T cell preparation compositions and methods

Abstract

Highly specific and beneficial T cell therapies can be provided by the generation of antigen-specific T cells by controlled ex vivo induction or expansion. The present disclosure provides T cell preparation methods and therapeutic T cell compositions useful for antigen-specific T cell therapy in subjects, individuals having cancer and other conditions, diseases, and conditions.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080050310.8, filed on May 7, 2020, entitled "Composition and Method for T Cell Preparation" (the corresponding PCT application was filed on May 7, 2020, and has the application number PCT / US2020 / 031898).

[0002] Cross-referencing

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 845,251, filed May 8, 2019, which is incorporated herein by reference in its entirety. Background Technology

[0004] Tumor vaccines typically consist of tumor antigens and immunostimulatory molecules (such as adjuvants, cytokines, or TLR ligands) that work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. These vaccines contain a mixture of common tissue-restricted tumor antigens or patient-specific antigens in the form of common antigens or whole tumor cell products. Common tissue-restricted tumor antigens are ideal immunogenic proteins selectively expressed in tumors in many individuals and are typically delivered to patients as synthetic peptides or recombinant proteins. In contrast, whole tumor cell products are delivered to patients as autologous irradiated cells, cell lysates, cell fusions, heat shock protein products, or total mRNA. Because whole tumor cells are isolated from an autologous patient, these cells can include both patient-specific tumor antigens and common tumor antigens. Finally, there is a third class of tumor antigens—neoantigens rarely used in vaccines—that consist of proteins with tumor-specific mutations (which can be patient-specific or common) that cause changes in their amino acid sequences. These mutant proteins are: (a) unique to tumor cells as mutations, and their corresponding proteins are found only in tumors; (b) avoid central tolerance and are therefore more likely to be immunogenic; and (c) provide excellent targets for immune recognition, including recognition by humoral and cellular immunity.

[0005] Adoptive immunotherapy, or adoptive cell therapy (ACT), involves transferring lymphocytes to a subject to treat a disease. Adoptive immunotherapy has not yet realized its potential to treat a wide range of diseases, including cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiency. However, most (if not all) adoptive immunotherapies require T-cell activation and expansion steps to generate a clinically effective therapeutic dose of T cells. Current technologies for generating therapeutic doses of T cells (including engineered T cells) remain limited by cumbersome T-cell preparation processes due to the inherent complexity of live cell culture and patient-to-patient variability. Existing T-cell preparation processes are not easily scaled up, are not reproducible, are unreliable, or are inefficient, and often produce inferior T-cell products that may be easily depleted and lose effector immune cell function. To date, engineered T-cell adoptive immunotherapy has achieved only limited success and routinely exhibits variable clinical activity. Therefore, this type of therapy is not suitable for widespread clinical use. Thus, there remains a need to develop compositions and methods for expanding and inducing antigen-specific T cells with favorable phenotypes and functions. Summary of the Invention

[0006] This disclosure provides novel and improved T-cell therapeutic agents for clinical development and application. While autologous T-cell therapeutic agents are generally safe to use, significant improvements are needed to meet treatment standards, and development in this field is both rapid and challenging. A previously published application by the applicant (WO2019 / 094642) represents a landmark advancement in compositions and methods for cancer T-cell therapy. This application stems from a surprising discovery that consuming certain cells expressing specific markers at different stages of ex vivo immune cell preparation yields highly immunogenic cellular compositions. This disclosure also stems in part from the discovery of novel and improved methods for antigen stimulation, leading to improved cellular compositions for therapeutic agent development. Novel methods and compositions are provided herein, wherein, at least in part, the selective consumption of certain immune cells from ex vivo stimulation and cell expansion environments provides novel therapeutic compositions and improved methods.

[0007] This article provides an improved ex vivo method for preparing tumor antigen-specific T cells, comprising: consuming CD14+ cells and / or CD25+ cells from an immune cell population containing antigen-presenting cells (APCs) and T cells to form a CD14 and / or CD25-depleted immune cell population containing APCs and T cells, wherein the immune cell population is derived from a biological sample of a human subject; and incubating the CD14 and / or CD25-depleted immune cell population containing the first population of APCs and T cells for a first time period in the presence of: FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) containing at least one substance expressed by cancer cells from a human subject with cancer. (a) a polypeptide containing a tumor antigen epitope sequence, or (b) a polynucleotide encoding the polypeptide; thereby forming a cell population comprising stimulated T cells; expanding the cell population comprising stimulated T cells to form an expanded cell population comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific to a complex comprising: (i) at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by cancer cells or APCs of the human subject in (b)(ii); and administering the expanded cell population comprising tumor antigen-specific T cells to the human subject, wherein the expanded cell population comprising tumor antigen-specific T cells comprises 1 x 102 8 Up to 1x10 11 Total cells.

[0008] This article provides an improved ex vivo method for preparing tumor antigen-specific T cells, comprising: consuming CD14+ cells and / or CD25+ cells from an immune cell population containing antigen-presenting cells (APCs) and T cells to form a CD14 and / or CD25-depleted immune cell population containing APCs and T cells, wherein the immune cell population is derived from a biological sample of a human subject; and incubating the CD14 and / or CD25-depleted immune cell population containing the first population of APCs and T cells for a first time period in the presence of: FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) a polypeptide containing at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a cell population containing stimulated T cells. Cell population; expanding the cell population containing stimulated T cells to form an expanded cell population containing tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific to a complex comprising: (i) at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by cancer cells or APCs of the human subject in (b)(ii); and administering the expanded cell population containing tumor antigen-specific T cells to the human subject, wherein the human subject: has unresectable melanoma, has previously received a PD-1 inhibitor or PD-L1 inhibitor and a regimen containing a CTLA-4 inhibitor and has disease progression, or has received or is currently receiving a PD-1 inhibitor or PD-L1 inhibitor for at least 3 months and has stable disease or disease progression without symptoms.

[0009] This article provides an improved ex vivo method for preparing tumor antigen-specific T cells, comprising: consuming CD14+ cells and / or CD25+ cells from an immune cell population containing antigen-presenting cells (APCs) and T cells to form a CD14 and / or CD25-depleted immune cell population containing APCs and T cells, wherein the immune cell population is derived from a biological sample of a human subject; and incubating the CD14 and / or CD25-depleted immune cell population containing the first population of APCs and T cells for a first time period in the presence of FMS-like tyrosine kinase 3 receptor. The ligand (FLT3L) and mRNA encoding a polypeptide comprising at least two different tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer; thereby forming a cell population comprising stimulated T cells; and expanding the cell population comprising stimulated T cells to form an expanded cell population comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific to a complex comprising: (i) the at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by cancer cells or APCs of the human subject (b)(ii).

[0010] This article provides an improved ex vivo method for preparing tumor antigen-specific T cells, comprising: consuming CD14+ cells and / or CD25+ cells from: (i) directly from washed and / or cryopreserved peripheral blood mononuclear cell (PBMC) samples from human subjects; (ii) from PBMC samples from human subjects containing a percentage of immature dendritic cells (DCs) approximately the same as the percentage of immature DCs in the peripheral blood of the human subject; (iii) from PBMC samples from human subjects containing a percentage of mature DCs approximately the same as the percentage of mature DCs in the peripheral blood of the human subject; and (iv) from human subjects... (v) PBMC samples from human subjects, wherein the ratio of immature DCs to mature DCs is approximately the same as that in the peripheral blood of the human subjects; (vi) PBMC samples from human subjects, wherein the ratio of immature DCs to mature DCs is approximately the same as that in the peripheral blood of the human subjects; (vii) PBMC samples from human subjects, wherein the ratio of APCs to total cell population is approximately the same as that in the peripheral blood of the human subjects; (vii) PBMC samples from human subjects, wherein the ratio of DCs to total cell population is approximately the same as that in the peripheral blood of the human subjects; (viii) PBMC samples from human subjects, wherein the ratio of DCs to total cell population is approximately the same as that in the peripheral blood of the human subjects; (ix) PBMC samples from human subjects in which the percentage of CD303+ cells in the total cell population is approximately the same as the percentage of CD303+ cells in the total cell population in the peripheral blood of the same human subject; (x) PBMC samples from human subjects in which the percentage of CD141+ cells in the total cell population is approximately the same as the percentage of CD141+ cells in the total cell population in the peripheral blood of the same human subject; or (xi) PBMC samples from human subjects in which CD19+ cells in the total cell population... The percentage of cells is approximately the same as the percentage of CD19+ in the total cell population in the peripheral blood of the human subject; thereby forming a CD14 and / or CD25 depleted PBMC population comprising APCs and a first population of T cells; and (b) incubating the CD14 and / or CD25 depleted immune cell population comprising APCs and a first population of T cells for a first time period in the presence of: FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a cell population comprising stimulated T cells;And to expand the cell population containing the stimulated T cells to form an expanded cell population containing tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific to a complex comprising: (i) at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by cancer cells or APCs of the human subject in (b)(ii).

[0011] In some embodiments, the method further includes administering the expanded cell population containing tumor antigen-specific T cells to the human subject.

[0012] In some embodiments, incubation includes incubating the CD14 and / or CD25 depleted immune cell population comprising a first population of APCs and T cells for a first time period in the presence of: (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) mRNA encoding a polypeptide comprising at least two different tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer.

[0013] In some embodiments, electroporation or nuclear transfection is introduced. In some embodiments, the electroporation or nuclear transfection is performed without separating the APCs and T cells from the first population of T cells from step (a).

[0014] In some embodiments, the method further includes administering the expanded cell population containing tumor antigen-specific T cells to the human subject. In some embodiments, incubation includes incubating the CD14 and / or CD25-depleted immune cell population containing a first population of APCs and T cells for a first time period in the presence of: (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) mRNA encoding a polypeptide containing at least two different tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer.

[0015] In some embodiments, the mRNA includes a 5' cap. In some embodiments, the 5' cap is CAP-1. In some embodiments, the mRNA includes a 3' poly-A tail. In some embodiments, the poly-A tail is 120 to 135 nucleotides in length. In some embodiments, a first tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences is linked to a second tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences via a linker sequence. In some embodiments, the 5' cap is operatively linked to a sequence encoding the at least two different tumor antigen epitope sequences via a linker sequence. In some embodiments, the at least two different tumor antigen epitope sequences are expressed as a single polypeptide chain. In some embodiments, incubation includes incubating a CD14 and / or CD25 depleted immune cell population containing APCs and a first T cell population in the presence of LPS and IFNγ.

[0016] In some embodiments, the length of each of the at least two different tumor antigen epitope sequences is 8 to 12 amino acids. In some embodiments, the length of each of the at least two different tumor antigen epitope sequences is 15 to 25 amino acids. In some embodiments, the polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or more different tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer.

[0017] In some embodiments, the expanded cell population comprising tumor antigen-specific T cells comprises 1x102 8 Up to 1x10 11 Total cells. In some embodiments, the expanded cell population comprising tumor antigen-specific T cells comprises 1 x 102 cells. 8 Up to 1x10 11 One CD3+ cell.

[0018] In some embodiments, the human subject has unresectable melanoma. Unlike resectable melanoma, tumor-infiltrating lymphocytes (TILs) cannot be obtained from unresectable melanoma; therefore, TILs cannot be used to treat unresectable melanoma. One advantage of the methods and compositions provided herein is that they can be used to treat unresectable melanoma.

[0019] In some implementations, human subjects had previously received PD-1 inhibitors or PD-L1 inhibitors and regimens containing CTLA-4 inhibitors and had experienced disease progression.

[0020] In some implementation schemes, human subjects have received or are currently receiving PD-1 inhibitors or PD-L1 inhibitors for at least 3 months and have stable conditions or asymptomatic disease progression.

[0021] In some embodiments, the percentage of CD3+ cells in the expanded cell population containing tumor antigen-specific T cells is at least 40%, 50%, or 60% of the total cell population.

[0022] In some embodiments, the percentage of CD107a+ cells in the expanded cell population containing tumor antigen-specific T cells is at least 10% of the tumor antigen-specific T cell population.

[0023] In some embodiments, the percentage of TNFα+ cells in the expanded cell population containing tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population.

[0024] In some embodiments, the percentage of IFNγ+ cells in the expanded cell population containing tumor antigen-specific T cells is at least 15% of the tumor antigen-specific T cell population.

[0025] In some embodiments, the percentage of TNFα+ and IFNγ+ cells in the expanded cell population containing tumor antigen-specific T cells is at least 2% of the tumor antigen-specific T cell population.

[0026] In some embodiments, the percentage of TNFα+ and CD107a+ cells in the expanded cell population containing tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cell population.

[0027] In some embodiments, the percentage of IFNγ+ and CD107a+ cells in the expanded cell population containing tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population.

[0028] In some embodiments, the percentage of TNFα+, IFNγ+, and CD107a+ cells in the expanded cell population comprising tumor antigen-specific T cells is at least 0.1% of the tumor antigen-specific T cell population.

[0029] In some embodiments, the percentage of CD4+ T cells (CD62L+ and CD45RA+) in the expanded cell population containing tumor antigen-specific T cells is at most 15%.

[0030] In some embodiments, the percentage of CD4+ T cells (CD62L- and CD45RA-) as effector memory T cells in the expanded cell population comprising tumor antigen-specific T cells is at least 60%.

[0031] In some embodiments, the percentage of CD4+ T cells (CD62L- and CD45RA+) as effector T cells in the expanded cell population containing tumor antigen-specific T cells is at most 5%.

[0032] In some embodiments, the percentage of CD4+ T cells (CD62L+ and CD45RA-) as central memory T cells in the expanded cell population containing tumor antigen-specific T cells is at least 10%.

[0033] In some embodiments, the percentage of CD8+ T cells as naive T cells (CD62L+CD45RA+) in the expanded cell population containing tumor antigen-specific T cells is at most 25%.

[0034] In some embodiments, the percentage of CD8+ T cells (CD62L-CD45RA-) that are effector memory T cells in the expanded cell population containing tumor antigen-specific T cells is at least 60%.

[0035] In some embodiments, the percentage of CD8+ T cells (CD62L-CD45RA+) as effector T cells in the expanded cell population containing tumor antigen-specific T cells is at most 10%.

[0036] In some embodiments, the percentage of CD8+ T cells (CD62L+CD45RA-) as central memory T cells in the expanded cell population comprising tumor antigen-specific T cells is at least 15%.

[0037] In some embodiments, the expanded cell population comprising tumor antigen-specific T cells produces cytokines and causes degranulation upon recognition of target cells.

[0038] In some implementations, the human subjects are refractory to anti-checkpoint inhibitor therapy.

[0039] In some implementations, the human subjects are between 18 and 75 years old.

[0040] In some implementations, the human subject has a mutation in the BRAF gene and has previously received a B-raf inhibitor or a B-raf / MEK combination therapy.

[0041] In some implementations, consumption includes consuming CD14+ and CD25+ cells from peripheral blood mononuclear cell (PBMC) samples from human subjects that have not yet undergone the step of monocyte maturation into mature dendritic cells (DCs).

[0042] In some implementations, consumption further includes consuming CD11b+ cells from a peripheral blood mononuclear cell (PBMC) sample from a human subject, which has not yet undergone the step of monocyte maturation into mature dendritic cells (DCs).

[0043] In some implementations, steps (b) and (c) are performed in less than 28 days.

[0044] In some embodiments, the proportion of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in the expanded cell population containing tumor antigen-specific T cells is at least twice the proportion of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in the biological sample.

[0045] In some embodiments, the proportion of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in the expanded cell population containing tumor antigen-specific T cells is at least twice the proportion of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in the biological sample.

[0046] In some implementations, at least 0.1% of the CD8+ T cells in the expanded cell population containing tumor antigen-specific T cells are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells.

[0047] In some implementations, at least 0.1% of the CD4+ T cells in the expanded cell population containing tumor antigen-specific T cells are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

[0048] In some embodiments, the expansion includes (A) contacting a cell population containing stimulated T cells with a second mature APC population, wherein the second mature APC population (i) has been incubated with FLT3L and (ii) presents at least one tumor antigen epitope sequence; and (B) expanding the cell population containing stimulated T cells for a second time period to form an expanded T cell population.

[0049] In some implementations, the second mature APC population has been incubated with FLT3L for at least 1 day before the cell population containing stimulated T cells is brought into contact with the second mature APC population.

[0050] In some embodiments, the biological sample is a peripheral blood sample, a leukapheresis sample, or an apheresis sample.

[0051] In some embodiments, the method further includes harvesting the expanded cell population containing tumor antigen-specific T cells, cryopreserving the expanded cell population containing tumor antigen-specific T cells, or preparing a pharmaceutical composition containing the expanded cell population containing tumor antigen-specific T cells.

[0052] In some embodiments, incubation includes incubating a first time period of a CD14 / CD25-depleted immune cell population containing APCs and a first population of T cells in the presence of FLT3L and RNA encoding the polypeptide.

[0053] In some implementations, the human subject with cancer is a human subject from whom the biological sample was obtained.

[0054] In some embodiments, the polypeptide has a length of 8 to 50 amino acids.

[0055] In some embodiments, the polypeptide comprises at least two tumor antigen epitope sequences, each expressed by cancer cells from a human subject with cancer.

[0056] In some implementations, consuming CD14+ cells and / or CD25+ cells from an immune cell population containing APCs and a first population of T cells includes contacting the immune cell population containing APCs and a first population of T cells with a CD14 binder and / or a CD25 binder.

[0057] In some implementations, consumption further includes consuming CD19+ cells from an immune cell population comprising APCs and a first population of T cells.

[0058] This article provides an ex vivo method for preparing tumor antigen-specific T cells, comprising: consuming CD11b+ cells from an immune cell population containing antigen-presenting cells (APCs) and T cells to form a CD11b-depleted immune cell population containing APCs and a first population of T cells, wherein the immune cell population is derived from a biological sample of a human subject; and incubating the CD11b-depleted immune cell population containing APCs and the first population of T cells for a first time period in the presence of: FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) containing (a) At least one polypeptide containing a tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (b) a polynucleotide encoding the polypeptide; thereby forming a cell population comprising stimulated T cells; and expanding the cell population comprising stimulated T cells to form an expanded cell population comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific to a complex comprising: (i) the at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by cancer cells or APCs of the human subject of (b)(ii).

[0059] This article provides a pharmaceutical composition comprising an expanded cell population containing tumor antigen-specific T cells generated by the methods described herein; and a pharmaceutically acceptable carrier.

[0060] This document provides a pharmaceutical composition comprising: (a) a population of immune cells derived from a biological sample, wherein the immune cell population comprises antigen-presenting cells (APCs) stimulated T cells, the T cells comprising T cell receptors (TCRs) specific to the epitopes of the polypeptide, wherein (i) the number of immune cells expressing CD11b in the immune cell population is proportionally lower than the number of immune cells expressing CD11b in the biological sample, and / or (ii) the number of immune cells expressing CD11c in the immune cell population is proportionally higher than the number of immune cells expressing CD11c in the biological sample; and (b) a pharmaceutically acceptable excipient.

[0061] This article provides a pharmaceutical composition comprising: (a) a population of immune cells derived from a biological sample, wherein the population of immune cells comprises antigen-presenting cells (APCs) stimulated T cells, the T cells comprising T cell receptors (TCRs) specific to the epitopes of peptides, wherein the APC-stimulated T cells have been incubated with cytokines; (b) the cytokines; and (c) a pharmaceutically acceptable excipient.

[0062] This article provides a pharmaceutical composition comprising: (a) a population of immune cells from a biological sample of a subject who has been administered fms-like tyrosine kinase 3 ligand (FLT3L), wherein the immune cell population comprises antigen-presenting cells (APCs) stimulated T cells, the T cells comprising T cell receptors (TCRs) specific to the epitopes of the peptide; and (b) a pharmaceutically acceptable excipient.

[0063] In some implementations, the immune cell population is derived from a biological sample of the subject.

[0064] In some implementations, the immune cell population is derived from biological samples of subjects who have been administered fms-like tyrosine kinase 3 ligand (FLT3L).

[0065] In some embodiments, the APC-stimulated T cells have been incubated with cytokines, and the pharmaceutical composition further comprises cytokines.

[0066] In some embodiments, the number of immune cells expressing CD11b in the immune cell population is proportionally lower than the number of immune cells expressing CD11b in the biological sample.

[0067] In some embodiments, the number of immune cells expressing CD11c in the immune cell population is proportionally higher than the number of immune cells expressing CD11c in the biological sample.

[0068] In some implementations, the number of CD14-expressing immune cells in the population is proportionally lower than the number of CD14-expressing immune cells in the biological sample.

[0069] In some implementations, the number of CD25-expressing immune cells in the population is proportionally lower than the number of CD25-expressing immune cells in the biological sample.

[0070] In some implementations, the number of CD19-expressing immune cells in the population is proportionally lower than the number of CD19-expressing immune cells in the biological sample.

[0071] In some embodiments, the APC is an APC stimulated by FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

[0072] In some implementations, the APC-stimulated T cells are APC-stimulated T cells that are stimulated with FLT3L.

[0073] In some implementations, the cytokine is IL-7, IL-15, or IL-21.

[0074] In some embodiments, the APC-stimulated T cells comprise T cells stimulated by an APC loaded with an antigen that presents an epitope on an MHC class I or MHC class II molecule.

[0075] In some embodiments, the antigen-loaded APCs include plasma cell-like dendritic cells (pDCs), CD11c+DCs, CD1c+DCs, or CD141+DCs.

[0076] In some embodiments, the CD11b cells comprise CD16+ mononuclear cells.

[0077] In some embodiments, the pharmaceutical composition further comprises agents that promote and maintain cell growth in vitro, including growth factors, cytokines, amino acids, supplements, or combinations thereof.

[0078] In some embodiments, the number of immune cells expressing CD1c in the immune cell population is proportionally higher than the number of immune cells expressing CD1c in the biological sample.

[0079] In some embodiments, the amount of immune cells or APCs expressing CD141 in the immune cell population is proportionally higher than the amount of immune cells or APCs expressing CD141 in the biological sample.

[0080] In some embodiments, the cell population containing the antigen-loaded APC comprises more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 60%, or more than 70% CD11c+ cells.

[0081] In some embodiments, the APC-stimulated T cells comprise T cells stimulated by a cell population containing less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% CD11b+ cells.

[0082] In some embodiments, the APC-stimulated T cells comprise T cells stimulated by a cell population containing more than 90% CD11c+ cells.

[0083] In some embodiments, the pharmaceutical composition described herein comprises T cells stimulated by a cell population containing more than 70% neoantigen peptide-expressing cells, namely CD11c+, CD1lc+, or CD141+ cells.

[0084] In some embodiments, the pharmaceutical composition contains at least 60% T cells that are specific to the epitope.

[0085] In some embodiments, the pharmaceutical compositions described herein contain a higher proportion of naive T cells that are induced or converted into neoantigen-inducing T cells, compared to cell compositions obtained by contacting isolated T cells with antigen-loaded APCs without reducing or consuming CD11b+ and / or CD19+ cells.

[0086] In some embodiments, the pharmaceutical composition described herein comprises more than 35% naive T cells, which are induced or converted into antigen-specific activated T cells that are specific to the epitope.

[0087] In some embodiments, the pharmaceutical compositions described herein contain a higher proportion of cancer neoantigen-specific CD8+ T cells compared to cell compositions obtained by contacting isolated T cells with antigen-loaded APCs without reducing or consuming CD11b+ and / or CD19+ cells.

[0088] In some embodiments, the pharmaceutical composition described herein comprises at least 30% CD8+ T cells.

[0089] In some embodiments, the pharmaceutical compositions described herein contain a higher proportion of memory T cells compared to cell compositions obtained by contacting isolated T cells with antigen-loaded APCs without reducing or consuming CD11b+ and / or CD19+ cells.

[0090] This article provides a method for treating cancer in a subject in need, comprising administering the pharmaceutical composition described herein to the subject.

[0091] This article provides a method for preparing T cells containing T cell receptors (TCRs) specific to an epitope of a polypeptide, the method comprising (a) consuming CD11b-expressing cells from an immune cell population comprising antigen-presenting cells and T cells to form a CD11b-depleted immune cell population containing T cells; and (b) incubating or expanding the CD11b-depleted immune cell population containing T cells; wherein memory T cells containing TCRs specific to the epitope are expanded, or naive T cells containing TCRs specific to the epitope are induced.

[0092] This article provides a method for preparing T cells containing epitope-specific T cell receptors (TCRs), the method comprising (a) enriching an immune cell population containing APCs and T cells against cells expressing CD11c, thereby forming a CD11c-enriched immune cell population containing T cells; and (b) incubating or expanding the CD11c-enriched immune cell population containing T cells; wherein memory T cells containing epitope-specific TCRs are expanded, or naive T cells containing epitope-specific TCRs are induced. In some embodiments, the APC preparation method comprises APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

[0093] In some embodiments, the method further includes preparing an APC article.

[0094] In some embodiments, the method for preparing the APC article includes incubating APC with FLT3L.

[0095] In some embodiments, the method for preparing the APC article includes incubating the APC with the polypeptide or a polynucleotide encoding the polypeptide.

[0096] This article provides a method for treating cancer in a subject in need, comprising administering to the subject a population of immune cells from a biological sample, wherein the population of immune cells comprises T cells stimulated by antigen-presenting cells (APCs), the T cells comprising T cell receptors (TCRs) specific to antigen peptide sequences, and wherein the subject has been administered fms-like tyrosine kinase 3 ligand (FLT3L).

[0097] This article provides a method for treating cancer in a subject in need, comprising: (a) administering an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) to the subject; and (b) administering to the subject a population of immune cells from a biological sample, wherein the population of immune cells comprises T cells stimulated by antigen-presenting cells (APCs) and the T cells comprise T cell receptors (TCRs) that are specific to antigen peptide sequences.

[0098] This article provides a method for treating cancer in a subject in need, comprising: (a) administering to the subject a population of immune cells derived from a biological sample, wherein the population of immune cells comprises T cells stimulated by antigen-presenting cells (APCs) and the T cells comprise T cell receptors (TCRs) specific to an antigenic peptide sequence; and (b) administering to the subject a polypeptide comprising the antigenic peptide sequence or a polynucleotide encoding the antigenic peptide sequence.

[0099] In some embodiments, the method further includes administering an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) to the subject prior to the administration of the immune cell population. Attached Figure Description

[0100] Figure 1A An example schematic diagram depicts an antigen-specific T cell preparation protocol.

[0101] Figure 1B An example schematic diagram depicts an antigen-specific T cell preparation protocol.

[0102] Figure 1C An example alternative schematic diagram depicts an antigen-specific T cell preparation protocol.

[0103] Figure 2 Example results are depicted, showing antigen-specific CD8 induced by long or short peptides. + The fraction of memory T cells. “Bulk” indicates that the sample containing T cells for induction is a complete peripheral blood mononuclear cell (PBMC). “Treg” indicates that the sample containing T cells for induction is a PBMC depleted of CD25-expressing cells.

[0104] Figure 3 An example flow cytometry analysis was described, showing antigen-specific CD8 induced by GAS 7 peptide. + The score of naive T cells.

