CD25 or CD14 and CD25, and wherein the APCs express at least one antigen-specific CD4 + T cells are antigen-specific CD4 T cells expanded or induced using a method comprising incubating APCs with a population of immune cells derived from a biological sample depleted of cells expressing CD14. +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 fold expansion or induction of antigen-specific CD4 T cells. + In one embodiment, the amount of T cells includes at least one antigen-specific CD8 T cell that contains a T cell receptor (TCR) specific for at least one antigen peptide sequence. + A method for preparing T cells is presented, the method comprising incubating APCs with a population of immune cells derived from a biological sample depleted of cells expressing CD25 or CD14 and CD25, and + The T cells are antigen-specific CD8 T cells expanded or induced using a method comprising incubating APCs with a population of immune cells derived from a biological sample depleted of cells expressing CD14. + 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 fold expansion or induction of antigen-specific CD8 T cells. + Includes the amount of T cells.

[0021]

[0023] 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 APCs are FLT3L-stimulated APCs. In some embodiments, incubating the population of immune cells is performed in a medium containing IL-7, IL-15, or a combination thereof. In some embodiments, the medium further comprises an indoleamine 2,3-dioxygenase 1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof. In some embodiments, the IDO inhibitor is epacadostat, navoximod, 1-methyltryptophan, or a combination thereof.

[0022]

[0024] In one aspect, 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 is provided, the method comprising: incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample for a first period of time; thereafter incubating at least one T cell of the biological sample with an APC, wherein 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, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.6, 7, 7.5, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, , 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 fold expanded or induced. In one embodiment, the method comprises administering to a subject a dose of at least one antigen-specific CD4 T cell that comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence. + A method for preparing a pharmaceutical composition comprising T cells is presented, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample for a first period of time; thereafter, detecting at least one CD4 T cell in the biological sample. + incubating T cells with APCs, + The T cells are isolated from a population of immune cells derived from a biological sample, the population comprising: a first period of incubation of the T cells with a FLT3L-like tyrosine kinase 3 receptor ligand (FLT3L) and a second period of incubation of the T cells with a population of immune cells derived from the biological sample; and a second period of incubation of the T cells with at least one CD4 + Antigen-specific CD4 T cells expanded or induced using a method comprising incubating T cells with APCs + 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 fold expansion or induction of antigen-specific CD4 T cells. + In one embodiment, the amount of T cells includes at least one antigen-specific CD8 T cell that contains a T cell receptor (TCR) specific for at least one antigen peptide sequence. +A method for preparing a pharmaceutical composition comprising T cells is presented, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample for a first period of time; thereafter, detecting at least one CD8 T cell in the biological sample. + incubating T cells with APCs, + The T cells include incubating a FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample for a first period of time; thereafter, detecting at least one CD8 T cell in the biological sample. + Expanded or induced antigen-specific CD8 T cells using a method comprising incubating T cells with APCs + 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 fold expansion or induction of antigen-specific CD8 T cells. + Includes the amount of T cells.

[0023]

[0025] In one aspect, 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 is provided, the method comprising incubating APCs with a population of immune cells derived from a biological sample depleted of cells expressing CD14 and / or CD25.

[0024]

[0026] In one embodiment, 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 is provided, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs with a population of immune cells derived from a biological sample.

[0025]

[0027] In one embodiment, 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 is provided. The method includes incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample for a first period of time; and then incubating at least one T cell of the biological sample with an APC.

[0026]

[0028] In one aspect, a method is provided for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating a population of immune cells derived from a biological sample with a first APC preparation of one or more APC preparations, followed by incubating the population of immune cells with the one or more APC preparations for one or more separate periods of less than 28 days, to expand at least one antigen-specific memory T cell or induce at least one antigen-specific naive T cell.

[0027]

[0029] In one aspect, a method is provided for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating a population of immune cells derived from a biological sample with no more than three APC preparations for no more than three separate periods of time, to expand at least one antigen-specific memory T cell or induce at least one antigen-specific naive T cell.

[0028]

[0030] In some embodiments, the population of immune cells is derived from a biological sample that has been depleted of cells expressing CD14 and / or CD25.

[0031] In some embodiments, the APCs are FLT3L-stimulated APCs. In some embodiments, at least one of the APC preparations comprises FLT3L-stimulated APCs. In some embodiments, at least two of the APC preparations comprise FLT3L-stimulated APCs. In some embodiments, at least three of the APC preparations comprise FLT3L-stimulated APCs. In some embodiments, each of the APC preparations comprises FLT3L-stimulated APCs.

[0029]

[0032] In some embodiments, the APC comprises one or more APC preparations. In some embodiments, the APC preparation comprises three or fewer APC preparations. In some embodiments, the APC preparations are incubated with the immune cells sequentially within one or more separate time periods. In some embodiments, the biological sample is from a subject. In some embodiments, the subject is human. In some embodiments, the subject has a disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is selected from the group consisting of ovarian cancer, lung cancer, and melanoma.

[0030]

[0033] 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 + Contains T cells.

[0031]

[0034] In some embodiments, at least one antigenic peptide sequence comprises a mutation selected from (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a read-through mutation, (E) a gene fusion mutation, and combinations thereof. In some embodiments, at least one antigenic peptide sequence comprises a point mutation and binds to an HLA protein of interest with greater affinity than the corresponding wild-type peptide. In some embodiments, at least one antigenic peptide sequence binds to an HLA protein of interest 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 binds to the peptide-HLA complex with a K of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in non-cancer cells of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or expressed gene in cancer cells of the subject. In some embodiments, one or more of the at least one antigenic peptide sequences has a length of at least 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 40; 50; 60; 70; 80; 90; 100; 150; 200; 250; 300; 350; 400; 450; 500; 600; 700; 800; 900; 1,000; 1,500; 2,000; 2,500; 3,000; 4,000; 5,000; 7,500; or 10,000 naturally occurring amino acids. In some embodiments, one or more of the at least one antigenic peptide sequences binds to proteins encoded by class I HLA alleles and has a length of 8 to 12 naturally occurring amino acids. In some embodiments, one or more of the at least one antigenic peptide sequences binds to proteins encoded by class II HLA alleles and has a length of 16 to 25 naturally occurring amino acids. In some embodiments, the 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 neoantigen, a tumor-associated antigen, a viral antigen, a minor histocompatibility antigen, or a combination thereof.

[0032]

[0035] In some embodiments, the method includes depleting cells expressing CD14 and / or CD25 from the biological sample. In some embodiments, depleting cells expressing CD14 and / or CD25 includes binding an agent that binds CD14 and / or CD25 to an APC or an APC of an APC preparation. In some embodiments, the agent that binds CD14 and / or CD25 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 agent that binds CD14 and / or CD25. In some embodiments, the agent that binds CD14 and / or CD25 is conjugated to a solid support.

[0033]

[0036] In some embodiments, the APCs or the APCs of the APC preparation comprise APCs loaded with one or more antigenic peptides comprising one or more of at least one antigenic peptide sequence. In some embodiments, the APCs or the APCs of the APC preparation are autologous APCs or allogeneic APCs. In some embodiments, the APCs or the APCs of the APC preparation comprise dendritic cells (DCs). In some embodiments, the APCs or the APCs of the APC preparation comprise CD14 + In some embodiments, the APCs or APCs of the APC preparation are derived from monocytes. In some embodiments, the APCs or APCs of the APC preparation 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.

[0034]

[0037] In some embodiments, the APCs or APCs of the APC preparation are derived from a second biological sample. In some embodiments, the second biological sample is derived from the same subject. In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0035]

[0038] 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 is a percentage of total CD4 + T cells, total CD8 + In some embodiments, at least one antigen-specific CD8 T cell 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. + The percentage of T cells is the total CD4 + T cells, total CD8 + In some embodiments, 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. + The percentage of T cells 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 percentage of at least one antigen-specific T cell in the biological sample is determined by measuring the percentage of total CD4 + T cells, total CD8 + In some embodiments, the 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%, or 0.5% of the total T cells or total immune cells. + The percentage of T cells is the total CD4 + T cells, total CD8 + In some embodiments, the at least one antigen-specific CD4 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%, or 0.5% of the total T cells or total immune cells. + The percentage of T cells is the 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.

[0036]

[0039] In some embodiments, the method further comprises administering to the subject one or more of the at least one antigen-specific T cell.

[0040] In some embodiments, the total duration of the separate periods is less than 28 days. In some embodiments, incubating comprises incubating a first APC preparation of the APCs in a T In some embodiments, the method comprises incubating the APCs or one or more of the APC preparations with a first medium comprising at least one cytokine or growth factor for a first period of time. In some embodiments, the at least one cytokine or growth factor comprises 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 comprises incubating the one or more of the APC preparations with at least one peptide for a second period of time. In some embodiments, the method comprises incubating the APCs or one or more of the APC preparations with a second medium comprising one or more cytokines or growth factors for a third period of time, thereby obtaining mature APCs. In some embodiments, the one or more cytokines or growth factors comprise 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 comprises removing the one or more cytokines or growth factors of the second culture medium after the third period of time and prior to the initiation of the fourth period of time.

[0037]

[0041] In some embodiments, the method is performed ex vivo.

[0042] In some embodiments, the biological sample is freshly obtained from a subject or is a frozen sample.

[0038]

[0043] In some embodiments, the method includes obtaining a biological sample from the subject, the biological sample comprising at least one APC and at least one PBMC.

[0044] In some embodiments, the methods comprise depleting cells expressing CD14 and / or CD25 from the biological sample, thereby obtaining a sample depleted of CD14 and / or CD25 cells.

[0039]

[0045] In some embodiments, the method comprises incubating a sample depleted of CD14 and / or CD25 cells with FLT3L for a first period of time.

[0046] In some embodiments, the method includes incubating at least one peptide with the CD14 and / or CD25 cell-depleted sample for a second period of time, thereby obtaining a first peptide-loaded mature APC sample.

[0040]

[0047] In some embodiments, the method includes incubating the first peptide-loaded mature APC sample with at least one PBMC for a third period of time, thereby obtaining a first stimulated PBMC sample.

[0041]

[0048] In some embodiments, the method includes incubating PBMCs of the first stimulated PBMC sample with FLT3L-stimulated APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample.

[0042]

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

[0043]

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

[0044]

[0051] In some embodiments, the method comprises: subjecting the PBMCs of the second stimulated PBMC sample to The mature APC sample is incubated with FLT3L-stimulated APCs for a fifth period of time, thereby obtaining a third stimulated PBMC sample.

[0045]

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

[0046]

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

[0047]

[0054] In some embodiments, the method comprises administering at least one T cell of a first stimulated PBMC sample to a subject in need thereof. In some embodiments, the method comprises administering at least one T cell of a second stimulated PBMC sample to a subject in need thereof. In some embodiments, the method comprises administering at least one T cell of a third stimulated PBMC sample to a subject in need thereof.

[0048]

[0055] In some embodiments, incubating the PBMCs of the first stimulated PBMC sample is performed in the presence of IL-7, IL-15, or a combination thereof. In some embodiments, incubating the PBMCs of the first stimulated PBMC sample is performed 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, incubating the PBMCs of the second stimulated PBMC sample is performed in the presence of IL-7, IL-15, or a combination thereof. In some embodiments, incubating the PBMCs of the second stimulated PBMC sample is performed in the presence of an indoleamine 2,3-dioxygenase 1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof.

[0049]

[0057] In one embodiment of the present invention, a biological sample from a subject is obtained, the biological sample comprising at least one antigen-presenting cell (APC); and cells expressing CD14 are enriched from the biological sample, thereby obtaining CD14. + Obtaining cell-enriched samples; CD14 + The cell-enriched sample is incubated with at least one cytokine or growth factor for a first period of time; and at least one peptide is administered to a subject, such as a subject, a subject, or a group of subjects ... a subject, or a group of subjects, such as a subject, a subject, a subject, a subject, a subject, a subject + A method is presented that includes incubating the PBMCs with the cell-enriched sample for a second period of time, thereby obtaining a peptide-loaded APC sample; incubating the peptide-loaded APC sample with one or more cytokines or growth factors for a third period of time, thereby obtaining a mature APC sample; incubating APCs from the mature APC sample with a CD14- and / or CD25-depleted sample comprising PBMCs for a fourth period of time; incubating the PBMCs with APCs from the mature APC sample for a fifth period of time; incubating the PBMCs with APCs from the mature APC sample for a sixth period of time; and administering at least one T cell from the PBMCs to a subject in need thereof.

[0050]

[0058] In one embodiment herein, a biological sample from a subject is obtained, the biological sample comprising at least one APC and at least one PBMC; cells expressing CD14 and / or CD25 and / or CD19 are depleted from the biological sample, thereby obtaining a sample depleted of CD14 and / or CD25 and / or CD19 cells; the sample depleted of CD14 and / or CD25 and / or CD19 cells is incubated with FLT3L for a first period of time; at least one peptide is incubated with the sample depleted of CD14 and / or CD25 and / or CD19 cells for a second period of time, thereby obtaining a peptide-loaded APC sample; and the peptide-loaded APC sample is co-cultured with at least one PBMC. incubating PBMCs from the first stimulated PBMC sample with APCs from the mature APC sample for a third period of time, thereby obtaining a first stimulated PBMC sample; optionally incubating PBMCs from the second stimulated PBMC sample with APCs from the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and administering at least one T cell from the first, second, or third stimulated PBMC sample to a subject in need thereof.

[0051]

[0059] In one embodiment herein, a biological sample from a subject is obtained, the biological sample comprising at least one APC and at least one PBMC; cells expressing CD14 and / or CD25 and / or CD19 are depleted from the biological sample, thereby obtaining a sample depleted of CD14 and / or CD25 and / or CD19 cells; the sample depleted of CD14 and / or CD25 and / or CD19 cells is incubated with FLT3L for a first period of time; at least one peptide is incubated with the sample depleted of CD14 and / or CD25 and / or CD19 cells for a second period of time, thereby obtaining a peptide-loaded APC sample; and the peptide-loaded AP sample. C sample with at least one PBMC for a third period of time, thereby obtaining a first stimulated PBMC sample; optionally, incubating PBMCs of the first stimulated PBMC sample with FLT3L-stimulated APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; optionally, incubating PBMCs of the second stimulated PBMC sample with FLT3L-stimulated APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and administering at least one T cell of the first, second, or third stimulated PBMC sample to a subject in need thereof.

[0052]

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

[0053]

[0061] In one embodiment herein, a biological sample from a subject is obtained, the biological sample comprising at least one APC and at least one PBMC; cells expressing CD14 and / or CD25 and / or CD19 are depleted from the biological sample, thereby obtaining a sample depleted of CD14 and / or CD25 and / or CD19 cells; the sample depleted of CD14 and / or CD25 and / or CD19 cells is incubated with FLT3L for a first period of time; at least one peptide is incubated with the sample depleted of CD14 and / or CD25 and / or CD19 cells for a second period of time, thereby obtaining a first peptide-loaded APC sample; and the first peptide-loaded APC sample is loaded with at least one peptide. optionally, incubating PBMCs of the second stimulated PBMC sample with FLT3L and FLT3L-stimulated APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; optionally, incubating PBMCs of the second stimulated PBMC sample with FLT3L and FLT3L-stimulated APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and administering at least one T cell of the first, second, or third stimulated PBMC sample to a subject in need thereof.

[0054]

[0062] In one aspect of the present specification, a method is provided, comprising: determining the expression of one or more cell markers of at least one immune cell of a stimulated immune cell sample; and determining binding of at least one immune cell of the stimulated immune cell sample to a peptide-MHC complex, wherein determining expression and determining binding are performed simultaneously. In some embodiments, the stimulated immune cell sample is a population of immune cells stimulated by APCs containing peptide-MHC complexes. In some embodiments, the population of immune cells is derived from a biological sample.

[0055]

[0063] In one embodiment herein, a method is provided, comprising: incubating a population of immune cells derived from a biological sample with APCs comprising peptide-MHC complexes, thereby obtaining a stimulated immune cell sample; determining expression of one or more cell markers of at least one immune cell of the stimulated immune cell sample; and determining binding of at least one immune cell of the stimulated immune cell sample to the peptide-MHC complex, wherein determining expression and determining binding are performed simultaneously.

[0056]

[0064] In some embodiments, the one or more cellular markers comprise TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, Granzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3, or any combination thereof. In some embodiments, the one or more cellular markers comprise a cytokine. In some embodiments, the one or more cellular markers comprise a degranulation marker. In some embodiments, the one or more cellular markers comprise a cell surface marker. In some embodiments, the one or more cellular markers comprise a protein. In some embodiments, determining binding of at least one immune cell of the stimulated immune cell sample to a peptide-MHC complex comprises determining binding of at least one immune cell of the stimulated immune cell sample to an MHC tetramer comprising the peptide and MHC of the peptide-MHC complex. In some embodiments, the MHC is a class I MHC or a class II MHC. In some embodiments, the peptide-MHC complex comprises one or more labels. In some embodiments, the population of immune cells derived from a biological sample comprises two or more samples, each comprising a population of immune cells derived from one or more biological samples. In some embodiments, the two or more samples are labeled with two or more sample labels. In some embodiments, determining expression and determining binding comprises fluorescence activated cell sorting (FACS). In some embodiments, determining expression and determining binding comprises single cell analysis. In some embodiments, determining expression and determining binding comprises determining the percentage of immune cells that express any of one or more cell markers and bind to the peptide-MHC complex. In some embodiments, the label comprises a fluorophore. In some embodiments, the population of immune cells comprises a population of immune cells representative of a population of immune cells of a composition described herein.

[0057]

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

[0066] All publications, patents, and patent applications mentioned herein are incorporated herein in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. For example, all publications and patents mentioned herein are incorporated herein in their entirety for the purpose of describing and disclosing kits, compositions, and methods described in the publications that may be used in connection with the methods, kits, and compositions described herein. The documents discussed herein are presented solely for their disclosure prior to the filing date of the present application. Nothing herein shall be construed as an admission that the inventors described herein are entitled to antedate such disclosure by virtue of prior invention or for any other reason. [Brief explanation of the drawings]

[0058] [Figure 1A]

[0067] FIG. 1A depicts an exemplary outline for a manufacturing protocol for antigen-specific T cells. [Figure 1B]

[0068] FIG. 1B depicts an exemplary outline for a manufacturing protocol for antigen-specific T cells. [Figure 2]

[0069] Figure 2 depicts exemplary results showing the fraction of antigen-specific CD8+ memory T cells induced by long or short peptides. "Bulk" indicates that the sample containing T cells used for induction is whole peripheral blood mononuclear cells (PBMCs). "Treg-" indicates that the sample containing T cells used for induction is PBMCs depleted of cells expressing CD25. [Figure 3]

[0070] FIG. 3 depicts an exemplary flow cytometry analysis showing the fraction of antigen-specific CD8+ naive T cells induced by GAS7 peptide. [Figure 4]

[0071] Figure 4 depicts exemplary results showing antigen-specific CD8+ T cell responses to a peptide pool of HIV short peptides, short PIN (previously identified neoantigen), or long PIN. "Total PBMC" indicates that the sample containing T cells used for induction is total PBMC. "CD25-PBMC" indicates that the sample containing T cells used for induction is PBMC depleted of CD25+ cells. [Figure 5A]

[0072] FIG. 5A depicts an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to a single previously identified neoantigen (PIN) under the indicated conditions. [Figure 5B]

[0073] FIG. 5B depicts an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to a single previously identified neoantigen (PIN) under the indicated conditions. [Figure 6]

[0074] FIG. 6 depicts exemplary results showing antigen-specific CD8+ T cell responses to the indicated peptides using PBMC samples from two human donors. [Figure 7]

[0075] FIG. 7 depicts exemplary flow cytometry plots of antigen-specific CD8+ T cell responses to the indicated mutant epitopes in healthy donors before and after up to three rounds of stimulation. [Figure 8A]

[0076] Figure 8A depicts an exemplary bar graph showing the results of antigen-specific memory CD8+ T cell responses to viral antigens. After up to three rounds of stimulation, approximately 50% of all CD8+ T cells were specific for the indicated viral epitopes (CMV pp65, EBV YVL, EBV BMLF1, and Mart-1). [Figure 8B]

[0077] Figure 8B depicts exemplary results of a recall assay of antigen-specific memory CD8+ T cell responses to peptide-loaded antigen-presenting cells and subsequent responses when incubated with viral antigen-loaded and non-viral antigen-loaded APCs. The figure depicts the fraction of CD8+ T cells releasing the indicated cytokines at two time points. [Figure 9]

[0078] Figure 9 depicts exemplary results of a cytotoxicity assay used to assess whether induced T cell cultures can kill antigen-expressing tumor lines. The fraction of live and dead caspase-3-positive tumor cells relative to total tumor cells is shown. Live caspase-3-positive tumor cells indicate cells undergoing early cell death. [Figure 10]