[0105] Figure 4 Example results are depicted, showing antigen-specific CD8. + T cell responses to HIV short peptides, previously identified short neoantigens (PINs), or long PIN peptide pools. "Complete PBMC" indicates that the sample containing T cells used for induction is a complete PBMC. "CD25 PBMC" indicates that the sample containing T cells used for induction has depleted CD25+ cells. Short, short peptides, or shortmers; long, long peptides, or longmers.

[0106] Figure 5A The antigen-specific CD8 was depicted under the conditions shown. + An example flow cytometry analysis of the response of naive T cells to a single previously identified neoantigen (PIN).

[0107] Figure 5B The antigen-specific CD8 was depicted under the conditions shown. + An example flow cytometry analysis of the response of naive T cells to a single previously identified neoantigen (PIN).

[0108] Figure 6Example results are depicted, showing antigen-specific CD8 using PBMC samples from two human donors. + The response of T cells to the peptide shown.

[0109] Figure 7 The study depicted antigen-specific CD8 in healthy donors before stimulation and after up to three rounds of stimulation. + Example flow cytometry plot of T cell response to the mutated epitope shown.

[0110] Figure 8A An exemplary bar chart is depicted, showing the antigen-specific memory CD8. + The result of T cell response to viral antigens. After up to three rounds of stimulation, all CD8 cells... + Approximately 50% of T cells are specific to the indicated viral epitopes (CMV pp65, EBV YVL, EBV BMLF1, and Mart-1).

[0111] Figure 8B Example results of a recall assay involving antigen-specific memory CD8 are depicted. + T cell responses to antigen-presenting cells loaded with peptides were then incubated with APCs loaded with and unloaded viral antigens. The figure depicts CD8 at two time points of release of the indicated cytokines. + T cell fraction.

[0112] Figure 9 Example results of a cytotoxicity assay used to assess whether induced T cell cultures can kill tumor lines expressing antigens are depicted. The fractions of live and dead caspase-3 positive tumor cells relative to total tumor cells are shown. Live caspase-3 positive tumor cells indicate that the cells are undergoing early cell death.

[0113] Figure 10 An example flow cytometry analysis involving antigen-specific CD4 is described. + T cell responses to antigen-presenting cells loaded with peptides were then observed, followed by incubation with APCs loaded with and without PIN. CD4+ release of IFNγ was also demonstrated. + The percentage of T cells.

[0114] Figure 11 The antigen-specific CD4 cells that release IFNγ upon restimulation with mutant or wild-type peptides were described. + Example results of the percentage of T cells.

[0115] Figure 12 An example flow cytometry analysis was described, which showed antigen-specific CD8. +The response of naive T cells to the short HTV5 peptide. Short-term and long-term induction were demonstrated.

[0116] Figure 13 An exemplary flow cytometry analysis is depicted, showing antigen-specific CD8 using a complete PBMC sample from a human donor. + The fraction of the response of naive T cells to the short ME1 peptide.

[0117] Figure 14 An example flow cytometry analysis is depicted, showing antigen-specific CD8 using a complete PBMC sample from a human donor. + The response of naive T cells to the short HTV3 peptide.

[0118] Figure 15 An example flow cytometry analysis is depicted, showing antigen-specific CD8 using a complete PBMC sample from a human donor. + The response of naive T cells to the long CSNK1A1 peptide.

[0119] Figure 16 An example flow cytometry analysis is depicted, showing antigen-specific CD8 antigens in a PBMC sample from a human donor using depleted CD25+ cells. + The response of naive T cells to the long CSNK1A1 peptide.

[0120] Figure 17 An example flow cytometry analysis is depicted, showing the use of depleted CD25. + Cellular PBMC samples from human donors, antigen-specific CD8 + The response of naive T cells to short GAS7 peptide.

[0121] Figure 18 An example flow cytometry analysis is depicted, showing the use of depleted CD25. + Cellular PBMC samples from human donors, antigen-specific CD8 + The response of naive T cells to the short ACTN4 peptide.

[0122] Figure 19A An example flow cytometry analysis is depicted, showing the use of depleted CD25. + Cellular PBMC samples from human donors, antigen-specific CD8 + The response of naive T cells to the short ACTN4 peptide. This demonstrates short-term induction.

[0123] Figure 19B An example flow cytometry analysis is depicted, showing the use of depleted CD25. + Cellular PBMC samples from human donors, antigen-specific CD8+ The response of naive T cells to a short HIV 3 peptide. Long-term induction was observed.

[0124] Figure 20 The study depicted antigen-specific CD8 antigens using complete PBMC samples from human donors. + Example flow cytometry analysis of the response of naive T cells to a short HIV5 peptide. Short-term and long-term induction are shown.

[0125] Figure 21 An example flow cytometry analysis is depicted, showing antigen-specific CD8 using a complete PBMC sample from a human donor. + The response of naive T cells to a short HIV 3 peptide. This demonstrates short-term induction.

[0126] Figure 22 An example flow cytometry analysis is depicted, showing the use of depleted CD25. + Cellular PBMC samples from human donors, antigen-specific CD8 + The response of naive T cells to the short PRDX5 peptide. Both very short-term and long-term induction were demonstrated.

[0127] Figure 23 An example flow cytometry analysis is depicted, showing the use of depleted CD25. + Cellular PBMC samples from human donors, antigen-specific CD8 + The response of naive T cells to a short HIV5 peptide. Short-term and long-term induction were demonstrated.

[0128] Figure 24 A schematic diagram depicts an example of a method for producing a therapeutic T-cell composition, the method comprising the expansion of memory T cells and the induction of naive T cells.

[0129] Figure 25 Exemplary methods for testing the functionality, phenotype, and / or function and / or T cell response of T cells are described.

[0130] Figure 26 Examples of recall assays used to test T cell functionality, phenotype, and / or function and / or T cell response are described.

[0131] Figure 27A An example flow cytometry analysis is described, demonstrating the ability of labeled samples, acquired individually or as mixtures, to deconvolve multiplexed samples in recall assays. Uniquely labeled samples are resolved with little or no cross-contamination with other barcodes.

[0132] Figure 27BAn example flow cytometry analysis is depicted, demonstrating the detection of antigen-specific CD8 in a recall assay by multimer staining of a mixture of samples with nine uniquely labeled samples. + T cells.

[0133] Figure 28A An example flow cytometry analysis of recall assays using samples labeled with six unique barcodes is depicted, in which recall was performed with unloaded DCs and DCs loaded with neoantigens.

[0134] Figure 28B The image depicts CD44 cells with functional numbers incubated with DCs loaded with the peptide at the indicated concentration in a recall response assay. + Example bar chart of the percentage of T cells. Analysis of de novo CD4 cells alone or in combination with unrelated samples without barcode labeling. + Samples of two inducible cultures for T cell responses. Barcoding did not alter detectable functionality. The number of functions induced from cells and the magnitude of the response were not significantly altered with sample barcoding.

[0135] Figure 29A An exemplary bar chart is depicted, showing the antigen-specific memory CD8. + The result of T cell response to viral antigens. CD8 responses to CMV pp65, MART-1, and EBV BRLF1 and BMLF1 epitopes. + Memory responses can be generated from CD8 in initial healthy donor material. + The percentage of T cells increased from 0.23% to >60%.

[0136] Figure 29B Example results of a recall assay involving antigen-specific memory CD8 are depicted. + T cell responses to viral antigens were then recalled using dendritic cells (DCs) loaded with and unloaded with viral antigens. The figure depicts CD8 at two time points of release of the indicated cytokines. + T cell fraction.

[0137] Figure 30A The study depicted the de novo induction of CD4 with multiple specificities in the same culture. + Example results for hit identification were obtained by detecting and functionally characterizing the response. In the example shown, induction was performed in four replica cultures targeting 10 HIV-derived epitopes, which were the primary targets of HIV-negative healthy donors. Antigen-specific responses were detected in 4 / 4 biological replicates, with varying magnitudes of response.

[0138] Figure 30BThe study depicted the de novo induction of CD4 with multiple specificities in the same culture. + Example results of pooling deconvolution performed on response detection and functional characterization. Multiple responses were detected in each test repetition, and in each case, the same two epitopes (HIV#5 and HIV#7) produced the highest order of response.

[0139] Figure 30C The study depicted the de novo induction of CD4 with multiple specificities in the same culture. + Exemplary results from response detection and functional characterization to determine sensitivity. In pooling deconvolution analysis, similar magnitudes were observed for each response. Responses to HIV#5, HIV#6, and HIV#4 showed ECGs. 50 The values ​​were 0.45 μM, 0.43 μM, and 9.1 μM, respectively.

[0140] Figure 31 An example schematic diagram depicts an antigen-specific T cell preparation protocol.

[0141] Figure 32 An example schematic diagram depicting a T cell induction protocol is shown.

[0142] Figure 33 An example schematic diagram depicting a dendritic cell generation scheme.

[0143] Figure 34 An exemplary pMHC multimer map was depicted, showing the pMHC multimers derived from the patient-specific epitope SRSF1. E>K ARAP1 Y>H and PKDREJ G>R Melanoma patients and patient-specific epitopes (AASDH neoORF and seven model neoantigens: ACTN4) K>N CSNK1A1 S>L DHX40neoORF, GLI3 P>L QARSR >W FAM178B P>L and RPS26 P>L CD8+ T cell responses induced in leukocyte isolates from melanoma patients. The first image in the first and second rows indicates the memory response, while the remaining images indicate the de novo response.

[0144] Figure 35 SRSF1 was depicted E>K and ARAP1 Y>H Example data of pMHC multimer plots before and after peptide stimulation (left panel). The pie chart depicts the functionality of neoantigen-specific T cells after re-attack with neoantigen-loaded DCs; pMHC multimers + CD8+ or CD4 + T cells are gated. The multifunctional spectrum of CD8+ memory, CD8+ de novo and CD4+ de novo responses induced in melanoma patients is shown by a combination of 1, 2 or 3 functions (e.g., the generation of one or more factors selected from IFNγ, TNFα, CD107a and 4-1BB).

[0145] Figure 36 The specificity of memory and de novo responses induced by mutant and wild-type peptides was described in melanoma patients. SRSF1 E>K and ARAP1 Y>H Specific T cell responses were assessed by challenging dendritic cells (DCs) loaded 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 the IFN-γ+ and / or TNFα+ and / or CD107a+ (Y-axis) of total CD8+ T cells in the samples were measured. Both responses showed significant differences from the 0 μM concentration, while no response was observed to the wild-type neoantigen peptide. Statistical analysis: FDR adjusted p-value, P-value: *≤0.05, ***≤0.001, ****≤0.0001.

[0146] Figure 37A Depicting according to CD8 + CD107a + The cytotoxicity profile of the induced memory response quantified by T cell frequency in melanoma patients. It also depicts the target cell killing of these T cell responses, as quantified according to the frequency of aCAS3+ tumor cells. The cytotoxic capacity of the induced CD8+ T cell response was assessed by re-attack with tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. This construct contained either mutant or wild-type sequences with a central mutation. Upregulation of CD107a on CD8+ T cells and upregulation of active cysteine ​​3 on tumor cells were measured after co-culture. Target ratio: 3.3:1 (SRSF1) E>K ).

[0147] Figure 37B Depicting according to CD8 + CD107a +Another example of the cytotoxicity profile of induced memory responses in melanoma patients, quantified by the frequency of T cells. It also depicts the target cell killing of these T cell responses, as quantified based on the frequency of aCAS3+ tumor cells. The cytotoxic capacity of the induced CD8+ T cell responses was assessed by re-attack with tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. This construct contained either mutant or wild-type sequences with a central mutation. Upregulation of CD107a on CD8+ T cells and upregulation of active cysteine ​​3 on tumor cells were measured after co-culture. The pMHC+ portion is highlighted in red circles. Effector:Target ratio: 5:1 (SRSF1) E>K Statistical analysis: Unpaired t-test, **≤0.01, ****≤0.0001.

[0148] Figure 37C Depicting according to CD8 + CD107a + The cytotoxicity profile of the induced de novo response quantified by T cell frequency in melanoma patients. It also depicts the target cell killing of these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The cytotoxic capacity of the induced CD8+ T cell response was assessed by re-attack with tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. This construct contained either mutant or wild-type sequences with a central mutation. Upregulation of CD107a on CD8+ T cells and upregulation of active cysteine ​​3 on tumor cells were measured after co-culture. Circles highlight the pMHC+ portion. Effect:target ratio: 0.66:1 (ARAP1Y>H). Statistical analysis: unpaired t-test, **≤0.01, ****≤0.0001.

[0149] Figure 38A The identification of neoantigen-specific CD4+ T cell responses in melanoma patients was depicted. Responses were identified based on the production of IFN-γ and TNFα (Y-axis) upon re-challenge with DCs (0.8 μM) loaded with a mutant neoantigen peptide. MKRN1 S>L CREBBP S>L TPCN1K>E was identified as a positive response.

[0150] Figure 38B The effects of targeting the mutant and wild-type peptides shown were described. Figure 38A The specificity of the CD4+ T cell response described in the study. In confirmatory studies, Figure 38AThe CD4 T cell responses shown were challenged with different concentrations (X-axis - 0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM) of mutant and wild-type neoantigen peptides, and the total CD4+ IFNγ+ and / or TNFα+ in the samples were measured (Y-axis). Two of the CD4+ T cell responses (MKRN1) were also measured. S>L and CREEBP S>L It showed significant differences at a concentration of 10 μM and was unresponsive to the wild-type neoantigen peptide, but TPCN1 K>E The response was reactive to both mutant and wild-type neoantigen peptides. Statistical analysis: FDR adjusted p-value, p-value < 0.05;

[0151] Figure 38C The versatility profile of these CD4+ T cell responses was depicted, as shown by combinations of 1, 2, 3, or 4 functions (e.g., one or more functions being the production of one or more factors selected from IFNγ, TNFα, CD107a, and 4-1BB). The versatility of the identified CD4+ T cell responses was assessed by re-challenge with DCs (0.8 μm) loaded with mutant neoantigen peptides. The percentages in the pie charts represent the percentage of functional CD4+ T cells (1, 2, and / or 3 functions). The representative data depicted were generated from CD4+ T cell responses induced in patients following stimulation.

[0152] Figure 39 The functionality of memory responses induced in two healthy donors with or without the addition of Epacadostat was depicted as a combination of 1, 2, or 3 functions (e.g., the generation of one or more factors selected from IFNγ, TNFα, and CD107α).

[0153] Figure 40 The de novo CD8 induction induced in six repeated inductions was depicted with or without the addition of Epacadostat. + T-cell response percentage (“hit rate”, averaged across four healthy donors).

[0154] Figure 41A The absolute number of antigen-specific cells from healthy donors after induction using the T-cell preparation protocol described herein is depicted with or without the addition of PD-1 blocking antibodies.

[0155] Figure 41B The absolute number of antigen-specific cells from healthy donors after induction using the T-cell preparation protocol described herein is depicted with or without the addition of PD-1 blocking antibodies.

[0156] Figure 42AThe study depicted CD8+ T cell compartments as derived from de novo CD8+ T cells with or without the addition of IL-12. + The percentage of T cells with multimer positivity frequency.

[0157] Figure 42B An exemplary graphical representation depicting the percentage of CD8+ T cells from de novo CD8+ T cell compartments with or without the addition of IL-12.

[0158] Figure 43 An exemplary graphical representation of the percentage hit rate of naive CD8 cells responding to high- and low-immunogenic antigens after different antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors is also depicted. An exemplary graphical representation of the absolute number of antigen-specific cells after different antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors with Mart-1 peptide or high- and low-immunogenic antigens is also depicted.

[0159] Figure 44A Exemplary flow cytometry results of CD123-positive cells are depicted after antigen-presenting cell enrichment and antigen loading protocols were performed using PBMCs from three different healthy donors.

[0160] Figure 44B An exemplary graphical representation of the absolute number of CD11c+ cell subgroups is depicted after three antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Treatments are: basal FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment and consumption of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and consumption of both CD11b-expressing and CD19-expressing cells.

[0161] Figure 45 An exemplary graphical representation of the total number of CD8 T cells and the indicated cell ratios after three antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors is depicted. Treatments are: basal FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment and consumption of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and consumption of both CD11b-expressing and CD19-expressing cells.

[0162] Figure 46 Exemplary flow cytometry results of CD11b-positive cells are depicted after antigen-presenting cell enrichment and antigen loading protocols were performed using PBMCs from three different healthy donors.

[0163] Figure 47Exemplary flow cytometry results of CD19-positive cells are depicted after antigen-presenting cell enrichment and antigen loading protocols were performed using PBMCs from three different healthy donors.

[0164] Figure 48 Exemplary graphical representations of cell expansion folds following three antigen-presenting cell enrichment and antigen loading protocols are depicted. The treatments are: basal FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment and consumption of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and consumption of CD11b-expressing cells and CD19-expressing cells.

[0165] Figure 49A Exemplary data are depicted illustrating the number of specific antigens responded to by naive CD8 T cells after three antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors. Results are averaged across the three healthy donors. Treatments were: basal FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment and consumption of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and consumption of CD11b-expressing cells and CD19-expressing cells. An exemplary graphical representation of the data is shown in the figure below.

[0166] Figure 49B An exemplary graphical representation of the percentage hit rates of highly immunogenic (left) and low immunogenic (right) antigens in naïve CD8 cells after three antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors is presented. Results are averaged across the three healthy donors. Treatments were: basal FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment and consumption of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and consumption of CD11b-expressing cells and CD19-expressing cells.

[0167] Figure 50 An exemplary graphical representation of the number of antigen-specific cells in T-cell-responsive populations of highly immunogenic and low immunogenic antigens is depicted after three antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors. Treatments included: basal FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment and consumption of CD11b-expressing cells; and CD11b- / CD19-, FLT3L treatment and consumption of both CD11b-expressing and CD19-expressing cells.

[0168] Figure 51AAn exemplary graphical representation of the percentage of viable cells after three antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors is depicted. The treatments are: basal, FLT3L treatment alone; basal + CD11b- / CD19-, FLT3L treatment and consumption of CD11b- and CD19-expressing cells; and +APC, adding an additional portion of PBMC to basal + CD11b- / CD19-, where the additional portion depletes cells expressing CD3, CD19, CD11b, CD25, and CD14.

[0169] Figure 51B An exemplary graphical representation of the percentage of viable cells after three antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors is depicted. The treatments are: basal, FLT3L treatment alone; basal + CD11b- / CD19-, FLT3L treatment and consumption of CD11b- and CD19-expressing cells; and +APC, adding an additional portion of PBMC to basal + CD11b- / CD19-, where the additional portion depletes cells expressing CD3, CD19, CD11b, CD25, and CD14.

[0170] Figure 51C An exemplary graphical representation of the percentage of viable cells after three antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors is depicted. The treatments are: basal, FLT3L treatment alone; basal + CD11b- / CD19-, FLT3L treatment and consumption of CD11b- and CD19-expressing cells; and +APC, adding an additional portion of PBMC to basal + CD11b- / CD19-, where the additional portion depletes cells expressing CD3, CD19, CD11b, CD25, and CD14.

[0171] Figure 51D Exemplary data are depicted, showing the number of specific antigens responded to by CD8 cells from each donor using an exemplary antigen-presenting cell enrichment protocol.

[0172] Figure 51E An exemplary graphical representation depicts the percentage hit rate of the indicated peptide in CD8 cell response, averaged across three healthy donors.

[0173] Figure 52A Exemplary flow cytometry analysis results from an experiment in which cell populations added to the culture process at different times were treated with membrane-permeable amine-reactive dyes (e.g., carboxyfluorescein succinimide ester or TagIT Violet). TMThe cells were labeled with one dye and then stimulated with antigen-loaded APCs. When applied for the second stimulation, a cell population that had been cultured for 14 days was labeled with one dye, while another cell population containing antigen-loaded APCs and fresh T cells was labeled with another dye. The two cell populations were then mixed together for restimulation or expansion. The relative contribution of each of these populations to the total antigen-specific T cell pool was recorded by the presence and dilution of each dye. In all cases, the cell population was cultured for 14 days (first stimulation), labeled with one dye, and then added to another cell population labeled with another dye that had been antigen-stimulated 1 day earlier (standard protocol), 4 days earlier (5-day head start), or 6 days earlier (7-day head start).

[0174] Figure 52B Exemplary schematic diagrams of three different T-cell expansion protocols are shown, each with two stimuli, including a lead start of antigen loading APC 2, 5, or 7 days prior to contact with T cells.

[0175] Figure 52C Showing the use Figure 52B The illustration shows the changes in antigen-specific T cell numbers over time for three different T cell expansion protocols. 1, Standard protocol; 2, 5-day lead start; 3, 7-day lead start.

[0176] Figure 53 An exemplary illustration shows the fold expansion of cultures treated with the indicated neoantigen peptide (pep) or neoantigen RNA. PBMCs depleted of CD14 / CD25 were stimulated with an antigen (peptide or mRNA encoding the antigen) after CD3 lymphocytes were isolated or removed. CD3 lymphocytes were reintroduced and stimulated for 14 days.

[0177] Figure 54 An exemplary illustration shows the number of multimeric positive antigen-specific cells in a culture nuclearly transfected with the indicated neoantigen peptide (pep) or neoantigen RNA. Nuclear transfection of the culture was performed in the presence or absence of T cells (-CD3). Irr, irradiated.

[0178] Figure 55 Exemplary flow cytometry analyses are depicted, demonstrating antigen-specific CD8+ memory responses using viral peptides or RNA encoding such peptides in short-term induction protocols and naivety using neoantigen-encoded peptides or RNA. answer.

[0179] Figure 56A A schematic diagram depicts an exemplary process for generating RNA containing sequences encoding neoantigens and using them to load PBMCs and activate T cells.

[0180] Figure 56B A schematic diagram depicts an exemplary process for generating sequences containing encoding neoantigens and using them to load PBMCs and activate T cells.

[0181] Figure 57A A schematic diagram depicts an exemplary RNA multiply construct encoding a neoantigen string.

[0182] Figure 57B Depicting in Figure 57A The diagram shows an exemplary arrangement of neoantigen strings in vivo in a 5'-3' direction.

[0183] Figure 58A A schematic diagram of an exemplary mRNA sequence is depicted, used to incorporate a 5'-cap structure into an mRNA encoding a tandem neoantigen string for expression in PBMCs. The addition of an "A" nucleotide to the mRNA string is for... Technically compatible.

[0184] Figure 58B An exemplary graphical representation of the percentage of live cells 24 hours after expression of mRNA encoding a tandem neoantigen string with different 5'-cap structures in PBMCs is depicted.

[0185] Figure 58C An exemplary graphical representation of the total number of GFP-positive cells 24 hours after expression of mRNA encoding a tandem neoantigen string with different 5'-cap structures in PBMCs is depicted.

[0186] Figure 59A Exemplary results illustrating the preparation of mRNA using modified nucleotides are depicted. The mRNA was modified by replacing all or part of the uridine (U) and cytidine (C) residues within it. For example, 30% of the C residues in the Part C group were replaced with methylcytidine. The results show the effect over time on the expression of the mRNA-encoded peptide in transfected PBMCs.

[0187] Figure 59B Exemplary data are depicted comparing the effects of commercial and in-house preparations of mRNA containing substituted uridine and / or cytidine on the generation of multimeric specific T cells stimulated by PBMCs loaded with mRNA.

[0188] Figure 59C Exemplary data is depicted, which compares to, for example Figure 59B The resulting expansion of stimulated T cells.

[0189] Figure 60AAn exemplary schematic diagram depicts mRNA constructs using short-polymer (9-10 amino acids, top) and long-polymer (25 amino acids, bottom) for expression in cells.

[0190] Figure 60B An exemplary illustration depicts multimer-specific CD8+ cells as a percentage of total CD8+ cells. The antigen used for the multimer assay is shown.

[0191] Figure 60C An exemplary flow cytometry analysis for detecting multimeric positive CD8+ T cells was described, comparing APCs stimulated with short-peptide (9-10 amino acids) and long-peptide (25 amino acids) peptides with APCs containing peptides encoding the same short-peptide (9-10 amino acids) and long-peptide (25 amino acids) peptides.

[0192] Figure 61A Describing the transfection Figure 61B-61D A schematic diagram of an exemplary RNA construct for the cells in the experiment shown.

[0193] Figure 61B An exemplary graphical representation of the results from the multimer assay is depicted. Under all three conditions of PBMC treatment, RNA-transfected PBMCs were superior to peptide-loaded PBMCs in generating antigen-specific T cells. For the Gli3 antigen, a more than 10-fold increase in multimer-positive cells was noted compared to peptide-loaded PBMCs.

[0194] Figure 61C Exemplary flow cytometry data are depicted, showing the detection of Gli3 multimer-positive T cells in each illustrated group with and without CD3 cell consumption. Direct transfection of CD25+ PBMCs yielded more multimer-positive cells compared to PBMCs that consumed CD14 and CD25 cells or PBMCs thawed from frozen stock.

[0195] Figure 61D An exemplary graphical representation of the results from the multimer assay is depicted. PBMCs treated overnight with FTL3L cells or CD25-depleted PBMCs were electroporated with RNA encoding a neoantigen sequence of 25 amino acids (longmer) or a neoantigen sequence of epitope length (shortmer). The percentage of neoantigen-positive cells in the culture was determined using the multimer technique.

[0196] Figure 61E Depicting from Figure 61DAn exemplary graphical representation of the cell expansion results of the experiment. PBMCs treated overnight with FTL3L cells or CD25-depleted PBMCs were electroporated with RNA encoding a neoantigen sequence of 25 amino acids (longmer) or a neoantigen sequence of epitope length (shortmer). The cell expansion folds after 26 days of culture and two stimulations are depicted.

[0197] Figure 62A Describing transfection Figure 62B-62D A schematic diagram of an exemplary RNA construct for the cells in the experiment shown.

[0198] Figure 62B An exemplary graphical representation depicting the number of ACTN4 and Gli3 responsive live T cells from the two donors on day 26 post-maturation, as shown on the X-axis.

[0199] Figure 62C Exemplary data depicting the percentage of Gli3-responsive T cells from live cells grown in the presence of the indicated mature mixture.

[0200] Figure 62D Exemplary flow cytometry data are depicted, showing the detection of Gli3 multimer-positive T cells grown in the presence of the indicated mature mixture.

[0201] Figure 63A Representative mass spectrometry data were depicted, showing the presentation of the Gli3 epitope by PBMCs using radioisotope incorporation detection. Expression of PBMCs transfected with mRNA encoding multiple epitopes (including the Gli3 epitope) and peptides was detected using a reference peptide labeled with a heavier isotope.

[0202] Figure 63B An exemplary graphical representation depicts the percentage of maximum presentation of the indicated epitopes by HLA-A02:01 over time after transfection of PBMCs with mRNA encoding each epitope. Epitopes labeled with each isotope were detected by mass spectrometry. Maximum surface presentation was observed at 6 hours post-transfection.

[0203] Figure 64A An exemplary graphical representation from a recall assay is depicted, showing the percentage change in the production of TNFα and / or IFNγ or the percentage of CD107a-positive cells (left) or the percentage of CD107a-positive cells for neoantigen-specific CD8 T cells attacked with gradually increasing concentrations of the indicated peptides for loading APC.

[0204] Figure 64BAn exemplary graphical representation from the multimer assay is depicted, showing the percentage change in the production of TNFα and / or IFNγ or the percentage of CD107a-positive cells (left) or the percentage of CD107a-positive cells for neoantigen-specific CD8 T cells attacked with gradually increasing concentrations of the indicated peptides for loading APC.

[0205] Figure 65 An exemplary Venn diagram depicts the criteria considered for producing optimal individual T-cell therapeutic agents using mRNA as an immunogen.

[0206] Figure 66 An exemplary flowchart is depicted, illustrating the steps involved in selecting peptide sequences to prepare patient-specific T-cell products.

[0207] Figure 67 Examples illustrate the use of... Figure 1A The clinical application of T cells prepared by the method shown has several advantages.

[0208] Figure 68 Exemplary representative flow cytometry data are depicted, illustrating the characterization of patient-specific T cell products prepared through multiple engineered runs. The percentage of CD3+ cells in live cells is depicted (top panel), as are the percentages of CD8+ and CD4+ cells in live CD3+ T cells (bottom panel).

[0209] Figure 69A An exemplary graphical representation of the data is depicted, showing the characterization of patient-specific T-cell products prepared through multiple engineered runs. The percentage of multimer-positive CD8-positive cells is shown.

[0210] Figure 69B Exemplary representative flow cytometry data are depicted, showing the characterization of patient-specific T-cell products prepared through multiple engineered runs. The percentages of multimeric A-positive and multimeric B-positive CD8 cells targeting the indicated epitopes are shown.

[0211] Figure 69C An exemplary pie chart is depicted, showing the pMHC identified after re-challenge with DCs loaded with a mutant neoantigen compared to unloaded DCs. + CD8 + T cell pluripotency.

[0212] Figure 70 Representative data were depicted, indicating the CD4 content of patient-specific T-cell products prepared through multiple engineering runs. + Changes in IFNγ and / or TNFα production by cells were also depicted. Exemplary representative data were also described, showing IFNγ levels in patient-specific T cell products prepared through multiple engineered runs. + and / or TNFα+ and / or CD107a + CD4 + Cell characterization.

[0213] Figure 71 An exemplary graphical representation is depicted, showing central memory T cells (T cells) in a patient-specific T cell product prepared through multiple engineered runs. cm ), effector memory T cells (T em ), effector T cells (T eff ) and naive T cells The score. Central memory T cells (T cm ): CD62L + CD45RA - Effector memory T cells (T cells) em ): CD62L - CD45RA - effector T cells (T cells) eff ): CD62L - CD45RA + Immature T cells CD62L+CD45RA.

[0214] Figure 72 An exemplary graphical representation of the data from the multimer assay is shown, illustrating the IFN-γ measured in the sample after DC attack with different concentrations of loaded peptides. + and / or TNFα + and / or CD107a + Cells account for a certain percentage of total CD8 + Cells (above) or total CD4 + The percentage of T cells (see chart below). The peptides used in each chart are shown.

[0215] Figure 73 An exemplary graphical representation of the data is depicted, indicating CD8. + Upregulation of CD107a (top row) on T cells and active caspase 3 (bottom row) on tumor cells. Measurements were obtained after co-culturing A375 tumor cell lines that were untransduced or transduced with a 200-amino acid construct, or A375 tumor cell lines with or without peptide loading.

[0216] Figure 74 An exemplary graphical representation of the data is depicted, indicating that induced T cells are able to kill cells expressing antigens. The recognition of neoantigen-specific T cells by autologous tumors or autologous tumors loaded with peptides is tested by a recall response assay. Readout: pMHC + (CD8 + (%) and pMHC - (CD8+ % of T cells IFN-γ + and / or TNFα + and / or CD107a + (Y-axis). Significance was determined using one-way ANOVA, with P < 0.05.

[0217] Figure 75 An exemplary schematic diagram depicts the groups and dosages used in clinical studies (NEO-PTC-01). Detailed Implementation

[0218] T-cell therapeutics are expected to be relatively safe and well-tolerated adoptive T-cell products. However, based on assessments of product-related risks, there are generally three classes of potential toxicities associated with T-cell therapeutics: (a) treatment-related toxicities due to lymphatic depletion, cell infusion, or cytokine release syndromes; (b) extratumor and extratarget toxicities due to the expansion of autoreactive clones or the cross-reactivity of neoantigen-specific T cells; and (c) extratumor and on-target toxicities due to the presentation of neoantigens in non-tumor tissues. This document describes novel immunotherapeutic agents and their uses, based on the discovery of neoantigens arising from mutational events unique to individual tumors. Therefore, this disclosure provides methods and protocols for creating antigen-specific immune cells, such as T cells, for the treatment of diseases.

[0219] This article presents a composition of neoantigen-reactive T cells for cancer immunotherapy. While adoptive T-cell therapy is a promising new approach to cancer treatment, it still requires several improvements. Typically, T cells must possess sufficient cytotoxicity against cancer cells, must not affect non-cancerous cells in the body, must not lose immunogenicity in the tumor environment, and should provide long-term protection. Furthermore, the use of virus-transduced cells presents its own challenges. Therefore, achieving the appropriate balance to obtain a therapeutically effective composition that specifically targets cancer cells, does not affect healthy cells, slows disease progression, leads to improved or at least significant tumor regression, and prevents cancer recurrence requires improvements at almost every step of a complex process.

[0220] To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0221] Antigens are foreign substances that induce an immune response in the body. "Neoantigens" refer to a class of tumor antigens generated by tumor-specific alterations in proteins. Neoantigens include, but are not limited to, tumor antigens generated by, for example, protein sequence substitutions, frameshift mutations, fusion peptides, in-frame deletions, insertions, and the expression of endogenous retroviral peptides.

[0222] A "novel epitope" is an epitope that is not present in reference cells, such as non-pathological cells (e.g., non-cancer cells) or germline cells, but is found in pathological cells, such as cancer cells. This includes situations where a corresponding epitope is found in normal non-pathological cells or germline cells, but the sequence of that epitope is altered due to one or more mutations in pathological cells, such as cancer cells, thus creating a new epitope.

[0223] A “mutation” refers to a change or difference in the nucleic acid sequence compared to a reference nucleic acid (e.g., nucleotide substitution, addition, or deletion). Somatic mutations can occur in any cell of the body except germ cells (sperm and egg) and are not passed on to children. These changes can (but do not always) lead to cancer or other diseases. In some implementations, mutations are nonsynonymous mutations. A “nonsynonymous mutation” is a mutation that causes an amino acid change in the translated product, such as an amino acid substitution (e.g., nucleotide substitution). When a mutation disrupts the normal phase of the periodicity of a gene codon (also called a “reading frame”), a “frameshift” occurs, resulting in the translation of a non-natural protein sequence. Different mutations in a gene can achieve the same altered reading frame.

[0224] "Antigen processing" or "processing" refers to the degradation of a polypeptide or antigen into a processed product (which is a fragment of the polypeptide or antigen, e.g., a polypeptide being degraded into a peptide) and the association of one or more of these fragments (e.g., via binding) with an MHC molecule for presentation by a cell (e.g., an antigen-presenting cell) to a specific T cell.

[0225] "Antigen-presenting cells" (APCs) are cells that present peptide fragments of protein antigens associated with MHC molecules on their cell surface. This term includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).

[0226] The term "affinity" refers to a measure of the binding strength between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide with class I or II HLA, or a peptide-HLA complex with a T-cell receptor (TCR)). D K refers to the dissociation constant between the two members of a binding pair and has a molar concentration unit. A K refers to the affinity constant between the two members of a binding pair, and is the reciprocal of the dissociation constant. Affinity can be determined experimentally, for example, using commercially available Biacore SPR units via surface plasmon resonance (SPR). off This refers to the dissociation rate constant of the two members of a binding pair (e.g., the dissociation rate constant of an HLA-binding peptide with class I or II HLA, or the dissociation rate constant of a peptide-HLA complex with a TCR). Kon It refers to the association rate constant of the two members of the binding pair (e.g., the association rate constant of an HLA-binding peptide with class I or II HLA or a peptide-HLA complex with a TCR).

[0227] Throughout the publicly available content, the "combined data" results can be expressed as "IC". 50 Affinity can also be expressed as the inhibitory concentration (IC50). 50 ), or the concentration when 50% of the binding pair's first member (e.g., peptide) is substituted. Similarly, ln(IC) 50 ) refers to IC 50 The natural logarithm of IC. For example, IC 50 This can be the concentration of the test peptide at which 50% inhibition of binding to the labeled reference peptide is observed in the binding assay. These values ​​can be close to K, taking into account the conditions under which the assay is run (e.g., limiting HLA protein concentration and / or the concentration of the labeled reference peptide). DValues. Assays used to determine binding are well known in the art and are described in detail, for example, in PCT publications WO94 / 20127 and WO 94 / 03205 and other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney et al., J. Immunol. 154:247 (1995); and Sette et al., Mol. Immunol. 31:813 (1994). Alternatively, binding may be expressed relative to the binding of a reference standard peptide. The binding can also be determined using other assay systems, including those 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)), and immobilized purified MHC (e.g., Hill et al., J. Immunol. 152,2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)). ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high-throughput soluble phase determination (Hammer et al., J. Exp. Med. 180:2353 (1994)) and measurements of type I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

[0228] When used to discuss epitopes, the term "derived" is synonymous with "prepared." Derived epitopes can be isolated from natural sources or synthesized according to standard protocols in the art. Synthetic epitopes may contain artificial amino acid residues, "amino acid mimics," such as the D isomer of a naturally occurring L amino acid residue or a non-natural amino acid residue such as cyclohexylalanine. Derived or prepared epitopes may be analogs of natural epitopes. The term "derived from" means 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 immature APCs (e.g., immature APCs). For example, APCs may be derived from monocytes (e.g., CD14 cells). + Monocytes). For example, dendritic cells can originate from monocytes (e.g., CD14). + (Monocytes). For example, APCs can originate from bone marrow cells.

[0229] An epitope is a set of characteristic features of a molecule (e.g., the charge of a peptide and its primary, secondary, and tertiary peptide structures) that together form a site for recognition by another molecule (e.g., an immunoglobulin, T-cell receptor, HLA molecule, or chimeric antigen receptor). For example, an epitope can be a set of amino acid residues involved in recognition by a specific immunoglobulin; a major histocompatibility complex (MHC) receptor; or, in the case of T cells, those residues recognized by T-cell receptor proteins and / or chimeric antigen receptors. Epitopes can be prepared by isolating from natural sources, or they can be synthesized according to standard protocols in the art. Synthetic epitopes can contain artificial amino acid residues—amino acid mimics (such as the D isomer of a naturally occurring L-amino acid residue or a non-naturally occurring amino acid residue). Throughout this disclosure, an epitope may be referred to as a peptide or peptide epitope in some instances. In some embodiments, there are limitations on the length of the peptides of this disclosure. Length-limited embodiments occur when the protein or peptide containing the epitope described herein contains a region that is 100% identical to the natural sequence (i.e., a continuous sequence of amino acid residues). To avoid defining epitopes, for example, by reading across the entire natural molecule, the length of any region that is 100% identical to the natural peptide sequence is limited. Therefore, for a peptide containing the epitopes described herein and a region that is 100% identical to the natural peptide sequence, the region that is 100% identical to the natural sequence typically has the following lengths: less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues. In some embodiments, the “epitope” described herein is contained in a peptide having a region of less than 51 amino acid residues in any increment of up to 5 amino acid residues, the region being 100% identical to the native peptide sequence; having, 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.

[0230] "T cell epitopes" are peptide sequences that are bound to MHC molecules in the form of peptide-MHC (pMHC) complexes. Peptide-MHC complexes can be recognized and bound by the TCR of T cells (e.g., cytotoxic T lymphocytes or T helper cells).

[0231] "T cells" include CD4 +T cells and CD8 + T cells. The term T cells also includes T helper type 1 T cells and T helper type 2 T cells. T cells can be generated by the methods described in this application for clinical use. The T cells or adoptive T cells mentioned herein, such as those used for clinical use, are cells isolated from biological sources, manipulated in vitro, cultured, and prepared into drug candidates for a specific therapy (e.g., cancer, such as melanoma). A drug candidate may be designated as a pharmaceutical product when the drug candidate cells pass specific qualitative and quantitative criteria suitable for clinical use. In some cases, a pharmaceutical product is selected from a number of drug candidates. In the context of this application, a pharmaceutical product is a T cell, more specifically, a population of T cells, or more specifically, a population of T cells with heterogeneous characteristics and subtypes. For example, a pharmaceutical product as disclosed herein may have a population of T cells comprising CD8+ T cells, CD4+ T cells, at least a certain number of cells exhibiting antigen specificity, a certain percentage of each cell type exhibiting a memory phenotype, and so on.

[0232] "Immune cells" refer to cells that play a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0233] An "immunogenic" peptide or "immunogenic" 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 that peptide.

[0234] A “protective immune response” or “therapeutic immune response” refers to a CTL and / or HTL response against an antigen derived from a pathogenic antigen (e.g., a tumor antigen) that somehow prevents or at least partially prevents disease symptoms, side effects, or progression. Immune responses may also include antibody responses promoted by stimulating helper T cells.

[0235] A T-cell receptor ("TCR") is a naturally occurring or partially or fully synthetic molecule found on the surface of T lymphocytes (T cells) that recognize antigens that bind to major histocompatibility complex (MHC) molecules. The ability of T cells to recognize antigens associated with a variety of diseases (e.g., cancer) or infectious organisms is conferred by their TCRs, which consist of alpha (α) and beta (β) chains or gamma (γ) and delta (δ) chains. The proteins that make up these chains are encoded by DNA, which generates the vast diversity of TCRs through unique mechanisms. This multi-subunit immune recognition receptor is associated with the CD3 complex and binds to peptides presented by MHC class I and II proteins on the surface of antigen-presenting cells (APCs). The binding of the TCR to peptides on APCs is a central event in T-cell activation.

[0236] As used herein, "chimeric antigen receptor" or "CAR" refers to an antigen-binding protein that includes an immunoglobulin antigen-binding domain (e.g., an immunoglobulin variable domain) and a T-cell receptor (TCR) constant domain. As used herein, the "constant domain" of a TCR polypeptide includes a proximal TCR constant domain, a TCR transmembrane domain, and / or a TCR cytoplasmic domain or fragments thereof. For example, in some embodiments, the CAR is a monomer comprising a polypeptide containing an immunoglobulin heavy chain variable domain linked to a TCRβ constant domain. In some embodiments, the CAR is a dimer comprising: a first polypeptide containing an immunoglobulin heavy chain or light chain variable domain linked to a TCRα or TCRβ constant domain, and a second polypeptide containing an immunoglobulin heavy chain or light chain variable domain (e.g., a κ or λ variable domain) linked to a TCRβ or TCRα constant domain.

[0237] The "major histocompatibility complex" or "MHC" is a cluster of genes that plays a role in controlling cell interactions that lead to physiological immune responses. The term "major histocompatibility complex" and the abbreviation "MHC" can include any class of MHC molecules, such as MHC class I and MHC class II molecules, and refers to a complex of genes present in all vertebrates. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. Therefore, "human leukocyte antigen" or "HLA" refers to human major histocompatibility complex (MHC) proteins (see, for example, Stites et al., *Immunology*, 8th ed., Lange Publishing, Los Altos, Calif. (1994)). For a detailed description of the MHC and HLA complexes, see Paul, *Fundamental Immunology*, 3rd ed., Raven Press, New York (1993).

[0238] The major histocompatibility complex (MHC) in the genome contains genetic regions whose gene products, expressed on the cell surface, are essential for binding and presenting endogenous and / or exogenous antigens, and thus for regulating immune processes. MHC proteins or molecules are crucial for signaling between lymphocytes and antigen-presenting cells or diseased cells in the immune response. MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. Proteins encoded by the MHC can be expressed on the cell surface and present T cells with self-antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments from invading microorganisms). MHC-binding peptides can be produced by the proteolytic cleavage of protein antigens and represent potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes). The MHC can transport peptides to the cell surface and present them there to specific cells, such as cytotoxic T lymphocytes, T helper cells, or B cells. MHC regions can be divided into three subgroups: class I, class II, and class III. MHC class I proteins may contain α chains and β2-microglobulins (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T cells. MHC class II proteins may contain α and β chains, and they can present antigen fragments to T helper cells. MHC class III regions may encode other immune components, such as complement components and cytokines. MHC can be either multigenic (with several MHC class I and MHC class II genes) or polymorphic (each gene has multiple alleles).

[0239] "Receptor" refers to a biomolecule or group of molecules capable of binding a ligand. Receptors can be used to transmit information in cells, cell formation, or organisms. A receptor contains at least one receptor unit, for example, where each receptor unit may consist of a protein molecule. The receptor has a structure complementary to that of the ligand and can complex with the ligand as a binding partner. Information is transmitted specifically through conformational changes in the receptor following complexation of the ligand on the cell surface. In some embodiments, a receptor should be understood, in particular, to be a MHC class I and II protein capable of forming a receptor / ligand complex with a ligand (particularly a peptide or peptide fragment of suitable length). "Ligand" refers to a molecule having a structure complementary to that of the receptor and capable of forming a complex with the receptor. In some embodiments, a ligand should be understood to mean a peptide or peptide fragment having a suitable length and a suitable binding motif in its amino acid sequence 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 implementations, "receptor / ligand complex" should also be understood to mean "receptor / peptide complex" or "receptor / peptide fragment complex", which includes MHC molecules that present peptides or peptide fragments, such as MHC class I or II molecules.

[0240] A “natural” 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, peptides or nucleic acids that are present in organisms (including viruses) and can be isolated from natural sources and have not been intentionally modified artificially in a laboratory are naturally occurring.

[0241] The terms "peptide" and "peptide epitope" are used interchangeably with "oligopeptide" in this specification, referring to a series of residues typically linked together by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues. "Synthetic peptide" refers to a peptide obtained from a non-natural source, such as one that is artificially created. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. "Synthetic peptides" include "fusion proteins."

[0242] The term "motif" refers to a pattern of residues in an amino acid sequence of a defined length, such as a peptide less than about 15 amino acid residues or less than about 13 amino acid residues. For example, for class I HLA motifs, they have about 8 to about 13 (e.g., 8, 9, 10, 11, 12, or 13) amino acid residues, while for class II HLA motifs, they have about 6 to about 25 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acid residues that are recognized by a specific HLA molecule. The motif is typically different for each HLA protein encoded by a given human HLA allele. The patterns of primary and secondary anchoring residues in these motifs differ. In some embodiments, MHC class I motifs recognize peptides of 7, 8, 9, 10, 11, 12, or 13 amino acid residues in length. In some implementations, MHC class II motifs recognize peptides with a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acid residues. "Cross-reactive binding" peptides are peptides that bind to more than one member of a binding pair member class (e.g., peptides that bind to both class I and class II HLA molecules).

[0243] The term "residue" refers to an amino acid residue or amino acid-mimicking residue incorporated into a peptide or protein via an amide bond or an amide bond mimic, 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 to the left (amino terminus or N-terminus) and the carboxyl group to the right (carboxyl terminus or C-terminus) of each amino acid residue. When referring to the position of an amino acid residue in a peptide epitope, the amino acid residues are numbered in the amino-to-carboxyl direction, with the first position being the residue at the amino terminus of the peptide or protein to which the epitope may be part. In the general formula representing a specific embodiment of the invention, unless otherwise stated, the amino-terminal and carboxyl-terminal groups (although not specifically shown) are the forms they present at physiological pH. In the amino acid structural formula, each residue is typically represented by a standard three-letter or one-letter nomenclature. The L-form of an amino acid residue is represented by a capital single letter or a three-letter symbol with the first letter capitalized, while the D-form of those amino acid residues is represented by a lowercase single letter or a lowercase three-letter symbol. However, they can also refer to L amino acid residues when using three-letter symbols or full names without capital letters. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or "G". The amino acid sequences of peptides described herein are generally represented using standard single-letter symbols. (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine).

[0244] A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), 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, replacing tyrosine with phenylalanine is a conserved substitution. Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate peptide function are well known in the art.

[0245] "Pharmaceutical acceptable" refers to compositions or components that are generally non-toxic, inert, and / or physiologically compatible. "Pharmaceutical excipients" or "excipients" include materials such as adjuvants, carriers, pH adjusters and buffers, tension modifiers, wetting agents, and preservatives. "Pharmaceutical excipients" are pharmaceutically acceptable excipients.

[0246] According to this disclosure, the term "vaccine" refers to a pharmaceutical article (pharmaceutical 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 to prevent or treat diseases. The terms "personalized cancer vaccine" or "individualized cancer vaccine" refer to a specific cancer patient and mean that the cancer vaccine is tailored to the needs or special circumstances of an individual cancer patient.

[0247] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to nucleotide polymers of any length, including DNA and RNA (e.g., mRNA). Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase. In some embodiments, the polynucleotide and nucleic acid can be in vitro transcribed mRNA. In some embodiments, the polynucleotide applied using the methods of the present invention is mRNA.

[0248] The terms "isolated" or "biologically pure" refer to materials that are substantially free of, or substantially free of, the components that are typically associated with the material when it is found in its natural state. Therefore, isolated peptides described herein do not contain some or all of the substances that are typically associated with the peptide in its in situ environment. For example, an "isolated" epitope can be an epitope that does not include the full sequence of the protein from which the epitope is derived. For example, naturally occurring polynucleotides or peptides present in living organisms are not isolated, but the same polynucleotides or peptides isolated from some or all of the coexisting substances in a natural system are isolated. Such polynucleotides may be part of a carrier, and / or such polynucleotides or peptides may be part of a composition and are still "isolated" because such carriers 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 also include such molecules produced synthetically. In some embodiments, isolated polypeptides, antibodies, polynucleotides, carriers, cells, or compositions are substantially pure. As used herein, the term “substantially pure” means a substance that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0249] In the context of two or more nucleic acids or peptides, the term "identical" or percentage "identity" means that two or more sequences or subsequences are identical or have a specific percentage of identical nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps if necessary), regardless of any conserved amino acid substitutions as part of sequence identity. Percentage identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining amino acid or nucleotide sequence alignments are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, the two nucleic acids or peptides described herein are substantially identical, meaning that when compared and aligned for maximum correspondence, as measured by sequence comparison algorithms or by visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. In some embodiments, identity exists in sequence regions of at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues, or any integer number of residues in between. In some embodiments, identity exists in regions longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical across the full length of the compared sequences, such as the coding regions of an amino acid sequence of a peptide or a nucleotide sequence.

[0250] The term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that will be the recipient of a particular treatment. Generally, when referring to human subjects, the terms "subject" and "patient" are used interchangeably in this document.

[0251] The terms "effective dose," "therapeutic effective dose," or "therapeutic effect" refer to the amount of a therapeutic agent that is effective in treating a disease or condition in a subject or mammal. A therapeutically effective dose of a drug has a therapeutic effect and therefore can prevent the development of a disease or condition; slow the development of a disease or condition; slow the progression of a disease or condition; alleviate, to some extent, one or more symptoms associated with a disease or condition; reduce morbidity and mortality; improve quality of life; or a combination of these effects.

[0252] The terms “treatment” or “relief” refer to: (1) measures of treatment that cure, alleviate, or reduce the symptoms of a diagnosed pathological condition or symptom, and / or halt the progression of the pathological condition or symptom; and (2) preventive or preventative measures that prevent or slow the development of a target pathological condition or symptom. Therefore, subjects requiring treatment include those who already have the condition; those who are susceptible to the condition; and those who wish to prevent the condition.

[0253] When used to describe cell samples (e.g., peripheral blood mononuclear cell (PBMC) samples), the term "depleted" refers to a cell sample in which cell subsets have been removed or consumed. For example, an immune cell sample depleted of CD25-expressing cells refers to an immune cell sample in which CD25-expressing cells have been removed or depleted. For example, one or more binding agents can be used to remove or consume one or more cells or cell types from a sample. For example, CD14 can be consumed or removed from a PBMC sample, for example, by using an antibody that binds to CD14. + cell.

[0254] "Stimulation" refers to a response induced by the binding of a stimulating molecule to its homologous ligand, thereby mediating a signal transduction event. For example, stimulation of T cells could refer to the binding of the T cell's TCR to the peptide-MHC complex. For example, stimulation of T cells could refer to the step in Protocol 1 or Protocol 2, where PBMCs are cultured together with APCs loaded with the peptide.

[0255] The term "enrichment" refers to a composition or fraction in which the target species has been partially purified, resulting in a concentration of the target species that is significantly higher than the level of naturally occurring species in the unenriched final product. The term "induced cell" refers to cells, cells, or cell populations that have been treated with inducing compounds that affect protein expression, gene expression, differentiation state, shape, morphology, viability, etc.

[0256] "Reference" can be used to correlate and / or compare results obtained by the methods of this disclosure with disease specimens. Typically, "reference" can be obtained based on one or more normal specimens, particularly specimens unaffected by disease, obtained from an individual or one or more different individuals (e.g., healthy individuals), such as individuals of the same species. "Reference" can be determined empirically by testing a sufficiently large number of normal specimens.

[0257] As used herein, unless otherwise stated, a tumor is a cancerous tumor, and the terms cancer and tumor are used interchangeably throughout the document. While a tumor is a cancer of solid tissue, several compositions and methods described herein are applicable in principle to hematologic malignancies, including leukemia.

[0258] Overview of T-cell therapy

[0259] The controlled in vitro induction or expansion of T cells (e.g., autologous T cells) to generate antigen-specific T cells can provide highly specific and beneficial T cell therapies (e.g., adoptive T cell therapy). This disclosure provides methods for preparing T cells and therapeutic T cell compositions that can be used to treat subjects with cancer and other conditions, diseases, and symptoms. The aim is to expand and induce antigen-specific T cells with a favorable phenotype and function. This disclosure provides compositions and methods for preparing T cells that can be used for antigen-specific T cell therapies (e.g., personal or personalized T cell therapies). The T cell compositions provided herein can be for personal antigen-specific T cell therapies. Figure 1 graphically illustrates an overview of a process associated with T cell therapy: on one hand, it includes identifying cancer and cancer-specific antigens in a subject with cancer, leading to the production of neoantigen peptides; on the other hand, preparing activated antigen-specific cells for immunotherapy and administering cell products.