[0079] Figure 10 depicts an exemplary flow cytometry analysis of antigen-specific CD4+ T cell responses to peptide-loaded antigen-presenting cells incubated with PIN-loaded and non-loaded APCs. The percentage of CD4+ T cells releasing IFNγ is shown. [Figure 11]

[0080] FIG. 11 depicts exemplary results for the percentage of antigen-specific CD4+ T cells releasing IFNγ after restimulation with mutant or wild-type peptides. [Figure 12]

[0081] Figure 12 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV5 peptides, demonstrating both short-term and long-term induction. [Figure 13]

[0082] Figure 13 depicts an exemplary flow cytometry analysis showing the fraction of antigen-specific CD8+ naive T cell responses to short ME1 peptides using a whole PBMC sample from a human donor. Both short-term and long-term induction is shown. [Figure 14]

[0083] Figure 14 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV3 peptides using a whole PBMC sample from a human donor. Both short-term and long-term induction is shown. [Figure 15]

[0084] 15 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to long CSNK1A1 peptides using a whole PBMC sample from a human donor. Both short-term and long-term induction is shown. [Figure 16]

[0085] 16 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to long CSNK1A1 peptides using a PBMC sample from a human donor depleted of CD25+ cells. Both short-term and long-term induction is shown. [Figure 17]

[0086] Figure 17 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short GAS7 peptides using a PBMC sample from a human donor depleted of CD25+ cells. Both short-term and long-term induction is shown. [Figure 18]

[0087] Figure 18 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short ACTN4 peptides using a PBMC sample from a human donor depleted of CD25+ cells. Both short-term and long-term induction is shown. [Figure 19A]

[0088] Figure 19A depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short ACTN4 peptides using a PBMC sample from a human donor depleted of CD25+ cells. Short-term induction is shown. [Figure 19B]

[0089] Figure 19B depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV3 peptides using a PBMC sample from a human donor depleted of CD25+ cells. Long-term induction is shown. [Figure 20]

[0090] Figure 20 depicts an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to short HIV5 peptides using a whole PBMC sample from a human donor, showing both short-term and long-term induction. [Figure 21]

[0091] 21 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV3 peptides using a whole PBMC sample from a human donor. Short-term induction is shown. [Figure 22]

[0092] Figure 22 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short PRDX5 peptides using a PBMC sample from a human donor depleted of CD25+ cells, demonstrating both very short-term and long-term induction. [Figure 23]

[0093] Figure 23 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV5 peptides using a PBMC sample from a human donor depleted of CD25+ cells. Both short-term and long-term induction is shown. [Figure 24]

[0094] FIG. 24 depicts a schematic of an example method for generating a therapeutic T cell composition, which method includes the expansion of memory T cells and the induction of naive T cells. [Figure 25]

[0095] FIG. 25 depicts an example of a method for examining T cell functionality, phenotype, and / or function and / or T cell responses. [Figure 26]

[0096] FIG. 26 depicts an example of a recall assay that examines T cell functionality, phenotype, and / or function and / or T cell responses. [Figure 27A]

[0097] Figure 27A depicts an exemplary flow cytometry analysis demonstrating the ability to disentangle multiplexed samples with labeled samples acquired individually or as a mixture in a recall assay. Uniquely labeled samples were resolved with minimal to no cross-contamination with other barcodes. [Figure 27B]

[0098] FIG. 27B depicts an exemplary flow cytometry analysis showing the detection of antigen-specific CD8+ T cells by multimer staining of a mixture of nine uniquely labeled samples in a recall assay. [Figure 28A]

[0099] FIG. 28A depicts an exemplary flow cytometry analysis of a recall assay using six uniquely barcoded samples recalled by non-neoantigen-loaded and neoantigen-loaded DCs. [Figure 28B]

[0100] Figure 28B depicts an exemplary bar graph showing the percent of CD4+ T cells incubated with DCs loaded with the indicated concentrations of peptide in a recall response assay, along with functional counts. Samples of two induced cultures containing de novo CD4+ T cell responses were analyzed alone without barcoding or mixed with an unrelated sample. Barcoding did not alter detectable functionality. The functional counts and magnitude of responses elicited from the cells were not significantly altered by barcoding the samples. [Figure 29A]

[0101] Figure 29A depicts an exemplary bar graph showing the results of antigen-specific memory CD8+ T cell responses to viral antigens. CD8+ memory responses to epitopes of CMV pp65, MART-1, and EBVBRLF1 and BMLF1 could be boosted from 0.23% of CD8+ T cells in the starting healthy donor material to >60%. [Figure 29B]

[0102] Figure 29B depicts exemplary results of a recall assay in which antigen-specific memory CD8+ T cell responses to viral antigens were recalled by viral antigen-loaded and non-viral antigen-loaded DCs. The figure depicts the fraction of CD8+ T cells at two time points releasing the indicated cytokines. [Figure 30A]

[0103] Figure 30A depicts exemplary results of hit identification by detecting and functionally characterizing de novo CD4+ responses induced with multiple specificities in the same culture. In the example shown, induction was performed in four replicate cultures targeting 10 HIV-derived epitopes that are naive targets in HIV-negative healthy donors. Antigen-specific responses were detected in four of the four biological replicates, although the magnitude of the responses varied. [Figure 30B]

[0104] Figure 30B depicts exemplary results of pooled deconvolution by detecting and functionally characterizing de novo CD4 responses induced with multiple specificities in the same culture. Multiple responses were detected in each replicate, and in each case the same two epitopes (HIV#5 and HIV#7) produced the largest responses. [Figure 30C]

[0105] Figure 30C depicts exemplary results of sensitivity determination by detecting and functionally characterizing de novo CD4+ responses induced with multiple specificities in the same culture. Similar magnitudes were observed for each response in the pooled deconvolution assay. Responses to HIV#5, HIV#6, and HIV#4 showed EC50s of 0.45 μM, 0.43 μM, and 9.1 μM, respectively. [Figure 31]

[0106] FIG. 31 depicts an exemplary outline for a manufacturing protocol for antigen-specific T cells. [Figure 32]

[0107] FIG. 32 depicts an exemplary outline for a T cell induction protocol. [Figure 33]

[0108] FIG. 33 depicts an exemplary outline for a dendritic cell generation protocol. [Figure 34]

[0109] Figure 34 depicts exemplary pMHC multimer plots showing CD8+ T cell responses induced in leukapheresis material from a melanoma patient targeting the patient-specific epitopes: SRSF1E>K, ARAP1Y>H, and PKDREJG>R, and targeting the patient-specific epitope: AASDHneoORF and seven model neoantigens: ACTN4K>N, CSNK1A1S>L, DHX40neoORF, GLI3P>L, QARSR>W, FAM178BP>L, and RPS26P>L. The first panel plots in the first and second rows indicate memory responses, and the remaining plots indicate de novo responses. [Figure 35-1]

[0110] Figure 35 depicts exemplary data for pMHC multimer plots before and after SRSF1E>K and ARAP1Y>H peptide stimulation, with data gated on pMHC multimer+ CD8+ T cells. Multifunctional profiles for CD8+ memory responses, CD8+ de novo responses induced in melanoma patients are shown, with a combination of one, two, or three functions (e.g., one or more functions being production of one or more factors selected from IFNγ, TNFα, CD107a, and 4-1BB). [Figure 35-2]Figure 35 shows exemplary data for pie charts depicting functionality of neoantigen-specific T cells upon rechallenge with neoantigen-loaded DCs, with data gated on pMHC multimer+ CD4+ T cells. The multifunctional profile for CD4+ de novo responses induced in melanoma patients is shown by a combination of one, two, or three functions (e.g., one or more functions is production of one or more factors selected from IFNγ, TNFα, CD107a, and 4-1BB). [Figure 36]

[0111] Figure 36 depicts the specificity of memory and de novo responses induced against mutant and wild-type peptides in melanoma patients. SRSF1E>K and ARAP1Y>H-specific T cell responses were assessed by challenging DCs loaded with mutant or wild-type neoantigen peptides at different concentrations (X-axis: 0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM) and measuring IFN-γ and / or TNFα and / or CD107a (Y-axis) among total CD8 T cells in the sample; all responses showed significant differences compared to the 0 μM concentration, indicating no responsiveness to the wild-type neoantigen peptide. Statistical analysis: FDR for corrected p-values, P-values: *≦0.05, ***≦0.001, ****≦0.0001. [Figure 37A]

[0112] Figure 37A depicts the cytotoxicity profile of memory responses induced in melanoma patients, as quantified by the frequency of CD8+CD107a+ T cells. Figure 37A also depicts target cell killing by these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The cytotoxic potential of the induced CD8+ T cell responses was assessed by rechallenging tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200-amino acid construct were used. The constructs contained either the mutant or wild-type sequence, with the mutation centrally located. Upon coculture, upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells was measured. The target ratio was 3.3:1 (SRSF1E>K). [Figure 37B]

[0113] Figure 37B depicts another example of the cytotoxicity profile of a memory response induced in a melanoma patient, quantified by the frequency of CD8+CD107a+ T cells. Figure 37B also depicts target cell killing by these T cell responses, quantified by the frequency of aCAS3+ tumor cells. The cytotoxic potential of the induced CD8+ T cell response was assessed by rechallenging tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200-amino acid construct were used. The constructs contained either the mutant or wild-type sequence, with the mutation centrally located. Upon coculture, upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells was measured. Red circles highlight the pMHC+ fraction. The effector:target ratio was 5:1 (SRSF1E>K). Statistical analysis: Unpaired T-test: P-value **≦0.01, ****≦0.0001. [Figure 37C]

[0114] Figure 37C depicts the cytotoxicity profile of de novo responses induced in melanoma patients, as quantified by the frequency of CD8+CD107a+ T cells. Figure 37C also depicts target cell killing by these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The cytotoxic potential of the induced CD8+ T cell responses was assessed by rechallenging tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200-amino acid construct were used. The constructs contained either the mutant or wild-type sequence, with the mutation centrally located. Upon coculture, upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells was measured. Red circles highlight the pMHC+ fraction. The effector:target ratio was 0.66:1 (ARAP1Y>H). Statistical analysis: Unpaired T-test: P-value **≦0.01, ****≦0.0001. [Figure 38A]

[0115] Figure 38A depicts the identification of neoantigen-specific CD4+ T cell responses in melanoma patients. Responses are identified based on IFN-γ and TNFα production (Y-axis) upon rechallenge with DCs loaded with mutant neoantigen peptides (0.8 μM). MKRN1S>L, CREBBPS>L, and TPCN1K>E were identified as positive responses. [Figure 38B]

[0116] Figure 38B depicts the specificity of the CD4+ T cell responses depicted in Figure 38A to the indicated mutant and wild-type peptides. In a validation study, the CD4+ T cell responses depicted in Figure 38A were challenged with different concentrations (X-axis: 0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM) of mutant or wild-type neoantigen peptides and assessed by measuring IFN-γ and / or TNFα (Y-axis) among total CD4+ T cells in the samples. Two of the CD4+ T cell responses (MKRN1S>L and CREEBPS>L) showed significant differences relative to the 0 μM concentration and were not responsive to the wild-type neoantigen peptide, whereas the TPCN1K>E response was reactive to both the mutant and wild-type neoantigen peptides. Statistical analysis: FDR for corrected p-values, p-value <0.05. [Figure 38C]

[0117] Figure 38C depicts the polyfunctional profile of these CD4+ T cell responses, as indicated by a combination of one, two, three, or four functions (e.g., one or more functions are production of one or more factors selected from IFNγ, TNFα, CD107a, and 4-1BB). The polyfunctionality of the identified CD4+ T cell responses was assessed by rechallenge of DCs loaded with mutant neoantigenic peptides (0.8 μM). The percentages within the pie chart represent the percentage of functional CD4+ T cells (one, two, and / or three functions). The representative data depicted were generated from post-stimulation CD4+ T cell responses induced in patients. [Figure 39]

[0118] Figure 39 depicts the functionality of memory responses induced in two healthy donors (e.g., HD66 and HD63) with or without the addition of epacadostat, as indicated by a combination of one, two, or three functions (e.g., the one or more functions being the production of one or more factors selected from IFNγ, TNFα, and CD107a). [Figure 40]

[0119] FIG. 40 depicts the percentage of de novo CD8+ T cell responses ("hit rate" averaged over four healthy donors) induced in six repeated inductions with or without the addition of epacadostat. [Figure 41A]

[0120] Figure 41A depicts the absolute number of antigen-specific cells from donor HD55 after induction by the T cell manufacturing protocol presented herein, with or without the addition of a PD-1 blocking antibody. [Figure 41B]

[0121] Figure 41B depicts the absolute number of antigen-specific cells from donor HD67 after induction by the T cell manufacturing protocol presented herein, with or without the addition of a PD-1 blocking antibody. [Figure 42A]

[0122] FIG. 42A depicts the fraction of pMHC+CD8+ T cells among de novo CD8+ T cell responses with or without the addition of IL-12. [Figure 42B]

[0123] FIG. 42B depicts the percentage of CD8+ T cells among the de novo CD8+ T cell responses with or without the addition of IL-12. [Figure 43]

[0124] Figure 43 depicts an example of the method described herein. A bioinformatics engine is used to predict patient-specific neoantigens. Synthetic long peptides covering the predicted neoantigens are used as immunogens in a stimulation protocol to evaluate their immunogenic potential. The stimulation protocol involves delivering peptides encoding these neoantigens to patient-derived APCs, which are then co-cultured with patient-derived T cells to prime the neoantigen-specific T cells. DETAILED DESCRIPTION OF THE INVENTION

[0059]

[0125] Described herein are novel immunotherapeutic agents and their uses based on the discovery of neoantigens that arise from mutational events unique to an individual's tumor. Accordingly, the disclosure described herein presents methods and protocols for generating antigen-specific immune cells, e.g., T cells, for use in treating disease. definition

[0126] The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are used unless the context requires otherwise. Unless expressly indicated otherwise, plural forms are also intended to be included. Furthermore, to the extent the terms "comprising," "including," "having," "having," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0060]

[0127] Terms such as "comprises," "comprised," "comprising," and the like may have the meanings ascribed to them in U.S. patent law, e.g., these terms may mean "includes," "included," "including," etc.; Terms such as "consisting essentially of" and "consisting essentially of" have the meaning ascribed to them in U.S. patent law, e.g., they allow for elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of the invention. Nothing in this specification is intended as a warranty.

[0061]

[0128] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include A and B; both A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass 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).

[0062]

[0129] The terms "about" or "approximately" can mean within an acceptable range of error for a particular value as 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, "about" can mean within one or more standard deviations, in accordance with practice in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold, of a value. When particular values ​​are described in this application and claims, unless otherwise qualified, the term "about" should be assumed to mean within an acceptable range of error for the particular value.

[0063]

[0130] To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0131] "Neoantigens" refer to a class of tumor antigens that arise from tumor-specific alterations within proteins, including, but not limited to, tumor antigens that arise from protein sequence substitutions, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, and expression of endogenous retroviral polypeptides.

[0064]

[0132] A "neoepitope" refers to an epitope that is not present in a reference non-diseased cell, e.g., a non-cancerous cell or a germline cell, but is found in a diseased cell, e.g., a cancer cell. This includes situations where the corresponding epitope is found in a normal non-diseased cell or a germline cell, but due to one or more mutations in the diseased cell, e.g., a cancer cell, the sequence of the epitope has changed, resulting in a neoepitope.

[0065]

[0133] A "reference" can be used to correlate and / or compare results obtained from diseased samples using the methods of the present disclosure. Typically, a "reference" can be obtained based on one or more normal samples obtained from an individual, particularly non-diseased samples, or one or more different individuals (e.g., healthy individuals), such as individuals of the same species. A "reference" can be empirically determined by testing a sufficiently large number of normal samples.

[0066]

[0134] A "mutation" refers to a change or difference (e.g., a nucleotide substitution, addition, or deletion) in a nucleic acid sequence compared to a reference nucleic acid. A "somatic mutation" can occur in somatic cells, excluding germ cells (sperm and eggs), and is not passed on to offspring. These changes can (but do not always) cause cancer or other diseases. In some embodiments, the mutation is a non-synonymous mutation. A "non-synonymous mutation" refers to a mutation (e.g., a nucleotide substitution) that results in an amino acid change, such as an amino acid substitution, in the translation product. A "frameshift" occurs when a mutation disrupts the normal phase of the codon periodicity of a gene (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. It is possible for different mutations within a gene to achieve the same reading frame change.

[0067]

[0135] The term "affinity" refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide and a class I or class II HLA, or a peptide-HLA complex and a T cell receptor (TCR)).D K denotes the dissociation constant between two members of a binding pair and has units of molar concentration. A K represents 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 is the off-rate constant for the two members of a binding pair (e.g., an HLA-binding peptide and a class I HLA or class II HLA). A, or the off-rate constant between the peptide-HLA complex and TCR). on represents the on-rate constant for the two members of a binding pair (e.g., the on-rate constant between an HLA-binding peptide and class I or class II HLA, or the on-rate constant between a peptide-HLA complex and a TCR).

[0068]

[0136] Throughout this disclosure, "combined data" results are referred to as "IC 50 The affinity can be expressed in terms of the 50% inhibitory concentration (IC 50 ) or the concentration at which 50% of the first member of the binding pair (e.g., a peptide) is displaced. Similarly, ln(IC 50 ) is an IC 50 represents the natural logarithm of. For example, IC 50 K can be the concentration of test peptide in a binding assay at which 50% inhibition of binding of the labeled reference peptide is observed. Given the conditions under which the assay is run (e.g., limiting HLA protein concentration and / or labeled reference peptide concentration), these values ​​can be used to calculate K DThe value may be approximated. Assays for determining binding are well known in the art and are described in detail in, for example, PCT Publication Nos. WO94 / 20127 and WO94 / 03205, as well as in 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 may be expressed relative to binding by a reference peptide. Binding can be measured using live cells (e.g., Ceppellini et al., Nature, 339:392 (1989); Christnick et al., Nature, 352:67 (1991); Busch et al., Int. Immunol., 2:443 (1990); Hill et al., J. Immunol., 147:189 (1991); del Guercio et al., J. Immunol., 154:685 (1995)), cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol., 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol., 152, 2890 (1994); Marshall et al., J. Immunol., 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBOJ., 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem., 268:15425 (1993)); high-flux soluble phase assays (Hammer et al., J. Exp. Med., 180:2353 (1994)), and class I Other assay systems may also be used to determine 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)).

[0069]

[0137] The term "derived" when used to discuss epitopes is synonymous with "prepared." Derived epitopes may be isolated from natural sources or synthesized according to standard protocols in the art. Synthetic epitopes may include "amino acid mimetics," which are artificial amino acid residues such as D-isomers of naturally occurring L-amino acid residues or unnatural amino acid residues such as cyclohexylalanine. Derived or prepared epitopes may be analogs of natural epitopes. The term "derived from" refers to origin or source and may include naturally occurring, recombinant, unpurified, purified, or differentiated molecules or cells. For example, expanded or induced antigen-specific T cells may be derived from T cells. For example, expanded or induced antigen-specific T cells may be derived from antigen-specific T cells in a biological sample. For example, mature APCs (e.g., professional APCs) may be derived from non-mature APCs (e.g., immature APCs). For example, APCs can be 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.

[0070]

[0138] An "epitope" is the collective characteristics of a molecule (e.g., the charge of a peptide, as well as the primary, secondary, and tertiary structure) that together form the 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 the set of amino acid residues involved in recognition by a particular immunoglobulin; a major histocompatibility complex (MHC) receptor; or, in the context of T cells, the residues recognized by the T cell receptor protein and / or a chimeric antigen receptor. Epitopes can be prepared by isolation from natural sources or synthesized according to standard protocols in the art. Synthetic epitopes can include artificial amino acid residues, amino acid mimetics (such as D-isomers of naturally occurring L-amino acid residues, or non-natural amino acid residues). Throughout this disclosure, epitopes are sometimes referred to as peptides or peptide epitopes. In certain embodiments, the peptides of the present disclosure are limited in length. Length-restricted embodiments occur when a protein or peptide containing an epitope described herein contains a region (i.e., a series of amino acid residues) that has 100% identity to a native sequence. The definition of an epitope places a limit on the length of any region that has 100% identity to a native peptide sequence, e.g., to avoid read-through of the entire native molecule. Thus, for a peptide containing an epitope described herein and a region with 100% identity to a native peptide sequence, the region with 100% identity to the native sequence generally has a length of 600 amino acid residues or less, 500 amino acid residues or less, 400 amino acid residues or less, 250 amino acid residues or less, 100 amino acid residues or less, 85 amino acid residues or less, 75 amino acid residues or less, 65 amino acid residues or less, and 50 amino acid residues or less.In certain embodiments, an "epitope" as described herein consists of a peptide having a region with 100% identity to a native peptide sequence, in any increment of up to 5 amino acid residues, but less than 51 amino acid residues in length; 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 residues.

[0071]

[0139] A "T cell epitope" refers to a peptide sequence bound by an MHC molecule in the form of a peptide-MHC (pMHC) complex that can be recognized and bound by the TCR of a T cell (e.g., a cytotoxic T lymphocyte or a helper T cell).

[0072]

[0140] "T cells" are CD4 + T cells and CD8 + The term T cell also includes both T helper type 1 T cells and T helper type 2 T cells.

[0141] "Immune cells" refer to cells that play a role in the immune response. Immune cells are derived from hematopoietic cells and include lymphocytes such as B cells and T cells; natural killer cells; and myeloid cells such as monocytes, macrophages, neutrophils, mast cells, basophils, and granulocytes.

[0073]

[0142] An "immunogenic" peptide or "immunogenic" epitope or "immunogenic" peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated or humoral response, such as a cytotoxic T lymphocyte (CTL) response, a helper T lymphocyte (HTL) response, and / or a B lymphocyte response. The immunogenic peptides described herein are capable of binding to an HLA molecule and subsequently inducing a cell-mediated or humoral response (e.g., a CTL (cytotoxic) response or an HTL response) against the peptide.

[0074]

[0143] 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 arrests the symptoms, side effects, or progression of a disease. The immune response can also include an antibody response facilitated by stimulation of helper T cells.

[0075]

[0144] "T cell receptor" ("TCR") refers to a molecule found on the surface of T lymphocytes (T cells) that recognizes antigens bound to major histocompatibility complex (MHC) molecules, whether naturally occurring or partially or completely synthetically produced. The ability of a T cell to recognize antigens associated with various diseases (e.g., cancer) or infectious organisms is conferred by its TCR, which is composed of both α and β chains or γ and δ chains. The proteins that make up these chains are encoded by DNA using a unique mechanism to generate the great diversity of TCRs. This multisubunit immune recognition receptor associates with the CD3 complex and binds peptides presented by MHC class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of the TCR to peptides on APCs is a central event in T cell activation.

[0076]

[0145] As used herein, a "chimeric antigen receptor" or "CAR" refers to an antigen-binding protein comprising an antigen-binding domain of an immunoglobulin (e.g., an immunoglobulin variable domain) and a constant domain of a T cell receptor (TCR). As used herein, the "constant domain" of a TCR polypeptide includes a TCR membrane-proximal constant domain, a TCR transmembrane domain, and / or a TCR cytoplasmic domain, or fragments thereof. For example, in some embodiments, a CAR is a monomer comprising a polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCR β constant domain. In some embodiments, a CAR is a dimer comprising a first polypeptide comprising an immunoglobulin heavy chain variable domain or light chain variable domain linked to a TCR α or TCR β constant domain, and a second polypeptide comprising an immunoglobulin heavy chain or light chain variable domain (e.g., a kappa or lambda variable domain) linked to a TCR β or TCR α constant domain.

[0077]

[0146] A "major histocompatibility complex" or "MHC" is a cluster of genes that plays a role in regulating cellular interactions that contribute to physiological immune responses. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes that can include any class of MHC molecule, such as MHC class I molecules and MHC class II molecules, and occurs in all vertebrates. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. Thus, "human leukocyte antigen" or "HLA" refers to human major histocompatibility complex (MHC) proteins (see, e.g., Stites et al., "Immunology," 8th ed., Lange Publishing, Los Altos, Calif. (1994)). For a detailed description of the MHC complex and the HLA complex, see Paul, "Fundamental Immunology," 3rd ed., Raven Press, New York (1993).

[0078]

[0147] The major histocompatibility complex in the genome contains gene regions whose gene products, expressed on the cell surface, are important for binding and presenting endogenous and / or foreign antigens, thereby regulating immunological processes. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or diseased cells in immune responses. MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments derived from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. MHC-binding peptides can result from proteolytic cleavage of protein antigens and can represent potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes). MHC transports peptides to the cell surface, where they can be presented to specific cells such as cytotoxic T lymphocytes, helper T cells, or B cells. The MHC region can be divided into three subgroups: class I, class II, and class III. MHC class I proteins consist of the α chain MHC class I proteins may contain α- and β-chains and may present antigen fragments to cytotoxic T cells. MHC class II proteins may contain α and β chains and may present antigen fragments to helper T cells. The MHC class III region may encode other immune components, such as complement components and cytokines. The MHC can be both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).

[0079]

[0148] "Antigen processing" or "processing" refers to the breakdown of a polypeptide or antigen into processing products, which are fragments of the polypeptide or antigen (e.g., breakdown of a polypeptide into peptides), and the association (e.g., via binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by a cell, e.g., an antigen-presenting cell.

[0080]

[0149] "Antigen-presenting cells" (APCs) refer to cells that present peptide fragments of protein antigens in association with MHC molecules on their 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).

[0081]

[0150] "Receptor" refers to a biological molecule or group of molecules capable of binding to a ligand. Receptors are used to transmit information in cells, cell formations, or organisms. A receptor comprises at least one receptor unit, where each receptor unit may be, for example, a protein molecule. A receptor has a structure complementary to that of a ligand and can complex with the ligand as a binding partner. The information is transmitted by a conformational change of the receptor, particularly on the surface of a cell, after complexation with the ligand. In some embodiments, receptors are understood to refer to, in particular, MHC class I and II proteins that are capable of forming a receptor / ligand complex with a ligand, in particular a peptide or peptide fragment of an appropriate length. "Ligand" refers to a molecule that has a structure complementary to that of a receptor and is capable of forming a complex with the receptor. In some embodiments, ligands are understood to refer to peptides or peptide fragments of an appropriate length and with an appropriate binding motif in their amino acid sequence, such that the peptide or peptide fragment can form a complex with an MHC protein, such as an MHC class I or MHC class II protein. In some embodiments, "receptor / ligand complex" shall also be understood to mean a "receptor / peptide complex" or a "receptor / peptide fragment complex" comprising a peptide-presenting MHC molecule or a peptide fragment-presenting MHC molecule, such as an MHC class I molecule or an MHC class II molecule.

[0082]

[0151] 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 exists in an organism (including viruses), may be isolated from a natural source, and has not been intentionally modified by man in a laboratory, is naturally occurring.

[0083]

[0152] As used herein, the terms "peptide" and "peptide epitope" are typically used interchangeably with "oligopeptide" to refer to a series of residues connected to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues. A "synthetic peptide" refers to a peptide obtained from a non-natural source, e.g., an artificial peptide. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. A "synthetic peptide" includes a "fusion protein."

[0084]

[0153] The term "motif" refers to a peptide of a defined length, e.g., less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, e.g., about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13) for a class I HLA motif, and about 6 to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for a class II HLA motif, that is a pattern of residues within the peptide that is recognized by a particular HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs differ in their patterns of primary and secondary anchor residues. In some embodiments, MHC class I motifs identify peptides that are 7, 8, 9, 10, 11, 12, or 13 amino acid residues in length. In some embodiments, MHC class II motifs identify peptides that are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acid residues in length. A "cross-reactive binding" peptide refers to a peptide that binds to more than one member of a class of binding pair member (e.g., a peptide that is bound by both class I and class II HLA molecules).

[0085]

[0154] The term "residue" refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or encoded by a nucleic acid (DNA or RNA). The nomenclature used to describe peptides or proteins follows conventional practice. The amino group is presented at the left (amino- or N-terminus) of each amino acid residue, and the carboxyl group is presented at the right (carboxy- or C-terminus). When referring to the position of an amino acid residue within a peptide epitope, the amino acid residues are numbered in the amino-to-carboxyl direction, with the first position being the residue located at the amino terminus of the epitope or the peptide or protein of which it may be a part. In formulas representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups are not specifically shown, but are in the form they assume at physiological pH values ​​unless otherwise specified. In amino acid structural formulas, each residue is generally represented by its standard three-letter or one-letter designation. The L-form of an amino acid residue is represented by a single capital letter or the first letter of a three-letter symbol, and the D-form of an amino acid residue is represented by a single lowercase letter or a lowercase three-letter symbol. However, the three-letter symbol or universal designation can be used without the capital letter to refer to an L-amino acid residue. Glycine has no asymmetric carbon atom and is referred to simply as "Gly" or "G." The amino acid sequences of peptides set forth 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; and Y, tyrosine).

[0086]

[0155] A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. The art defines families of amino acid residues with similar side chains, 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine with phenylalanine is a conservative substitution. Substitutions that do not eliminate peptide function are known. Methods for identifying conservative nucleotide and amino acid substitutions are well known in the art.

[0087]

[0156] "Pharmaceutically acceptable" generally refers to a composition or component of a composition that is non-toxic, inert, and / or physiologically compatible. "Pharmaceutical excipients" or "excipients" include materials such as adjuvants, carriers, pH adjusting / buffering agents, tonicity adjusting agents, humectants, preservatives, and the like. A "pharmaceutical excipient" is a pharmaceutically acceptable excipient.

[0088]

[0157] According to the present disclosure, the term "vaccine" relates to a pharmaceutical preparation (composition) or product that, upon administration, induces an immune response, e.g., a cellular or humoral immune response that recognizes and attacks pathogens or diseased cells, such as cancer cells. Vaccines can be used for the prevention or treatment of diseases. The terms "individualized cancer vaccine" or "personalized cancer vaccine" and "individualized cancer vaccine" refer to a specific cancer patient and mean that the cancer vaccine is adapted to the needs or special circumstances of the individual cancer patient.

[0089]

[0158] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to polymers of nucleotides of any length, including DNA and RNA, e.g., mRNA. The 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, the polynucleotides and nucleic acids can be mRNA transcribed in vitro. In some embodiments, the polynucleotide administered using the methods of the invention is mRNA.

[0090]

[0159] The terms "isolated" or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its natural state. Thus, the isolated peptides described herein are free of some or all of the materials normally associated with the peptide in their in situ environment. For example, an "isolated" epitope may be an epitope that does not include the entire sequence of the protein from which it is derived. For example, a naturally occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide separated from some or all of the coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector and / or such polynucleotides or peptides may be part of a composition, but are still "isolated" in that such vectors or compositions are not part of their natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein and further include such molecules produced synthetically. In some embodiments, an isolated polypeptide, antibody, polynucleotide, vector, cell, or composition is substantially pure. As used herein, the term "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0091]

[0160] "Identical" or percent "identity," in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be determined using sequence comparison software or algorithms, or by visual inspection. In the art, methods used to align amino acid or nucleotide sequences are well known. A variety of algorithms and software are known that can be used. 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 they have at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% nucleotide or amino acid residue identity, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequence that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 residues, such as at least about 80-100 residues. In some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the amino acid sequence of a peptide or the coding region of a nucleotide sequence.

[0092]

[0161] The term "subject" refers to any animal (e.g., mammal), including but not limited to, a human, non-human primate, dog, cat, rodent, etc., who is the recipient of a particular treatment. Typically, the terms "subject" and "patient," which refer to a human subject, are used interchangeably herein.

[0093]

[0162] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of a therapeutic agent effective to "treat" a disease or disorder in a subject or mammal. A therapeutically effective amount of a drug has a therapeutic effect and thus may prevent the onset of a disease or disorder; may delay the onset of a disease or disorder; may slow the progression of a disease or disorder; may alleviate to some extent one or more of the symptoms associated with a disease or disorder; may reduce morbidity and mortality; may improve quality of life; or may have a combination of such effects.

[0094]

[0163] The terms "treating" or "treatment" or "treating" or "alleviating" or "alleviating" refer to both (1) therapeutic measures that cure, slow, reduce the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Accordingly, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.

[0095]

[0164] The term "depleted," when used to describe a cell sample (e.g., a peripheral blood mononuclear cell (PBMC) sample), refers to a cell sample in which a subpopulation of cells has been removed or depleted. For example, an immune cell sample depleted of CD25-expressing cells 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 +Cells can be depleted or removed from the PBMC sample, such as by using an antibody that binds to CD14.

[0096]

[0165] "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 can refer to the binding of the TCR of a T cell to a peptide-MHC complex. For example, stimulation of a T cell can refer to the step in Protocol 1 or Protocol 2 when PBMCs are cultured with peptide-loaded APCs.

[0097]

[0166] The term "enriched" refers to a composition or fraction in which a species of interest has been partially purified such that the concentration of the species of interest is substantially greater than the naturally occurring level of the species in the final product without enrichment. The term "induced cells" refers to cells that have been treated with an inducing compound, cell, or population of cells that affects the protein expression, gene expression, differentiation state, shape, morphology, survival, etc. of the cell. Overview of T-cell therapy and its manufacturing

[0167] The generation of antigen-specific T cells by controlled induction or expansion of T cells (e.g., autologous T cells) ex vivo can result in highly specific and beneficial T cell therapy (e.g., adoptive T cell therapy). The present disclosure provides T cell manufacturing methods and therapeutic T cell compositions that can be used to treat subjects with cancer and other conditions, diseases, and disorders. The goal is to expand and induce antigen-specific T cells with a suitable phenotype and function. The present disclosure provides compositions and methods for producing T cells that can be used for antigen-specific T cell therapy (e.g., individualized T cell therapy or personalized T cell therapy). The T cell compositions provided herein can be personalized antigen-specific T cell therapy.

[0098]

[0168] 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 stimulate antigen-specific CD8 + For example, the methods presented herein can be used to expand memory T cells. + For example, the methods presented herein can be used to induce naive T cells. + For example, the methods presented herein can be used to expand memory T cells. + The therapeutic compositions may be used to induce naive T cells. Also provided herein are therapeutic compositions comprising antigen-specific T cells. For example, the therapeutic compositions may comprise antigen-specific memory T cells. For example, the therapeutic compositions may comprise antigen-specific naive T cells. Also provided herein are methods of use or treatment using the therapeutic compositions described herein. T cell composition

[0169] Provided herein is a composition (e.g., a pharmaceutical composition) comprising a population of immune cells. The composition may comprise at least one antigen-specific T cell comprising a T cell receptor (TCR). The composition may comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence.

[0099]

[0170] In some embodiments, the compositions provided herein comprise T cells stimulated by APCs, such as APCs preloaded with antigenic peptides. The composition can comprise a population of immune cells, including T cells derived from a sample (e.g., a biological sample), where the T cells comprise APC-stimulated T cells. In some embodiments, the composition comprises a population of immune cells incubated with one or more cytokines, growth factors, or ligands, such as ligands that bind to cell surface receptors of 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 polyI:C. In some embodiments, the composition comprises a population of immune cells incubated with one or more APCs or APC preparations. For example, the composition can comprise APCs stimulated with one or more cytokines, growth factors, and / or ligands, or a population of immune cells incubated with an APC preparation stimulated with cytokines, growth factors, and / or ligands. For example, the composition may comprise APCs stimulated by one or more cytokines or cytokine-stimulated APCs. For example, the composition may comprise a population of immune cells incubated with APCs stimulated with one or more growth factors or a growth factor-stimulated APC preparation. For example, the composition may comprise a population of immune cells incubated with APCs stimulated with one or more ligands or a ligand-stimulated APC preparation.

[0100]

[0171] In some embodiments, the APCs are autologous APCs, allogeneic APCs, or artificial APCs. In some embodiments, the APCs comprise dendritic cells (DCs). In some embodiments, the APCs comprise CD14 + In some embodiments, APCs may be obtained from the skin, spleen, bone marrow, thymus, lymph nodes, peripheral blood, or umbilical cord blood. +The monocytes are derived from a biological sample containing PBMCs from a subject. For example, CD14 + Monocytes may be isolated, enriched, or purified from a biological sample derived from a subject, including PBMCs. + The monocytes are stimulated with one or more cytokines or growth factors. In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, 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 that contains PBMCs.

[0101]

[0172] In some embodiments, CD14 + The isolated population of APCs can be enriched or substantially enriched. In some embodiments, the isolated population of APCs can be enriched for CD14 + The isolated population of APCs is at least 30%, at least 50%, at least 75%, or at least 90% homogeneous. + The isolated population of APCs is at least 60%, at least 75%, or at least 90% homogeneous. + APCs, such as APCs, can include APCs derived from monocytic dendritic cell precursors, for example, 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.

[0102]

[0173] CD14 + APC and CD14 + Substantially enriched cell populations of APCs can be isolated by methods also provided by the present invention. The methods generally involve obtaining a population of cells containing APC precursors, differentiating the APC precursors into immature or mature APCs, and isolating CD14 from the differentiated population of immature or mature APCs. + It may also include isolation of APCs.

[0103]

[0174] APC precursor cells can be obtained by methods known in the art. APC precursors can be isolated, for example, by density gradient separation, fluorescence-activated cell sorting (FACS), immunological cell separation methods such as panning, complement lysis, rosetting, magnetic cell separation, nylon wool separation, and combinations of such methods. Methods for immunoselective APC 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.

[0104]

[0175] Enriched populations of APC precursors can also be obtained. Methods for obtaining such enriched precursor populations are known in the art. For example, enriched populations of APC precursors can be isolated from tissue sources by selectively removing cells that are adhered to a substrate. For example, using tissue sources such as bone marrow or peripheral blood, adherent monocytes can be removed from cell preparations using commercially available plastic substrates (e.g., beads or magnetic beads) to obtain a population enriched for non-adherent APC precursors.

[0105]

[0176] Monocyte APC precursors can also be obtained from tissue sources by using APC precursor adhesive substrates. For example, monocytic APC precursors isolated by leukopheresis can be obtained from tissue sources by using APC precursor adhesive substrates. In this method, peripheral blood leukocytes are contacted with a monocytic APC precursor adhesive substrate having a high surface area to volume ratio, and adherent monocytic APC precursors are isolated. In a further embodiment, the coupled substrate can be a particulate or fibrous substrate having a high surface area to volume ratio, such as microbeads, microcarrier beads, pellets, granules, powders, capillaries, microvillous membranes, etc. Furthermore, the particulate or fibrous substrate can be glass, polystyrene, plastic, glass-coated polystyrene microbeads, etc.

[0106]

[0177] APC precursors can also be cultured in vitro for differentiation and / or expansion. Methods for differentiating / expanding APC precursors are known in the art. Generally, expansion can be achieved by culturing the precursors in the presence of at least one cytokine that induces differentiation / proliferation of APCs (e.g., dendritic cells). 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 culture, thereby further enriching the population of APC precursors. Typically, such agents include cytokines such as IL-13, IL-4, or IL-15.

[0107]

[0178] The isolated population of APC precursors can be cultured and differentiated to obtain immature or mature APCs. Suitable tissue culture media include, for example, AIM-V®, RPMI, or other media. Examples of tissue culture media include, but are not limited to, 1640, DMEM, X-VIVO 15®, etc. Tissue culture media are typically supplemented with amino acids, vitamins, divalent cations, and cytokines that promote differentiation of precursors towards an APC phenotype. Typically, the differentiation-promoting cytokines are GM-CSF and / or IL-4.

[0108]

[0179] Furthermore, upon expansion, differentiation, and maturation to the APC phenotype, cultures of APC precursors express CD14 + Plasma may be included to promote APC development. A typical plasma concentration is about 5%. In addition, for example, when APC precursors are isolated by adhesion to a substrate, plasma may promote CD14 expression during the early adhesion step of culture. + It can be included in the culture medium to promote the phenotype. Typical plasma concentrations at the time of attachment are about 1% or higher.

[0109]

[0180] The monocytic APC precursors can be cultured for any suitable period of time. In certain embodiments, the culture period suitable for differentiation of the precursors into immature APCs can be about 1 to about 10 days, for example, about 4 to about 7 days. The differentiation of the immature APCs from the precursors can be achieved by detecting cell surface markers (e.g., CD11c + , CD83 low , CD86 - / low , HLA - DR + Immature APCs can be monitored by methods known to those skilled in the art, such as by the presence or absence of antigens. Immature APCs can also be cultured in tissue culture medium appropriate to maintain the immature APCs in a state for further differentiation or for antigen uptake, processing, and presentation. For example, immature APCs can be maintained in the presence of GM-CSF and IL-4.

[0110]

[0181] After differentiation from APC precursors, CD14 + The cells are CD14 + APCs can be isolated to obtain an isolated population of APCs. Typically, CD14 + If APCs are enriched or isolated from substantially enriched APCs prior to maturation, the isolated population will contain immature CD14 + APCs are enriched or substantially enriched. Generally, CD14 + Isolation of APCs + The method includes contacting the cell population from which the cells are isolated with a CD14-specific probe. In one 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 with an unlabeled antibody specific for CD14 and a labeled second antibody specific for the first antibody. CD14 + Cells were also sorted by FACS to identify CD14 low and CD14 - It can also be isolated from CD14 cells. high Gating for positivity of, for example, CD1 on PBMC-derived monocytes The CD14-specific binding agent can be determined by reference to IgG4 staining. Typically, the CD14-specific binding agent is, for example, an anti-CD14 antibody (e.g., a monoclonal antibody or an antigen-binding fragment thereof). Those skilled in the art will be familiar with many anti-CD14 antibodies suitable for use in the present invention, and many anti-CD14 antibodies can be purchased commercially.