[0260] New antigens for T-cell-based therapies

[0261] Traditional antigen-targeted immunotherapy focuses on tumor-associated antigens (TAAs), including cancer testis antigens (typically germline-restricted gene products aberrantly expressed in tumors) or antigens derived from genes exhibiting tissue-specific expression. However, tumors also display protein products of mutated genes known as neoantigens. The number and type of mutations can be readily determined using next-generation sequencing methods, including single-amino acid missense mutations, fusion proteins, and novel open reading frames (neoORFs) ranging in length from one to 100 or more amino acids. Neoantigens are antigens containing non-silenced mutations in epitopes, and the same antigen is not expressed in non-cancerous cells within the same individual. Mutation-based antigens are particularly valuable because they bypass central tolerance (a process that occurs during normal thymic development, which removes self-reactive T cells) and exhibit fine tumor specificity. Each non-synonymous (i.e., protein-coding) mutation has the ability to produce neoantigens that can be recognized by the patient's T cells. T cells recognizing these neoantigens can either directly kill tumor cells or catalyze a broader immune response against the tumor. The methods described herein aim to induce and expand such neoantigen-reactive T cells in a patient-specific manner and to use these cells for adoptive cell therapy.

[0262] In some implementations, the neoantigen used herein contains point mutations.

[0263] In some implementations, the neoantigen used herein contains frameshift mutations.

[0264] In some implementations, the neoantigen used herein contains crossover mutations.

[0265] In some implementations, the neoantigen used herein contains insertion mutations caused by the insertion of one or more nucleotides.

[0266] In some implementations, the neoantigen used herein contains deletion mutations caused by the deletion of one or more nucleotides.

[0267] In some implementations, the neoantigen may be caused by an insertion-deletion mutation.

[0268] In some embodiments, the antigen or neoantigen peptide binds to an HLA protein (e.g., HLA class I or HLA class II). In specific embodiments, the antigen or neoantigen peptide binds to the HLA protein with a greater affinity than the corresponding wild-type peptide. In specific embodiments, the IC50 of the antigen or neoantigen peptide... 50 or K D At least below 5000 nM, at least below 500 nM, at least below 100 nM, at least below 50 nM or lower.

[0269] In some embodiments, the length of the antigen or neoantigen peptide may be about 8 to about 50 amino acid residues, or about 8 to about 30, about 8 to about 20, about 8 to about 18, about 8 to about 15, or about 8 to about 12 amino acid residues. In some embodiments, the length of the antigen or neoantigen peptide may be about 8 to about 500 amino acid residues, or about 8 to about 450, about 8 to about 400, about 8 to about 350, about 8 to about 300, about 8 to about 250, about 8 to about 200, about 8 to about 150, about 8 to about 100, about 8 to about 50, or about 8 to about 30 amino acid residues.

[0270] In some embodiments, the length of the antigen or neoantigen peptide may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues. In some embodiments, the length of the neoantigen peptide may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acid residues. In some implementations, the length of the antigen or neoantigen peptide can be up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or fewer amino acid residues. In some implementations, the length of the antigen or neoantigen 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.

[0271] In some embodiments, the total length of the antigen or neoantigen peptide is 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.

[0272] In some implementations, the total length of the antigen or neoantigen peptide is 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, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids.

[0273] In some embodiments, the neoantigen peptide 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 neoantigen peptide may have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or higher. In some embodiments, the neoantigen peptide may have a pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or lower.

[0274] In some embodiments, the antigen or neoantigen peptide may have an HLA binding affinity of about 1 pM to about 1 mM, about 100 pM to about 500 μM, about 500 pM to about 10 μM, about 1 nM to about 1 μM, or about 10 nM to about 1 μM. In some embodiments, the antigen or neoantigen peptide may have an HLA binding affinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 μM or higher. In some implementations, the antigen or neoantigen peptide may have an HLA binding affinity 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, or 900 μM.

[0275] In some embodiments, the antigens or neoantigen peptides described herein may include carriers such as those known in the art, such as thyroglobulin, albumin such as human serum albumin, tetanus toxoid, polyamino acid residues (such as poly-L-lysine, poly-L-glutamic acid), influenza virus proteins, hepatitis B virus core proteins, etc.

[0276] In some embodiments, the antigen or neoantigen peptide described herein can be acylated via a terminal -NH2 group (e.g., via an alkyl acyl group (C1-C)). 20 Modifications can be made by thiohydroxyacetylation or terminal carboxyl amidation (e.g., ammonia, methylamine, etc.). In some embodiments, these modifications can provide sites for attachment to a support or other molecules.

[0277] In some embodiments, the antigen or neoantigen peptide described herein may contain modifications, such as, but not limited to, glycosylation, side-chain oxidation, biotinylation, phosphorylation, addition of a surfactant (e.g., lipids), or may be chemically modified, such as acetylation. Furthermore, the bonds in the peptide can be bonds other than peptide bonds, such as covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0278] In some embodiments, the antigen or neoantigen peptide described herein may include substitutions to alter the physical properties (e.g., stability or solubility) of the resulting peptide. For example, an antigen or neoantigen peptide may be modified by replacing cysteine ​​(C) with α-aminobutyric acid (“B”). Due to its chemical properties, cysteine ​​has a tendency to form disulfide bonds and structurally alters the peptide to reduce its binding capacity. Replacing C with α-aminobutyric acid not only alleviates this problem but, in some cases, actually improves binding and cross-binding capacity. Replacing cysteine ​​with α-aminobutyric acid can occur at any residue of the antigen or neoantigen peptide, such as at anchored or unanchored sites on an endopeptide epitope or similar site, or at other sites on the peptide.

[0279] In some embodiments, the antigenic peptide or neoantigen peptide described herein may comprise amino acid mimics or non-natural amino acid residues, such as D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1, 2, 3, or 4-pyrenealanine; D- or L-3-thienylalanine; D- or L-(2-pyridyl)-alanine; D- or L-(3-pyridyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenyl Glycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoro-methyl)-phenylalanine; D-ρ-fluorophenylalanine; D- or L-ρ-biphenyl-phenylalanine; D- or L-ρ-methoxybiphenylalanine; D- or L-2-indole(allyl)alanine; and D- or L-alkylalanine, wherein the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, isopentyl, or non-acidic amino acid residues. Aromatic rings of non-natural amino acids include, for example, thiazolyl, thiophene, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrroleyl, and pyridyl aromatic rings. Modified peptides with various amino acid mimics or non-natural amino acid residues are particularly useful because they tend to exhibit increased in vivo stability. Such peptides may also have improved shelf life or preparative properties.

[0280] In some embodiments, the peptide is contacted with immune cells to activate the cells and make them antigen-reactive.

[0281] In some implementations, the peptide is in contact with immune cells in vitro.

[0282] In some implementations, the peptide comes into contact with immune cells in a living system, such as the human body.

[0283] In some implementations, the immune cells are antigen-presenting cells.

[0284] In some implementations, the immune cells are T cells.

[0285] This disclosure relates to a method for preparing T cells that are specific to immunogenic antigens.

[0286] This disclosure also relates to compositions comprising antigen-specific T cells stimulated by APCs. In some embodiments, one or more antigenic peptides are loaded onto an APC, wherein T cells are then stimulated with the peptide-loaded APC to generate antigen-specific T cells. In some embodiments, the antigen is a neoantigen. In some embodiments, the APC used for peptide loading is a dendritic cell.

[0287] In some embodiments, the peptide sequence contains a mutation not present in the non-cancer cells of the subject. In some embodiments, the peptide is encoded by a gene of the subject's cancer cells or a gene expressed thereon. In some embodiments, the peptide sequence is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 or more naturally occurring amino acids.

[0288] In some embodiments, the peptide sequence binds to a protein encoded by a class I HLA allele and has a length of 8-12 naturally occurring amino acids. In some embodiments, the peptide sequence 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 sequence comprises multiple antigenic peptide sequences. In some embodiments, the multiple antigenic peptide sequences comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 antigenic peptide sequences.

[0289] In some embodiments, the antigen described herein is a neoantigen. Candidate immunogenic neoantigen sequences can be identified by any suitable method known in the art. The methods disclosed herein can be used, for example, to generate a specific therapy for a subject's disease or to generate a vaccine against a disease. Candidate immunogenic neoantigens may be previously identified neoantigens. In some embodiments, candidate immunogenic neoantigens may not have been previously identified. Candidate immunogenic neoantigens used in the methods and compositions described herein can be specific to a subject. In some embodiments, candidate neoantigens used in the methods and compositions described herein can be specific to multiple subjects.

[0290] In animals and humans, mutated epitopes can be effective in inducing immune responses or activating T cells. In one embodiment, potential immunogenic epitopes of infectious agents such as viruses can be identified in a subject. In one embodiment, potential immunogenic mutated epitopes can be identified in a subject with a disease such as cancer. In some embodiments, the potential immunogenic antigens or neoantigens used in the methods described herein can be differentiation antigens expressed in tumors and cells of the tissue types that produce them. In some embodiments, the potential immunogenic antigens or neoantigens used in the methods described herein can be cancer / germline antigens not expressed in another differentiation tissue. In some embodiments, the potential immunogenic antigens or neoantigens used in the methods described herein can be mutated antigens. For example, candidate immunogenic antigens or neoantigen peptides used in the methods described herein may comprise antigens or neoantigens of missense point mutations or fusion proteins resulting from tumor-specific translocations of gene segments. In some embodiments, the potential immunogenic antigens or neoantigens used in the methods described herein can be overexpressed antigens. In some embodiments, potential immunogenic antigens or neoantigens can be found in tumors. For example, potential immunogenic antigens or neoantigens used in the methods described herein may include proteins whose expression is tightly regulated in cells of differentiated normal tissues.

[0291] Next-generation sequencing technologies can be used to identify potential immunogenic mutation epitopes by performing genomic or exome sequencing on tumor tissue and healthy tissue from cancer patients. For example, next-generation sequencing technologies can be used to sequence genes selected based on their mutation frequency and ability to act as antigens or neoantigens. In one embodiment, sequencing data can be analyzed to identify peptides with potential immunogenic mutations that can bind to a subject's HLA molecules. In one embodiment, a computer can be used to analyze the data. In another embodiment, the presence of antigenic or neoantigen peptides in the sequence data can be analyzed. In one embodiment, the affinity of a potential immunogenic antigen or neoantigen peptide for an MHC molecule can be used to determine this.

[0292] Potential immunogenic antigens or neoantigenic peptides can be identified by direct protein sequencing. For example, protein sequencing of enzymatic protein digests using multidimensional mass spectrometry techniques (e.g., tandem mass spectrometry (MS / MS)) can be used to identify potential immunogenic antigens or neoantigenic peptides for use with the methods described herein.

[0293] High-throughput methods for de novo sequencing of unknown proteins can be used to identify potential immunogenic antigens or neoantigenic peptides. For example, high-throughput methods for de novo sequencing of unknown proteins, such as meta-shotgun protein sequencing, can be used to analyze the proteome of a subject's tumor to identify potentially immunogenic neoantigens.

[0294] MHC multimers can also be used to identify potential immunogenic antigens or neoantigenic peptides to identify antigen-specific T-cell responses. For example, MHC tetramer-based screening techniques can be used for high-throughput analysis of antigen-specific T-cell responses in patient samples. Tetramer-based screening techniques can be used for the preliminary identification of potential immunogenic tumor-specific antigens, or alternatively as a secondary screening protocol to assess which potential immunogenic antigens a patient may have been exposed to, thereby aiding in the selection of potential immunogenic antigens for use in the methods described herein.

[0295] In some embodiments, a specific neoantigen is targeted for immunotherapy. In some embodiments, a neoantigen peptide is synthesized. The neoantigen peptides used herein are designed such that each peptide is specific for an HLA antigen and can bind to an HLA antigen with high binding affinity and specificity. In some embodiments, the peptides used herein are designed based on a high-performance HLA binding prediction model generated by the inventors and described, for example, in the following patent applications / publications: WO2011143656, WO2017184590, and U.S. Provisional Applications 62 / 783,914 and 62 / 826,827; all of which are incorporated herein by reference. NetMHCIIpan may be the current predictive standard, but may be considered inaccurate. Data may only exist for certain common alleles of HLA-DR in the three class II loci (DR, DP, and DQ). In short, the newly generated predictive model helps identify immunogenic antigenic peptides and can be used to develop drugs, such as personalized medicine, as well as for the isolation and characterization of antigen-specific T cells. The machine learning HLA-peptide presentation predictive model includes: at least several predictive variables determined based on training data, wherein the training data includes: sequence information of peptides presented by HLA proteins expressed in cells and identified by mass spectrometry; training peptide sequence information containing amino acid position information, wherein the training peptide sequence information is associated with HLA proteins expressed in cells; and a function representing the relationship between the amino acid position information received as input and the presentation probability generated as output based on the amino acid position information and the predictive variables. CD4+ T cell responses can have anti-tumor activity. In existing predictive methods, high CD4+ T cell response rates can be observed without using class II predictions (e.g., 60% of SLP epitopes in the NeoVax study (49% in NT-001) and 48% of mRNA epitopes in the BioNTech study). It may be unclear whether these epitopes are typically presented naturally (by tumors or phagocytic dendritic cells). Therefore, it is hoped that improved identification of naturally presented class II epitopes will translate high CD4+ T response rates into therapeutic effects. The roles of gene expression, enzyme cleavage, and pathway / localization bias may not yet be strongly quantified. It may be unclear whether autophagy (class II presentation by tumor cells) or phagocytosis (class II presentation of tumor epitopes by APCs) is a more relevant pathway, although most existing MS data are likely presumed to originate from autophagy. Different data generation methods may exist to learn the rules of class II presentation, including field criteria and proposed approaches. Field criteria could include affinity measurements, which could form the basis of the NetMHCIIpan predictor, offering low throughput and requiring radioactive reagents, and omitting the role of processing.The new approach includes mass spectrometry, where data from cell lines / tissues / tumors can help determine autophagy processing rules (most of which are already published), while monoallelic MS allows for the determination of allele-specific binding rules (assuming multiallelic MS data is too complex for effective learning). The newly generated prediction method includes training a machine learning HLA-peptide presentation prediction model, wherein training involves using a computer processor to input the amino acid position information sequences of HLA-peptides isolated from one or more HLA-peptide complexes from cells expressing HLA class II alleles into the HLA-peptide presentation prediction model; the machine learning HLA-peptide presentation prediction model includes: at least a plurality of predictor variables determined based on training data, which includes: sequence information of peptides presented by HLA proteins expressed in cells and identified by mass spectrometry; training peptide sequence information containing amino acid position information of the training peptides, wherein the training peptide sequence information is associated with HLA proteins expressed in cells; and amino acids representing those received as input. The presentation model is a function of the relationship between positional information and the presentation probability generated as output based on the amino acid positional information and predictor variables. In some embodiments, the presentation model has a positive predictive value of at least 0.25 with a recall of 0.1%-10%. In some embodiments, the presentation model has a positive predictive value of at least 0.4 with a recall of 0.1%-10%. In some embodiments, the presentation model has a positive predictive value of at least 0.6 with a recall of 0.1%-10%. In some embodiments, the mass spectrometry method is monoallelic mass spectrometry. In some embodiments... The peptide is presented by HLA proteins expressed in the cell via autophagy. In some embodiments, the peptide is presented by HLA proteins expressed in the cell via phagocytosis. In some embodiments, the quality of training data is improved by using multiple quality metrics. In some embodiments, the multiple quality metrics include removal of common contaminant peptides, high score peak intensity, high score, and high quality accuracy. In some embodiments, the score peak intensity is at least 50%. In some embodiments, the score peak intensity is at least 70%. In some embodiments, the peptide presented by the HLA protein expressed in the cell is a peptide presented by a single immunoprecipitated HLA protein expressed in the cell. In some embodiments, the multiple predictor variables include a peptide-HLA affinity predictor variable. In some embodiments, the multiple predictor variables include a source protein expression level predictor variable. In some embodiments, the multiple predictor variables include a peptide cleavability predictor variable. In some embodiments, the peptide presented by the HLA protein includes peptides identified by searching a peptide database using a reverse database search strategy. In some embodiments, the HLA protein is HLA-DR and HLA-DP or HLA-DQ protein.In some embodiments, the HLA protein is an HLA-DR protein selected from HLA-DR and HLA-DP or HLA-DQ proteins. In some embodiments, the HLA protein is an HLA-DR protein selected from the group consisting of: HLA-DPB1*01:01 / HLA-DPA1*01:03, HLA-DPB1*02:01 / HLA-DPA1*01:03, HLA-DPB1*03:01 / HLA-DPA1*01:03, HLA-DPB1*04:01 / HLA-DPA1*01:03, HLA-DPB1*04:02 / HLA-DPA1*01:03, HLA-DPB1*06:01 / HLA-DPA1*01:03, HLA -DQB1*02:01 / HLA-DQA1*05:01,HLA-DQB1*02:02 / HLA-DQA1*02:01, HLA-DQB1*06:02 / HLA-DQA1*01:02,HLA-DQB1*06:04 / HLA-D QA1*01:02, HLA-DRB1*01:01, HLA-DRB1*01:02, HLA-DRB1*03:01, HLA-DRB1*03:02, HLA-DRB1*04:01, HLA-DRB1*04:02, HLA-DRB 1*04:03, HLA-DRB1*04:04, HLA-DRB1*04:05, HLA-DRB1*04:07, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*08:02, HLA-DRB1* 08:03, HLA-DRB1*08:04, HLA-DRB1*09:01, HLA-DRB1*10:01, HLA-DRB1*11:01, HLA-DRB1*11:02, HLA-DRB1*11:04, HLA-DRB1*12 HLA-DRB1*12:02, HLA-DRB1*13:01, HLA-DRB1*13:02, HLA-DRB1*13:03, HLA-DRB1*14:01, HLA-DRB1*15:01, HLA-DRB1*15:02, HLA-DRB1*15:03, HLA-DRB1*16:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB3*03:01, HLA-DRB4*01:01, and HLA-DRB5*01:01. In some embodiments, the peptides presented by HLA proteins include peptides identified by comparing the MS / MS spectra of HLA-peptides with the MS / MS spectra of one or more HLA-peptides in a peptide database.

[0296] In some implementations, the mutation is selected from point mutations, splice site mutations, frameshift mutations, readthrough mutations, and gene fusion mutations.

[0297] In some embodiments, the peptide presented by the HLA protein has a length of 15-40 amino acids. In some embodiments, the peptide presented by the HLA protein includes a peptide identified by: (a) isolating one or more HLA complexes from a cell line expressing a single HLA class II allele; (b) isolating one or more HLA-peptides from said one or more isolated HLA complexes; (c) obtaining MS / MS profiles of said one or more isolated HLA-peptides; and (d) obtaining peptide sequences corresponding to the MS / MS profiles of said one or more isolated HLA-peptides from a peptide database; wherein the sequence of said one or more isolated HLA-peptides is identified from the one or more sequences obtained in step (d).

[0298] Various antigenic peptides can be used to induce or expand T cells. Various antigenic peptides can be used to activate antigen-presenting cells (APCs), which in turn activate T cells by bringing them into contact with antigen-loaded APCs.

[0299] In some embodiments, the peptide contains a mutation selected from the following: (A) point mutation, (B) splice site mutation, (C) frameshift mutation, (D) readthrough mutation, (E) gene fusion mutation, and combinations thereof. In some embodiments, the peptide contains a point mutation and binds to the subject's HLA protein with a greater affinity than the corresponding wild-type peptide.

[0300] In some embodiments, the peptide is expressed at IC50 values ​​of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 Binds to the subject's HLA protein. In some embodiments, the peptide is expressed at an IC50 concentration of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K D The peptide binds to the subject's HLA protein. In some embodiments, each peptide binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, the TCR of induced or amplified antigen-specific T cells is reduced to an IC50 of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K D The TCR binds to the peptide-HLA complex. In some embodiments, the TCR is expressed at IC50 values ​​of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K DThe binding peptide-HLA complex. In some embodiments, each of the at least one antigenic peptide sequence contains a mutation not present in the subject's non-cancer cells. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or expressed gene of the subject's cancer cells.

[0301] In some embodiments, the peptide has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 or more naturally occurring amino acids. In some embodiments, the peptide binds to a protein encoded by a class I HLA allele and has a length of 8-12 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 multiple peptides. In some embodiments, the multiple peptides comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 or more antigenic peptides.

[0302] In some respects, this disclosure provides peptides (e.g., peptides with tumor-specific mutations, viral peptides, or peptides associated with non-cancerous diseases) or polynucleotides encoding such peptides, identified using the methods briefly described above.

[0303] In some embodiments, optical methods are used to select or identify immunogenic antigens. In some embodiments, barcode probes are used to select or identify immunogenic antigens. In some embodiments, barcode probes comprising a target-specific region and a barcode-coded region are used to select or identify immunogenic antigens. In some embodiments, the target-specific region comprises a nucleic acid sequence that hybridizes to a target polynucleotide or has at least about 90%, 95%, or 100% sequence complementarity.

[0304] Preparation of activated antigen-specific T cells

[0305] This article provides 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 induce or activate T cells. For example, the methods provided herein can be used to expand activated T cells. For example, the methods provided herein can be used to induce naive T cells. For example, the methods provided herein can be used to expand antigen-specific CD8+ cells. + T cells. For example, the methods presented in this article can be used to expand antigen-specific CD4 cells. + T cells. For example, the methods presented in this article can be used to expand antigen-specific CD8 cells with memory phenotypes. + T cells. For example, a therapeutic composition may contain antigen-specific CD8+ T cells. For example, a therapeutic composition may contain antigen-specific memory T cells.

[0306] T cells can be activated in vitro using a composition containing a neoantigen peptide or a polynucleotide encoding a neoantigen peptide.

[0307] T cells can be activated in vitro using a composition containing antigen-presenting cells loaded with antigens.

[0308] In some implementations, APCs and / or T cells are derived from biological samples obtained from the subject.

[0309] In some implementations, APCs and / or T cells are derived from biological samples as peripheral blood mononuclear cells (PBMCs).

[0310] In some implementations, FLT3L is administered to the subject prior to obtaining a biological sample for preparing APCs and / or T cells.

[0311] In some implementations, APCs and / or T cells are derived from biological samples used as leukocyte isolates.

[0312] In some embodiments, antigen-presenting cells are first loaded with neoantigen peptides in vitro and used to prepare neoantigen-activated T cells. In some embodiments, the compositions provided herein comprise T cells stimulated by an APC, such as an APC pre-loaded with an antigen peptide. The compositions may comprise an immune cell population containing T cells from a sample (e.g., a biological sample), wherein said T cells include APC-stimulated T cells. In some embodiments, mRNA encoding one or more neoantigen peptides is introduced into the APC for neoantigen peptide expression. Such APCs are used to stimulate or activate T cells.

[0313] In some embodiments, the biological sample comprises at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total cell count in a biological sample derived from peripheral blood or leukocyte ablation, comprising less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or less than 10% of antigen-activated T cells. In some embodiments, the biological sample comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% of the total cell count in a biological sample derived from peripheral blood or leukocyte ablation, of antigen-activated T cells.

[0314] In some embodiments, the biological sample contains antigen-naïve T cells. In some embodiments, the biological sample comprises antigen-immature cells of a total cell count greater than about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% from a biological sample derived from peripheral blood or leukocyte ablation.

[0315] In some embodiments, the composition contains at least one antigen-specific CD8. + The percentage of T cells is less than approximately 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, and 5% in biological samples derived from peripheral blood or leukocyte apheresis. In some embodiments, the composition contains at least one antigen-specific CD4. +The percentage of T cells is at least approximately 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% in biological samples derived from peripheral blood or leukocyte apheresis.

[0316] In some embodiments, the percentage of the at least one antigen-specific T cells in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total immune cells. In some embodiments, the biological sample contains at least one antigen-specific CD8+. + The percentage of T cells is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of total immune cells. In some embodiments, the biological sample contains at least one antigen-specific CD4. + The percentage of T cells is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of total immune cells. In some embodiments, the percentage of antigen-specific T cells in the biological sample is at most about 0.5%. In some embodiments, the biological sample contains neoantigen-specific CD8+. + The percentage of T cells is at most about 0.5%. In some embodiments, the biological sample contains antigen-specific CD4. + The percentage of T cells was at most about 0.5% of the biological sample.

[0317] Preparation of APC loaded with neoantigen

[0318] In some embodiments, the composition comprises a population of immune cells that has been 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, IL-7, FLT3L, TNF-α, IL-1β, IL-15, PGE1, IL-6, IFN-α, R848, LPS, ss-rna40, and poly(I:C). In some embodiments, the composition comprises a population of immune cells that has been incubated with one or more APCs or APC products. For example, the composition may comprise a population of immune cells that has been incubated with APCs or APC products stimulated by one or more cytokines, growth factors, and / or ligands. For example, the composition may comprise a population of immune cells that has been incubated with APCs or APC products stimulated by one or more cytokines. For example, the composition may comprise a population of immune cells that has been incubated with APCs stimulated by one or more growth factors or with an APC product stimulated by growth factors. Alternatively, the composition may comprise a population of immune cells that has been incubated with APCs stimulated by one or more ligands or with an APC product stimulated by ligands.

[0319] In some implementations, the APC is an autologous APC, an allogeneic APC, or an artificial APC.

[0320] Immune cells are characterized by cell surface molecules. In some embodiments, immune cells are preferably selected based on cell surface markers, for example, by selecting from a biological sample using antibodies capable of binding to cell surface receptors. In some embodiments, some cells are negatively selected to enrich one or more cell types that do not express the cell surface molecules to which they are negatively selected.

[0321] In some embodiments, antigen-presenting cells (APCs) are prepared from biological samples by selection from APCs or precursor cells, said cells being cultured in the presence of neoantigen peptides to produce neoantigen-loaded APCs for activating T cells. Some relevant cell surface markers for selecting and / or enriching a group of cells are described below.

[0322] CD1 (differentiation cluster 1) is a family of glycoproteins expressed on the surface of various human antigen-presenting cells. They are associated with class I MHC molecules and are involved in the presentation of lipid antigens to T cells.

[0323] CD11b, or integrin αM (ITGAM), is a heterodimeric integrin α-Mβ-2 (α MThe β2) protein subunit, also known as macrophage-1 antigen (Mac-1) or complement receptor 3 (CR3). ITGAM is also called CR3A and differentiation cluster molecule 11b (CD11b). α M The second chain of β2 is the common integrin β2 subunit called CD18, therefore integrin α M β2 belongs to the β2 subfamily (or leukocyte) integrin. α M β2 is expressed on the surface of many leukocytes involved in the innate immune system, including monocytes, granulocytes, macrophages, and natural killer cells. It mediates inflammation by regulating leukocyte adhesion and migration and participates in various immune processes such as phagocytosis, cell-mediated cytotoxicity, chemotaxis, and cell activation. It is also involved in the complement system due to its ability to bind inactivated complement component 3b (iC3b). Integrin α M The ITGAM(α) subunit of β2 is directly involved in inducing cell adhesion and diffusion, but cannot mediate cell migration in the absence of the β2(CD18) subunit.

[0324] CD11c, also known as integrin αX (complement component 3 receptor 4 subunit) (ITGAX), is the gene encoding CD11c. CD11c is an integrin αX chain protein. Integrins are heterodimer membrane proteins composed of α and β chains. This protein combines with the β2 chain (ITGB2) to form a leukocyte-specific integrin called inactivated C3b (iC3b) receptor 4 (CR4). The αXβ2 complex appears to overlap with the properties of the αMβ2 integrin in the adhesion of neutrophils and monocytes to stimulated endothelial cells and in the phagocytosis of complement-coated granules. CD11c is a type I transmembrane protein found at high levels on most dendritic cells, but also on monocytes, macrophages, neutrophils, and some B cells. It induces cell activation and helps trigger the respiratory burst of neutrophils; it is expressed in hairy cell leukemia, acute non-lymphocytic leukemia, and some B-cell chronic lymphocytic leukemias.