[0111]

[0182] In another embodiment, the CD14-specific probe is coupled to a substrate and detects CD14 + The cells can be isolated by affinity selection. + A cell population containing CD14 cells is exposed to the coupled substrate and + The cells are allowed to adhere specifically. Then, non-adherent CD14 - The cells are washed from the substrate and the adherent cells are then transfected with CD14 + The APCs are eluted to obtain a substantially enriched isolated cell population. 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 beads or magnetic beads). Affinity isolation of cell populations using substrate-coupled antibodies specific for surface markers is generally known.

[0112]

[0183] During culture, immature APCs (CD14 -The isolated population of immature APCs, or all immature APCs prior to isolation, can optionally be exposed to a predetermined antigen. Suitable predetermined antigens can include any antigen for which modulation by T cells is desired. In one embodiment, immature APCs are cultured in the presence of prostate-specific membrane antigen (PSMA) for cancer immunotherapy and / or tumor growth inhibition. Other antigens can include, for example, bacterial cells, viruses, partially purified or purified bacterial or viral antigens, tumor cells, tumor-specific or tumor-associated antigens (e.g., tumor cell lysates, tumor cell membrane preparations, antigens isolated from tumors, fusion proteins, liposomes, etc.), recombinant cells expressing antigens on their surface, autoantigens, and any other antigens. Any of the antigens can also be presented as peptides or recombinantly produced proteins or portions thereof. After contact with the antigen, the cells can be cultured for any suitable time to allow for antigen uptake and processing, such as to expand the population of antigen-specific APCs.

[0113]

[0184] For example, in one embodiment, immature APCs can be cultured after antigen uptake to promote maturation of the immature APCs into mature APCs that present the antigen in the context of MHC molecules. Methods for APC maturation are known. Such maturation can be carried out by culture in the presence of known maturation factors, such as cytokines (e.g., TNF-α, IL-1β, or CD40 ligand), bacterial products (e.g., LPS or BCG), etc. Maturation of immature APCs into mature APCs can be monitored by methods known in the art, such as, for example, measuring the presence or absence of cell surface markers (e.g., upregulation of CD83, CD86, and MHC molecules) or testing for expression of mRNA or proteins specific for mature APCs, for example, using oligonucleotide arrays.

[0114]

[0185] Optionally, immature APCs can be cultured in tissue culture medium appropriate 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, for example, anti-inflammatory cytokines (e.g., IL-10 and TGF-β), to inhibit maturation of immature APCs.

[0115]

[0186] In another embodiment, CD14 + The isolated population of APCs is enriched for mature APCs. + The isolated population of mature APCs is CD14 + An isolated population of immature APCs can be obtained by culturing them in the presence of maturation factors (e.g., bacterial products and / or pro-inflammatory cytokines) as described above, thereby inducing maturation. 4 + and CD14 - A mixed population of immature APCs (differentiated from APC precursors) may be cultured to induce maturation, with the maturation stage monitored as described above, and at the appropriate stage of mature APC enrichment, CD14 + The cells are CD14 + They can be separated as described above to obtain an isolated population enriched or substantially enriched for mature APCs.

[0116]

[0187] According to yet another embodiment of the present invention, APCs can be preserved, for example, by cryopreservation, before or after exposure to a prostate cancer antigen. Cryopreservatives that can be used include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate, and inorganic salts. A controlled, slow cooling rate can be crucial. Different cryoprotectants and different cell types typically have different optimal cooling rates. The heat of the fusion phase, in which water turns to ice, is typically minimal. The cooling procedure can be performed, for example, using a programmable freezing device or a methanol bath procedure. A programmable freezing device allows for the determination of the optimal cooling rate and facilitates standard, reproducible cooling. Programmable controlled rate freezers, such as Cryomed or Planar, allow the freezing regimen to be fine-tuned according to the desired cooling rate profile.

[0117]

[0188] After complete freezing, the APCs can be quickly transferred to a long-term cryogenic storage container. In a typical embodiment, the samples can be cryogenically stored in liquid nitrogen (-196°C) or its vapor (-165°C). In particular, the considerations and procedures for the manipulation, cryopreservation, and long-term storage of hematopoietic stem cells derived from bone marrow or peripheral blood are largely applicable to the APCs of the present invention.

[0118]

[0189] Frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37-41°C) and immediately cooled upon thawing. It may be desirable to treat cells in the 20-digit range to prevent cell aggregation upon thawing. Various procedures can be used to prevent aggregation, including, but not limited to, the addition of DNase, low molecular weight dextran, and citric acid, hydroxyethyl starch, etc., before and / or after freezing. If cryoprotectants are toxic in humans, they should be removed before therapeutic use of thawed APCs. One method for removing cryoprotectants is by dilution to a low concentration. Once frozen APCs are thawed and recovered, they can be used to activate T cells as described herein for unfrozen APCs.

[0119]

[0190] In some embodiments, the composition comprises a population of immune cells depleted of one or more types of immune cells. For example, the composition can comprise a population of immune cells depleted of one or more types of immune cells, where the population of immune cells expresses one or more proteins, such as one or more cell surface receptors. In some embodiments, the composition comprises a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, where the amount of immune cells expressing CD14 and / or CD25 in the population is different relative to the amount of immune cells expressing CD14 and / or CD25 in the biological sample. For example, the composition can comprise a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, where the amount of immune cells expressing CD14 in the population is different relative to the amount of immune cells expressing CD14 in the biological sample. For example, the composition can comprise a population of immune cells derived from a biological sample that includes at least one antigen-specific T cell that comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence, where the population The amount of immune cells expressing CD25 in the composition is different relative to the amount of immune cells expressing CD25 in the biological sample. For example, the composition can include a population of immune cells derived from a biological sample that includes at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, where the amount of immune cells expressing CD14 and CD25 in the population is different relative to the amount of immune cells expressing CD14 and CD25 in the biological sample. For example, the composition can include a population of immune cells derived from a biological sample, where the amount of immune cells expressing CD14 and CD25 in the population is relatively less than the amount of immune cells expressing CD14 and CD25 in the biological sample.

[0120]

[0191] In some embodiments, a composition comprises a population of immune cells comprising T cells derived from a sample (e.g., a biological sample), where the T cells comprise APC-stimulated T cells and the APC is a FLT3L-stimulated APC. For example, a composition can comprise a population of immune cells comprising T cells derived from a sample (e.g., a biological sample), where 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, and the APC is a FLT3L-stimulated APC. In some embodiments, a composition comprises a population of immune cells comprising T cells derived from a biological sample, where the T cells are APC-stimulated T cells and comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, and the APC is a FLT3L-stimulated APC, and the amount of antigen-specific T cells in the population is relatively greater than the amount of antigen-specific T cells in the biological sample. In some embodiments, the T cells comprise a plurality of antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence. In some embodiments, the T cells comprise a plurality of antigen-specific T cells comprising a plurality of T cell receptors (TCR) specific for at least one antigenic peptide sequence. In some embodiments, the T cells comprise a plurality of antigen-specific T cells comprising a plurality of T cell receptors (TCR) specific for a plurality of antigenic peptide sequences.For example, the plurality of antigen-specific T cells in the composition may be 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, 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, 1 x 10. 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9, 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9 x 10 12 For example, the plurality of T cell receptors (TCRs) specific for at least one antigenic peptide sequence can 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, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 850 ... For example, a plurality of T cell receptors (TCRs) specific for multiple 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 multiple antigenic peptide sequences. For example, the plurality of antigenic peptide sequences can 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 antigenic peptide sequences.

[0121]

[0192] In some embodiments, the composition or pharmaceutical composition comprises a population of immune cells derived from a biological sample. In some embodiments, the immune cells comprise a plurality of antigen-specific T cells. In some embodiments, each of the antigen-specific T cells comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence. In some embodiments, the composition or pharmaceutical composition comprises a population of immune cells, wherein the amount of immune cells expressing CD14 and / or CD25 in the population 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.

[0122]

[0193] In some embodiments, the compositions provided herein comprise a population of immune cells derived from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, and a pharmaceutically acceptable excipient, wherein the amount of immune cells expressing CD14 and / or CD25 in the population differs relative to the amount of immune cells expressing CD14 and / or CD25 in the biological sample.

[0123]

[0194] In some embodiments, the compositions provided herein comprise a population of immune cells derived from a biological sample, wherein the amount of immune cells in the population that express CD14 and CD25 is relatively less than the amount of immune cells that express CD14 and CD25 in the biological sample.

[0124]

[0195] In some embodiments, the compositions provided herein comprise a population of immune cells, including T cells, derived from a biological sample, wherein the T cells are APC-stimulated T cells and comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, and the APC is a FLT3L-stimulated APC; and a pharmaceutically acceptable excipient. 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 population is relatively 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 population is relatively greater than the amount of immune cells expressing CD14 and / or CD25 in the biological sample.

[0125]

[0196] In some embodiments, the pharmaceutical composition comprises a CD4 + T cells, in this case CD4 + The percentage of antigen-specific T cells among the 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 is directed against naive CD8 + T cells, in this case naive CD8 + The percentage of antigen-specific T cells among the 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, in this case memory CD8 +The percentage of antigen-specific T cells among the T cells is at least about 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 1%, 2% or 3% of the T cells. 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0126]

[0197] In some embodiments, the pharmaceutical composition comprises a population of immune cells derived from a biological sample, the population of immune cells comprising T cells, 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, and the APC is a FLT3L-stimulated APC; and a pharmaceutically acceptable excipient.

[0127]

[0198] In some embodiments, a pharmaceutical composition comprises a population of immune cells derived from a biological sample, the population of immune cells comprising T cells, the T cells comprising a plurality of neo-antigen T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence; and a pharmaceutically acceptable excipient; wherein the percentage of the T cells that are antigen-specific 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%.

[0128]

[0199] In some embodiments, the neo-antigen-specific T cells comprise APC-stimulated T cells. In some embodiments, the percentage of immune cells in the population that express CD14 and / or CD25 is less than the percentage of immune cells in the biological sample that express CD14 and / or CD25. In some embodiments, the biological sample is derived from a subject. In some embodiments, the subject is human. In some embodiments, the subject has a disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the neo-antigen-specific T cells are CD4 + and / or CD8 + In some embodiments, the neo-antigen-specific T cells comprise CD4-enriched T cells and / or CD8-enriched T cells. For example, CD4 + T cells or CD8+ T cells can be isolated, enriched, or purified from a biological sample derived from a subject, including PBMCs. In some embodiments, neo-antigen-specific T cells are naive CD4 + and / or naive CD8 + In some embodiments, the neoantigen peptides are T cells. In some embodiments, the naive T cells are characterized by surface expression of L-selectin (CD62L). In some embodiments, the naive T cells are characterized by the absence of one or more of the activation markers CD25, CD44, or CD69. In some embodiments, the naive T cells are characterized by the absence of memory CD45RO isoforms. In some embodiments, the naive T cells are characterized by expression of a functional IL-7 receptor, consisting of the IL-7 receptor alpha, which is CD127, and the common gamma chain subunit, which is CD132. In some embodiments, at least one neoantigen peptide sequence comprises a mutation selected from (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a read-through mutation, (E) a gene fusion mutation, and combinations thereof, and the cancer neoantigen peptide binds to an HLA protein of interest with an IC of less than 500 nM. 50 , and binds with greater affinity than the corresponding wild-type peptide. In some embodiments, each of the at least one neo-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 neo-antigenic peptide sequence comprises a mutation that is not present in non-cancer cells of the subject. In some embodiments, each of the at least one neo-antigenic peptide sequence is encoded by an expressed gene in a cancer cell of the subject.

[0129]

[0200] In some embodiments, one or more of the at least one neo-antigenic peptide sequence has a length of 8 to 50 naturally occurring amino acids. In some embodiments, the at least one neo-antigenic peptide sequence comprises a plurality of neo-antigenic peptide sequences. In some embodiments, the plurality of neo-antigenic peptide sequences comprises 2 to 50, 3 to 50, 4 to 50, 5 to 5, 6 to 50, 7 to 50, 8 to 50, 9 to 50, or 10 to 50 neo-antigenic peptide sequences.

[0130]

[0201] In some embodiments, the APCs are a preparation of one or more APCs. In some embodiments, the APCs include APCs loaded with one or more neo-antigenic peptides comprising one or more of the at least one neo-antigenic peptide sequences. In some embodiments, the APCs are autologous APCs or allogeneic APCs.

[0131]

[0202] In some embodiments, the APCs comprise dendritic cells (DCs). In some embodiments, the APCs comprise CD14 + Derived from monocytes. In some embodiments, CD14 + Monocytes are enriched from a biological sample containing PBMCs derived from a subject. For example, CD14 + can be isolated, enriched, or purified from a biological sample derived from a subject, including PBMCs.

[0132]

[0203] In some embodiments, 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, or a combination thereof. In some embodiments, CD14 + The monocytes are derived from a second biological sample comprising PBMCs. In some embodiments, the second biological sample is from the same subject.

[0133]

[0204] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the percentage of at least one antigen-specific T 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% of total T cells or total immune cells. In some embodiments, at least one antigen-specific CD8 + The percentage of T cells is the total CD4 + T cells, total CD8 + In some embodiments, the composition comprises at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the T cells, total T cells, or total immune cells. + The percentage of T cells 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 percentage of at least one antigen-specific T cell in the biological sample is determined by measuring the percentage of total CD4 + T cells, total CD8 + In some embodiments, the biological sample contains 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. In some embodiments, the biological sample contains at least one antigen-specific CD8 + The percentage of T cells is the total CD4 + T cells, total CD8 + In some embodiments, the biological sample contains 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. In some embodiments, the biological sample contains at least one antigen-specific CD4 + The percentage of T cells is the total CD4 + T cells, total CD8 + At most, approximately 0.00001% of T cells, total T cells, or total immune cells, 0.0 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 + In some embodiments, the percentage of T cells is at most about 0.5%. + The percentage of T cells is at most about 0.5%.

[0134]

[0205] In some embodiments, the percentage of antigen-specific T cells in the pharmaceutical composition is greater than or equal to the total CD4 + T cells, total CD8 + In some embodiments, the antigen-specific CD8 T cells or total T cells or total immune cells in the pharmaceutical composition are at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the T cells, total T cells, or total immune cells. + The percentage of T cells is the total CD4 + T cells, total CD8 + In some embodiments, the antigen-specific naive CD8 T cells or CD8 T cells are 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 immune cells in the pharmaceutical composition. + The percentage of T cells is the total CD4 + T cells, total CD8 + In some embodiments, the antigen-specific memory CD8 T cells or total T cells or total immune cells are at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the T cells, total T cells, or total immune cells in the pharmaceutical composition. + The percentage of T cells is the total CD4 + T cells, total CD8 +In some embodiments, the antigen-specific CD4 T cells or total T cells or total immune cells are at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the T cells, total T cells, or total immune cells in the pharmaceutical composition. + The percentage of T cells 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. Manufacturing method

[0206] Provided herein are methods for producing antigen-specific T cells. Provided herein are methods for preparing T cell compositions, such as therapeutic T cell compositions. For example, the methods can include expanding or inducing antigen-specific T cells. Preparing (e.g., inducing or expanding) T cells can also refer to producing T cells, and can refer to any type of T cell (e.g., CD4 + T cells and CD8 + The present invention broadly encompasses procedures for isolating, stimulating, culturing, inducing, and / or expanding antigen-specific T cells (T cells). In a first aspect of the present specification, there is provided 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: activating an APC with a cell expressing CD14 and / or CD25; A method is presented that includes incubating with a population of immune cells derived from a depleted biological sample.

[0135]

[0207] In a second aspect herein, there is provided 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 FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs with a population of immune cells derived from a biological sample.

[0136]

[0208] In a third aspect herein, there is provided a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample for a first period of time; and thereafter incubating at least one T cell of the biological sample with an APC.

[0137]

[0209] In a fourth aspect herein, there is provided 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 the population of immune cells with a first APC preparation of one or more APC preparations followed by incubating the population of immune cells derived from a biological sample with the one or more APC preparations for one or more separate periods of less than 28 days, wherein at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is induced.

[0138]

[0210] In a fifth aspect herein, there is provided 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 a population of immune cells derived from a biological sample with no more than three APC preparations for no more than three separate periods, whereby at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is induced.

[0139]

[0211] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells derived from a biological sample with one or more APC preparations for one or more separate periods, thereby stimulating the T cells to become antigen-specific T cells, wherein the percentage of antigen-specific T cells is greater than or equal to the total CD4 + T cells, total CD8 + At least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of T cells, total T cells, or total immune cells. 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 derived from a biological sample with no more than three APC preparations for no more than three separate periods of time, thereby stimulating the T cells to become antigen-specific T cells. 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 derived from a biological sample with no more than two APC preparations for no more than two separate periods of time, thereby stimulating the T cells to become antigen-specific T cells.

[0140]

[0212] In some embodiments, the method includes (a) obtaining a biological sample from a subject that includes at least one antigen-presenting cell (APC); and (b) enriching cells that express CD14 from the biological sample, thereby detecting CD14. + Obtaining cell-enriched samples; (c) CD14 + (d) incubating the cell-enriched sample with at least one cytokine or growth factor for a first period of time; (e) incubating the cell-enriched sample with at least one peptide to inhibit the CD14 + (e) incubating the peptide-loaded APC sample with one or more cytokines or growth factors for a third period of time, thereby obtaining a mature APC sample; (f) incubating APCs from the mature APC sample with a CD14 and / or CD25-depleted sample comprising PBMCs for a fourth period of time; (g) incubating the PBMCs with APCs from the mature APC sample for a fifth period of time; (h) incubating the PBMCs with APCs from the mature APC sample for a sixth period of time; and (i) administering at least one T cell from the PBMCs to a subject in need thereof.

[0141]

[0213] In some embodiments, the method includes (a) obtaining a biological sample from a subject comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD14 and / or CD25 from the biological sample, thereby obtaining a CD14- and / or CD25-depleted sample; (c) incubating the CD14- and / or CD25-depleted sample with FLT3L for a first period of time; (d) incubating at least one peptide with the CD14- and / or CD25-depleted sample of (c) for a second period of time, thereby obtaining a peptide-loaded APC sample; and (e) incubating the peptide-loaded APC sample with FLT3L for a second period of time. (f) incubating the peptide-loaded APC sample with at least one PBMC for a third period of time, thereby obtaining a first stimulated PBMC sample; (f) incubating the PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; (g) incubating the PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and (h) administering at least one T cell of the third stimulated PBMC sample to a subject in need thereof.

[0142]

[0214] In some embodiments, a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating APCs with a population of immune cells derived from a biological sample depleted of cells expressing CD14 and / or CD25.

[0143]

[0215] Provided herein, in some embodiments, 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 a population of immune cells derived from a biological sample with a first APC preparation of one or more APC preparations, followed by incubating the population of immune cells with the one or more APC preparations for one or more separate periods of less than 28 days, to expand at least one antigen-specific memory T cell or induce at least one antigen-specific naive T cell. Provided herein, in some embodiments, 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 a population of immune cells derived from a biological sample with no more than three APC preparations for no more than three separate periods, to expand at least one antigen-specific memory T cell or induce at least one antigen-specific naive T cell.

[0144]

[0216] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells (e.g., PBMCs) with APCs. A method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for an original peptide sequence includes incubating a population of immune cells (e.g., PBMCs) with APCs for a period of time. In some embodiments, the population of immune cells is derived from a biological sample. In some embodiments, the population of immune cells is derived from a sample (e.g., a biological sample) that has been depleted of cells expressing CD14. In some embodiments, the population of immune cells is derived from a sample (e.g., a biological sample) that has been depleted of cells expressing CD25. In some embodiments, the population of immune cells is derived from a sample (e.g., a biological sample) that has been depleted of cells expressing CD14 and cells expressing CD25.

[0145]

[0217] 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 FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs with a population of immune cells derived from a biological sample. Presented herein, in some embodiments, is a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample for a first period of time; and thereafter incubating at least one T cell of the biological sample with APCs.

[0146]

[0218] 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 derived from a sample (e.g., a biological sample) with FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells derived from a sample (e.g., a biological sample) with FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells derived from a sample (e.g., a biological sample) with FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs. In some embodiments, a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample (e.g., for a period of time); and then contacting the T cells of the biological sample with APCs. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells derived 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 derived from a sample (e.g., a biological sample) with one or more APC preparations for one or more separate periods of time.In some embodiments, a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells derived from a sample (e.g., a biological sample) with one or more APC preparations for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate periods of time. In some embodiments, the one or more separate periods are less than 28 days, calculated from incubating the population of immune cells with a first APC preparation of the one or more APC preparations.

[0147]

[0219] 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: In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells with APCs for a period of time, wherein the population of immune cells is derived from a biological sample that has been depleted of cells expressing CD14 and / or CD25.

[0148]

[0220] 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 derived from a biological sample with FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs for a period of time.

[0149]

[0221] 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 FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample; and then contacting the T cells of the biological sample with APCs.

[0150]

[0222] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells derived from a biological sample with one or more APC preparations for one or more distinct periods of time, thereby inducing or expanding the antigen-specific T cells, wherein the one or more distinct periods are less than 28 days, calculated from incubating the population of immune cells with a first APC preparation of the one or more APC preparations. In some embodiments, incubating the population of immune cells derived from the biological sample with one or more APC preparations for one or more distinct periods of time is performed in a medium containing IL-7, IL-15, or a combination thereof. In some embodiments, the medium further comprises an indoleamine 2,3-dioxygenase 1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof. The IDO inhibitor can be epacadostat, navoximod, 1-methyltryptophan, or a combination thereof. In some embodiments, the IDO inhibitor inhibits antigen-specific CD8 + In some embodiments, IDO inhibitors may maintain the functional profile of memory CD8 cell responses. PD-1 antibodies may increase the number of antigen-specific memory CD8 + The absolute number of T cell responses may be increased. PD-1 antibodies may increase the proliferation rate of cells treated with such antibodies. The additional presence of IL-12 may increase the expansion of antigen-specific cells and / or CD8 + This can result in an increase in the frequency of T cells.

[0151]

[0223] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells derived from a biological sample with one or more APC preparations for one or more separate periods of time, thereby inducing or expanding the antigen-specific T cells, wherein the antigen-specific T cells, antigen-specific CD4 + T cells, or antigen-specific CD8 + T cell percentages are total T cells, total CD4 + T cells, total CD8+ At least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of T cells, total immune cells, or total cells.

[0152]

[0224] In some embodiments, the method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises: The method comprises incubating a population of cells with up to three APC preparations for up to three separate periods of time, thereby stimulating the T cells to become antigen-specific T cells.

[0153]

[0225] In some embodiments, the population of immune cells is derived from a biological sample that has been depleted of cells expressing CD14 and / or CD25. In some embodiments, the APCs are FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs. In some embodiments, the APCs comprise one or more APC preparations. In some embodiments, the APC preparation comprises three or fewer APC preparations. In some embodiments, the APC preparations are incubated with the immune cells sequentially within one or more separate time periods.

[0154]

[0226] In some embodiments, the biological sample is derived 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 disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the antigen-specific T cells are CD4 + and / or CD8 + In some embodiments, the antigen-specific T cells include CD4-enriched and / or CD8-enriched T cells. For example, CD4+ and / or CD8 + T cells can be isolated, enriched, or purified from a biological sample derived from a subject, including PBMCs. In some embodiments, antigen-specific T cells are naive CD4 + and / or naive CD8 + In some embodiments, the antigen-specific T cells are memory CD4 + and / or Memory CD8 + T cells.

[0155]

[0227] In some embodiments, at least one antigenic peptide sequence comprises a mutation selected from (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a read-through mutation, (E) a gene fusion mutation, and combinations thereof, and the cancer antigenic peptide binds to an HLA protein of interest with an IC of less than 500 nM. 50 and binds with greater affinity than the corresponding wild-type peptide. In some embodiments, each of the at least one antigenic peptide sequence binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in non-cancer cells of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by an expressed gene in cancer cells of the subject. In some embodiments, one or more of the at least one antigenic peptide sequence has a length of 8 to 50 naturally occurring amino acids. In some embodiments, the at least one antigenic peptide sequence comprises a plurality of antigenic peptide sequences. In some embodiments, the plurality of antigenic peptide sequences comprises 2 to 50, 3 to 50, 4 to 50, 5 to 50, 6 to 50, 7 to 50, 8 to 50, 9 to 50, or 10 to 50 antigenic peptide sequences.

[0156]

[0228] In some embodiments, the APCs comprise APCs loaded with one or more antigenic peptides comprising one or more of the at least one antigenic peptide sequences. In some embodiments, the APCs are autologous or allogeneic APCs. In some embodiments, the APCs comprise dendritic cells (DCs).

[0157]

[0229] In some embodiments, the method comprises depleting cells that express CD14 and / or CD25 from the biological sample. + Depleting the cells comprises contacting the APC with an agent that binds to CD14. In some embodiments, the APC is CD14 + In some embodiments, APCs are enriched from a biological sample. For example, APCs can be isolated, enriched, or purified from a biological sample derived from a subject that contains PBMCs.

[0158]

[0230] In some embodiments, the APCs are stimulated with one or more cytokines or growth factors. In some embodiments, the one or more cytokines or growth factors include GM-CSF, I 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.

[0159]

[0231] In some embodiments, the APCs are derived from a second biological sample. In some embodiments, the second biological sample is derived from the same subject.

[0232] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the percentage of antigen-specific T cells in the methods 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 methods is about 0.1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to 65%, or about 65% to about 70% of T cells or total immune cells. + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total T cells or total immune cells. +The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells. In some embodiments, the percentage of antigen-specific T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the percentage of antigen-specific CD8 T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the percentage of antigen-specific naive CD8 T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the percentage of T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the antigen-specific CD4 + 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%.

[0160]

[0233] In some embodiments, the method includes stimulating the T cells with IL-7, IL-15, or a combination thereof. In some embodiments, the method further comprises stimulating the T cells with IL-7, IL-15, or a combination thereof in the presence of antibody 1 or IL-12. In some embodiments, the method further comprises administering the antigen-specific T cells to the subject.

[0161]

[0234] In some embodiments, the first period of the one or more periods is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 days.

[0235] In some embodiments, the total duration of the separate periods is less than 28 days, in some embodiments, the total duration of the separate periods is 20 to 27 days, in some embodiments, the total duration of the separate periods is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 days.

[0162]

[0236] In some embodiments, the method comprises incubating a first one of the APC preparations with the T cells for more than 7 days. In some embodiments, the method comprises incubating a first one of the APC preparations with the T cells for more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the method comprises incubating a first one of the APC preparations with the T cells for 7-20, 8-20, 9-20, 10-20, 11-20, or 12-20 days. In some embodiments, the method comprises incubating a first one of the APC preparations with the T cells for about 10-15 days.

[0163]

[0237] In some embodiments, the method comprises incubating a second one of the APC preparations with the T cells for 5 to 9 days, hi some embodiments, the method comprises incubating a second one of the APC preparations with the T cells for 5, 6, 7, 8, or 9 days.

[0164]

[0238] In some embodiments, the method comprises incubating a third one of the APC preparations with the T cells for 5 to 9 days, hi some embodiments, the method comprises incubating a third one of the APC preparations with the T cells for 5, 6, 7, 8, or 9 days.

[0165]

[0239] In some embodiments, the method comprises incubating a first one of the APC preparations with the T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days, incubating a second one of the APC preparations with the T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days, and incubating a third one of the APC preparations with the T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days.

[0166]

[0240] In some embodiments, the biological sample is obtained freshly from a subject or from a frozen sample.

[0241] In some embodiments, the method comprises incubating one or more of the APC preparations with a first medium comprising at least one cytokine or growth factor for a first period of time. In some embodiments, the first period of time is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days. In some embodiments, the first period of time is no longer 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 longer 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.

[0167]

[0242] In some embodiments, the method includes incubating one or more of the APC preparations with at least one peptide for a second period of time, hi some embodiments, the second period of time is 1 hour or less.

[0168]

[0243] In some embodiments, the method includes incubating one or more of the APC preparations with a second medium comprising one or more cytokines or growth factors for a third period of time, thereby obtaining mature APCs. In some embodiments, the one or more cytokines or growth factors comprise 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 period of time is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days or less. In some embodiments, the third period of time is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days. In some embodiments, the third period of time is no longer than 2, 3, 4, or 5 days. In some embodiments, the third period of time is at least 1, 2, 3, or 4 days.

[0169]

[0244] In some embodiments, the method further comprises removing one or more cytokines or growth factors of the second culture medium after the third period of time and before the start of the fourth period of time.

[0245] In some embodiments, the method is performed ex vivo.

[0170]

[0246] In some embodiments, the method for preparing T cells includes obtaining a biological sample from a subject that includes APCs. + Cells were enriched from biological samples, which allowed the identification of CD14 + In some embodiments, the method comprises obtaining an enriched sample. + In some embodiments, the method comprises incubating the enriched sample with a first medium comprising at least one cytokine or growth factor for a first period of time. + In some embodiments, the method comprises incubating the peptide-loaded APC sample with a second medium comprising one or more cytokines or growth factors for a third period of time, thereby obtaining a mature APC sample. In some embodiments, the method comprises contacting the APCs of the mature APC sample with peripheral blood mononuclear cells (PBMCs) and a third medium comprising at least one cytokine or growth factor for a fourth period of time. In some embodiments, the method comprises incubating the PBMCs with the APCs of the mature APC sample for a fifth period of time. In some embodiments, the method comprises incubating the PBMCs with the APCs of the mature APC sample for a sixth period of time. In some embodiments, the method comprises administering T cells from the PBMCs to a subject in need thereof.

[0171]

[0247] In some other embodiments, the method for preparing T cells includes obtaining a biological sample from a subject that includes APCs. + Cells were enriched from biological samples, which allowed the identification of CD14 + In some embodiments, the method comprises obtaining an enriched sample. +the enriched sample is treated with a first medium containing at least one cytokine or growth factor for at least or at most or about 30, 40, or 50 minutes; or 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.

[0172]

[0248] In some embodiments, the method comprises administering at least one peptide to a subject comprising: +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, thereby obtaining a peptide-loaded APC sample. In some embodiments, the method comprises incubating the peptide-loaded APC sample with medium comprising one or more cytokines or growth factors for at least or up to 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, thereby obtaining a mature APC sample. In some embodiments, the method comprises contacting APCs of the mature APC sample with PBMCs and a medium comprising at least one cytokine or growth factor for at least or up to 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 PBMCs with APCs of the mature APC sample for at least or up to 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 PBMCs with APCs from the mature APC sample for at least or up to 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 from the PBMCs to a subject in need thereof.

[0173]

[0249] In some embodiments, the method includes (a) obtaining a biological sample from a subject that includes at least one antigen-presenting cell (APC); and (b) enriching cells that express CD14 from the biological sample, thereby detecting CD14. + Obtaining cell-enriched samples; (c) CD14 + (d) incubating the cell-enriched sample with at least one cytokine or growth factor for a first period of time; (e) incubating the cell-enriched sample with at least one peptide to inhibit the CD14 + (e) incubating the peptide-loaded APC sample with one or more cytokines or growth factors for a third period of time, thereby obtaining a mature APC sample; (f) incubating the APCs of the mature APC sample with a CD14- and / or CD25-depleted sample comprising PBMCs for a fourth period of time; (g) incubating the PBMCs with the APCs of the mature APC sample for a fifth period of time; (h) incubating the PBMCs with the APCs of the mature APC sample for a sixth period of time; and (i) administering at least one T cell of the PBMCs to a subject in need thereof. In some embodiments, the first period of time is at least or at most 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, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 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 period of time is at least or at most 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 period of time is at least or at most 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 period of time is at least or at most 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 period of time is at least or at most 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.

[0174]

[0250] In some embodiments, the method includes (a) obtaining a biological sample from a subject comprising at least one APC and at least one PBMC; (b) depleting cells expressing 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; and (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 peptide-loaded APCs. (e) incubating the peptide-loaded AP sample with at least one PBMC for a third period of time, thereby obtaining a first stimulated PBMC sample; (f) incubating PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth period of time, thereby obtaining a second stimulated PBMC sample; (g) optionally, incubating PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth period of time, thereby obtaining a third stimulated PBMC sample; and (h) administering at least one T cell of the 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 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 period of time is at least or at most 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 period of time is at least or at most 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 period of time is at least or at most 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 period of time is at least or at most 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.

[0175]

[0251] In some other embodiments, a method of preparing T cells includes: (a) obtaining a biological sample from a subject, the biological sample comprising APCs and T cells; (b) incubating the biological sample with a first medium comprising at least one cytokine or growth factor for a first period of time; (c) incubating at least one peptide with the biological sample of (b) for a second period of time, thereby obtaining a peptide-loaded APC sample; (d) incubating the peptide-loaded APC sample with a second medium comprising one or more cytokines or growth factors for a third period of time, thereby obtaining a mature APC sample; (e) after the third period of time, incubating the mature APC sample with human serum for a fourth period of time; (f) incubating the biological sample with one or more cytokines for a fifth period of time; and (g) administering the T cells of the biological sample to a subject in need thereof. In some embodiments, the at least one cytokine or growth factor comprises FLT3L. In some embodiments, the first period of time 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 period of time is at least 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, or 3 hours. In some embodiments, the third period of time 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 period of time is about 2, 3, or 4 days. In some embodiments, the fifth period of time 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 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 period of time is at least or at most 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 period of time is at least or at most 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 period of time is at least or at most 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 period of time is at least or at most 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.

[0176]

[0252] The induced or expanded T cells can 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 +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 + Contains T cells.

[0177]

[0253] A variety of antigenic peptides can be used to induce or expand T cells. In some embodiments, the peptide comprises a mutation selected from (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a read-through mutation, (E) a gene fusion mutation, and combinations thereof. In some embodiments, the peptide comprises a point mutation and binds to an HLA protein of interest with greater affinity than the corresponding wild-type peptide. In some embodiments, the peptide has an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 In some embodiments, the peptide binds to an HLA protein of interest with an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K D In some embodiments, each peptide binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, the TCR of the induced or expanded antigen-specific T cells binds to an HLA protein of the subject with an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K DIn some embodiments, the TCR binds to a peptide-HLA complex with an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K D In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in non-cancer cells of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or expressed gene in cancer cells of the subject. In some embodiments, the peptide has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 or more naturally occurring amino acids. In some embodiments, the peptide binds to a protein encoded by an HLA class I allele and has a length of 8 to 12 naturally occurring amino acids. In some embodiments, the peptide binds to a protein encoded by a class II HLA allele and has a length of 16-25 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.

[0178]

[0254] In various embodiments, APCs are used to stimulate / induce T cells. In some embodiments, the APCs or APCs of the APC preparation are loaded with one or more antigenic peptides. In some embodiments, the APCs or APCs of the APC preparation are autologous or allogeneic APCs. In some embodiments, the APCs or APCs of the APC preparation comprise dendritic cells (DCs). In some embodiments, the method comprises depleting cells expressing CD14 and / or CD25 from the biological sample. In some embodiments, the method comprises depleting cells expressing CD19 from the biological sample. In some embodiments, depleting cells expressing CD14 and / or CD25 comprises binding an agent that binds CD14 and / or CD25 to the APCs or APCs of the APC preparation. In some embodiments, the agent that binds CD14 and / or CD25 is biotinylated. In some embodiments, depleting cells expressing CD14 and / or CD25 comprises binding an anti-biotin reagent on a solid support to the agent that binds CD14 and / or CD25. In some embodiments, the agent that binds CD14 and / or CD25 is conjugated to a solid support. In some embodiments, depleting cells expressing CD19 comprises conjugating an agent that binds CD19 to an APC or an APC of an APC preparation. In some embodiments, the agent that binds CD19 is biotinylated. In some embodiments, depleting cells expressing CD19 comprises conjugating an anti-biotin reagent on a solid support to the agent that binds CD19. In some embodiments, the agent that binds CD19 is conjugated to a solid support. In some embodiments, the APC or an APC of an APC preparation comprises conjugating an anti-biotin reagent on a solid support to the agent that binds CD19. + Derived from monocytes. In some embodiments, the APCs or APCs of the APC preparation are CD141-enriched APCs or CD141-enriched dendritic cells.

[0179]

[0255] In some embodiments, the APCs or APCs of the APC preparation are enriched from a biological sample. In some embodiments, the APCs or APCs of the APC preparation 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. In some embodiments, the APCs or APCs of the APC preparation are derived from a second biological sample. In some embodiments, the second biological sample is derived from the same subject. In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the biological sample is obtained fresh from the subject or from a frozen sample.

[0180]

[0256] In some embodiments, the percentage of at least one antigen-specific T cell is greater than or equal to the total CD4 + T cells, total CD8 + In some embodiments, at least one antigen-specific CD8 T cell 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. + The percentage of T cells is the total CD4 + T cells, total CD8 +In some embodiments, 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. + The percentage of T cells is the total CD4 + T cells, total CD8 + At least about 0.00001%, 0.00002%, 0.00 T cells, total T cells, or total immune cells 0.005%, 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%.

[0181]

[0257] In some embodiments, the percentage of at least one antigen-specific T cell in the biological sample is determined by measuring the percentage of total CD4 + T cells, total CD8 + In some embodiments, the 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% of the total T cells or total immune cells. + The percentage of T cells is the total CD4 + T cells, total CD8 +In some embodiments, the at least one antigen-specific CD4 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% of the total T cells or total immune cells. + The percentage of T cells is the 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.

[0182]

[0258] In some embodiments, the method further comprises administering one or more of the at least one antigen-specific T cells to the subject. In some embodiments, the total duration of the separate periods is less than 28 days. In some embodiments, the incubating comprises incubating one APC preparation of the plurality of APC preparations with the T cells for a period of more than 7 days. In some embodiments, the incubating comprises incubating a first, second, third, or fourth APC preparation of the plurality of APC preparations with the T cells for a period of more than 7 days. In some embodiments, the method comprises incubating the APC or one or more of the 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 or at most 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 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. In some embodiments, the method comprises incubating one or more preparations of APCs with at least one peptide for a second period of time. In some embodiments, the second period of time is at least or at most 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 or one or more APCs with a second medium comprising one or more cytokines or growth factors for a third period of time, thereby obtaining mature APCs.In some embodiments, the third period of time is at least or at most 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 .... In some embodiments, the one or more cytokines or growth factors comprise GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-rna40, poly I:C, or a combination thereof. In some embodiments, the method further comprises removing the one or more cytokines or growth factors of the second culture medium after the third period of time and prior to the start of a fourth period of time. In some embodiments, the fourth period of time is at least or at most 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 neoantigen, a tumor-associated antigen, a viral antigen, a minor histocompatibility antigen, or a combination thereof. In some embodiments, the method is performed ex vivo. In some embodiments, the at least one antigen-specific T cell comprises a plurality of antigen-specific T cells. Antigen-presenting cells (APCs) and preparation methods

[0259] In some embodiments, the method comprises inducing, stimulating, or expanding T cells with antigen-presenting cells (APCs). The APCs can be pre-loaded with antigenic peptides before contacting the T cells. In some embodiments, the method of expanding or inducing antigen-specific T cells comprises stimulating a population of immune cells comprising T cells with APCs. In some embodiments, the population of immune cells is derived from a biological sample depleted of cells expressing CD14 and / or CD25. In some embodiments, the APCs are FLT3L-stimulated APCs. In some embodiments, the APCs comprise one or more APC preparations. In some embodiments, at least one of the one or more APC preparations comprises FLT3L-stimulated APCs. In some embodiments, the one or more APC preparations comprise no more than three APC preparations. In some embodiments, the one or more APC preparations are incubated with the immune cells sequentially within one or more separate time periods.

[0183]

[0260] Antigen-presenting cells (APCs) present peptide fragments of protein antigens in association with MHC molecules on their cell surface. The presented peptides are associated with MHC molecules as peptide-MHC complexes (pMHC) on the cell surface of APCs. Processing and presentation of peptide-MHC complexes can involve a series of sequential steps, including protein protease-mediated digestion; transport of the peptide to the endoplasmic reticulum (ER) mediated by transporters associated with antigen processing (TAPs); formation of peptide-MHC I molecules using newly synthesized MHC molecules; and transport of the peptide-MHC molecules to the cell surface.

[0184]

[0261] 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 upon formation of TCR-pMHC. In some embodiments, the MHC of an antigen-presenting cell (e.g., class I MHC or class II MHC) is loaded with peptide and presented by the APC by introducing into the APC nucleic acid (e.g., RNA) encoding an antigenic peptide or polypeptide, including the peptide sequence to be presented.

[0185]

[0262] From a biological perspective, for a somatic mutation to generate an immune response, several criteria must be met: the allele containing the mutation must be expressed by the cell; the mutation must be within the coding region of a protein and be non-synonymous; the translated protein must be cleaved by the proteasome or other intracellular proteolytic pathway; the epitope containing the mutation must be presented by the MHC complex; the presented epitope must be recognized by the TCR; and finally, the TCR-pMHC complex must initiate a signaling cascade that activates the T cell. It is.