[0325] CD14 is a surface antigen preferentially expressed on monocytes / macrophages. It works in conjunction with other proteins to mediate an innate immune response to bacterial lipopolysaccharide (LPS). Alternative splicing results in multiple transcriptomorphs encoding the same protein. CD14 exists in two forms: one anchored to the membrane via a glycosylphosphatidylinositol tail (mCD14), and the other is a soluble form (sCD14). Soluble CD14 either appears after the shedding of mCD14 (48 kDa) or is secreted directly from intracellular vesicles (56 kDa). CD14 acts as a co-receptor (along with Toll-like receptors TLR4 and MD-2) for the detection of bacterial LPS. CD14 binds to LPS only in the presence of lipopolysaccharide-binding protein (LBP). Although LPS is considered its primary ligand, CD14 also recognizes other pathogen-associated molecular patterns, such as lipoteichoic acid.

[0326] CD25 is expressed by conventional T cells upon stimulation, and it has been shown that only CD4 is expressed in human peripheral blood. + CD25 hi T cells are "inhibitors".

[0327] In some implementations, APCs comprise dendritic cells (DCs). In some implementations, APCs are derived from CD14. + Monocytes. In some embodiments, APCs may be obtained from skin, spleen, bone marrow, thymus, lymph nodes, peripheral blood, or umbilical cord blood. In some embodiments, CD14... + Monocytes are derived from biological samples from subjects containing PBMCs. For example, CD14 can be isolated, enriched, or purified from biological samples from subjects containing PBMCs. + Monocytes. In some implementations, CD14 is stimulated with one or more cytokines or growth factors. + Monocytes. In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, R848, LPS, ss-rna40, poly-I:C, or combinations thereof. In some embodiments, CD14 + The mononuclear cells were derived from a second biological sample containing PBMCs.

[0328] In some embodiments, the isolated APC population may be enriched or substantially enriched. In some embodiments, the isolated APC population is at least 30%, at least 50%, at least 75%, or at least 90% homogeneous. In some embodiments, the isolated APC population is at least 60%, at least 75%, or at least 90% homogeneous. APCs, such as APCs, may include, for example, APCs derived in culture from mononuclear dendritic precursors, as well as endogenously derived APCs present in tissues such as peripheral blood, umbilical cord blood, skin, spleen, bone marrow, thymus, and lymph nodes.

[0329] APCs and cell populations substantially enriched with APCs can be isolated using methods further provided by this invention. These methods typically involve obtaining a cell population comprising APC precursors, differentiating the APC precursors into immature or mature APCs, and may further include isolating APCs from the differentiated immature or mature APC population.

[0330] APC precursor cells can be obtained using methods known in the art. APC precursors can be isolated using, for example, density gradient separation, fluorescence-activated cell sorting (FACS), immunocellular separation techniques such as panning, complement lysis, rosette, magnetic cell separation, nylon hair separation, and combinations of these methods. Methods for immunoselecting APCs include, for example, using antibodies against cell surface markers associated with APC precursors, such as anti-CD34 and / or anti-CD14 antibodies conjugated to the substrate.

[0331] 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 adhering to a substrate. Using tissue sources such as bone marrow or peripheral blood, adhering mononuclear cells can be removed from cell products using commercially treated plastic substrates (e.g., beads or magnetic beads) to obtain non-adherent populations of enriched APC precursors.

[0332] Monocyte APC precursors can also be obtained from tissue sources using an APC precursor adhesion substrate. For example, peripheral blood leukocytes separated by, for example, leukocyte ablation are contacted with a mononuclear APC precursor adhesion substrate having a high surface area to volume ratio, and the adhered mononuclear APC precursors are separated. In another embodiment, the coupled substrate can be a granular or fibrous substrate with a high surface area to volume ratio, such as microbeads, microcarrier beads, pellets, granules, powders, capillaries, microporous membranes, etc. Furthermore, the granular or fibrous substrate can be glass, polystyrene, plastic, glass-coated polystyrene microbeads, etc.

[0333] APC precursors can also be cultured in vitro for differentiation and / or expansion. Methods for differentiating / expanding APC precursors are known in the art. Typically, expansion can be achieved by culturing the precursors in the presence of at least one cytokine that induces APC differentiation / proliferation (e.g., dendritic cells). These cytokines are typically granulocyte colony-stimulating factor (G-CSF) or granulocyte / phage colony-stimulating factor (GM-CSF). Additionally, other agents can be used to inhibit the proliferation and / or maturation of non-APC cell types in the culture, thereby further enriching the APC precursor population. Typically, such agents include cytokines such as IL-13, IL-4, or IL-15.

[0334] The isolated APC precursor population is cultured and differentiated to obtain immature or mature APCs. Suitable tissue culture media include, but are not limited to, those mentioned above. RPMI 1640, DMEM, X-VIVO, etc. Tissue culture media are typically supplemented with amino acids, vitamins, divalent cations, and cytokines to promote the differentiation of precursors into the APC phenotype. Common differentiation-promoting cytokines are GM-CSF and / or IL-4.

[0335] Furthermore, during the expansion, differentiation, and maturation of the APC phenotype, cultures of APC precursors can include plasma to promote APC development. Typical plasma concentrations are approximately 5%. Additionally, in cases where APC precursors are isolated via adhesion to a substrate, plasma can be included in the culture medium during the adhesion step to promote early CD14 development. + Phenotype. Typical plasma concentrations during the adhesion process are approximately 1% or higher.

[0336] Monocyte APC precursors can be cultured for any suitable time. In some embodiments, a suitable culture time for differentiating the precursor into immature APCs can be from about 1 day to about 10 days, for example from about 4 days to about 7 days. This can be achieved by methods known to those skilled in the art, such as by the presence or absence of cell surface markers (e.g., CD11c). + CD83 low, CD86 - / Low HLA-DR + This can be used to monitor the differentiation of immature APCs from precursors. Immature APCs can also be cultured in appropriate tissue media to maintain them in a state of further differentiation or antigen uptake, processing, and presentation. For example, immature APCs can be maintained in the presence of GM-CSF and IL-4.

[0337] In some embodiments, APC precursors can be isolated prior to differentiation. In some embodiments, the isolated population can be enriched or substantially enriched with APC precursors. In some embodiments, APC precursors are isolated using a CD14-specific probe. In an exemplary embodiment, CD14-expressing cells are detected by FACS using a CD14-specific probe directly conjugated to a fluorescent molecule (e.g., FITC or PE), or with an unlabeled antibody specific to CD14 and a labeled second antibody specific to the first antibody. CD14 can also be sorted by FACS. + Cells and CD14 低 and CD14 - Cell isolation. CD14 staining can be used as a reference, for example, on monocytes derived from PBMCs, to determine CD14 levels. 高 Positive gating. Typically, CD14-specific binders are, for example, anti-CD14 antibodies (e.g., their monoclonal or antigen-binding fragments). Many anti-CD14 antibodies suitable for use in this invention are well known to those skilled in the art, and many are commercially available. Differentiation into immature APCs (CD14-negative) can occur after isolation.

[0338] In another embodiment, a CD14-specific probe is coupled to a substrate, and CD14 is isolated by affinity selection. + Cells. Will include CD14 + The cell population is exposed to the coupled substrate, and CD14 is activated. + Cell-specific adhesion. Unadhered CD14 was then washed away from the substrate. - Cells are then eluted to obtain a separated cell population substantially enriched with APC precursors. The CD14-specific probe may be, for example, an anti-CD14 antibody. The substrate may be, for example, a commercially available tissue culture plate or beads (e.g., glass or magnetic beads). Methods using substrate-conjugated surface marker-specific antibodies to affinity-separate cell populations are well known.

[0339] During culture, immature APCs may optionally be exposed to a predetermined antigen. Suitable predetermined antigens may include any antigens on which T-cell modulation is desired. In one embodiment, immature APCs are cultured in the presence of prostate-specific membrane antigen (PSMA) for use in cancer immunotherapy and / or tumor growth inhibition. Other antigens may include, for example, bacterial cells, viruses, partially purified or purified bacterial or viral antigens, tumor cells, tumor-specific or tumor-associated antigens (e.g., tumor cell lysates, tumor cell membrane products, antigens isolated from tumors, fusion proteins, liposomes, etc.), recombinant cells expressing antigens on their surface, autoantigens, and any other antigens. Any antigen may also be presented as a peptide or a portion of a recombinantly generated protein. After contact with the antigen, cells may be cultured for any suitable time to allow antigen uptake and processing to expand the antigen-specific APC population, etc.

[0340] For example, in one embodiment, immature APCs can be cultured after antigen uptake to promote their maturation into mature APCs that present antigens in the context of MHC molecules. Methods for APC maturation are known. For example, such maturation can be performed by culturing in the presence of known maturation factors such as cytokines (e.g., TNF-α, IL-1β, or CD40 ligands), bacterial products (e.g., LPS or BCG), etc. The maturation of immature APCs to mature APCs can be monitored by methods known in the art, such as measuring the presence or absence of cell surface markers (e.g., upregulation of CD83, CD86, and MHC molecules) or by using, for example, oligonucleotide arrays to detect the expression of mature APC-specific mRNAs or proteins.

[0341] Optionally, immature APCs can be cultured in a suitable tissue culture medium to expand the cell population and / or maintain immature APCs in a state suitable for further differentiation or antigen uptake. For example, immature APCs can be maintained and / or expanded in the presence of GM-CSF and IL-4. Immature APCs can also be cultured in the presence of anti-inflammatory molecules such as anti-inflammatory cytokines (e.g., IL-10 and TGF-β) to inhibit the maturation of immature APCs.

[0342] On the other hand, the isolated APC population is enriched with mature APCs. A population of isolated mature APCs can be obtained by inducing maturation in the presence of differentiated immature APC populations in the presence of maturation factors (e.g., bacterial products and / or pro-inflammatory cytokines) as described above. Immature APCs can be isolated by removing CD14+ cells.

[0343] According to another aspect of the invention, APCs can be preserved by cryopreservation, for example, before or after exposure to a suitable antigen. Cryopreservatives that can be used include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, isoebasol, D-ribitol, D-mannitol, D-sorbitol, inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glyceryl monoacetate, and inorganic salts. A controlled, slow cooling rate may be critical. Different cryoprotectants and different cell types typically have different optimal cooling rates. The heat generated during the melting phase of water turning into ice should generally be minimized. The cooling process can be performed using, for example, a programmable freezing device or a methanol bath procedure. Programmable freezing devices allow for the determination of the optimal cooling rate and facilitate standard, repeatable cooling. Cryostats with programmable rate control, such as Cryomed or Planar, allow the freezing protocol to be tuned to the desired cooling rate profile.

[0344] After complete freezing, APCs can be rapidly transferred to long-term cryogenic storage containers. In a typical embodiment, samples can be stored cryogenically in liquid nitrogen (-196°C) or its vapor (-165°C). The considerations and procedures for handling, cryopreservation, and long-term preservation of hematopoietic stem cells, particularly those derived from bone marrow or peripheral blood, are largely applicable to the APCs of this invention.

[0345] Frozen cells are preferably thawed rapidly (e.g., in a water bath maintained at 37-41°C) and cooled immediately after thawing. Cell processing may be necessary to prevent clumping during thawing. Various procedures can be used to prevent clumping, including but not limited to adding DNase, low molecular weight dextran and citrate, hydroxyethyl starch, etc., before and / or after freezing. If the cryoprotectant is toxic to humans, it should be removed before the thawed APCs are used for therapeutic purposes. One method of removing the cryoprotectant is to dilute it to an extremely low concentration. Once the frozen APCs have been thawed and recovered, they can be used to activate T cells as described herein with respect to unfrozen APCs.

[0346] In one aspect, compositions for T-cell activation comprise a population of immune cells that have been depleted of one or more types of immune cells. For example, the composition may comprise a population of immune cells that have been depleted of one or more types of immune cells expressing one or more proteins, such as one or more cell surface receptors. In some embodiments, the composition comprises a population of immune cells from a biological sample containing at least one antigen-specific T cell that contains a T-cell receptor (TCR) specific to at least one antigenic peptide sequence, wherein the amount of immune cells expressing CD14 and / or CD25 in the population is proportionally different from the amount of immune cells expressing CD14 and / or CD25 in the biological sample. For example, the composition may comprise a population of immune cells from a biological sample containing at least one antigen-specific T cell that contains a T-cell receptor (TCR) specific to at least one antigenic peptide sequence, wherein the amount of immune cells expressing CD14 in the population is proportionally different from the amount of immune cells expressing CD14 in the biological sample. For example, the composition may comprise a population of immune cells from a biological sample containing at least one antigen-specific T cell that includes a T cell receptor (TCR) specific to at least one antigenic peptide sequence, wherein the amount of CD25-expressing immune cells in the population is proportionally different from the amount of CD25-expressing immune cells in the biological sample. For example, the composition may comprise a population of immune cells from a biological sample containing at least one antigen-specific T cell that includes a T cell receptor (TCR) specific to at least one antigenic peptide sequence, wherein the amount of CD14- and CD25-expressing immune cells in the population is proportionally different from the amount of CD14- and CD25-expressing immune cells in the biological sample. For example, the composition may comprise a population of immune cells from a biological sample wherein the amount of CD14- and CD25-expressing immune cells in the population is proportionally less than the amount of CD14- and CD25-expressing immune cells in the biological sample.

[0347] This article provides a method for preparing a cellular composition for cancer immunotherapy, comprising: I. preparing antigen-loaded antigen-presenting cells (APCs), comprising: (a) obtaining peripheral blood mononuclear cells (PBMCs) from a subject pretreated with fms-like tyrosine kinase 3 ligand (FLT3L); (b) contacting the PBMCs in vitro with: (i) a plurality of cancer neoantigen peptides, or one or more polynucleotides encoding the plurality of cancer neoantigen peptides, wherein each cancer neoantigen peptide or a portion thereof binds to a protein encoded by an HLA allele expressed in the subject; (ii) a stimulant for activating the cells; (iii) an agent for promoting and maintaining the in vitro growth of the cells, thereby obtaining a cell population; and (iv) an agent for reducing or consuming CD11b+ cells from the cell population to obtain CD11b+ cells. 低 Or CD11b-depleted antigen-loaded APCs; II. Incorporate isolated T cells with the CD11b... 低 Or, in vitro contact of antigen-loaded APCs with depleted CD11b; III. Preparation of antigen-induced T cells for use in cell compositions for cancer immunotherapy.

[0348] This article provides an improved method for the ex vivo preparation of tumor antigen-specific T cells, comprising: (a) consuming CD14+ cells and / or CD25+ cells from an immune cell population containing antigen-presenting cells (APCs) and T cells to form a CD14 and / or CD25-depleted immune cell population containing APCs and T cells, wherein the immune cell population is derived from a biological sample of a human subject; (b) incubating the APCs and T cells from step (a) for a first time period in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) containing to (a) a polypeptide containing a tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (b) a polynucleotide encoding the polypeptide; thereby forming a cell population comprising stimulated T cells; (c) expanding the stimulated T cells from step (b) to form an expanded cell population comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific to a complex comprising: (i) at least one tumor antigen epitope sequence from steps (b)(ii), and (ii) an MHC protein expressed by cancer cells or APCs of the human subject from step (b)(ii). This document provides a method comprising administering the expanded cell population from step (c) to the human subject, wherein the expanded cell population from step (c) comprises 1 x 102 8 Up to 1x10 11 Total cells.

[0349] In some implementations, subjects are pretreated with FLT3L for at least approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week prior to PBMC or leukocyte ablation.

[0350] In some embodiments, the cell population is enriched for CD11c+ cells. In some embodiments, the antigen-loaded APCs comprise dendritic cells (DCs). In some embodiments, the antigen-loaded APCs comprise plasmacytoid dendritic cells (pDCs). In some embodiments, the antigen-loaded APCs comprise CD1c+ DCs. In some embodiments, the antigen-loaded APCs comprise CD141+ DCs. In some embodiments, the cell population comprises macrophages. In some embodiments, the method further comprises reducing or consuming CD19+ cells from the cell population for activation or enrichment of neoantigen-activated T cells. In some embodiments, the method further comprises reducing or consuming both CD11b+ and CD19+ cells from the cell population for activation or enrichment of neoantigen-activated T cells.

[0351] In some embodiments, the method further includes reducing or consuming CD14+ cells from the cell population for the preparation and enrichment of antigen-activated T cells. In some embodiments, the method further includes reducing or consuming CD25+ cells from the cell population for the preparation and enrichment of antigen-activated T cells. In some embodiments, the method further includes reducing or consuming one or more of CD19+, CD14+, CD25+, or CD11b+ cells from the cell population for the activation or enrichment of neoantigen-activated T cells.

[0352] In some implementations, the stimulants used to activate cells include FL3TL.

[0353] In some implementations, agents that promote the growth and maintenance of cells in vitro include growth factors, cytokines, amino acids, supplements, or combinations thereof.

[0354] In some implementations, antigen-loaded APCs can stimulate T cells for 2, 3, 4, 5, 6, or 7 days.

[0355] In some implementations, each of the multiple cancer neoantigen peptides is 8-30 amino acids long.

[0356] In some embodiments, each of the plurality of neoantigen peptides contains a neoantigen epitope. In some embodiments, the plurality of cancer neoantigen peptides comprises 2, 3, 4, 5, 6, 7, or 8 neoantigen peptides; and each of the plurality of neoantigen peptides has the neoantigen peptide properties as described in the previous section.

[0357] In some embodiments, the neoantigen peptide used to prepare the antigen-loaded APC is a long peptide containing at least 20 amino acids, or at least 30 amino acids, or at least 40 amino acids, or at least 50 amino acids, or any number of amino acids in between. In some embodiments, the neoantigen peptide used to prepare the antigen-loaded APC contains amino acids flanking either side of the mutation, which promotes endogenous processing of the neoantigen peptide to increase the rate of presentation to T cells.

[0358] Longer immunogenic peptides can be designed in several ways. In some embodiments, when an HLA-binding peptide is predicted or known, the longer immunogenic peptide may consist of: (1) a single binding peptide extending 2-5 amino acids to the N-terminus and C-terminus of each corresponding gene product; or (2) tandem of some or all binding peptides with the extended sequence of each binding peptide. In other embodiments, when sequencing reveals the presence of long (>10 residues) epitope sequences in the tumor, such as novel epitopes (e.g., due to frameshifts, readthroughs, or intron inclusions leading to new peptide sequences), the longer neoantigen peptide may consist of the entire novel tumor-specific amino acid segment as a single longer peptide or several overlapping longer peptides. In some embodiments, it is speculated that the use of longer peptides allows for endogenous processing by patient cells and may lead to more effective antigen presentation and T-cell response induction. In some embodiments, two or more peptides may be used, wherein the peptides overlap and are layered on top of the longer neoantigen peptide.

[0359] In some embodiments, each of the plurality of neoantigen peptides contains the same neoantigen epitope. In some embodiments, the plurality of neoantigen peptides contains more than one neoantigen epitope.

[0360] In some implementations, one or more polynucleotides encoding the various cancer neoantigen peptides are DNA.

[0361] In some implementations, one or more polynucleotides encoding the various cancer neoantigen peptides are inserted into one or more mammalian expression vectors.

[0362] In some implementations, one or more polynucleotides encoding the various cancer neoantigen peptides are messenger RNAs.

[0363] In some embodiments, the present invention provides RNA, oligonucleotides, and polynucleotide molecules comprising modified nucleosides.

[0364] In some embodiments, the present invention provides a gene therapy vector comprising the said RNA, oligonucleotides, and polynucleotides.

[0365] In some embodiments, the present invention provides gene therapy methods and gene transcription silencing methods that include the above.

[0366] In some implementations, the polynucleotide encodes a neoantigen peptide.

[0367] In some implementations, the polynucleotide encodes more than one neoantigen peptide.

[0368] In some embodiments, the polynucleotide is a messenger RNA. In some embodiments, each messenger RNA contains coding sequences for two or more tandem neoantigen peptides.

[0369] In some embodiments, each messenger RNA contains coding sequences for two, three, four, five, six, seven, eight, nine, or ten or more tandem neoantigenic peptides. Typically, the mRNA contains a 5'-UTR, a protein-coding region, and a 3'-UTR. The mRNA has only a limited half-life in cells and in vitro. In some embodiments, the mRNA is a self-amplifying mRNA. In the context of this invention, the mRNA can be generated by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0370] RNA stability and translation efficiency can be altered. For example, RNA can be stabilized, and its translation can be increased by one or more modifications that have stabilizing effects and / or improve RNA translation efficiency. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. To increase the expression of the RNA used according to the invention, the RNA can be modified within the coding region (i.e., the sequence encoding the expressed peptide or protein) without altering the sequence of the expressed peptide or protein, thereby increasing GC content to increase mRNA stability and codon optimization, thereby enhancing translation in the cell.

[0371] In some embodiments, the mRNA may include multiple neoantigen epitopes. In some embodiments, long polynucleotide sequences that encode neo-ORFs, such as mutant GATA3 sequences encoding neo-ORFs, may be used. In some cases, mRNA containing a large portion or even the entire coding region of a gene encoding a neoantigen peptide is delivered to immune cells for endogenous antigen processing and presentation.

[0372] In some implementations, the coding sequence for each neoantigen peptide is 24-120 nucleotides long.

[0373] In some embodiments, the mRNA is 50-10,000 nucleotides long. In some embodiments, the mRNA is 100-10,000 nucleotides long. In some embodiments, the mRNA is 200-10,000 nucleotides long. In some embodiments, the mRNA is 50-5,000 nucleotides long. In some embodiments, the mRNA is 100-5,000 nucleotides long. In some embodiments, the mRNA is 100-1,000 nucleotides long. In some embodiments, the mRNA is 300-800 nucleotides long. In some embodiments, the mRNA is 400-700 nucleotides long. In some embodiments, the mRNA is 450-600 nucleotides long. In some embodiments, the mRNA is at least 200 nucleotides long. In some embodiments, the length of the mRNA is greater than 250 nucleotides, greater than 300 nucleotides, greater than 350 nucleotides, greater than 400 nucleotides, greater than 450 nucleotides, greater than 500 nucleotides, greater than 550 nucleotides, greater than 600 nucleotides, greater than 650 nucleotides, greater than 700 nucleotides, greater than 750 nucleotides, greater than 800 nucleotides, greater than 850 nucleotides, greater than 900 nucleotides, greater than 950 nucleotides, greater than 1000 nucleotides, greater than 2000 nucleotides, greater than 3000 nucleotides, greater than 4000 nucleotides, or greater than 5000 nucleotides.

[0374] In some embodiments, mRNA encoding one or more neoantigenic peptides is modified, wherein the modification involves the 5'-UTR. In some embodiments, the modification involves providing RNA having a 5'-cap or a 5'-cap analogue in the 5'-UTR. The term "5'-cap" refers to a cap structure found at the 5' end of an mRNA molecule and is typically composed of a guanosine nucleotide linked to the mRNA via an unusual 5' to 5' triphosphate bond. In some embodiments, this guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, a 7-methylguanosine cap (mG). In the context of this invention, the term "5'-cap" includes 5'-cap analogues that resemble RNA cap structures and are modified to have the ability to stabilize RNA and / or enhance RNA translation in vivo and / or in cells (if attached to RNA). In some embodiments, the mRNA is co-transcribedly capped.

[0375] In some embodiments, the mRNA encoding one or more neoantigenic peptides includes a 3'-UTR containing a poly-A tail. In some embodiments, the poly-A tail is 100-200 bp in length. In some embodiments, the poly-A tail is longer than 20 nucleotides. In some embodiments, the poly-A tail is longer than 50 nucleotides. In some embodiments, the poly-A tail is longer than 60 nucleotides. In some embodiments, the poly-A tail is longer than 70 nucleotides. In some embodiments, the poly-A tail is longer than 80 nucleotides. In some embodiments, the poly-A tail is longer than 90 nucleotides. In some embodiments, the poly-A tail is longer than 100 nucleotides. In some embodiments, the poly-A tail is longer than 110 nucleotides. In some embodiments, the poly-A tail is longer than 120 nucleotides. In some embodiments, the poly-A tail is longer than 130 nucleotides. In some embodiments, the poly-A tail is longer than 140 nucleotides. In some embodiments, the poly-A tail is longer than 150 nucleotides. In some embodiments, the poly-A tail is longer than 160 nucleotides. In some embodiments, the poly-A tail is longer than 170 nucleotides. In some embodiments, the poly-A tail is longer than 180 nucleotides. In some embodiments, the poly-A tail is longer than 190 nucleotides. In some embodiments, the poly-A tail is longer than 200 nucleotides. In some embodiments, the poly-A tail is longer than 210 nucleotides. In some embodiments, the poly-A tail is longer than 220 nucleotides. In some embodiments, the poly-A tail is longer than 230 nucleotides. In some embodiments, the poly-A tail is longer than 100 nucleotides. In some embodiments, the poly-A tail is longer than 240 nucleotides. In some embodiments, the poly-A tail is longer than 100 nucleotides. In some embodiments, the poly-A tail is approximately 250 nucleotides.

[0376] In some embodiments, the poly-A tail comprises 100-250 adenosine units. In some embodiments, the poly-A tail comprises 120-130 adenine units. In some embodiments, the poly-A tail comprises 120 adenine units. In some embodiments, the poly-A tail comprises 121 adenine units. In some embodiments, the poly-A tail comprises 122 adenine units. In some embodiments, the poly-A tail comprises 123 adenine units. In some embodiments, the poly-A tail comprises 124 adenine units. In some embodiments, the poly-A tail comprises 125 adenine units. In some embodiments, the poly-A tail is 129 bases long.

[0377] In some implementations, the coding sequences of two consecutive neoantigen peptides are separated by a spacer region or a linker.

[0378] In some embodiments, the spacer or linker contains up to 5000 nucleotide residues. An exemplary spacer sequence is GGCGGCAGCGGCGGCGGCGGCAGCGGCGGC. Another exemplary spacer sequence is GGCGGCAGCCTGGGCGGCGGCGGCAGCGGC. Another exemplary spacer sequence is GGCGTCGGCACC. Another exemplary spacer sequence is CAGCTGGGCCTG. Another exemplary spacer sequence is a sequence encoding lysine, such as AAA or AAG. Another exemplary spacer sequence is CAACTGGGATTG.

[0379] In some implementations, the mRNA includes one or more additional structures to enhance the processing and presentation of antigenic epitopes by APCs.

[0380] In some embodiments, the adapter or spacer region may contain cleavage sites. Cleavage sites ensure that the protein product containing the epitope sequence string is cleaved into individual epitope sequences for presentation. Preferably, the cleavage sites are placed adjacent to certain epitopes to avoid unintentionally cleaving epitopes within the sequence. In some embodiments, the epitopes and cleavage regions on the mRNA encoding the epitope string are designed non-randomly.

[0381] In some embodiments, the mRNA encoding the neoantigen peptide of the present invention is administered to a subject in need. In some embodiments, the mRNA to be administered contains at least one modified nucleoside-phosphate.

[0382] In some embodiments, T cells are activated by artificial antigen-presenting cells using neoantigen peptides. In some embodiments, T cells are activated using artificial scaffolds loaded with neoantigen peptides conjugated to MHC antigens, which can bind to the MHC antigens with high affinity.

[0383] In some embodiments, the additional structure includes a specific domain encoding a protein selected from MITD, SP1, and the 10th fibronectin domain: 10FnIII.

[0384] In some embodiments, cells derived from peripheral blood or leukocyte ablation are contacted once or more with a variety of cancer neoantigen peptides or one or more polynucleotides encoding said cancer neoantigen peptides to prepare antigen-loaded APCs.

[0385] In some embodiments, the method includes incubating APCs from one or more APC products with a first medium containing at least one cytokine or growth factor for a first time period.

[0386] In some embodiments, the method includes incubating one or more APC products with at least one peptide for a second time period.

[0387] In some implementations, the enriched cells further include CD1c+ cells.

[0388] In some implementations, the cell population is enriched for CD11c+ and CD141+ cells.

[0389] In some embodiments, the cell population containing the antigen-loaded APC comprises more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more of CD11c+ cells.

[0390] In some embodiments, the cell population containing the antigen-loaded APC comprises fewer than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, 4% or less of cells expressing CD11b+.

[0391] In some embodiments, the cell population containing the antigen-loaded APC comprises more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of CD11c+ neoantigen peptide-expressing cells.

[0392] In some embodiments, the cell population containing antigen-loaded APCs comprises more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of cells expressing neoantigen peptides as CD11c+CD1c+ or CD141+ cells.

[0393] In some implementations, the APC loaded with the neoantigen includes a mature APC.

[0394] In some embodiments, the method includes obtaining a biological sample from a subject, the biological sample containing at least one APC and at least one PBMC or at least one T cell.

[0395] In some embodiments, the method includes consuming cells expressing CD14 and / or CD25 and / or CD19 from a biological sample to obtain a sample depleted of CD14 and / or CD25 and / or CD19 cells.

[0396] In some embodiments, the method includes incubating a sample depleted of CD14 and / or CD25 and / or CD19 cells with FLT3L for a first time period.

[0397] In some embodiments, the method includes incubating at least one peptide with a sample depleted of CD14 and / or CD25 and / or CD19 cells for a second time period to obtain a sample loaded with a first mature APC peptide.

[0398] Neoantigen-activated T cells were prepared using APCs loaded with neoantigens.

[0399] In some embodiments, APCs loaded with neoantigens prepared by the above method are incubated with T cells to obtain antigen-activated T cells. This method may include generating at least one antigen-specific T cell, wherein the antigen is a neoantigen. In some embodiments, generating at least one antigen-specific T cell includes generating multiple antigen-specific T cells.

[0400] In some implementations, T cells are obtained from biological samples from the subject.

[0401] In some embodiments, T cells are obtained from a biological sample from the same subject from whom the APC was derived. In some embodiments, T cells are obtained from a biological sample from a different subject from whom the APC was derived.

[0402] In some embodiments, APCs and / or T cells are derived from biological samples as peripheral blood mononuclear cells (PBMCs). In some embodiments, APCs and / or T cells are derived from biological samples as leukocyte isolates.

[0403] In some implementations, the APC includes dendritic cells (DCs).

[0404] In some implementations, the APC is derived from CD14+ monocytes, or CD14-enriched APCs, or CD141-enriched APCs.

[0405] In some implementations, the CD14+ monocytes are enriched from a biological sample from a subject containing peripheral blood mononuclear cells (PBMCs).

[0406] In some embodiments, the APC is a PBMC. In some embodiments, the PBMC is a freshly isolated PBMC. In some embodiments, the PBMC is a frozen PBMC. In some embodiments, the PBMC is an autologous PBMC isolated from a subject or patient.

[0407] In some embodiments, PBMCs are loaded with antigens, wherein the antigens may be peptides or polypeptides or polynucleotides, such as mRNA, encoding said peptides and polypeptides. PBMCs (monocytes, dendritic cells, phagocytes) can absorb antigens through phagocytosis, process them, and present them on their surface for T cell activation. The peptides or polypeptides loaded on PBMCs may be supplemented with adjuvants to increase immunogenicity. In some embodiments, PBMCs are loaded with nucleic acid antigens. Nucleic acid antigens may be in the form of mRNA containing sequences encoding one or more antigens. In some embodiments, mRNA antigen loading does not require supplemental adjuvants because, for example, RNA itself can act as an adjuvant.

[0408] In some embodiments, PBMCs are directly isolated or thawed from frozen samples and incubated with one or more antigens, such as neoantigens, or compositions containing neoantigens, or one or more nucleic acids or polynucleotides encoding said one or more antigens. In some embodiments, the PBMC samples are not further cultured for differentiation or further maturation of one or more cellular components within the PBMCs (e.g., maturation of antigen-presenting cells, or differentiation of monocytes into dendritic cells) before exposing the PBMCs to one or more antigens or nucleic acids encoding said one or more antigens. In some embodiments, one or more cell types are consumed or removed from freshly isolated or freshly thawed PBMC populations before exposing the cells to one or more antigens or nucleic acids encoding said one or more antigens or incubating with them. In some embodiments, CD14+ cells are consumed from PBMCs. In some embodiments, CD25+ cells are consumed from PBMCs. In some embodiments, CD11b+ cells are consumed from PBMCs. In some embodiments, both CD14+ and CD25+ cells are consumed from PBMCs before incubating with one or more antigens or one or more nucleic acids encoding said one or more antigens. In some embodiments, CD11b+ and / or CD14+ and / or CD25+ cells are consumed from PBMCs. In some embodiments, the methods provided herein include preparing tumor antigen-specific T cells by consuming CD14+ and / or CD25+ cells from a PBMC sample from a human subject, wherein the percentage of immature dendritic cells (DCs) in the sample is substantially the same as the percentage of immature DCs in the peripheral blood of the human subject. In some embodiments, the methods provided herein include preparing tumor antigen-specific T cells by consuming CD14+ and / or CD25+ cells from a PBMC sample from a human subject, wherein the percentage of mature DCs in the sample is substantially the same as the percentage of mature DCs in the peripheral blood of the human subject. In some embodiments, the methods provided herein include preparing tumor antigen-specific T cells by consuming CD14+ and / or CD25+ cells from a PBMC sample from a human subject, wherein the ratio of immature DCs to mature DCs in the sample is substantially the same as the ratio of immature DCs to mature DCs in the peripheral blood of the human subject. In some implementations, the methods provided herein include preparing tumor antigen-specific T cells by consuming CD14+ and / or CD25+ cells from PBMC samples from human subjects, which have not undergone the step of maturing immature DCs into mature DCs.

[0409] In some implementations, CD14+ monocytes are stimulated with one or more cytokines or growth factors.

[0410] In some embodiments, 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, polyI:C, or combinations thereof.

[0411] In some implementations, CD14+ monocytes are derived from a second biological sample containing PBMCs.

[0412] In some implementations, the second biological sample comes from the same subject.

[0413] In some implementations, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0414] In some embodiments, the 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.

[0415] In some implementations, the IDO inhibitor is epacadostat, navoximod, 1-methyltryptophan, or a combination thereof.

[0416] In some implementations, FLT3L is administered to the subject prior to obtaining a biological sample for preparing APCs and / or T cells.

[0417] In some implementations, as described in the preceding sections of this disclosure, T cells are obtained from biological samples from a subject.

[0418] In some implementations, the biological sample is obtained fresh from the subject or is a frozen sample.

[0419] In some embodiments, the incubation is carried out in the presence of at least one cytokine or growth factor, including GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, IL-15, R848, LPS, ss-rna40, polyI:C, or any combination thereof.

[0420] In some embodiments, the method includes stimulating T cells with IL-7, IL-15, or a combination thereof. In some embodiments, the method includes stimulating T cells with IL-7, IL-15, or a combination thereof in the presence of an IDO inhibitor, a PD-1 antibody, or IL-12. In some embodiments, stimulated T cells are expanded under suitable in vitro T cell growth conditions in the presence of the one or more tumor antigen epitope sequences or APCs loaded with the one or more tumor antigen epitope sequences or loaded with (e.g., expressing) a nucleic acid sequence (e.g., mRNA sequence) encoding the one or more tumor antigen epitope sequences, and one or more cytokines or growth factors (including GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, R848, LPS, ss-rna40, poly-I:C, FLT3L, or combinations thereof). In some embodiments, the method further includes administering antigen-specific T cells to a subject.

[0421] In some embodiments, the method includes incubating APCs prepared as described in the previous section with T cells in the presence of a culture medium containing at least one cytokine or growth factor to generate neoantigen-activated T cells.

[0422] In some embodiments, the incubation includes incubating the T cells with a first APC product for more than 7 days. In some embodiments, the incubated T cells are stimulated T cells that have been expanded in vitro for more than 7 days in the presence of the APC product, cytokines, and growth factors.

[0423] In some embodiments, the incubation includes incubating a first APC product with T cells for more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.

[0424] In some implementations, the first time period of the one or more time periods is approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 days.

[0425] In some implementations, the total duration of the individual time periods is less than 28 days. In some implementations, the total duration of the individual time periods is 20-27 days. In some implementations, the total duration of the individual time periods is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 days.

[0426] In some embodiments, the method includes incubating the first APC product with T cells for more than 7 days. In some embodiments, the method includes incubating the first APC product with 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 includes incubating the first APC product with T cells for 7-20, 8-20, 9-20, 10-20, 11-20, or 12-20 days. In some embodiments, the method includes incubating the first APC product with T cells for approximately 10-15 days.

[0427] In some embodiments, the method includes incubating a second APC product with T cells for 5-9 days. In some embodiments, the method includes incubating a second APC product with T cells for 5, 6, 7, 8, or 9 days. In some embodiments, the method further includes removing the one or more cytokines or growth factors of the second medium after the third time period and before the start of the fourth time period.

[0428] In some embodiments, the method includes incubating a third APC product with T cells for 5-9 days. In some embodiments, the method includes incubating a third APC product with T cells for 5, 6, 7, 8, or 9 days.

[0429] In some embodiments, the method includes incubating a first APC product with T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days; incubating a second APC product with T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days; and incubating a third APC product with T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days.

[0430] In some embodiments, the method is performed in vitro. In some embodiments, T cells are cultured in a medium containing cytokines. In some embodiments, examples of cytokines include IL-7. In some embodiments, examples of cytokines include IL-15. In some embodiments, examples of cytokines include both IL-7 and IL-15. In some embodiments, T cells are cultured in a medium containing IL-7 and / or IL-15. In some embodiments, the final concentration of cytokines in the T cell culture or culture medium is at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some implementations, the final concentration of IL-7 in the T cell culture or culture medium is at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the final concentration of IL-15 in the T cell culture or culture medium is at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the T cells are cultured in a medium further containing FLT3L. In some implementations, the final concentration of FLT3L in the T cell culture or culture medium is at least 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, or 200 ng / mL.In some embodiments, T cells are incubated, induced, or stimulated in a medium containing FLT3L for a first time period. In some embodiments, T cells are incubated, induced, or stimulated in a medium containing additional FLT3L for a second time period. In some embodiments, T cells are incubated, induced, or stimulated in a medium containing additional FLT3L for a third time period. In some embodiments, T cells are incubated, induced, or stimulated in a medium containing additional FLT3L for a fourth, fifth, or sixth time period, each time with freshly added FLT3L.

[0431] In some implementations, T cells are cultured in the presence of neoantigens, such as those presented by APCs, wherein the culture medium contains a high potassium [K] concentration. + Content. In some embodiments, a high [K] content is present in the culture medium during incubation with APCs or T cells. + T cells are cultured at a concentration of [K] for at least a certain period of time. In some embodiments, the [K] in the culture medium is added during incubation with APCs or T cells. + The content changes for at least a period of time. In some embodiments, the content in the culture medium remains constant during T cell in vitro culture. In some embodiments, the [K] content in the T cell culture medium... + The concentration is ≥5 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥6 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥7 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥8 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥9 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥10 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥11 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥12 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥13 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥14 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥15 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥16 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥17 mM. In some embodiments, the [K] in the T cell culture medium... +The concentration is ≥18 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥19 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥20 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥22 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥25 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥30 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥35 mM. In some embodiments, the [K] in the T cell culture medium... + The concentration is ≥40 mM. In some embodiments, the [K] in the T cell culture medium... + The content is approximately 40 mM.

[0432] In some implementations, [K] in the T cell culture medium is present for at least a certain period during the incubation of T cells with the neoantigen. + The concentration is approximately 40 mM. In some embodiments, the neoantigen may be presented by an APC loaded with the neoantigen. In some embodiments, in [K] + The T-cell effector function, CD8+ cytotoxicity, cytokine production, and memory phenotype were tested in the presence of [K]. In some implementations, [K] was used to test T-cell effector function, CD8+ cytotoxicity, cytokine production, and memory phenotype. + T cells grown in the presence of [K] express effector T cell phenotypes. In some implementations, in high [K]... + T cells grown in the presence of [K] express memory cell markers. In some implementations, in high [K]... + T cells grown in the presence of [a specific substance] do not express T cell depletion markers.

[0433] In some embodiments, the stimulated T cells are a population of immune cells comprising activated T cells stimulated with an APC containing a neoantigen peptide-MHC complex. In some embodiments, the method may include incubating a population of immune cells from a biological sample with an APC containing a peptide-MHC complex to obtain a stimulated immune cell sample; determining the expression of one or more cell markers in at least one immune cell in the stimulated immune cell sample; and determining the binding of at least one immune cell in the stimulated immune cell sample to the peptide-MHC complex; wherein the determination of the expression of certain cell surface markers or other determinant markers such as intracellular factors or released substances such as cytokines and the determination of binding to the neoantigen peptide-MHC complex are performed simultaneously. In some embodiments, the one or more cell markers include TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, enzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3, or any combination thereof. In some embodiments, the one or more cell markers include cytokines. In some embodiments, the one or more cell markers include degranulation markers. In some embodiments, the one or more cell markers include cell surface markers. In some embodiments, the one or more cell markers include proteins. In some embodiments, determining the binding of at least one immune cell in a stimulated immune cell sample to a peptide-MHC complex includes determining the binding of at least one immune cell in the stimulated immune cell sample to an MHC tetramer comprising the peptide and the peptide-MHC complex. In some embodiments, the MHC is a class I or class II MHC. In some embodiments, the peptide-MHC complex comprises one or more markers.

[0434] In some embodiments, T cell activation is verified by detecting the release of cytokines from activated T cells. In some embodiments, the cytokine is one or more of the following: TNF-α, IFN-γ, or IL-2. In some embodiments, T cell activation is verified by its specific antigen binding and cytokine release. In some embodiments, T cell activation is verified by its ability to kill tumor cells in vitro. Samples of activated T cells can be used to verify the activation status of T cells. In some embodiments, samples from T cells are taken from T cell cultures to determine cell composition and activation status by flow cytometry.

[0435] In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of total T cells or total immune cells. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 5%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 7%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 10%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 12%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 15%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 20%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 25%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 30%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 40%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 50%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 60%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 70%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 80%. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is about 90%.

[0436] In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is about 5%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is about 7%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is about 10%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is approximately 12%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is approximately 15%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is approximately 20%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is approximately 25%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is approximately 30%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is approximately 40%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is approximately 50%. In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the composition is approximately 70% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0437] In some embodiments, the percentage of at least one antigen-specific CD4+ T cells in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0438] In some embodiments, the percentage of the at least one antigen-specific T cell in the biological sample is up to about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0439] In some embodiments, the percentage of the at least one antigen-specific CD8+ T cells in the biological sample is up to about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0440] In some embodiments, the percentage of the at least one antigen-specific CD4+ T cells in the biological sample is up to about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0441] 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.

[0442] In some embodiments, the number of at least one antigen-specific CD8+ T cells in the composition is at least about 1x10^6, 2x10^6, 5x10^6, 1x10^7, 2x10^7, 5x10^7, 1x10^8, 2x10^8 or 5x10^8 antigen-specific CD8+ T cells.

[0443] In some embodiments, the number of at least one antigen-specific CD4+ T cells in the composition is at least about 1x10^6, 2x10^6, 5x10^6, 1x10^7, 2x10^7, 5x10^7, 1x10^8, 2x10^8 or 5x10^8 antigen-specific CD4+ T cells.

[0444] Pharmaceutical Composition

[0445] This document provides compositions (e.g., pharmaceutical compositions) comprising a population of immune cells. The compositions may comprise at least one antigen-specific T cell containing a T cell receptor (TCR). The compositions may comprise at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigenic peptide sequence.

[0446] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, said carriers comprising excipients and adjuvants that facilitate the processing of the active agent into a pharmaceutically acceptable article. Suitable formulations may depend on the chosen route of administration. Any known techniques, carriers, and excipients may be used as suitable techniques, carriers, and excipients as understood in the art.

[0447] In some embodiments, the pharmaceutical composition is formulated as a cell-based therapeutic agent, such as a T-cell therapeutic agent. In some embodiments, the pharmaceutical composition comprises a peptide-based therapy, a nucleic acid-based therapy, an antibody-based therapy, and / or a cell-based therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic agent or a nucleic acid-based therapeutic agent, wherein the nucleic acid encodes a polypeptide. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic agent or a nucleic acid-based therapeutic agent, wherein the nucleic acid encodes a polypeptide; wherein the peptide-based therapeutic agent or the nucleic acid-based therapeutic agent is contained in a cell, wherein the cell is a T cell. In some embodiments, the pharmaceutical composition comprises an antibody-based therapeutic agent. The composition may comprise T cells specific to two or more immunogenic antigens or neoantigen peptides.

[0448] In one aspect, this article provides a pharmaceutical composition comprising (a) an immune cell population comprising T cells derived from a biological sample, wherein the T cells comprise at least one antigen-specific T cell, the antigen-specific T cell being an APC-stimulated T cell and comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence, wherein the APC is an FLT3L-stimulated APC; and (b) a pharmaceutically acceptable excipient.

[0449] In one aspect, this document provides a pharmaceutical composition comprising: (a) a population of immune cells from a biological sample containing at least one antigen-specific T cell, the antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigenic peptide sequence; and (b) a pharmaceutically acceptable excipient; wherein the amount of immune cells expressing CD14 and / or CD25 in the population is proportionally different from the number of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the at least one antigen-specific T cell comprises at least one APC-stimulated T cell. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the population is proportionally less than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the population is proportionally greater than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the at least one antigen-specific T cell comprises at least one CD4+ T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD8+ T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD4-enriched T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD8-enriched T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory CD4+ T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory CD8+ T cell. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of total T cells or total immune cells. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0450] In addition to the active ingredient, the pharmaceutical composition may also contain pharmaceutically acceptable excipients, carriers, buffer solutions, stabilizers, or other substances known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance will depend on the route of administration.

[0451] Acceptable carriers, excipients, or stabilizers are those that are non-toxic to the recipient at the dose and concentration used, and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). Low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as... Or polyethylene glycol (PEG).

[0452] An acceptable carrier is physiologically acceptable to the patient to whom it is administered and maintains the therapeutic properties of the administered compound. Acceptable carriers and their formulations are generally described, for example, in Remington's Pharmaceutical Sciences (18th edition, A. Gennaro, Mack Publishing Co., Easton, PA 1990). An example of a carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable substance, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting the subject compound from one site of administration to another, or in an in vitro assay system. An acceptable carrier is compatible with other components of the formulation and is harmless to the subject to which it is administered. An acceptable carrier should also not alter the specific activity of the neoantigen.

[0453] In one respect, this article provides pharmaceutically acceptable or physiologically acceptable compositions comprising solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coating materials, isotonic agents, and absorption enhancers or delay agents compatible with drug administration. Therefore, a pharmaceutical composition or pharmaceutical formulation refers to a composition suitable for use as a drug in a subject. Compositions may be formulated to be compatible with a specific route of administration (i.e., systemic or local). Therefore, compositions comprise carriers, diluents, or excipients suitable for administration via various routes.

[0454] In some embodiments, the composition may further comprise acceptable additives 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, sorbitol, 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 used to increase the stability of peptides and reduce gelation of solutions, such as polysorbate 20 or polysorbate 80. Surfactants may be added to the composition in amounts from 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0455] Pharmaceutical compositions can be administered, for example, by injection. Compositions for injection include aqueous solutions (which are water-soluble) or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, or phosphate-buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or suitable mixtures thereof. For example, in the case of dispersions, flowability can be maintained by using a coating such as lecithin, by maintaining the desired particle size, and by using surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, such as sugars, polyols like mannitol, sorbitol, and sodium chloride, may be included in the composition. The resulting solution can be packaged for use as is or lyophilized; the lyophilized product can then be combined with a sterile solution prior to administration. For intravenous or site-specific injection, the active ingredient will be in the form of a parenteral acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic media such as sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. A sterile injectable solution can be prepared by incorporating the desired amount of the active ingredient, along with one or a combination of the ingredients listed above (if desired), into a suitable solvent, followed by sterile filtration. Typically, a dispersion is prepared by incorporating the active ingredient into a sterile medium containing an alkaline dispersion medium and any other desired ingredients listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredients from a previously sterilized filtered solution.

[0456] For example, the composition can be routinely administered intravenously, such as by injecting a unit dose. For injection, the active ingredient can be in the form of a parenteral-acceptable aqueous solution that is substantially pyrogen-free and has suitable pH, isotonicity, and stability. Suitable solutions can be prepared using, for example, isotonic media such as sodium chloride injection, Ringer's solution, lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. Alternatively, the composition can be administered via nebulization.

[0457] When considering the use of the composition in pharmaceuticals or any of the methods described herein, it is anticipated that the composition will be substantially pyrogen-free, thereby preventing inflammatory or unsafe allergic reactions when administered to human patients. Testing the composition for pyrogens and preparing substantially pyrogen-free compositions are well known to those skilled in the art and can be accomplished using commercially available kits.

[0458] Acceptable carriers may contain compounds that act as stabilizers, increase or delay absorption, or increase or delay clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, or dextran; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents may also be used to stabilize or increase or decrease the absorption of pharmaceutical compositions, including liposome carriers. To prevent digestion, compounds may be conjugated to the composition to make it resistant to acid and enzymatic hydrolysis, or compounds may be conjugated in a suitable resistant carrier such as liposomes. Means of protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135; and U.S. Patent 5,391,377).

[0459] The composition can be administered in a manner compatible with dosage forms and at a therapeutically effective amount. The amount to be administered depends on the subject being treated, the subject's immune system's ability to utilize the active ingredient, and the desired degree of binding capacity. The precise amount of active ingredient to be administered depends on the practitioner's judgment and is specific to each individual. Appropriate protocols for initial administration and booster injections also differ, but typically involve an initial administration followed by repeated administration at intervals of one or more hours via subsequent injections or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations can be considered.

[0460] In some embodiments, the present invention relates to an immunogenic composition, such as a pharmaceutical composition capable of eliciting a neoantigen-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic composition comprises a neoantigen therapeutic agent described herein (e.g., peptide, polynucleotide, TCR, CAR, TCR- or CAR-containing cells, peptide-containing dendritic cells, polynucleotide-containing dendritic cells, antibody, etc.) corresponding to a tumor-specific antigen or neoantigen.

[0461] In some embodiments, the pharmaceutical compositions described herein can induce specific cytotoxic T cell responses, specific helper T cell responses, or B cell responses.

[0462] In some embodiments, the antigenic peptide or polynucleotide may be provided as an antigen-presenting cell (e.g., dendritic cells) containing such a peptide or polynucleotide. In other embodiments, such antigen-presenting cells are used to stimulate T cells for a patient. In some embodiments, the antigen-presenting cell is a dendritic cell. In relevant embodiments, the dendritic cell is an autologous dendritic cell pulsed with a neoantigen peptide or nucleic acid. The neoantigen peptide may be any suitable peptide that produces an appropriate T cell response. In some embodiments, the T cell is a CTL. In some embodiments, the T cell is an HTL. Thus, one embodiment of this disclosure is an immunogenic composition containing at least one antigen-presenting cell (e.g., dendritic cells) pulsed or loaded with one or more of the neoantigen peptides or polynucleotides described herein. In some embodiments, such APCs are autologous (e.g., autologous dendritic cells). Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient may be loaded with neoantigen peptides or polynucleotides ex vivo. In relevant embodiments, such APCs or PBMCs are injected back into the patient. The polynucleotide can be any suitable polynucleotide capable of transducing dendritic cells, thereby leading to the presentation of neoantigen peptides and the induction of immunity. In some embodiments, such antigen-presenting cells (APCs) (e.g., dendritic cells) or peripheral blood mononuclear cells (PBMCs) are used to stimulate T cells (e.g., autologous T cells). In relevant embodiments, the T cell is a CTL. In other relevant embodiments, the T cell is an HTL. In some embodiments, the T cell is a CD8+ cell. + T cells. In some implementations, these T cells are CD4+ cells. + T cells. These T cells are then injected into the patient.

[0463] In some embodiments, CTL is injected into the patient. In some embodiments, HTL is injected into the patient. In some embodiments, both CTL and HTL are injected into the patient. The administration of either therapeutic agent can be simultaneous or sequential and in any order.

[0464] In some embodiments, the pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment may be formulated for parenteral, topical, nasal, oral, or external administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, such as intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the composition may be administered intratumorally. The composition may be administered at the surgical resection site to induce a local immune response against the tumor. In some embodiments, compositions for parenteral administration are described herein comprising a solution of a neoantigenic peptide, and the immunogenic composition is dissolved or suspended in an acceptable carrier, such as an aqueous carrier. Various aqueous carriers may be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions may be sterilized using conventional, known sterilization techniques or by filtration. The resulting aqueous solution may be packaged for use as is or lyophilized, with the lyophilized product combined with the sterile solution prior to administration. The composition may contain pharmaceutically acceptable adjuvants required to approximate physiological conditions, such as pH adjusters and buffers, tension regulators, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0465] The ability of adjuvants to enhance the immune response to antigens typically manifests as a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity can be reflected in a significant increase in antibody titers against antigens, and an increase in T cell activity can be reflected in increased cell proliferation, cytotoxicity, or cytokine secretion. Adjuvants can also alter immune responses, for example, by changing a primarily humoral or type 2 T helper cell response to a primarily cellular or type 1 T helper cell response.