[0186]

[0263] Monocytes circulate in the bloodstream and then migrate to tissues, where they can differentiate into macrophages and dendritic cells. Classical monocytes are typically characterized by high levels of CD14 cell surface receptor expression. Monocytes and B cells can be competent APCs, but their antigen-presenting capacity is thought to be limited to reactivating already primed T cells. These cell types may not be capable of directly activating functionally naive or unprimed T cell populations. Professional antigen-presenting cells are highly efficient at internalizing antigens by phagocytosis or receptor-mediated endocytosis and then presenting fragments of antigens bound to MHC molecules on their membranes. T cells recognize and interact with antigen-MHC molecule complexes on the membrane of antigen-presenting cells. Additional costimulatory signals are then provided by the antigen-presenting cells, resulting in T cell activation. The expression of costimulatory molecules is a typical feature of professional antigen-presenting cells.

[0187]

[0264] Professional antigen-presenting cells can be highly efficient at internalizing antigens by phagocytosis or receptor-mediated endocytosis and then presenting fragments of antigens bound to MHC molecules on their membranes. T cells can recognize and interact with antigen-MHC molecule complexes on the membrane of APCs. Further costimulatory signals can then be provided by APCs, resulting in T cell activation. The expression of costimulatory molecules can define the characteristics of professional antigen-presenting cells. Examples of professional APCs include, but are not limited to, dendritic cells (DCs), macrophages, and B cells. Professional APCs can express high levels of MHC class II, ICAM-1, and B7-2.

[0188]

[0265] One of the major types of professional antigen-presenting cells is the dendritic cell, which has the broadest range of antigen presentation. Other major types of professional antigen-presenting cells include macrophages, B cells, and certain activated epithelial cells. Dendritic cells are a population of leukocytes that present antigens (e.g., antigens captured in peripheral tissues) to T cells via the MHC class II and I antigen presentation pathway. Dendritic cells can activate both naive T cells and primed T cells (e.g., memory T cells). Dendritic cells (DCs) can be a population of leukocytes that present antigens captured in peripheral tissues to T cells via the MHC class I and II antigen presentation pathway. Dendritic cells can be potent inducers of immune responses, and activation of these cells can be a crucial step for inducing antitumor immunity. Dendritic cells can be potent inducers of immune responses, and activation of these cells can be a crucial step for inducing antitumor immunity.

[0189]

[0266] Dendritic cells can be categorized as "immature" and "mature" cells, which can be used as a convenient way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be considered to exclude all possible intermediate stages of differentiation. Immature dendritic cells can be characterized as antigen-presenting cells with a high capacity for antigen uptake and processing, which correlates with high expression of Fcγ receptors and mannose receptors. The mature phenotype is typically characterized by low expression of these markers, but also by high expression of cell surface molecules that contribute to T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB). Mature dendritic cells express CD11b and CD11b receptors. + , CD11c + , HLA-DR + , CD80 + , CD86 + , CD54 + , CD3 - , CD19 - , CD14- , CD141 + (BDCA-3), and / or CD1a + The maturation of dendritic cells can be a process by which such antigen-presenting dendritic cells prime T cells. The state of dendritic cells can be referred to as an activated state when dendritic cells produce a dendritic cell-specific antigen, whereas presentation by immature dendritic cells results in tolerance. Dendritic cell maturation can be triggered by biomolecules with microbial signatures detected by innate receptors (e.g., bacterial DNA, viral RNA, endotoxin, etc.), pro-inflammatory cytokines (e.g., TNF, interleukins, and interferons), ligand binding of CD40L to CD40 on the dendritic cell surface, and substances released from cells undergoing cell death. Additional non-limiting 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 derived in vitro by culturing bone marrow cells 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 dendritic cells isolated from PBMCs. + Cytokines or growth factors that can be used to induce 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 polyI:C.

[0190]

[0267] Non-professional antigen-presenting cells typically do not constitutively express MHC class II proteins, which are typically expressed only upon stimulation of the non-professional antigen-presenting cells with certain cytokines, such as IFN-γ.

[0191]

[0268] The source of antigen-presenting cells (APCs) can typically be a tissue source containing APCs or APC precursors capable of expressing and presenting antigenic peptides in vitro. In some embodiments, APCs are capable of proliferating and becoming professional APCs when loaded with target RNA and / or treated with the necessary cytokines or factors.

[0192]

[0269] In one embodiment, APC precursor cells can be expanded and matured in vitro into dendritic cells (DCs). While many tissue sources can be used, typical tissue sources include spleen, thymus, tissue biopsy, tumor, afferent lymphatic vessels, lymph nodes, and the like. Sources of APCs and / or precursor APCs may include lymph nodes, bone marrow, apheresis or leukapheresis products, and / or peripheral blood. In certain embodiments, apheresis products, bone marrow, and peripheral blood may be sources. Fetal tissue, fetal blood, or umbilical cord blood, which are also rich in growth factors, may also be used as a source of blood for obtaining APCs and / or precursor APCs. Examples of precursor cells include embryonic stem cells, CD34 + APCs include, but are not limited to, monocyte progenitor cells, monocytes, and pre-B cells. For example, APCs can be monocytes or CD34 + The cells may be derived from precursor cells, including the cells.

[0193]

[0270] In one aspect, the source of APCs and / or precursor APCs can be the product of apheresis or leukapheresis. 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)). The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, and other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In another embodiment of the invention, cells can be washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium, may lack magnesium, and may lack many, 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-automated "flow-through" centrifuge. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free PBS, Mg-free PBS, etc. Alternatively, the desired fraction of the apheresis sample can be resuspended in a suitable buffer. Any remaining components may be removed and the cells may be directly resuspended in culture medium.

[0194]

[0271] 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 the blood of humans or non-human vertebrates. APCs can also be isolated from enriched populations of leukocytes. The leukocyte population can be prepared by methods known to those skilled in the art. Such methods typically include collection of heparinized blood, apheresis or leukapheresis, buffy coat preparation, rosetting, centrifugation, density gradient centrifugation (e.g., using Ficoll, colloidal silica particles, and sucrose), differential lysis of non-leukocyte cells, and filtration. The leukocyte population can also be prepared by collecting blood from a subject, defibrinizing it to remove platelets, and lysing red blood cells. The leukocyte population can optionally be enriched for monocytic dendritic cell precursors.

[0195]

[0272] Blood cell populations can be obtained from various subjects depending on the desired use of the enriched leukocyte population. The subject can be a healthy subject. Alternatively, blood cells can be obtained from subjects in need of immune stimulation, such as cancer patients or other patients who would benefit from immune stimulation. Similarly, blood cells can be obtained from subjects in need of immunosuppression, such as patients with autoimmune disorders (e.g., rheumatoid arthritis, diabetes, systemic lupus erythematosus, multiple sclerosis, etc.). The leukocyte population can also be obtained from HLA-matched healthy individuals.

[0196]

[0273] When blood is used as a source of APCs, blood leukocytes can be obtained using conventional methods that maintain their viability. According to one embodiment of the present invention, blood can be diluted into medium, which may or may not contain heparin or other suitable anticoagulants. The volume of blood to the volume of medium can be approximately 1:1. Cells can be concentrated by centrifugation of the blood in medium at approximately 1,000 rpm (150 g) at 4°C. Platelets and red blood cells can be depleted by resuspending the cells in any number of solutions known in the art that lyse red blood cells, such as ammonium chloride. For example, the mixture can be approximately 1:1 by volume of medium and ammonium chloride. The cells can be concentrated by centrifugation and washed in the desired solution until a population of white blood cells substantially free of platelets and red blood cells is obtained. Any isotonic solution commonly used in tissue culture can be used as a medium to separate blood leukocytes from platelets and red blood cells. Examples of such isotonic solutions can be phosphate buffered saline, Hank's balanced salt solution, and complete growth medium. APCs and / or APC precursors can also be purified by elutriation.

[0197]

[0274] In one embodiment, isolation of APCs and / or precursor APCs is performed by dividing Ficoll-processed whole blood or apheresed peripheral blood into a batch of cells (typically about 5×10 cells). 8 ~about 2×10 10paramagnetic particles (approximately 1 vial of beads or 4 x 10 beads) coupled with one or more types of irrelevant antibodies or non-antibodies 9 This separation can be performed by pre-incubating the cells with paramagnetic particles (e.g., 22-37°C) for about 30 minutes to 2 hours, followed by magnetic removal of cells that have been conjugated to or phagocytosed the paramagnetic particles. Such separation can be performed using standard methods available in the art. For example, various commercially available magnetic separation methods (e.g., DYNAL® Magnetic Particle Concentrators (DYNAL®)) can be used. Any magnetic separation method can be used, including MPC®. Verification of isolation can be monitored by a variety of methods known to those skilled in the art, including flow cytometric analysis of cells before and after the isolation.

[0198]

[0275] APCs are cultured in a suitable culture medium in a suitable culture container or vessel. In certain embodiments, the culture medium may be The cells may be supplemented with one or more cytokines. The substrate can be any container with a tissue culture-compatible surface. Examples include various bags, flasks, roller bottles, Petri dishes, and multi-well plates made for use in tissue culture. Surfaces treated with substances, such as collagen or poly-L-lysine, or antibodies specific to a particular cell type, to promote cell adhesion can also be used, provided they allow for differential cell attachment as described below. Surfaces can also be chemically treated, for example, by ionization. Cells can be grown on a substrate of 1 cm. 2 Approximately 10 cells per 5 ~10 7 In one embodiment, the cells are seeded at an initial cell density of 1 cm. 2 10 cells per 6 Can be sown individually.

[0199]

[0276] In one embodiment, primary cultures derived from a selected tissue source are incubated at about 37°C under standard tissue culture conditions of humidity, CO2, and pH until a population of cells has adhered to the substrate sufficiently to allow separation of nonadherent cells. Some immature APCs in blood, particularly immature DCs, are initially nonadherent to plastic, in contrast to monocytes, so that precursors can be isolated after overnight culture. Monocytes and fibroblasts may comprise the majority of adherent cells and can typically adhere to the substrate within about 30 minutes to about 24 hours. In certain embodiments, nonadherent cells can be separated from adherent cells between about 1 and 16 hours. Nonadherent cells can be separated after about 1 to 2 hours. Any method that does not dislodge significant numbers of adherent cells can be used to separate adherent from nonadherent cells. In certain embodiments, cells can be dislodged by simple shaking or pipetting. In certain embodiments, pipetting may be most preferred.

[0200]

[0277] Adherent cells, including precursor APCs (e.g., monocytes) isolated according to the methods of the present invention, can be incubated at about 37°C under standard tissue culture conditions of humidity, CO2, and pH until the population of cells reaches the immature APC stage. In certain embodiments, according to the present disclosure, adherent cells can be incubated for 4 hours to 7 days. However, one of skill in the art will readily appreciate that incubation times and conditions can vary. Immature APCs can express CD14, CD25, CD30, CD45, CD50, CD60, CD85, CD90, CD100, CD110, CD120, CD130, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD149, CD149, CD149, CD149, CD149, CD141, CD149, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD149 ... - In some cases, CD14 + Immature APCs may also express CD1a, CD40, CD86, CD54, and intermediate levels of MHC class II (the level of marker expression on sample cells can be compared by flow cytometry analysis with the expression levels on MHC class II-negative cells and on cells known to express high levels of MHC class II). Immature APCs typically do not express CCR7.

[0201]

[0278] In certain aspects of the present disclosure, it is not necessary to separate the T cells from the APCs. For example, in one embodiment, PBMCs containing APCs and T cells may be exposed to an antigen as described herein, and the resulting antigen-specific T cells may be further expanded as described herein.

[0202]

[0279] Certain aspects of the invention do not require that the APCs or T cells described herein be derived from autologous sources. Thus, APCs and T cells can be obtained from matched or unmatched donors, or from cell lines, T cell lines, or other cells expanded in vitro. Methods for haplotype matching are known in the art. Furthermore, APCs and T cells, or supernatants derived therefrom, can be obtained from xenogeneic sources, e.g., mice, rats, non-human primates, and porcine cells can also be used.

[0203]

[0280] Suitable APC preparations include, for example, dendritic cells and monocytes. In other embodiments, APCs are activated non-nominal A cells, such as, for example, B cells, cells, or epithelial or endothelial cells. The APCs may be immature or mature APCs. The APCs and T cells are typically co-cultured for about 6 to about 48 hours, although longer and shorter times are within the scope of the present invention. The co-culture is typically carried out for a time sufficient to allow activation of the T cells, but may be carried out for less than the time required for differentiation and / or maturation of significant numbers of immature APCs or APC precursors.

[0204]

[0281] In certain embodiments, monocytic dendritic cell precursors can be isolated, for example, by contacting enriched leukocytes or monocytes with a monocytic dendritic cell precursor adhesive substrate. Briefly, when an enriched population of leukocytes or monocytes is contacted with a substrate, monocytic dendritic cell precursors or monocytes within the cell population can adhere to the substrate. Other leukocytes exhibit reduced binding affinity for the substrate, allowing monocytic dendritic cell precursors to be preferentially enriched on the surface of the substrate. Suitable substrates include, for example, particulate substrates such as glass particles, plastic particles, glass-coated plastic particles, glass-coated polystyrene particles, microcapillaries, and microvilli. The surface of the substrate can optionally be treated to enhance adhesion of monocytic dendritic cell precursors to the substrate. The surface of the substrate can be coated, for example, with proteins, cytokines, plasma, and / or monocyte-binding proteins. After contacting a leukocyte-enriched cell population or a monocyte-enriched cell population with a monocytic dendritic cell precursor adhesive substrate, the monocytic dendritic cell precursors adhere to the substrate and form complexes containing monocytic dendritic cell precursors on the substrate. Binding of the monocytic dendritic cell precursors can be monitored by antibody detection using an anti-cell surface marker antibody, such as an anti-CD14 antibody, for example, by FACS forward / side scatter analysis. In some embodiments, the leukocyte population can be contacted with the substrate for about 5 to about 300 minutes, more typically about 30 to about 120 minutes. The monocytic dendritic cell precursor complexes can optionally be washed with a suitable wash buffer to remove nonspecifically bound leukocytes. Suitable wash buffers include tissue culture medium, phosphate-buffered saline, Dulbecco's phosphate-buffered saline, etc. The medium can be supplemented with amino acids, vitamins, and / or hormones to promote the survival and / or proliferation of the monocytic dendritic cell precursors. The effectiveness of the washing may be monitored by FACS forward / side scatter analysis of the wash buffer, or by staining the eluted cells for cell surface markers, etc. Typically, the complexes may be washed several times to remove non-specifically bound leukocytes. Adhered monocytic dendritic cell precursors may be eluted from the substrate.For example, precursors can be eluted from the matrix by treatment with phosphate-buffered saline containing 0.4% EDTA or other non-toxic chelating agents. Monocytic dendritic cell precursors can typically be eluted from the matrix without the use of trypsin or other proteases.

[0205]

[0282] In other embodiments, dendritic cells can be isolated according to other methods known to those skilled in the art (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. Patent Nos. 5,994,126 and 5,851,756). Methods for immunosecretory dendritic cells include using antibodies against cell surface markers associated with dendritic cell precursors, such as anti-CD34 and / or anti-CD14 antibodies coupled to a substrate (e.g., Bernhard et al., Cancer Res., 55:1099-104 (1995); Caux et al., Nature 360:258-61 (1992)), or antibodies against cell surface markers associated with fully differentiated dendritic cells, such as CD11c, CD54, CD83, CD80, and CD86.

[0206]

[0283] In other embodiments, the APCs can be non-nominal APCs under inflammatory or other activation conditions. For example, non-nominal APCs can include epithelial cells stimulated with interferon-γ, T cells, B cells, 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.

[0207]

[0284] APCs can be cultured, expanded, differentiated, and / or matured as desired depending on the type of APC. APCs can be cultured in any suitable culture vessel, such as, for example, culture plates, flasks, culture bags, and bioreactors.

[0208]

[0285] In certain embodiments, APCs can be cultured in a culture or growth medium suitable for maintaining and / or expanding the number of APCs in the preparation. The culture medium can be selected according to the type of APCs being isolated. 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 also be included in the growth medium. For example, cytokines such as granulocyte / macrophage colony-stimulating factor (GM-CSF) and / or interleukin 4 (IL-4) can be added to maintain and / or expand mature dendritic cells. In other embodiments, immature APCs can be cultured and / or expanded. Immature dendritic cells can retain the ability to take up target mRNA and process new antigens. In some embodiments, immature dendritic cells can be cultured in a medium suitable for their maintenance and culture. The culture medium may be supplemented with amino acids, vitamins, antibiotics, divalent cations, etc. Additionally, cytokines, growth factors, and / or hormones may also be included in the growth medium.

[0209]

[0286] Other immature APCs can be similarly cultured or expanded. Preparations of immature APCs can be matured to form mature APCs. APC maturation can occur upon or after exposure to antigenic peptides. In certain embodiments, preparations of immature dendritic cells can be matured. Suitable maturation factors include, for example, cytokines such as TNF-α, bacterial products (e.g., BCG), and the like. In another aspect, isolated APC precursors can be used to prepare preparations of immature APCs. The APC precursors can be cultured, differentiated, and / or matured. In certain embodiments, monocytic dendritic cell precursors can be cultured in the presence of an appropriate culture medium supplemented with amino acids, vitamins, cytokines, and / or divalent cations to promote differentiation of the monocytic dendritic cell precursors into immature dendritic cells. In some embodiments, the APC precursors are isolated from PBMCs. PBMCs can be obtained from a donor, e.g., a human donor, and can be used fresh or frozen for future use. In some embodiments, the APCs are prepared from one or more APC preparations. In some embodiments, the APCs comprise APCs loaded with one or more antigenic peptides comprising one or more of at least one antigenic peptide sequence. In some embodiments, the APCs are autologous APCs, allogeneic APCs, or artificial APCs.

[0210]

[0287] In some embodiments, the APC precursor is a monocyte. In some embodiments, the monocyte is a CD14 + In some embodiments, the monocytes are isolated using an anti-CD14 antibody. In some embodiments, the isolated monocytes are cultured at 10 cells / well in 2 mL of medium. 5 ~10 7 In some embodiments, isolated monocytes are seeded at approximately 3 x 10 cells per well in 2 mL of medium. 6In some embodiments, the isolated monocytes are cultured in a medium containing cytokines or growth factors. In some embodiments, the isolated monocytes are cultured in a 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, 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 induced to become dendritic cells in ex vivo culture medium. In some embodiments, the derived dendritic cells are further matured ex vivo and loaded with antigenic peptides. In some embodiments, the derived dendritic cells are cultured in medium containing one or more antigenic peptides. In some embodiments, the antigenic peptide is a neo-antigenic peptide. Examples of neo-antigenic peptides include, but are not limited to, HIV short peptides, HIV long peptides, previously identified neoantigen (PIN) short peptides, and PIN long peptides. In some embodiments, the derived dendritic cells are cultured in medium containing one or more neo-antigenic peptides for at least 30 minutes, at least 50 minutes, at least 1 hour, or at least 2 hours. In some embodiments, the derived dendritic cells are further incubated with one or more cytokines after incubation with the antigenic peptides. In some embodiments, the one or more cytokines 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.

[0211]

[0288] In some embodiments, whole PBMCs are used to prepare APCs, which may 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, PBMCs are depleted of regulatory T cells (Treg cells) and then cultured in a medium containing FLT3L. In some embodiments, PBMCs are depleted of CD14 + The cells are depleted and then cultured in medium containing FLT3L. In some embodiments, the PBMCs are CD25 + The cells are depleted and then cultured in medium containing FLT3L. In some embodiments, the PBMCs are CD25 + and CD14 + The cells are depleted and then cultured in medium containing FLT3L. In some embodiments, the PBMCs are CD25 + cells, CD14 + cells, and CD19 + In some embodiments, PBMCs are cultured in medium containing FLT3L and then depleted of CD14 + In some embodiments, PBMCs are cultured in medium containing FLT3L and then depleted of CD25 + In some embodiments, PBMCs are cultured in medium containing FLT3L and then depleted of CD14 + Cells and CD25 + In some embodiments, isolated CD14 + Monocytes are cultured in a medium containing FLT3L. In some embodiments, PBMCs (total PBMCs, CD14 + CD25 depleted PBMCs + CD25 depleted PBMCs + / CD14 + Depleted PBMCs, or CD25 + / CD14 + / CD19 + depleted PBMCs) or isolated CD14 +The monocytes are cultured in a medium containing one or more antigens. In some embodiments, PBMCs or isolated CD14 + Monocytes are cultured in a 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. The mature cytokines can be added to the cell culture or medium at various concentrations. In some embodiments, the mature cytokine is added to the cell culture or medium at a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the mature cytokine is added to the cell culture or 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 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, mature cytokines are added to the cell culture or medium at a final concentration of at least 10 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 80 U / mL, 100 U / mL, 200 U / mL, 500 U / mL, 800 U / mL, 1000 U / mL, 1500 U / mL, 2000 U / mL, or 2500 U / mL (enzyme units as used herein are calculated according to the manufacturer's protocol). In some embodiments, PBMC cultures (whole PBMCs or PBMCs depleted of certain cells) used for APC preparation are subjected to further incubation or cytokine treatment for T cell induction or stimulation. In this case, APC preparation (e.g., maturation and peptide loading) and T cell induction or stimulation are performed using the same cell culture. In some other cases, the APC preparation is a separate cell population derived from the PBMC population used for T cell stimulation.