[0466] Suitable adjuvants are known in the art (see WO2015 / 095811) and include, but are not limited to, poly(I:C), poly-ICLC, STING agonists, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCMATRIX, JuvImmune, LipoVac, MF59, monophospholipid A, Montanide IMS1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, Vector systems, PLG microparticles, remiquimod, SRL172, viromas and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Pam3CSK4, saponin-derived Aquila QS21 stimulant (Aquila Biotech, Worcester, Mass., USA), mycobacterial extracts and synthetic bacterial cell wall mimics, as well as other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Several dendritic cell-specific immune adjuvants have been described (Dupuis M et al., Cell Immunol. 1998; 186(1):18-27; Allison AC; Dev Biol Stand. 1998; 92:3-11)(Mosca et al., Frontiers in Bioscience, 2007; 12:4050-4060)(Gamvrellis et al., Immunol & CellBiol. 2004; 82:506-516). Cytokines may also be used. Several cytokines are directly associated with influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into effective antigen-presenting cells (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1β, IL-4, IL-6, and CD40L) (US Patent 5,849,589, which is incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0467] The effects of CpG immunostimulatory oligonucleotides on adjuvants in a therapeutic setting have also been reported. Unbound by theory, CpG oligonucleotides function by activating the innate (adaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell immunogenic drug compositions, autologous cell immunogenic drug compositions, and polysaccharide conjugates of both prophylactic and therapeutic immunogenic drug compositions. Importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced TH1 cell activation and the generation of highly cytotoxic T lymphocytes (CTLs), even in the absence of CD4+ T cell assistance. Even in the presence of adjuvants such as alum or incomplete Freund's adjuvant (IFA), which typically promote TH2 bias, the TLR9-induced TH1 bias is maintained. When formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, CpG oligonucleotides exhibit even greater adjuvant activity, which is particularly useful for inducing strong responses when the antigen is relatively weak. They can also accelerate immune responses and allow for reduced antigen doses, showing comparable antibody responses in some studies to full-dose immunogenic drug compositions without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484). U.S. Patent 6,406,705 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. One commercially available CpG TLR9 antagonist is dSLIM (a dual-stem-ring immunomodulator) from Mologen (Berlin, DE), which is a component of the drug compositions described herein. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, may also be used.

[0468] Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g., CpR, Idera), poly(I and / or polyC) (e.g., polyI:CI2U), non-CpG bacterial DNA or RNA, ssRNA40 targeting TLR8, and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, trimemumab, and SC58175, which can act therapeutically and / or as adjuvants. The amount and concentration of adjuvants and additives that can be used in the context of this invention can be readily determined by those skilled in the art without requiring extensive experimentation. Additional adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, saxaglastine).

[0469] In some embodiments, the immunogenic composition according to this disclosure may contain more than one different adjuvant. Furthermore, the invention includes pharmaceutical compositions containing any adjuvant substance, including any of the adjuvants described above and combinations thereof. In some embodiments, the immunogenic composition contains a neoantigen therapeutic agent (e.g., peptide, polynucleotide, TCR, CAR, TCR- or CAR-containing cells, polypeptide-containing dendritic cells, polynucleotide-containing dendritic cells, antibodies, etc.), and the adjuvants may be administered alone in any suitable order.

[0470] Lipidification can be categorized into several different types, such as N-myristoylation, palmitoylation, GPI-anchoring, isopentenylation, and several other types of modification. N-myristoylation is the covalent attachment of a myristate ester (C14 saturated acid) to a glycine residue. Palmitoylation is the thioester linkage of a long-chain fatty acid (C16) to a cysteine ​​residue. GPI-anchoring is the linkage via an amide bond to glycosyl-phosphatidylinositol (GPI). Isopentenylation is the thioether linkage of an isoprene-like lipid (e.g., farnesyl (C-15), geraniol (C-20)) to a cysteine ​​residue. Other types of modification may include S-diacylglycerol attachment via the sulfur atom of cysteine, O-octanoyl conjugation via serine or threonine residues, S-archaeol conjugation to cysteine ​​residues, and cholesterol attachment.

[0471] Fatty acids used to generate lipotropic peptides may include C2 to C30 saturated, monounsaturated, or polyunsaturated fatty acyl groups. Exemplary fatty acids may include palmitoyl, myristoyl, stearoyl, and decanoyl. In some cases, a lipid moiety with adjuvant properties is attached to the target peptide to initiate or enhance immunogenicity in the absence of an exogenous adjuvant. Lipotropic peptides or lipopeptides may be referred to as self-adjuvanted lipopeptides. Any fatty acid described above and elsewhere herein may initiate or enhance the immunogenicity of the target peptide. Fatty acids that may initiate or enhance immunogenicity may include palmitoyl, myristoyl, stearoyl, lauroyl, capryloyl, and decanoyl.

[0472] Peptides such as naked peptides or liposomes can be incorporated into liposomes. Sometimes, liposomes can be incorporated into liposomes. For example, the lipid portion of a liposome can spontaneously integrate into the lipid bilayer of a liposome. Therefore, lipopeptides can be presented on the "surface" of the liposome. Exemplary liposomes suitable for incorporation into formulations include, but are not limited to, multilayer vesicles (MLV), oligolayer vesicles (OLV), monolayer vesicles (UV), small monolayer vesicles (SUV), medium-sized monolayer vesicles (MUV), large monolayer vesicles (LUV), giant monolayer vesicles (GUV), multivesicles (MVV), monolayer or oligolayer vesicles prepared by reverse-phase evaporation (REV), multilayer vesicles prepared by reverse-phase evaporation (MLV-REV), stable multilayer vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion (VET), vesicles prepared by Freund's crusher (FPV), vesicles prepared by fusion (FUV), dehydrated-rehydrated vesicles (DRV), and foam bodies (BSV).

[0473] Depending on the preparation method, liposomes can be monolayered or multilayered and can vary in size, ranging from about 0.02 μm to greater than about 10 μm in diameter. Liposomes can adsorb onto many types of cells and then release incorporated reagents (e.g., peptides described herein). In some cases, liposomes fuse with target cells, thereby subsequently emptying the contents of the liposome into the target cell. Liposomes can be endocytosed by phagocytes. Endocytosis can be followed by lysosomal degradation of liposomal lipids and release of the encapsulating agent.

[0474] The liposomes provided herein may also include carrier lipids. In some embodiments, the carrier lipid is a phospholipid. Carrier lipids capable of forming liposomes include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Other suitable phospholipids include distearylphosphatidylcholine (DSPC), myristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), myristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPA); myristoylphosphatidylglycerol (DMPA), distearylphosphatidylphosphatidylglycerol (DSPA), dipalmitoylphosphatidylserine (DPPS), myristoylphosphatidylserine (DMPS), distearylphosphatidylserine (DSPS), dipalmitoylphosphatidylethanolamine (DPPE), myristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), and combinations thereof. In some embodiments, the liposomes also contain sterols (e.g., cholesterol) that regulate liposome formation. The carrier lipid can be any known non-phosphorylated polar lipid.

[0475] Known techniques can be used to encapsulate pharmaceutical compositions within liposomes. Biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of this invention.

[0476] The pharmaceutical composition can be administered in liposomes or microspheres (or particles). Methods for preparing liposomes and microspheres for administration to patients are known to those skilled in the art. Essentially, the material is dissolved in an aqueous solution, appropriate phospholipids and lipids and surfactants (if desired) are added, and the material is dialyzed or sonicated as needed.

[0477] Microspheres formed from polymers or proteins are well known to those skilled in the art and can be adapted for direct entry into the bloodstream via the gastrointestinal tract. Alternatively, compounds can be incorporated into and implanted into microspheres or microsphere complexes for slow release over days to months.

[0478] Cell-based immunogenic pharmaceutical compositions can also be administered to subjects. For example, antigen-presenting cell (APC)-based immunogenic pharmaceutical compositions can be formulated using any known techniques, carriers, and excipients as understood in the art. APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells. Sometimes, APC-based immunogenic pharmaceutical compositions can be dendritic cell-based immunogenic pharmaceutical compositions.

[0479] Dendritic cell-based immunogenic pharmaceutical compositions can be prepared by any method known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared by in vitro or in vivo methods. In vitro methods may include using autologous dendritic cells (DCs) pulsed with the peptide described herein in vitro to activate or load DCs prior to administration to a patient. In vivo methods may include using an antibody conjugated to the peptide described herein to target a specific DC receptor. DC-based immunogenic pharmaceutical compositions may further comprise DC activators, such as TLR3, TLR-7-8, and CD40 agonists. DC-based immunogenic pharmaceutical compositions may further comprise adjuvants and pharmaceutically acceptable carriers.

[0480] Adjuvants can be used to enhance the immune response (humoral and / or cellular response) induced in patients receiving immunogenic drug compositions. Sometimes, adjuvants induce a Th1 response. At other times, they induce a Th2 response. A Th1 response is characterized by the production of cytokines such as IFN-γ, while a Th2 response is characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

[0481] In some respects, lipid-based adjuvants, such as MPLA and MDP, can be used with the immunogenic pharmaceutical compositions disclosed herein. For example, monophospholipid A (MPLA) is an adjuvant that induces increased presentation of liposomal antigens to specific T lymphocytes. Additionally, muramyl dipeptide (MDP) can also be used as a suitable adjuvant with the immunogenic pharmaceutical formulations described herein.

[0482] Adjuvants may also contain stimulating molecules, such as cytokines. Non-limiting examples of cytokines include: CCL20, α-interferon (IFNα), β-interferon (IFNβ), γ-interferon (IFNγ), platelet-derived growth factor (PDGF), TNFα, GM-CSF, epidermal growth factor (EGF), skin T-cell attraction chemokine (CTACK), epidermal thymus expression chemokine (TECK), mucosa-associated epithelial cell chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1-, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA. -1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, angiogenic factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, cysteine ​​ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-I, JNK, interferon response gene, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, and TAP2.

[0483] Other adjuvants include: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic factor, and fibroblast growth factor. IL-7, IL-22, Nerve Growth Factor, Vascular Endothelial Growth Factor, Fas, TNF Receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, Killer, Trail-R2, Trick2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, Inactive NIK, SAP K, SAP-1, JNK, interferon response gene, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[0484] In some respects, adjuvants can be modulators of Toll-like receptors. Examples of Toll-like receptor modulators include TLR9 agonists, and are not limited to small molecule modulators of Toll-like receptors, such as imiquimod. Sometimes, adjuvants are selected from bacterial toxoids, polyoxypropylene-polyethylene glycol block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or combinations thereof. Sometimes, the adjuvant is an oil-in-water emulsion. An oil-in-water emulsion may contain at least one oil and at least one surfactant, wherein the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion may have a diameter of less than 5 μm, or even submicron diameters; these small sizes are achieved via microfluidics to provide a stable emulsion. Droplets smaller than 220 nm can be used for filtration sterilization.

[0485] In some cases, immunogenic pharmaceutical compositions may include carriers and excipients (including, but not limited to, buffers, carbohydrates, mannitol, proteins, peptides or amino acids such as glycine, antioxidants, antibacterial agents, chelating agents, suspending agents, thickeners and / or preservatives), water, oils (including petroleum, animal, plant or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, etc.), salt solutions, aqueous solutions of dextran and glycerol, flavoring agents, coloring agents, anti-sticking agents and other acceptable additives, adjuvants or binders, and other pharmaceutically acceptable excipients required to achieve approximate physiological conditions, such as pH buffers, tension regulators, emulsifiers, wetting agents, etc. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. In another case, the pharmaceutical product is substantially preservative-free. In other cases, the pharmaceutical product may contain at least one preservative. It should be recognized that while any suitable carrier known to those skilled in the art may be used to administer the pharmaceutical compositions described herein, the type of carrier will vary depending on the mode of administration.

[0486] Immunogenic pharmaceutical compositions may include preservatives such as thimerosal or 2-phenoxyethanol. In some cases, immunogenic pharmaceutical compositions are substantially free of mercury (e.g., <10 μg / mL), for example, free of thimerosal. α-Tocopherol succinate can be used as a substitute for mercury compounds.

[0487] To control tension, immunogenic drug compositions may contain physiological salts, such as sodium salts. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride.

[0488] The immunogenic pharmaceutical composition may have a weight molar osmolality of 200 mOsm / kg to 400 mOsm / kg, 240-360 mOsm / kg or 290-310 mOsm / kg.

[0489] Immunogenic drug compositions may contain one or more buffer solutions, such as Tris buffer; borate buffer; succinate buffer; histidine buffer (especially containing aluminum hydroxide adjuvant); or citrate buffer. In some cases, the buffer solution is contained in 5-20 or 10-50 mM.

[0490] The pH of the immunogenic pharmaceutical composition may be from about 5.0 to about 8.5, from about 6.0 to about 8.0, from about 6.5 to about 7.5, or from about 7.0 to about 7.8.

[0491] The immunogenic pharmaceutical composition may be sterile. The immunogenic pharmaceutical composition may be pyrogen-free, for example, containing <1 EU (endotoxin unit, standard measure) per dose, and may be <0.1 EU per dose. The composition may be gluten-free.

[0492] Immunogenic pharmaceutical compositions may contain detergents, such as polyoxyethylene sorbitan ester surfactants (known as "Tween") or poly(oxyethylene styrene) alcohols (such as poly(oxyethylene styrene) alcohol-9 (Triton X-100) or tert-octylphenoxypolyethoxyethanol). The detergent may be present only in trace amounts. Immunogenic pharmaceutical compositions may contain less than 1 mg / mL of each of poly(oxyethylene styrene) alcohol-10 and polysorbate 80. Other trace residual components may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0493] Immunogenic pharmaceutical compositions can be formulated into sterile solutions or suspensions in suitable media known in the art. The pharmaceutical compositions can be sterilized using conventional, known sterilization techniques, or by filtration. The resulting aqueous solution can be packaged for use as is, or lyophilized, with the lyophilized product combined with the sterile solution prior to administration.

[0494] For example, a pharmaceutical composition comprising an active agent (such as the immune cells disclosed herein) can be formulated with one or more adjuvants to have a specific molar ratio. For instance, a molar ratio of approximately 99:1 to approximately 1:99 may be used between the active agent, the immune cells as described herein, and one or more adjuvants. In some cases, the range of the molar ratio of the active agent, the immune cells as described herein, and one or more adjuvants may be selected from approximately 80:20 to approximately 20:80; approximately 75:25 to approximately 25:75; approximately 70:30 to approximately 30:70; approximately 66:33 to approximately 33:66; approximately 60:40 to approximately 40:60; approximately 50:50; and approximately 90:10 to approximately 10:90. The molar ratio of the active agent, the immune cells as described herein, to one or more adjuvants may be approximately 1:9, and in some cases, it may be approximately 1:1. Active agents, such as immune cells as described herein, can be combined with one or more adjuvants and formulated together in the same dosage unit, such as a vial, suppository, tablet, capsule, or aerosol spray; or each agent, form, and / or compound can be formulated in a separate unit, such as two vials, two suppositories, two tablets, two capsules, one tablet and one vial, or aerosol spray.

[0495] In some cases, immunogenic pharmaceutical compositions may be administered in conjunction with adjuvant agents. The choice of adjuvant agents may depend at least in part on the condition being treated. Adjuvant agents may include, for example, checkpoint inhibitors such as anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 agents (e.g., anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 antibodies); or any agent that is effective against pathogen infection (e.g., viral infection), including, for example, drugs used to treat inflammatory conditions such as NSAIDs, such as ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. For example, checkpoint inhibitors can be PD-1 / PD-L1 agonists selected from: nivolumab (ONO-4538 / BMS-936558, MDX1106, OPDIVO), pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011), and MPDL328OA (ROCHE). As another example, the formulation may additionally contain one or more supplements, such as vitamin C, E, or other antioxidants.

[0496] Pharmaceutical compositions comprising an active agent, such as immune cells as described herein, combined with one or more adjuvants, can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients, diluents, and / or adjuvants, such as carriers that facilitate the processing of the active agent into an administerable article. Suitable formulations may depend at least in part on the chosen route of administration. The pharmaceutical agents described herein can be delivered to patients using a variety of routes or modes of administration, including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular administration, as well as inhalation.

[0497] The active agent can be formulated for parenteral administration (e.g., by injection, such as bolus or continuous infusion) and can be presented in unit dose form in ampoules, pre-filled syringes, small-volume infusions, or in multi-dose containers with added preservatives. The composition can be in the form of suspensions, solutions, or emulsions in oily or aqueous media, such as solutions in aqueous polyethylene glycol.

[0498] In some embodiments, the pharmaceutical composition comprises a preservative or stabilizer. In some embodiments, the preservative or stabilizer is selected from cytokines, growth factors, adjuvants, or chemical substances. In some embodiments, the composition comprises at least one agent that helps maintain cell viability through at least one freeze-thaw cycle. In some embodiments, the composition comprises at least one agent that helps maintain cell viability through at least one freeze-thaw cycle.

[0499] For injectable formulations, the carrier can be selected from those suitable in the art, including aqueous solutions or oily suspensions or emulsions (containing sesame oil, corn oil, cottonseed oil, or peanut oil), as well as elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical carriers. The formulation may also contain biocompatible, biodegradable polymer compositions, such as poly(lactic acid-co-glycolic acid). These materials can be formed into microspheres or nanospheres, loaded with the drug, and further encapsulated or derivatized to provide excellent sustained release properties. Carriers suitable for periocular or intraocular injection include, for example, suspensions of the therapeutic agent in injectable water, liposomes, and carriers suitable for lipophilic substances. Other carriers for periocular or intraocular injection are well known in the art.

[0500] In some cases, pharmaceutical compositions are formulated according to standard procedures to be suitable for intravenous administration to the human body. Generally, compositions for intravenous administration are solutions in sterile isotonic buffer solutions. When necessary, the composition may also contain solubilizers and local anesthetics such as lidocaine to reduce pain at the injection site. Typically, the components are supplied separately or mixed together in unit dosage forms, for example, as lyophilized powders or anhydrous concentrates in sealed containers such as ampoules or sachets indicating the amount of active agent. When administered by infusion, the composition can be dispensed using infusion bottles containing sterile pharmaceutical-grade water or saline. When administered by injection, ampoules containing sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0501] Preparation method

[0502] This article provides methods for preparing antigen-specific T cells. This article provides methods for preparing T cell compositions, such as therapeutic T cell compositions. For example, methods may include expanding or inducing antigen-specific T cells. Preparation (e.g., induction or expansion) of T cells can also refer to the preparation of T cells and broadly encompasses the isolation, stimulation, culture, induction, and / or expansion of any type of T cells (e.g., CD4+). + T cells and CD8 +A procedure for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence, the method comprising incubating the APC with a population of immune cells from a biological sample of depleted cells expressing CD14 and / or CD25. In some embodiments, the method comprises preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence, the method comprising incubating the APC with a population of immune cells from a biological sample of depleted cells expressing CD11b and / or CD19. In some embodiments, the method comprises incubating the APC with a population of immune cells from a biological sample of depleted cells expressing any CD11b and / or CD19 and / or CD14 and / or CD25 or any combination thereof.

[0503] In a second aspect, this article provides a method for preparing at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs together with a population of immune cells from a biological sample.

[0504] In a third aspect, this article provides a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising: incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample for a first time period; and subsequently incubating at least one T cell from the biological sample with an antigen-specific T cell (APC).

[0505] In a fourth aspect, this document provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating a population of immune cells from a biological sample with one or more APC products for one or more separate time periods, said time period being less than 28 days from the incubation of the immune cell population with the first of the one or more APC products, wherein at least one antigen-specific memory T cell is expanded, or at least one antigen-specific naive T cell is induced.

[0506] In a fifth aspect, this document provides a method for preparing at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating a population of immune cells from a biological sample with three or fewer APC products for three or fewer time periods, wherein at least one antigen-specific memory T cell is expanded, or at least one antigen-specific naive T cell is induced.

[0507] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with one or more APC products for one or more separate time periods to stimulate T cells to become antigen-specific T cells, wherein the percentage of antigen-specific T cells is 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 for preparing antigen-specific T cells comprising a T-cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with three or fewer APC products for three or fewer individual time periods, thereby stimulating the T cells to become antigen-specific T cells. In some embodiments, a method for preparing antigen-specific T cells comprising a T-cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with two or fewer APC products for two or fewer individual time periods, thereby stimulating the T cells to become antigen-specific T cells.

[0508] In some embodiments, this document provides a method comprising incubating a population of immune cells from a biological sample with one or more APC products for one or more separate time periods to stimulate T cells to become antigen-specific T cells, wherein the APC product is a population of PBMC cells, and consuming cells expressing one or more cell surface markers from the cell population prior to loading the APC population with antigen. In some embodiments, CD14+ cells are consumed prior to loading the APC population with antigen. In some embodiments, CD25+ cells are consumed prior to loading the APC population with antigen. In some embodiments, CD11b+ cells are consumed prior to loading the APC population with antigen. In some embodiments, CD19+ cells are consumed prior to loading the APC population with antigen. In some embodiments, CD3+ cells are consumed prior to loading the APC population with antigen. In some embodiments, both CD25+ and CD14+ cells are consumed prior to loading the APC population with antigen. In some embodiments, both CD11b+ and CD25+ cells are consumed prior to loading the APC population with antigen. In some embodiments, both CD11b+ and CD14+ cells are consumed prior to loading the APC population with antigen. In some embodiments, CD11b+, CD14+, and CD25+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+ and CD19+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+, CD19+, and CD25+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+, CD14+, CD19+, and CD25+ cells are consumed before the APC population is loaded with antigen. In some embodiments, the method includes adding a CD3+ cell-rich PBMC-derived population of APCs enriched by CD3+ cell depletion to any of the aforementioned consumed APC populations. In some embodiments, the APC-rich PBMC-derived population of cells is CD3+ depleted, and the cells are depleted by any one or more of CD11b+, CD14+, CD19+, or CD25+.

[0509] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the method includes adding a composition comprising one or more antigenic peptides or their encoded nucleic acids to the PBMC sample, thereby loading antigens onto the APCs within the PBMCs for antigen presentation to T cells in the PBMCs.

[0510] In some implementations, the method includes: (a) obtaining a biological sample from a subject, the biological sample containing at least one antigen-presenting cell (APC); and (b) enriching cells expressing CD11c from the biological sample to obtain CD11c. + (c) Samples enriched with cells; CD11c+ (d) Incubating the cell-enriched sample with at least one cytokine or growth factor for a first time period; (c) Adding at least one peptide to (d) CD11c + (e) Incubate the enriched sample together for a second time period to obtain an APC peptide-loaded sample; (f) Incubate the APC sample together with one or more cytokines or growth factors for a third time period to obtain a mature APC sample; (g) Incubate the APC of the mature APC sample together with a sample depleted of CD11b and / or CD14 and / or CD25 containing PBMCs for a fourth time period; (h) Incubate the PBMC together with the APC of the mature APC sample for a fifth time period; (i) Incubate the PBMC together with the APC of the mature APC sample for a sixth time period; and (ii) Administer at least one T cell from the PBMC to a subject in need.

[0511] In some implementations, the method includes: (a) obtaining a biological sample from a subject, the biological sample containing at least one antigen-presenting cell (APC); and (b) enriching cells expressing CD14 from the biological sample to obtain CD14. + (c) Samples enriched with cells; CD14 + (d) Incubating the cell-enriched sample with at least one cytokine or growth factor for a first time period; (c) Incubating at least one peptide with (d) CD14. + (e) Incubate the enriched sample together for a second time period to obtain an APC peptide-loaded sample; (f) Incubate the APC peptide-loaded sample together with one or more cytokines or growth factors for a third time period to obtain a mature APC sample; (g) Incubate the APC of the mature APC sample together with a sample depleted of CD14 and / or CD25 containing PBMCs for a fourth time period; (h) Incubate the PBMCs together with the APC of the mature APC sample for a fifth time period; (i) Incubate the PBMCs together with the APC of the mature APC sample for a sixth time period; and (ii) Administer at least one T cell from the PBMC to a subject in need.

[0512] In some embodiments, the method includes: (a) obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; (b) consuming cells expressing CD11b and / or CD19 from the biological sample to obtain a CD11b and / or CD19 cell-depleted sample; (c) incubating the CD11b and / or CD19 cell-depleted sample with FLT3L for a first time period; and (d) incubating at least one peptide with the CD11b and / or CD19 cell-depleted sample from (c) for a second time period to obtain an APC peptide-loaded sample. (e) Incubating the APC peptide-loaded sample with at least one PBMC for a third time period to obtain a first-stimulated PBMC sample; (f) Incubating the PBMCs of the first-stimulated PBMC sample with the APCs of a mature APC sample for a fourth time period to obtain a second-stimulated T cell sample; (g) Incubating the PBMCs of the second-stimulated PBMC sample with the APCs of a mature APC sample for a fifth time period to obtain a third-stimulated PBMC sample; (h) Administering at least one T cell from the third-stimulated PBMC sample to a subject in need.

[0513] In some embodiments, the method includes: (a) obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; (b) consuming cells expressing CD11b and / or CD19 and / or CD14 and / or CD25 from the biological sample to obtain a CD11b and / or CD19 cell-depleted sample; (c) incubating the CD11b and / or CD19 and / or CD14 and / or CD25 cell-depleted sample with FLT3L for a first time period; and (d) adding at least one peptide to the CD11b and / or CD19 and / or CD14 and / or CD25 cell-depleted sample from (c). (e) Incubate the APC peptide-loaded sample for a second time period to obtain a sample of APC peptide-loaded cells; (f) Incubate the APC peptide-loaded sample with at least one PBMC for a third time period to obtain a sample of PBMCs with first stimulation; (g) Incubate the PBMCs of ...

[0514] In some embodiments, the method includes: (a) obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; (b) consuming cells expressing CD14 and / or CD25 from the biological sample to obtain a sample depleted of CD14 and / or CD25 cells; (c) incubating the sample depleted of CD14 and / or CD25 cells with FLT3L for a first time period; and (d) incubating at least one peptide with the sample depleted of CD14 and / or CD25 cells from (c) for a second time period to obtain a sample loaded with APC peptide. (e) Incubate the APC peptide-loaded sample with at least one PBMC for a third time period to obtain a first-stimulated PBMC sample; (f) Incubate the PBMCs of the first-stimulated PBMC sample with the APCs of a mature APC sample for a fourth time period to obtain a second-stimulated T cell sample; (g) Incubate the PBMCs of the second-stimulated PBMC sample with the APCs of a mature APC sample for a fifth time period to obtain a third-stimulated PBMC sample; (h) Administer at least one T cell from the third-stimulated PBMC sample to a subject in need.

[0515] In some embodiments, a method for preparing at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigen peptide sequence includes incubating the APC with a population of immune cells from a biological sample of depleted cells expressing CD14 and / or CD25.

[0516] In some embodiments, this document provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence, the method comprising incubating a population of immune cells from a biological sample with one or more APC products for one or more separate time periods, said time period being less than 28 days from the incubation of the immune cell population with a first of the one or more APC products, wherein at least one antigen-specific memory T cell is amplified, or at least one antigen-specific naive T cell is induced. In some embodiments, this document provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence, the method comprising incubating a population of immune cells from a biological sample with three or fewer APC products for three or fewer separate time periods, wherein at least one antigen-specific memory T cell is amplified, or at least one antigen-specific naive T cell is induced.