[0212]

[0289] In some embodiments, the method includes incubating one or more APCs or one or more APC preparations with a peptide, thereby creating a peptide-loaded APC sample. For example, the method may include incubating one or more APCs or one or more APC preparations with one or more peptides at a concentration of 0.001 to 100 μM, thereby creating a peptide-loaded APC sample. For example, the method may 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 may 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 may 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 involve injecting one or more APCs or one or more APC preparations with one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty 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, or more of the peptides. M, 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 may include incubating one or more APCs or one or more APC preparations at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, The method may include incubating with 7, 18, 19, or 20 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, methods can include injecting one or more APCs or one or more APC preparations with one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more peptides at concentrations 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, The method may include incubating at a concentration of 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. T cells

[0290] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. T cells are CD4 + T cells (helper T cells) and CD8 + T cells (cytotoxic T cells) and CD4 + T cells can assist other white blood cells in immune processes, including the maturation of B cells and the activation of cytotoxic T cells and macrophages. CD4 + T cells are activated when presented with peptide antigens 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 are also involved 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 somatic cell. Most T cells possess a T cell receptor (TCR). The ability of T cells to recognize antigens associated with various diseases (e.g., cancer) or infectious organisms is conferred by their TCRs, which are composed of both α and β chains or γ and δ chains. The proteins that make up these chains are encoded by DNA using a unique mechanism to generate TCR diversity. This multisubunit immune recognition receptor can associate with the CD3 complex and bind to peptides presented by MHC class I and II proteins on the surface of antigen-presenting cells (APCs). The first signal in T cell activation can be provided by binding of the T cell receptor to a short peptide presented by MHC on another cell. This ensures that only T cells with a TCR specific for that peptide are activated. Although B cells and macrophages can also be important APCs, the partner cells are typically professional antigen-presenting cells, typically dendritic cells, in the case of naive responses. Binding of the TCR to an antigenic peptide on an APC can be a central event in T cell activation, occurring at the immunological synapse, the interface between the T cell and the APC.

[0213]

[0291] Each TCR contains variable complementarity-determining regions (CDRs), as well as framework regions (FRs) and constant regions. The amino acid sequences of the loops of the third complementarity-determining regions (CDR3s) of the α and β chain variable domains, respectively, determine the majority of the sequence diversity of αβ T cells, resulting from recombination between Vβ (variable), Dβ (diversity), and Jβ (joining) gene segments within the β chain locus, and between similar Vα and Jα gene segments within the α chain locus. The presence of multiple such gene segments within the α and β chain loci of a TCR allows a large number of significantly different CDR3 sequences to be encoded. During TCR gene rearrangement, independent addition and deletion of nucleotides at Vβ-Dβ, Dβ-Jβ, and Vα-Jα junctions further increases the diversity of CDR3 sequences. In this respect, immune competence is reflected in TCR diversity. The γδ TCR differs significantly from the αβ TCR in that it encodes a receptor that interacts closely with the innate immune system. TCRγδ is expressed early during development, has a specialized anatomical distribution, unique pathogen and small molecule specificities, and interacts with a broad spectrum of innate and acquired cells. A biased pattern of TCRγ V and J segment expression is established early during ontogeny, when a restricted subset of TCRγδ cells colonizes diverse tissues prenatally.

[0214]

[0292] The T cells can be prepared according to methods known in the art. The T cells can be an enriched T cell preparation, an APC-depleted cell preparation, or a substantially purified T cell preparation. The T cells can be a mixed T cell population or a purified T cell subset. The T cells can be an enriched T cell preparation containing an expanded number or percentage of T cells relative to an isolated population of T cells.

[0215]

[0293] T cells or subsets of T cells can be obtained from various lymphoid tissues. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, thymus, tissue biopsies, tumors, lymph node tissue, gastrointestinal tract-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen tissue, lymphoid tissue, and tumors. As used herein, the term "peripheral blood lymphocytes" (PBLs) and its grammatical equivalents may refer to lymphocytes circulating in the blood (e.g., peripheral blood). Peripheral blood lymphocytes may refer to lymphocytes that are not localized in an organ. Peripheral blood lymphocytes may include T cells, NK cells, B cells, or any combination thereof.

[0216]

[0294] The method may include isolating T cells from a subject. The method may include obtaining T cells isolated from a subject. The T cells may be obtained from a T cell line. The T cells may be obtained from an autologous source. The T cells may be obtained from an allogeneic source. The T cells may also be obtained from a xenogeneic source, such as mouse, rat, non-human primate, and pig.

[0217]

[0295] The T cells may be an APC-depleted cell preparation. The T cells may be substantially free of APCs. For example, the T cells may comprise T cells separated from more than 75% APCs. In some embodiments, peripheral blood mononuclear cells (PBMCs) may be obtained from blood, for example, in a heparinized vial. The PBMCs may be separated from red blood cells by centrifugation, and the PBMCs may be collected from the interface. The collected PBMCs may optionally be washed (e.g., with PBS).

[0218]

[0296] Purification of T cells can be achieved by positive or negative selection, including, but not limited to, the use of antibodies directed against, for example, CD2, CD3, CD4, CD5, CD8, CD14, CD16, CD19, and / or CD25. + , CD4 + , CD8 + , CD45RA + , and / or CD45RO +Specific T cell subsets, such as T cells, can be isolated by positive or negative selection methods. + , CD14 - , and / or CD25 - T cells can be isolated by positive or negative selection methods. For example, CD45RA + , CD14 - , and / or CD25 - T cells can be isolated by positive or negative selection methods. For example, CD3 + , CD14 - , and / or CD25 - T cells can be isolated by positive or negative selection methods. For example, CD28 + , CD14 - , and / or CD25 - T cells can be isolated by positive or negative selection methods. For example, CD4 + , CD14 - , and / or CD25 - T cells can be isolated by positive or negative selection methods. For example, CD8 + , CD14 - , and / or CD25 - T cells can be isolated by positive or negative selection methods. For example, CD14 - and / or C. D25 - T cells can be isolated by negative selection methods, e.g., CD19 - T cells can be isolated by negative selection methods, e.g., CD16 - T cells can be isolated by negative selection methods. For example, CD3 + and CD28 +T cells can be positively selected using CD3 / CD28 conjugated magnetic beads. In one embodiment of the present invention, enrichment of a T cell population by negative selection can be achieved by a combination of antibodies directed against surface markers specific to the cells being negatively selected. For example, enrichment of a T cell population can be achieved by negative selection using antibodies directed against CD19, CD16, CD14, CD25, or any combination thereof. For example, enrichment of a T cell population can be achieved by negative selection using a combination of antibodies directed against CD19, CD16, CD25, and / or CD14.

[0219]

[0297] For example, a T cell sample can include cells derived from a subject's circulating blood and can be obtained by apheresis or leukapheresis. A T cell sample can contain T cells, monocytes, granulocytes, B cells, lymphocytes, including other nucleated white blood cells, red blood cells, and / or platelets. Undesired components of the T cell sample can be removed, and the remaining T cells can be resuspended in culture medium. For example, cells can be washed to remove the plasma fraction. For example, T cells can be isolated from peripheral blood lymphocytes by lysis of red blood cells and centrifugation through a PERCOLL™ gradient.

[0220]

[0298] Various embodiments herein provide compositions and methods comprising T cells. In some embodiments, the T cells comprise a TCR having a TCR α chain and a TCR β chain. In some embodiments, the T cells comprise a TCR having a TCR γ chain and a TCR δ chain. 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 +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 include naive CD4 + In some embodiments, the antigen-specific T cells include memory CD8 + In some embodiments, the antigen-specific T cells are naive CD8 + In some embodiments, the antigenic peptide sequence comprises a mutation selected from (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a read-through mutation, (E) a gene fusion mutation, and combinations thereof. In some embodiments, the antigenic peptide sequence binds to an HLA protein of interest with greater affinity than the corresponding wild-type peptide. In some embodiments, the antigenic peptide sequence binds to an HLA protein of interest 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 binds to a peptide-HLA complex 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 Combine with.

[0221]

[0299] In some embodiments, T cells are cultured in a medium containing cytokines. Exemplary cytokines include IL-7 and IL-15. In some embodiments, the cytokines in the T cell culture or medium are 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, or 16 ng / mL. In some embodiments, IL-7 in the T cell culture or medium has a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, IL-15 in the T cell culture or medium has a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the T cells are cultured in medium further containing FLT3L. In some embodiments, FLT3L in the T cell culture or medium has a final concentration of at least 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, or 200 ng / mL. In some embodiments, T cells are incubated, induced, or stimulated for a first period in medium containing FLT3L. In some embodiments, T cells are incubated, induced, or stimulated for a second period in medium containing additionally added FLT3L. In some embodiments, T cells are incubated, induced, or stimulated for a third period in medium containing further added FLT3L. In some embodiments, the T cells are incubated, induced, or stimulated in medium containing further added FLT3L for a fourth, fifth, or sixth period, with FLT3L being newly added within each period. antigen

[0300] The present disclosure relates to a method for producing T cells specific to an immunogenic antigen. The present disclosure also relates to a composition comprising antigen-specific T cells stimulated by APCs. In some embodiments, one or more antigen peptides are loaded into APCs, and the peptide-loaded APCs are then used to stimulate T cells to generate antigen-specific T cells. In some embodiments, the antigen is a neoantigen. In some embodiments, the APCs used for peptide loading are dendritic cells.

[0222]

[0301] In some embodiments, the peptide sequence comprises a mutation that is not present in the subject's non-cancer cells. In some embodiments, the peptide is encoded by a gene or expressed gene in the subject's cancer cells. In some embodiments, the peptide sequence has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 or more naturally occurring amino acids. In some embodiments, the peptide sequence binds to proteins encoded by class I HLA alleles and has a length of 8-12 naturally occurring amino acids. In some embodiments, the peptide sequence binds to proteins encoded by class II HLA alleles and has a length of 16-25 naturally occurring amino acids. In some embodiments, the peptide sequence comprises a plurality of antigenic peptide sequences. In some embodiments, the plurality of antigenic peptide sequences comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 antigenic peptide sequences. Includes.

[0223]

[0302] In some embodiments, the antigens described herein are neoantigens. Candidate immunogenic neoantigen sequences may be identified by any suitable method known in the art. The methods of the present disclosure may be useful, for example, to provide a specific treatment for a disease in a subject or to create a vaccine against a disease. Candidate immunogenic neoantigens may be neoantigens that have already been identified. In some embodiments, candidate immunogenic neoantigens may not yet have been identified. Candidate immunogenic neoantigens for use in the methods and compositions described herein may be specific to a subject. In some embodiments, candidate neoantigens for use in the methods and compositions described herein may be specific to multiple subjects.

[0224]

[0303] In both animals and humans, mutant epitopes can potentially be effective in inducing an immune response or activating T cells. In one embodiment, potentially immunogenic epitopes of an infectious agent in a subject, such as a virus, can be determined. In one embodiment, potentially immunogenic mutant epitopes of a subject with a disease, such as cancer, can be determined. In some embodiments, potentially immunogenic antigens or neo-antigens for use in the methods described herein can be differentiation antigens expressed in tumors and in cells of the tissue type from which they originate. In some embodiments, potentially immunogenic antigens or neo-antigens for use in the methods described herein can be cancer antigens / germline antigens that are not expressed in other differentiated tissues. In some embodiments, potentially immunogenic antigens or neo-antigens for use in the methods described herein can be mutant antigens. For example, candidate immunogenic antigenic or neo-antigenic peptides for use in the methods described herein may include antigens or neo-antigens of fusion proteins created through missense point mutations or tumor-specific translocations of gene segments. In some embodiments, potentially immunogenic antigens or neo-antigens for use in the methods described herein may be overexpressed antigens. In some embodiments, potentially immunogenic antigens or neo-antigens may be found within tumors. For example, potentially immunogenic antigens or neo-antigens for use in the methods described herein may include proteins whose expression is tightly regulated in cells of differentiated normal tissues.

[0225]

[0304] Potentially immunogenic mutant epitopes can be determined by genome or exome sequencing of tumor and healthy tissues from cancer patients using next-generation sequencing technology. For example, genes selected based on their mutation frequency and ability to act as antigens or neo-antigens can be sequenced using next-generation sequencing technology. In one embodiment, the sequencing data can be analyzed to identify potentially immunogenic mutant peptides that can bind to the HLA molecule of interest. In one embodiment, the data can be analyzed using a computer. In another embodiment, the sequence data can be analyzed for the presence of antigenic peptides or neo-antigenic peptides. In one embodiment, potentially immunogenic antigenic peptides or neo-antigenic peptides can be determined by their affinity for MHC molecules.

[0226]

[0305] Potentially immunogenic antigenic or neo-antigenic peptides can be determined by direct protein sequencing. For example, protein sequencing of enzymatic protein digests using multidimensional mass spectrometry (e.g., tandem mass spectrometry (MS / MS)) can be used to identify potentially immunogenic antigenic or neo-antigenic peptides for use in the methods described herein.

[0227]

[0306] High-throughput methods for de novo sequencing of unknown proteins can be used to identify potentially immunogenic antigenic or neo-antigenic peptides. For example, high-throughput methods for de novo sequencing of unknown proteins, such as meta-shotgun protein sequencing, can be used to analyze a subject's tumor to identify expressed, potentially immunogenic neoantigens.

[0228]

[0307] Potentially immunogenic antigenic or neo-antigenic peptides can also be identified using MHC multimers to identify antigen-specific T cell responses. For example, high-throughput analysis of antigen-specific T cell responses in patient samples can be performed using MHC tetramer-based screening methods. Tetramer-based screening methods can be used for the initial identification of potentially immunogenic tumor-specific antigens, or alternatively, as a secondary screening protocol to assess which potentially immunogenic antigens a patient has already been exposed to, thereby facilitating the selection of potentially immunogenic antigens for use in the methods described herein.

[0229]

[0308] In some embodiments, immune cells may be analyzed or characterized. For example, immune cells of a composition described herein may 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 of a stimulated immune cell sample; and determining binding of at least one immune cell of the stimulated immune cell sample to a peptide-MHC complex; wherein the expression determination and the binding determination are performed simultaneously. In some embodiments, the stimulated immune cell sample is a population of immune cells stimulated by APCs comprising peptide-MHC complexes. In some embodiments, the population of immune cells is derived from a biological sample. In some embodiments, the method may include incubating a population of immune cells derived from the biological sample with APCs comprising peptide-MHC complexes, thereby obtaining a stimulated immune cell sample; determining the expression of one or more cell markers of at least one immune cell of the stimulated immune cell sample; and determining binding of at least one immune cell of the stimulated immune cell sample to a peptide-MHC complex; wherein the expression determination and the binding determination are performed simultaneously. In some embodiments, the one or more cellular markers comprise TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, Granzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3, or any combination thereof. In some embodiments, the one or more cellular markers comprise a cytokine. In some embodiments, the one or more cellular markers comprise a degranulation marker. In some embodiments, the one or more cellular markers comprise a cell surface marker. In some embodiments, the one or more cellular markers comprise a protein. In some embodiments, determining binding of at least one immune cell of the stimulated immune cell sample to a peptide-MHC complex comprises determining binding of at least one immune cell of the stimulated immune cell sample to an MHC tetramer comprising the peptide and MHC of the peptide-MHC complex. In some embodiments, the MHC is a class I MHC or a class II MHC. In some embodiments, the peptide-MHC complex comprises one or more labels.In some embodiments, the population of immune cells derived from a biological sample comprises two or more samples, each comprising a population of immune cells derived from one or more biological samples. In some embodiments, the two or more samples are labeled with two or more sample labels. In some embodiments, determining expression and determining binding comprises fluorescence activated cell sorting (FACS). In some embodiments, determining expression and determining binding comprises single cell analysis. In some embodiments, determining expression and determining binding comprises determining the percentage of immune cells that express one or more cell markers and bind to the peptide-MHC complex. In some embodiments, the label comprises a fluorophore. In some embodiments, the population of immune cells comprises a population of immune cells representative of a population of immune cells of a composition described herein. In some embodiments, the antibody may be selected from the group consisting 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, CD3, CD28, CD4, CD8, or any antibody. Immune cell populations that express any combination and / or do not express CD14, CD19, CD16, CD25, or any combination thereof can be analyzed or characterized. For example, the method can include analyzing or characterizing specific T cell subpopulations, such as T cell subpopulations that express 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 do not express CD14, CD19, CD16, CD25, or any combination thereof. For example, the method can include analyzing or characterizing immune cell populations that do not express CD14, CD25, CD19, CD16, or any combination thereof.

[0230]

[0309] In some embodiments, the expression of one or more cell markers within the immune cell population may be determined. For example, the method may 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 can include incubating a population of immune cells derived from the biological sample with APCs comprising peptide-MHC complexes, thereby obtaining a stimulated immune cell sample; determining 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 of the stimulated immune cell sample; and determining binding of at least one immune cell of the stimulated immune cell sample to the peptide-MHC complex, wherein the determining of expression and the determining of binding are performed simultaneously.

[0231]

[0310] Potentially immunogenic antigenic or neo-antigenic peptides for use in the methods described herein can be known antigenic or neo-antigenic sequences. For example, potentially immunogenic antigenic or neo-antigenic peptides for use in the methods described herein can be derived from a database of antigenic or neo-antigenic sequences.

[0232]

[0311] In some aspects, the disclosure presents peptides or polynucleotides encoding peptides (e.g., peptides with tumor-specific mutations, viral peptides, or peptides associated with non-cancer diseases) identified using the methods described herein.

[0233]

[0312] In some embodiments, optical methods are used to select or identify immunogenic antigens. In some embodiments, barcoded probes are used to select or identify immunogenic antigens. In some embodiments, barcoded probes comprising a target-specific region and the barcoded region are used to select or identify immunogenic antigens. In some embodiments, the target-specific region comprises a nucleic acid sequence that hybridizes with or has at least about 90%, 95%, or 100% sequence complementarity to a nucleic acid sequence of a target polynucleotide.

[0234]

[0313] In some embodiments, a sequencing method is used to identify immunogenic antigens. Any suitable sequencing method, for example, next generation sequencing (NGS) technology, can be used in accordance with the present invention. In the future, third generation sequencing methods may replace NGS technology and accelerate the sequencing step of the method. For the sake of clarity, the term "next generation sequencing" or "NGS" in the context of the present invention differs from "traditional" sequencing methods, known as Sanger chemistry, in that it involves randomly and parallelly reading nucleic acid templates along the entire genome by dividing the genome into smaller pieces. This term refers to all high-throughput sequencing technologies. Such NGS technologies (also known as massively parallel sequencing technologies) can deliver nucleic acid sequence information for a whole genome, exome, transcriptome (all transcribed sequences of a genome), or methylome (all methylated sequences of a genome) in a very short time, for example, within 1-2 weeks, for example, within 1-7 days, or even within less than 24 hours, essentially enabling single-cell sequencing. In the context of the present invention, several NGS platforms that are commercially available or mentioned in the literature, such as the NGS platform described in detail in WO2012 / 159643, can be used.

[0235]

[0314] In certain embodiments, the antigenic peptides or neo-antigenic peptides, or epitopes thereof, are 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 The immunogenic antigen or epitope thereof may include, but is not limited to, 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 a specific embodiment, the immunogenic antigen or epitope thereof is 100 amino acids or less.

[0236]

[0315] In some embodiments, for MHC class I, the antigenic or neo-antigenic peptides or epitopes thereof are 13 residues or less in length, typically consisting of about 8 to about 11 residues, particularly 9 or 10 residues. In some embodiments, for MHC class II, the immunogenic antigenic or neo-antigenic peptides or epitopes thereof are 9 to 24 residues in length.