[0517] In some embodiments, a method for preparing antigen-specific T cells comprising a T-cell receptor (TCR) specific to at least one antigenic peptide sequence includes contacting a population of immune cells (e.g., PBMCs) with an APC. In some embodiments, a method for preparing antigen-specific T cells comprising a T-cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells (e.g., PBMCs) with an APC for a period of time. In some embodiments, the immune cell population is derived from a biological sample. In some embodiments, the immune cell population is derived from a sample (e.g., a biological sample) depleted of cells expressing CD14. In some embodiments, the immune cell population is derived from a sample (e.g., a biological sample) depleted of cells expressing CD25. In some embodiments, the immune cell population is derived from a sample (e.g., a biological sample) depleted of both cells expressing CD14 and cells expressing CD25.

[0518] In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs with a population of immune cells from a biological sample. In some embodiments, a method is provided herein for preparing a pharmaceutical composition comprising at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigenic peptide sequence, the method comprising: incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample for a first time period; thereafter incubating at least one T cell from the biological sample with an APC.

[0519] In some embodiments, a method for preparing at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes contacting a population of immune cells from a sample (e.g., a biological sample) with an FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes contacting a population of immune cells from a sample (e.g., a biological sample) with an APC stimulated by an FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a sample (e.g., a biological sample) with an APC stimulated by an FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell (the at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence) includes incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample (e.g., for a period of time); then contacting the T cells of the biological sample with an APC. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes contacting a population of immune cells from a sample (e.g., a biological sample) with one or more APC products. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a sample (e.g., a biological sample) with one or more APC products for one or more separate time periods. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a sample (e.g., a biological sample) with one or more APC products for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate time periods. In some embodiments, the one or more separate time periods, calculated from the start of incubation of the immune cell population with the first of the one or more APC products, are less than 28 days.

[0520] In some embodiments, a method for preparing antigen-specific T cells comprising a T-cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells with APCs for a period of time, wherein the immune cell population is derived from a biological sample containing PBMCs. In some embodiments, a method for preparing antigen-specific T cells comprising a T-cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells with APCs for a period of time, wherein the immune cell population is derived from a biological sample depleted of cells expressing CD14 and / or CD25.

[0521] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with APCs stimulated by FMS-like tyrosine kinase 3 receptor ligand (FLT3L) for a period of time.

[0522] In some embodiments, a method for preparing a pharmaceutical composition comprising antigen-specific T cells (the antigen-specific T cells comprising T cell receptors (TCRs) that are specific to at least one antigenic peptide sequence) includes incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample; and then contacting the T cells of the biological sample with APCs.

[0523] In some embodiments, a method for preparing antigen-specific T cells comprising a T-cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with one or more APC products for one or more separate time periods to induce or expand antigen-specific T cells, wherein the one or more separate time periods, calculated from the start of incubation of the immune cell population with the first of the one or more APC products, are less than 28 days. In some embodiments, the incubation of the population of immune cells from a biological sample with one or more APC products for one or more separate time periods is carried out in a medium containing IL-7, IL-15, or a combination thereof. In some embodiments, the medium further comprises an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof. The IDO inhibitor may be epacadostat, navoximod, 1-methyltryptophan, or a combination thereof. In some embodiments, the IDO inhibitor may increase antigen-specific CD8. + The number of cells. In some implementations, IDO inhibitors can maintain memory CD8. +The functional spectrum of T cell responses. PD-1 antibodies can increase the absolute number of antigen-specific memory CD8+ T cell responses. PD-1 antibodies can increase the proliferation rate of cells treated with this antibody. The addition of IL-12 can lead to an increase in antigen-specific cells and / or CD8+ T cells. + The frequency of T cells increases.

[0524] In some embodiments, a method for preparing antigen-specific T cells comprising a T-cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with one or more APC products for one or more separate time periods to expand or induce antigen-specific T cells, wherein the antigen-specific T cells, antigen-specific CD4+, and other antigen-specific T cells are present. + T cells or antigen-specific CD8 + The percentage of T cells is the total number of T cells and 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 immune cells, or total cells.

[0525] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific to at least one antigenic peptide sequence includes incubating a population of immune cells from a biological sample with three or fewer APC products for three or fewer separate time periods, thereby stimulating the T cells to become antigen-specific T cells.

[0526] In some embodiments, the immune cell population is derived from a biological sample depleted of cells expressing CD14 and / or CD25. In some embodiments, the APC is an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) stimulated APC. In some embodiments, the APC comprises one or more APC products. In some embodiments, the APC product comprises three or fewer APC products. In some embodiments, the APC products are sequentially incubated with immune cells at one or more separate time intervals.

[0527] In some embodiments, the biological sample is derived from a subject. In some embodiments, the subject is a human being. For example, the subject may be a patient or a donor. In some embodiments, the subject suffers from a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the antigen-specific T cells contain CD4. + and / or CD8 + T cells. In some embodiments, the antigen-specific T cells comprise CD4-enriched T cells and / or CD8-enriched T cells. For example, CD4 can be isolated, enriched, or purified from a biological sample from a subject containing PBMCs. + T cells and / or CD8 + T cells. In some implementations, antigen-specific T cells are naive CD4+ cells. + and / or childish CD8 + T cells. In some implementations, antigen-specific T cells are memory CD4 cells. + and / or memorize CD8 + T cells.

[0528] In some embodiments, the at least one antigenic peptide sequence comprises a mutation selected from: (A) a point mutation, and the cancer antigen peptide having an IC50 of less than 500 nM. 50 The at least one antigenic peptide sequence binds to the subject's HLA protein with a greater affinity than the corresponding wild-type peptide, and includes (B) splice site mutations, (C) frameshift mutations, (D) read-through mutations, (E) gene fusion mutations, and combinations thereof. In some embodiments, each of the at least one 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 contains a mutation not present in the subject's non-cancer cells. In some embodiments, each of the at least one antigenic peptide sequence is encoded by an expressed gene of the subject's cancer cells. In some embodiments, one or more of the at least one antigenic peptide sequence has a length of 8-50 naturally occurring amino acids. In some embodiments, the at least one antigenic peptide sequence comprises multiple antigenic peptide sequences. In some embodiments, the multiple antigenic peptide sequences comprise 2-50, 3-50, 4-50, 5-50, 6-50, 7-50, 8-50, 9-50, or 10-50 antigenic peptide sequences.

[0529] In some embodiments, the APC includes an APC loaded with one or more antigenic peptides, said antigenic peptides comprising one or more of at least one antigenic peptide sequence. In some embodiments, the APC is an autologous APC or an allogeneic APC. In some embodiments, the APC includes dendritic cells (DCs).

[0530] In some embodiments, the method includes consuming cells expressing CD14 and / or CD25 from a biological sample. In some embodiments, CD14 is consumed. + The cell includes contacting the CD14 binding agent with APC. In some embodiments, the APC is derived from CD14. + Mononuclear cells. In some implementations, APCs are enriched from biological samples. For example, APCs can be isolated, enriched, or purified from biological samples from subjects containing PBMCs.

[0531] In some embodiments, APCs are stimulated with one or more cytokines or growth factors. In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, or combinations thereof. In some embodiments, the one or more cytokines or growth factors include IL-4, GM-CSF, TNF-α, IL-1β, PGE1, IL-6, IL-7, or combinations thereof.

[0532] In some implementations, the APC is derived from a second biological sample. In some implementations, the second biological sample is derived from the same subject.

[0533] In some embodiments, the percentage of antigen-specific T cells in the method is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells. In some embodiments, the percentage of antigen-specific T cells in the method is about 0.1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to 65%, or about 65% to about 70%. In some embodiments, the method uses antigen-specific CD8+. + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells. In some embodiments, the method uses antigen-specific naive CD8+. +The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells. In some embodiments, the method incorporates antigen-specific memory CD8. + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells. In some embodiments, antigen-specific CD4 is used in this method. + 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, antigen-specific CD4 is used in this method. + 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+ in the biological sample is... + 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 biological sample contains antigen-specific naive CD8+. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the biological sample contains antigen-specific memory CD8. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, antigen-specific CD4 in the biological sample +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%.

[0534] In some implementations, the biological samples are either freshly obtained from the subject or frozen samples.

[0535] In some embodiments, the method includes incubating one or more APC products together with a first medium containing at least one cytokine or growth factor for a first time period. In some embodiments, the first time period 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 time period is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days.

[0536] In some implementation schemes, the first time period is at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 days.

[0537] In some embodiments, the first time period does not exceed 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the at least one cytokine or growth factor includes GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-γ, LPS, IFN-α, R848, LPS, ss-rna40, poly-I:C, or any combination thereof.

[0538] In some embodiments, the method includes incubating one or more APC products with at least one peptide for a second time period. In some embodiments, the second time period does not exceed 1 hour.

[0539] In some embodiments, the method includes incubating one or more APC products with a second medium containing one or more cytokines or growth factors for a third time period to obtain mature APC. In some embodiments, the one or more cytokines or growth factors include GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-4, FLT3L, IFN-γ, LPS, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848 (requimod), LPS, ss-rna40, poly-I:C, CpG, or combinations thereof. In some embodiments, the third time period is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days. In some embodiments, the third time period is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days. In some implementations, the third time period is no more than 2, 3, 4, or 5 days. In other implementations, the third time period is at least 1, 2, 3, or 4 days.

[0540] In some embodiments, the method further includes removing one or more cytokines or growth factors from the second medium after the third time period and before the start of the fourth time period.

[0541] PBMCs loaded with antigens for in vitro T cell induction

[0542] In some embodiments, the methods provided herein include isolating PBMCs from a human blood sample and directly loading antigens onto the PBMCs. PBMCs in direct contact with the antigens can readily absorb the antigens through phagocytosis and present them to T cells that may be present in or added to the culture. In some embodiments, the methods provided herein include isolating PBMCs from a human blood sample and nuclear transfecting or electroporating the PBMCs with polynucleotides encoding one or more antigens, such as mRNA. In some embodiments, antigen delivery to PBMCs, rather than antigen-presenting cells that have matured into dendritic cells (DCs), provides significant advantages in terms of time and preparation efficiency. PBMCs may further deplete one or more cell types. In some embodiments, PBMCs may deplete CD3+ cells at the initial stage of antigen loading, and the CD3+ cells are returned to the PBMC culture to stimulate CD3+ T cells. In some embodiments, PBMCs may deplete CD25+ cells. In some embodiments, PBMCs may deplete CD14+ cells. In some embodiments, PBMCs may deplete CD19+ cells. In some embodiments, PBMCs may deplete both CD14 and CD25-expressing cells. In some embodiments, CD11b+ cells are consumed from the PBMC sample prior to antigen loading. In some embodiments, both CD11b+ and CD25+ cells are consumed from the PBMC sample prior to antigen loading.

[0543] In some implementations, PBMCs isolated from human blood samples can be processed to the lowest possible extent before loading the antigen. Increased processing of PBMCs, such as freezing and thawing cells, multiple cell consumption steps, etc., may impair cell health and viability.

[0544] In some implementations, PBMCs are allogeneic to the treatment subject. In some implementations, PBMCs are allogeneic to the subject receiving adoptive cell therapy with antigen-specific T cells.

[0545] In some implementations, the PBMCs are HLA-matched for the treatment subject. In other implementations, the PBMCs are allogeneic and HLA-matched for the subject's subtype, while the CD3+ T cells are autologous. The PBMCs are loaded with their respective antigens (e.g., determined from analysis by a peptide presentation analysis platform such as RECON) and co-cultured with the subject's T-cell-containing PBMCs to stimulate antigen-specific T cells.

[0546] In some embodiments, mRNA is used as an immunogen for both uptake and antigen presentation. One advantage of using mRNA over peptide antigen loading of PBMCs is that the RNA itself acts as an adjuvant, eliminating the need for additional adjuvants. Another advantage of using mRNA is that the peptide is endogenously processed and presented. In some embodiments, the mRNA comprises a shortmer construct encoding a peptide of 9-10 amino acids containing an epitope. In some embodiments, the mRNA comprises a longmer construct encoding a peptide of approximately 25 amino acids. In some embodiments, the mRNA comprises a tandem of multiple epitopes. In some embodiments, the polymer may contain one or more epitopes from the same antigenic protein. In some embodiments, the polymer may contain one or more epitopes from several different antigenic proteins. Several embodiments are described in the Examples section. Loading antigens into PBMCs via antigen loading can include various mechanisms for delivering and incorporating nucleic acids into the PBMCs. In some embodiments, the delivery or incorporation mechanisms include transfection, electroporation, nuclear transfection, chemical delivery such as lipid encapsulation or liposome-mediated delivery.

[0547] Using antigen-loaded PBMCs to stimulate T cells can save the maturation time required in methods for generating DCs from PBMC samples prior to T cell stimulation. In some embodiments, using antigen-loaded PBMCs, such as mRNA-loaded PBMCs as APCs, reduces the total preparation time by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, using antigen-loaded PBMCs as APCs reduces the total preparation time by 3 days. In some embodiments, using antigen-loaded PBMCs as APCs reduces the total preparation time by 4 days. In some embodiments, using antigen-loaded PBMCs as APCs reduces the total preparation time by 5 days. In some embodiments, using antigen-loaded PBMCs as APCs reduces the total preparation time by 6 days. In some embodiments, using antigen-loaded PBMCs as APCs reduces the total preparation time by 7 days.

[0548] In some embodiments, using mRNA as the antigen may be preferred because it is easy to design and prepare nucleic acids and easy to transfect PBMCs. In some embodiments, mRNA-loaded PBMCs can stimulate T cells and generate more antigen-specific T cells. In some embodiments, mRNA-loaded PBMCs can stimulate T cells and generate a higher yield of antigen-specific T cells. In some embodiments, mRNA-loaded PBMCs can stimulate T cells and generate antigen-specific T cells with a higher presentation of the input antigen, i.e., reactive to a wide variety of antigens. In some embodiments, mRNA-loaded PBMCs can stimulate T cells in an expanded cell pool that are reactive to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens. In some embodiments, mRNA-loaded PBMCs can stimulate T cells that are reactive to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens compared to conventional antigen-loaded APCs (such as peptide-loaded DCs).

[0549] Treatment

[0550] This article provides a method for treating cancer in a subject, comprising: I. contacting antigen-presenting cells (APCs) loaded with cancer neoantigens with isolated T cells in vitro, wherein the antigen-presenting cells (APCs) loaded with cancer neoantigens are CD11b depleted; II. preparing cancer neoantigen-induced T cells for use in a cell composition for ex vivo cancer immunotherapy; and III. administering the cell composition for cancer immunotherapy to a subject, wherein the administration alleviates or improves at least one or more cancer-related conditions or symptoms, thereby treating the subject, wherein the cancer neoantigen-loaded APCs and cancer neoantigen-induced T cells each express a protein encoded by an HLA allele expressed in the subject, and the neoantigen can specifically bind to the protein.

[0551] In some embodiments, the method further includes administering one or more of the at least one antigen-specific T cells to a subject. In some embodiments, the therapeutic composition containing T cells is administered by injection. In some embodiments, the therapeutic composition containing T cells is administered by infusion. When administered by injection, the active agent may be formulated in an aqueous solution, particularly in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or saline buffer. The solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. In another embodiment, the pharmaceutical composition does not contain adjuvants or any other substances added to enhance the peptide-stimulated immune response. In some embodiments, the method further includes administering one or more of the at least one antigen-specific T cells as the pharmaceutical composition described herein to a subject. In some embodiments, the pharmaceutical composition contains a preservative or stabilizer. In some embodiments, the preservative or stabilizer is selected from cytokines, growth factors, or adjuvants or chemicals. In some embodiments, at least one antigen-specific T cell is administered to the subject within 28 days from the date of collection of PBMC samples from the subject.

[0552] In addition to the formulations previously described, active agents can also be formulated as depot products. Such long-acting formulations can be administered via implantation or transdermal delivery (e.g., subcutaneous or intramuscular), intramuscular injection, or the use of transdermal patches. Thus, for example, the agent can be formulated with suitable polymeric or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or formulated as a slightly soluble derivative, such as a slightly soluble salt.

[0553] This document also provides methods for treating subjects suffering from a disease, condition, or illness. Treatment methods may include administering the compositions or pharmaceutical compositions disclosed herein to subjects suffering from a disease, condition, or illness.

[0554] This disclosure provides treatment methods including immunogenic therapies. Methods for treating diseases such as cancer or viral infections are provided. The methods may include administering to a subject an effective amount of a composition comprising immunogenic antigen-specific T cells according to the methods provided herein. In some embodiments, the antigen comprises a viral antigen. In some embodiments, the antigen comprises a tumor antigen.

[0555] Non-limiting examples of therapeutic agents that can be prepared include peptide-based therapeutic agents, nucleic acid-based therapeutic agents, antibody-based therapeutic agents, T-cell-based therapeutic agents, and antigen-presenting cell-based therapeutic agents.

[0556] In some other aspects, this document provides for the use of compositions or pharmaceutical compositions in the preparation of medicaments for treatment. In some embodiments, the treatment method includes administering to a subject an effective amount of T cells that specifically recognize an immunogenic neoantigen peptide. In some embodiments, the treatment method includes administering to a subject an effective amount of a TCR that specifically recognizes an immunogenic neoantigen peptide, such as a TCR expressed in T cells.

[0557] In some implementations, the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, colorectal cancer, rectal cancer, soft tissue sarcoma, Kaposi's sarcoma, B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma). NHL, small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunogenic NHL, high-grade lymphocytic NHL, high-grade small non-lytic cell NHL, large mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloma, hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorder (PTLD), abnormal angiogenesis associated with nevus hamartomatosis, edema, Meigs syndrome, and combinations thereof.

[0558] The methods described herein are particularly useful in personalized medicine settings, where immunogenic neoantigen peptides identified according to the methods described herein are used to develop therapeutic agents (such as vaccines or therapeutic antibodies) for the same individual. Therefore, methods for treating a subject's disease may include identifying an immunogenic neoantigen peptide in the subject according to the methods described herein; synthesizing said peptide (or a precursor thereof, such as a polynucleotide encoding the peptide (e.g., mRNA)); preparing T cells specific to the identified neoantigen; and administering the neoantigen-specific T cells to the subject. In some embodiments, methods for treating a subject's disease may include identifying an immunogenic neoantigen peptide in the subject according to the methods described herein; synthesizing a polynucleotide encoding said immunogenic neoantigen peptide or a precursor thereof, such as mRNA; preparing T cells specific to the identified neoantigen; and administering the neoantigen-specific T cells to the subject.

[0559] The agents and compositions described herein can be used alone or in combination with conventional treatment regimens such as surgery, radiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated). For example, the methods described herein can be used to identify a group of tumor antigens, and these can be used, for example, in most cancer patients.

[0560] In some embodiments, in addition to the composition comprising an immunogenic therapeutic agent, at least one or more chemotherapeutic agents may be administered. In some embodiments, the one or more chemotherapeutic agents may belong to different classes of chemotherapeutic agents.

[0561] When implementing the treatments or methods of use described herein, a therapeutically effective amount of the therapeutic agent may be administered to a subject suffering from a disease or condition. The therapeutically effective amount can vary widely depending on the severity of the disease, the subject's age and relative health status, the potency of the compound used, and other factors.

[0562] Subjects may be, for example, mammals, humans, pregnant women, older adults, adults, adolescents, pre-pubescent children, children, toddlers, infants, newborns, or newborn babies. Subjects may be patients. In some cases, subjects may be humans. In some cases, subjects may be children (i.e., young adults below puberty). In some cases, subjects may be infants. In some cases, subjects may be formula-fed infants. In some cases, subjects may be individuals joining a clinical study. In some cases, subjects may be laboratory animals, such as mammals or rodents. In some cases, subjects may be mice. In some cases, the subject may be obese or overweight.

[0563] In some embodiments, the subject has previously received one or more different cancer treatments. In some embodiments, the subjec...

Claims

1. A method for preparing 1x10 8 Up to 1x10 11 An in vitro method for an ex vivo cell population comprising tumor antigen-specific T cells, the method comprising: (a) CD25+ cells are consumed from an immune cell population containing antigen-presenting cells (APCs) and T cells to form a CD25-depleted immune cell population containing APCs and a first population of T cells, wherein the immune cell population is derived from a biological sample of a human subject with cancer. (b) Incubate the first population of APCs and T cells from step (a) for a first time period in the presence of the following substances: (A) a polypeptide containing at least one tumor antigen epitope sequence expressed by cancer cells of the human subject with said cancer, or (B) a polynucleotide encoding the polypeptide. This results in the formation of a cell population containing stimulated T cells; and (c) In vitro expansion of the stimulated T cells from step (b) to form a cell population containing tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific to a complex comprising: (i) at least one tumor antigen epitope sequence from step (b), and (ii) an MHC protein expressed by cancer cells or APCs of the subject in (b). At least 0.1% of the CD8+ T cells in the cell population are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells.

2. The in vitro method according to claim 1, wherein the immune cell population comprising antigen-presenting cells (APCs) and T cells is peripheral blood mononuclear cells (PBMCs).

3. The ex vivo method according to claim 1, wherein steps (b) and (c) are performed in less than 28 days.

4. The in vitro method according to claim 1, wherein the at least one tumor antigen epitope sequence expressed by cancer cells of the subject with cancer comprises at least two tumor antigen epitope sequences, wherein the at least two tumor antigen epitope sequences comprise a tumor antigen epitope sequence of 8 to 12 amino acids in length and a tumor antigen epitope sequence of 15 to 25 amino acids in length.

5. The in vitro method of claim 1, wherein at least one tumor antigen epitope sequence expressed by cancer cells of the subject with cancer is not expressed by non-cancer cells of the human subject with cancer.

6. The in vitro method of claim 1, wherein the consumption further comprises consuming CD14+ T cells from the immune cell population comprising antigen-presenting cells (APCs) and T cells.

7. The in vitro method according to claim 1, wherein the incubation of the first population of APCs and T cells from step (a) for a first time period further includes incubation in the presence of FLT3L.

8. The in vitro method according to claim 1, wherein the cell population containing tumor antigen-specific T cells comprises CD3+ cells, and the percentage of CD3+ cells in the cell population containing tumor antigen-specific T cells is at least 40% of the total cells in the cell population.

9. The in vitro method according to claim 1, wherein the cell population comprising tumor antigen-specific T cells includes: (i) The percentage of CD107a+ cells in the cell population containing tumor antigen-specific T cells is at least 10% of the tumor antigen-specific T cells in the cell population; (ii) The percentage of TNFα+ cells in the cell population containing tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cells in the cell population; (iii) The percentage of IFNγ+ cells in the cell population containing tumor antigen-specific T cells is at least 15% of the tumor antigen-specific T cells in the cell population; (iv) The percentage of TNFα+ and IFNγ+ cells in the cell population containing tumor antigen-specific T cells is at least 2% of the tumor antigen-specific T cells in the cell population; (v) The percentage of TNFα+ and CD107a+ cells in the cell population containing tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cells in the cell population; (vi) The percentage of IFNγ+ and CD107a+ cells in the cell population containing tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cells in the cell population; (vii) The percentage of TNFα+, IFNγ+ and CD107a+ cells in the cell population containing tumor antigen-specific T cells is at least 0.1% of the tumor antigen-specific T cells in the cell population; (viii) The percentage of CD4+ T cells (CD62L+ and CD45RA+) as naive T cells in the cell population containing tumor antigen-specific T cells is at most 15% of the total cells in the cell population; (ix) The percentage of CD4+ T cells (CD62L- and CD45RA-) as effector memory T cells in the cell population containing tumor antigen-specific T cells is at least 60% of the total cells in the cell population; (x) The percentage of CD4+ T cells (CD62L- and CD45RA+) as effector T cells in the cell population containing tumor antigen-specific T cells is at most 5% of the total cells in the cell population; (xi) The percentage of CD4+ T cells (CD62L+ and CD45RA-) as central memory T cells in the cell population containing tumor antigen-specific T cells is at least 10% of the total cells in the cell population; (xii) The cell population containing tumor antigen-specific T cells includes CD8+ cells. The percentage of CD8+ T cells (CD62L+ and CD45RA+) as naive T cells in the cell population containing tumor antigen-specific T cells is at most 25% of the total cells in the cell population; (xiii) The percentage of CD8+ T cells (CD62L- and CD45RA-) that are effector memory T cells in the cell population containing tumor antigen-specific T cells shall be at least 60%; (xiv) The percentage of CD8+ T cells (CD62L- and CD45RA+) as effector T cells in the cell population containing tumor antigen-specific T cells is at most 10%; or (xv) The percentage of CD8+ T cells (CD62L+ and CD45RA-) as central memory T cells in the cell population containing tumor antigen-specific T cells is at least 15%.

10. The in vitro method of claim 1, wherein the cell population comprising tumor antigen-specific T cells produces cytokines and induces degranulation upon recognition of target cells.

11. The ex vivo method of claim 1, wherein the subject is refractory to anti-checkpoint inhibitor therapy.

12. The ex vivo method of claim 1, wherein the subject has a mutation in the BRAF gene and has previously received B-raf inhibitor or B-raf / MEK combination therapy.

13. The ex vivo method of claim 1, wherein consumption comprises consuming CD25+ cells from a peripheral blood mononuclear cell (PBMC) sample from a subject, the sample having not yet undergone the step of monocyte maturation into mature dendritic cells (mature DCs).

14. The in vitro method according to claim 1, wherein the proportion of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in the cell population containing tumor antigen-specific T cells is at least twice the proportion of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in the biological sample.

15. The in vitro method according to claim 1, wherein the proportion of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in the cell population containing tumor antigen-specific T cells is at least twice the proportion of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in the biological sample.

16. The in vitro method according to claim 1, wherein at least 0.1% of the CD4+ T cells in the cell population are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

17. The method for ex vivo extraction according to claim 1, wherein: (I) The cancer is unresectable melanoma; (II) The human subject with the cancer has previously received a PD-1 inhibitor or PD-L1 inhibitor and a regimen containing a CTLA-4 inhibitor and has experienced disease progression; or (III) The human subject with the cancer has received or is currently receiving a PD-1 inhibitor or PD-L1 inhibitor for at least 3 months and has a stable condition or asymptomatic disease progression.

18. The in vitro method according to claim 1, wherein the polynucleotide encoding the polypeptide is mRNA.

19. The in vitro method of claim 1, wherein the polynucleotide encoding the polypeptide encodes at least 2, 3, 4, 5 or more tumor antigen epitope sequences, the tumor antigen epitope sequences being expressed by cancer cells of the human subject with cancer but not by non-cancer cells of the human subject.

20. Contains 1x10 8 Up to 1x10 11 Use of a cell population in the preparation of a medicament for treating cancer in a subject of need, wherein the cell population comprises tumor antigen-specific T cells, and wherein at least 0.1% of the CD8+ T cells in the cell population are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells; wherein: (I) The cancer is unresectable melanoma; (II) The subject has previously received a PD-1 inhibitor or PD-L1 inhibitor and a regimen containing a CTLA-4 inhibitor and has experienced disease progression; or (III) The subject has received or is currently receiving a PD-1 inhibitor or PD-L1 inhibitor for at least 3 months and has a stable condition or asymptomatic disease progression.

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