[0237]

[0316] Long immunogenic peptides can be designed in several ways. In some embodiments, when HLA-binding peptides are predicted or known, the long immunogenic peptide could consist of (1) individual binding peptides with 2-5 amino acid extensions to the N- and C-termini of each corresponding gene product; or (2) a concatenation of some or all of the binding peptides with the extension sequences, for each. In other embodiments, when sequencing reveals long (>10 residue) epitope sequences, such as neoepitopes present in tumors (e.g., due to frameshifts, readthrough, or intron inclusions resulting in novel peptide sequences), the long neoantigenic peptide could consist of the entire novel tumor-specific stretch of amino acids, either as a single long peptide or as several overlapping long peptides. In some embodiments, the use of long peptides is hypothesized to allow for endogenous processing by patient cells, resulting in more effective antigen presentation and induction of T cell responses. In some embodiments, two or more peptides may be used that overlap and align over a long neo-antigenic peptide.

[0238]

[0317] In some embodiments, the antigenic peptide or neo-antigenic peptide binds to an HLA protein (e.g., HLA class I or HLA class II). In specific embodiments, the antigenic peptide or neo-antigenic peptide binds to an HLA protein with greater affinity than the corresponding wild-type peptide. In specific embodiments, the antigenic peptide or neo-antigenic peptide has an IC of at least 5000 nM or less, at least 500 nM or less, at least 100 nM or less, or at least 50 nM or less. 50 or K D It has.

[0239]

[0318] In some embodiments, the antigenic peptide or neo-antigenic peptide can be about 8 to about 50 amino acid residues in length, or about 8 to about 30, about 8 to about 20, about 8 to about 18, about 8 to about 15, or about 8 to about 12 amino acid residues in length. The neo-antigenic peptide can be about 8 to about 500 amino acid residues in length, or about 8 to about 450, about 8 to about 400, about 8 to about 350, about 8 to about 300, about 8 to about 250, about 8 to about 200, about 8 to about 150, about 8 to about 100, about 8 to about 50, or about 8 to about 30 amino acid residues in length.

[0240]

[0319] In some embodiments, the antigenic peptide or neo-antigenic 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 or more amino acid residues in length. In some embodiments, the neo-antigenic peptides 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 antigenic peptide or neo-antigenic 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 length. In some embodiments, the antigenic peptide or neo-antigenic 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, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or fewer amino acid residues in length.

[0241]

[0320] In some embodiments, the antigenic peptide or neo-antigenic peptide has an overall length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

[0242]

[0321] In some embodiments, the antigenic peptide or neo-antigenic peptide has an overall length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids.

[0243]

[0322] In some embodiments, the neo-antigenic 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 neo-antigenic 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 neo-antigenic peptides may have a pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

[0244]

[0323] In some embodiments, an antigenic peptide or neo-antigenic peptide may have a binding affinity to HLA 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, an antigenic peptide or neo-antigenic peptide may have a binding affinity to HLA 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 antigenic peptide or neo-antigenic peptide may have a binding affinity for HLA of up to 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.

[0245]

[0324] In some embodiments, the antigenic peptides or neo-antigenic peptides described herein may comprise a carrier, such as a carrier known in the art, e.g., thyroglobulin, albumin, such as human serum albumin, tetanus toxin, polyamino acid residues such as poly-L-lysine, poly-L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, etc.

[0246]

[0325] In some embodiments, the antigenic peptides or neo-antigenic peptides described herein are acylated at the terminal NH2, e.g., by alkanoyl (C1-C 20 ) or thioglycolyl acetylation, amidation of the terminal carboxyl, e.g., with ammonia, methylamine, etc. In some embodiments, these modifications may provide sites for linkage to a support or other molecule.

[0247]

[0326] In some embodiments, the antigenic peptides or neo-antigenic peptides described herein may contain modifications, such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surface active materials, e.g., lipids, etc., or may be chemically modified, e.g., acetylated, etc. Furthermore, bonds within the peptide may be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0248]

[0327] In some embodiments, the antigenic peptides or neo-antigenic peptides described herein may contain substitutions that modify the physical properties (e.g., stability or solubility) of the resulting peptide. For example, an antigenic peptide or neo-antigenic peptide may be modified through the substitution of cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical nature, cysteine ​​has a tendency to form disulfide bridges and structurally alter the peptide sufficiently to reduce binding capacity. Substitution of C with α-aminobutyric acid not only alleviates this problem, but in certain cases may actually improve binding and cross-linking capacity. Substitution of cysteine ​​with α-aminobutyric acid may occur at any residue in the antigenic peptide or neo-antigenic peptide, for example, at anchor or non-anchor positions of an epitope or analog within the peptide, or at other positions in the peptide.

[0249]

[0328] In some embodiments, the antigenic peptides or neo-antigenic peptides described herein contain amino acid mimetics or unnatural amino acid residues, such as D-naphthylalanine or L-naphthylalanine; D-phenylglycine or L-phenylglycine; D-2-thienylalanine or L-2-thienylalanine; D-1, 2, 3, or 4-pyrenylalanine or L-1, 2, 3, or 4-pyrenylalanine; D-3-thienylalanine or L-3-thienylalanine; D-(2-pyridinyl)-alanine or L-(2-pyridinyl)-alanine. -pyridinyl)-alanine; D-(3-pyridinyl)-alanine or L-(3-pyridinyl)-alanine; D-(2-pyrazinyl)-alanine or L-(2-pyrazinyl)-alanine; D-(4-isopropyl)-phenylglycine or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-ρ-fluorophenylalanine; D-ρ-biphenyl-phenylalanine or L-ρ-biphenyl- Phenylanine; D-ρ-methoxybiphenylphenylalanine or L-ρ-methoxybiphenylphenylalanine; D-2-indole(allyl)alanine or L-2-indole(allyl)alanine; and D- or L-alkylalanine, where the alkyl group can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, isopentyl, or a non-acidic amino acid residue. Aromatic rings of unnatural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides containing various amino acid mimetics or unnatural amino acid residues are particularly useful because they tend to exhibit increased stability in vivo. Such peptides may also have improved shelf life or manufacturing properties.

[0250]

[0329] Peptide stability can be assayed in a number of ways. Various biological media, such as peptidases, human plasma, and human serum, have been used to examine 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 a 25% human serum (v / v) assay. The protocol is as follows: pooled human serum (type AB, non-heat-inactivated) is defatted by centrifugation prior to 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 6% aqueous trichloroacetic acid (TCA) or ethanol. The turbid reaction sample is cooled (4°C) for 15 minutes and then spun to pellet precipitated serum proteins. The presence of the peptide is then determined by reverse phase HPLC using stability specific chromatographic conditions.

[0251]

[0330] In some embodiments, the antigenic peptides or neo-antigenic peptides described herein may be in solution, lyophilized, or crystalline form.

[0331] In some embodiments, the antigenic peptides or neo-antigenic peptides described herein may be prepared synthetically, by recombinant DNA technology, or by chemical synthesis, or may be isolated from natural sources, such as naturally occurring tumors or pathogenic organisms. Epitopes may be synthesized separately or may be joined directly or indirectly within the peptide. The antigenic peptides or neo-antigenic peptides described herein are substantially free of other naturally occurring host cell proteins and fragments thereof, although in some embodiments, peptides may be synthetically conjugated to naturally occurring fragments or particles.

[0252]

[0332] In some embodiments, peptides can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols (see, e.g., Stewart and Young, "Solid Phase Peptide Synthesis," 2nd ed., Pierce Chemical Co., 1984). Additionally, individual peptides can be joined using chemical ligation to yield larger peptides, still within the scope of the present invention.

[0253]

[0333] Alternatively, a nucleotide sequence encoding the peptide may be inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression. Recombinant DNA techniques, such as those described herein, may also be used. These procedures are generally known in the art, as generally described in Sambrook et al., "Molecular Cloning," Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). Thus, recombinant peptides comprising or consisting of one or more of the epitopes described herein may be used to present appropriate T cell epitopes.

[0254]

[0334] In one aspect, the disclosure described herein also provides compositions comprising one, at least two, or more than two antigenic or neo-antigenic peptides. In some embodiments, the compositions described herein contain at least two significantly different peptides. In some embodiments, the at least two significantly different peptides are derived from the same polypeptide. By significantly different polypeptides, it is meant that the peptides vary in length, amino acid sequence, or both. The peptides are derived from any polypeptide known or found to contain tumor-specific mutations. In some embodiments, the isolated antigenic or neo-antigenic peptide is encoded by a gene with a point mutation that results in an amino acid substitution in the native peptide. Pharmaceutical Composition

[0335] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, including excipients and adjuvants, that facilitate the processing of the active agent into a pharmaceutically usable preparation. The appropriate formulation can depend on the chosen route of administration. Any of the well-known techniques, carriers, and excipients can be used as appropriate and as understood in the art.

[0255]

[0336] In some embodiments, the pharmaceutical composition is formulated as a cell-based therapy, e.g., a T cell therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapy, a nucleic acid-based therapy, an antibody-based therapy, and / or a cell-based therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapy or a nucleic acid-based therapy, wherein the nucleic acid encodes a polypeptide. In some embodiments, the pharmaceutical composition comprises an antibody-based therapy. The composition may comprise T cells specific for two or more immunogenic antigenic peptides or neo-antigenic peptides.

[0256]

[0337] In addition to the active ingredient, a pharmaceutical composition may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials will depend on the route of administration.

[0257]

[0338] Acceptable carriers, excipients, or stabilizers are those that are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 residues) 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 TWEEN®, PLURONICS®, or polyethylene glycol. (PEG), and other non-ionic surfactants.

[0258]

[0339] An acceptable carrier is physiologically acceptable to the patient to whom it is administered, and it preserves the therapeutic properties of the compound to which it is administered. Acceptable carriers and their formulations are generally described, for example, in "Remington's Pharmaceutical Sciences" (18th ed., A. Gennaro, Mack Publishing Co., Easton, PA 1990). One example of a carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting the compound of interest from one organ or part of the body at the site of administration to another organ or part of the body, or in an in vitro assay system. An acceptable carrier is compatible with the other ingredients of the formulation and is not toxic to the subject to which it is administered. An acceptable carrier should also not alter the specific activity of the neoantigen.

[0259]

[0340] In one aspect of the present specification, a pharmaceutically acceptable or physiologically acceptable composition is provided, which comprises a solvent (aqueous or non-aqueous), a solution, an emulsion, a dispersion medium, a coating, an isotonic agent, an absorption enhancer, or an absorption retarder, which is compatible with the administration of a pharmaceutical. Thus, a pharmaceutical composition or pharmaceutical preparation refers to a composition suitable for the use of a pharmaceutical in a subject. The composition can be formulated to be compatible with a specific administration route (i.e., a systemic route or a local route). Thus, the composition comprises a carrier, a diluent, or an excipient suitable for administration by various routes.

[0260]

[0341] In some embodiments, the composition may further comprise an acceptable additive to improve the stability of immune cells in the composition. Acceptable additives may not alter 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, which increase peptide stability and reduce gelling of the solution. Surfactants may be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0261]

[0342] Pharmaceutical compositions can be administered, for example, by injection. Injectable compositions include aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride can be included in the composition. The resulting solution may be packaged for immediate use or lyophilized; the lyophilized preparation may then be combined with a sterile solution prior to administration. For intravenous injection or injection at the affected site, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability. Those skilled in the art will appreciate that, for example, sodium chloride injection, Ringer's injection, lactated Ringer's injection It is possible to prepare a suitable solution using an isotonic medium such as a solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as required, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from the ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method can be vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients from the solution that has already been sterile-filtered.

[0262]

[0343] The composition can be conventionally administered intravenously, for example, by injection of a unit dose. For injection, the active ingredient can be in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has appropriate pH, isotonicity, and stability. For example, an isotonic vehicle such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection can be used to prepare a suitable solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as required. Additionally, the composition can be administered via aerosolization.

[0263]

[0344] When a composition is contemplated for use in medicine or any of the methods presented herein, it is envisioned that the composition may be substantially free of pyrogens so that the composition does not provoke an inflammatory or unsafe allergic reaction when administered to a human patient. Testing compositions for pyrogens and preparing compositions that are substantially pyrogen-free is well understood by those skilled in the art and can be achieved using commercially available kits.

[0264]

[0345] Acceptable carriers may contain compounds that stabilize, enhance, or delay absorption, or that increase or delay clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, or dextran; low molecular weight proteins; compositions that reduce peptide clearance or hydrolysis; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents may also be used to stabilize, enhance, or decrease absorption of pharmaceutical compositions containing liposomal carriers. To protect against digestion, the compound may be complexed with a composition that makes it resistant to acid and enzymatic hydrolysis, or may be complexed within a suitably resistant carrier, such as a liposome. Means for protecting compounds from digestion are known in the art (e.g., Fix (1996), Pharm Res., 13:17601764; Samanen (1996), J. Pharm. Pharmacol., 48:119135; and U.S. Pat. No. 5,391,377).

[0265]

[0346] Compositions may be administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The amount administered will depend on the subject being treated, capacity of the subject's immune system to utilize the active ingredient, and the desired degree of binding capacity. The precise amount of active ingredient required to be administered depends on the judgment of the health care practitioner and is peculiar to each individual. Suitable regimens for initial and booster administration are also variable, but typically include an initial administration followed by subsequent injections or other administrations at hourly or multiple hourly intervals. Alternatively, continuous intravenous infusion sufficient to maintain blood levels is contemplated.

[0266]

[0347] In some embodiments, the present invention is directed to immunogenic compositions, e.g., pharmaceutical compositions, capable of eliciting a neo-antigen-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic composition is a tumor-specific antigen or a tumor-specific neo-antigen. The present invention also includes neo-antigen therapeutic agents described herein (e.g., peptides, polynucleotides, TCRs, CARs, cells containing a TCR or CAR, dendritic cells containing a polypeptide, dendritic cells containing a polynucleotide, antibodies, etc.) corresponding to the above.

[0267]

[0348] 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.

[0349] In some embodiments, antigen polypeptides or polynucleotides can be presented as antigen-presenting cells (e.g., dendritic cells) containing such polypeptides or polynucleotides. In other embodiments, such antigen-presenting cells are used to stimulate T cells for use in a patient. In some embodiments, the antigen-presenting cells are dendritic cells. In rela...

Claims

1. 1. An ex vivo method for preparing cancer antigen-specific T cells suitable for T cell therapy in a human subject with cancer, the ex vivo method comprising: (a) CD25 ＋ depleting cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of depleted immune cells comprising a first population of APCs and T cells, wherein the population of immune cells is derived from a biological sample from a human subject with cancer; (b) treating the depleted population of immune cells for a first period of time with either (A) or (B) of the following: (A) a polypeptide comprising an epitope sequence of one or more cancer antigens expressed by cancer cells of a human subject with cancer; or (B) a polynucleotide encoding a polypeptide comprising an epitope sequence of one or more cancer antigens expressed by cancer cells of a human subject with cancer; to form a population of stimulated T cells; and (c) expanding the population of stimulated T cells, thereby forming an expanded population of cancer antigen-specific T cells; wherein expanding comprises: ＋ T cells or naive CD4 ＋ and expanding cancer antigen-specific T cells derived from the T cells; the expanded population of cancer antigen-specific T cells comprises at least 1 x 10 T cells specific for a complex comprising: (I) an epitope sequence of a first cancer antigen among the epitope sequences of one or more cancer antigens; and (II) an MHC protein expressed by a cancer cell or APC of the human subject with cancer; wherein: (i) Among the expanded population of cancer antigen-specific T cells, CD8 ＋ At least 0.1% of the T cells are naive CD8 T cells from the biological sample. ＋ derived from T cells, or (ii) among the expanded population of cancer antigen-specific T cells, CD4 ＋ At least 0.1% of the T cells are naive CD4 T cells from the biological sample. ＋ derived from T cells, The method, wherein steps (b) and (c) are carried out for less than 28 days.

2. CD8 in an expanded population of cancer antigen-specific T cells specific for the complex ＋ CD8 out of total T cells ＋ A fraction of T cells expressing CD8 in a biological sample specific for the complex ＋ CD8 out of total T cells ＋ The ex vivo method of claim 1 , wherein the fraction of cells is at least two times greater than the fraction of T cells.

3. CD4 in an expanded population of cancer antigen-specific T cells specific for the complex ＋ CD4 out of total T cells ＋ A fraction of T cells in a biological sample specific for the complex ＋ CD4 out of total T cells ＋ The ex vivo method of claim 1 , wherein the fraction of cells is at least two times greater than the fraction of T cells.

4. 2. The ex vivo method of claim 1, wherein the biological sample is peripheral blood mononuclear cells (PBMCs) from a human subject with cancer.

5. 2. The ex vivo method of claim 1, wherein the incubating step comprises incubating the depleted population of immune cells in the presence of a polynucleotide (B) encoding a polypeptide comprising an epitope sequence of one or more cancer antigens expressed by cancer cells of a human subject having cancer in the presence of FLT3L, wherein the polynucleotide encoding the polypeptide is RNA.

6. 6. The ex vivo method of claim 5, wherein the expanding further comprises contacting the population of stimulated T cells with a second population of mature APCs that have been incubated with FLT3L.

7. 7. The ex vivo method of claim 6, wherein the second population of mature APCs is incubated with FLT3L for at least one day before contacting the population of stimulated T cells with the second population of mature APCs.

8. 7. The ex vivo method of claim 6, wherein the expanding further comprises contacting the population of stimulated T cells with a third population of mature APCs, wherein the third population of mature APCs has been incubated with FLT3L prior to contacting the population of stimulated T cells with the third population of mature APCs.

9. 2. The ex vivo method of claim 1, wherein the polypeptide is 8 to 50 amino acids in length.

10. 2. The ex vivo method of claim 1, wherein each of the epitope sequences of the one or more cancer antigens contains a mutation and binds with greater affinity to an MHC protein expressed by cancer cells of the human subject with cancer than the corresponding wild-type epitope sequence.

11. 2. The ex vivo method of claim 1, wherein the epitope sequence of the one or more cancer antigens comprises epitope sequences of two or more cancer antigens expressed by cancer cells of a human subject with cancer.

12. The expanded population of cancer antigen-specific T cells comprises the following (A) and (B): (A) a first complex comprising: (I) an epitope sequence of a first cancer antigen among the epitope sequences of two or more cancer antigens; and (II) an MHC protein expressed by a cancer cell or APC of a human subject with cancer; and (B) a second complex comprising (I) an epitope sequence of a second cancer antigen among the epitope sequences of the two or more cancer antigens, and (II) an MHC protein expressed by a cancer cell or APC of a human subject with cancer; 12. The ex vivo method of claim 11, comprising T cells specific for the

13. 10. The ex vivo method of claim 1, wherein depleting comprises contacting the population of immune cells with a CD25-binding agent.

14. Depletion of CD14 from immune cell populations ＋ 10. The ex vivo method of claim 1, further comprising depleting the cells.

15. The expanded T cell population is at least 1 x 10 ６ Total CD8 ＋ T cells or at least 1 x 10 ６ Total CD4 ＋ 10. The ex vivo method of claim 1, comprising T cells.

16. 10. The ex vivo method of claim 1, wherein the ex vivo method further comprises harvesting the expanded population of cancer antigen-specific T cells, cryopreserving the expanded population of cancer antigen-specific T cells, or preparing a pharmaceutical composition for use in treating cancer comprising the expanded population of cancer antigen-specific T cells.

17. 2. The ex vivo method of claim 1, wherein the ex vivo method further comprises stimulating the population of one or more APCs with one or more cytokines or growth factors, thereby obtaining one or more APC preparations comprising mature APCs, and wherein expanding comprises contacting the stimulated population of T cells with one or more APC preparations comprising mature APCs.

18. 18. The ex vivo method of claim 17, 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.

19. CD8 specific for at least one epitope sequence of one or more cancer antigens ＋ The number of T cells is at least about 1 x 10 ６ , 2 × 10 ６ , 5 x 10 ６ , 1 x 10 ７ , 2 × 10 ７ , 5 x 10 ７ , 1×10 ８ , 2 × 10 ８ , or 5 x 10 ８ The ex vivo method of claim 1 , wherein the number of cells is 1 or more.

20. At least one of the epitope sequences of the one or more cancer antigens binds to a protein encoded by an HLA allele expressed by the subject with an IC of less than 500 nM. ５０ The ex vivo method of claim 1, wherein the binding is

21. 2. The ex vivo method of claim 1, wherein at least one of the epitope sequences of the one or more cancer antigens is encoded by an expressed gene in the subject's cancer cells and contains a mutation that is not present in the subject's non-cancer cells.

22. The ex vivo method of claim 1, further comprising simultaneously analyzing (i) binding of the expanded population of cancer antigen-specific T cells to peptide-MHC complexes of a cell sample, and (ii) determining the expression in the cell sample of one or more 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, and CD16.

23. The ex vivo method according to any one of claims 19 to 22, wherein the obtained cancer antigen-specific T cells are used for the manufacture of a medicament for use in treatment, and the treatment is for cancer.

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