T cell production composition and method

Abstract

To provide T cell manufacturing compositions and methods.SOLUTION: Generation of antigen-specific T cells by controlled ex vivo induction or expansion can provide highly specific and beneficial T cell therapies. The present disclosure provides T cell manufacturing methods and therapeutic T cell compositions which can be used for treating subjects with cancer and other conditions, diseases and disorders. Personal antigen-specific T cell therapies are also provided. The present disclosure is derived also in part from discovery of new and improved methods for antigenic stimulation, resulting in improved cell compositions for therapeutics development. Provided herein are new methods and compositions such that, at least in part, selective depletion of certain immune cells from the ex vivo stimulation and cell expansion milieu provides new therapeutic compositions and improved methods.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the interests of U.S. Provisional Patent Application No. 62 / 845,251, filed on 8 May 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Tumor vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., adjuvants, cytokines, or TLR ligands) that work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. Such vaccines contain either a mixture of co-existing and patient-specific antigens, either in the form of a co-existing tissue-restricted tumor antigen or a whole-tumor cell preparation. Co-existing tissue-restricted tumor antigens are, ideally, immunogenic proteins that are selectively expressed in tumors across many individuals and are generally delivered to patients as synthetic peptides or recombinant proteins. In contrast, whole-tumor cell preparations are delivered to patients as autologous irradiated cells, cell lysates, cell fusions, heat shock protein preparations, or whole mRNA. Since whole-tumor cells are isolated from the patient's own cells, the cells can contain patient-specific tumor antigens along with co-existing tumor antigens. Finally, there is a third class of tumor antigens, neoantigens, which have rarely been used in vaccines, consisting of proteins with tumor-specific mutations (which may be patient-specific or well-shared) resulting in altered amino acid sequences. Such mutated proteins (a) are specific to tumor cells (because the mutation and its corresponding protein are found only in tumors); (b) evade central tolerance and are therefore more likely to be immunogenic; and (c) provide excellent targets for immune recognition, including by both humoral and cellular immunity.

[0003] Adoptive immunotherapy, or adoptive cell therapy (ACT), is the transfer of lymphocytes into a target for the treatment of disease. It is yet to be seen how adoptive immunotherapy can realize its potential to treat a wide variety of diseases, including cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiency. However, most, if not all, adoptive immunotherapy strategies require T cell activation and augmentation steps to generate clinically effective, therapeutically administered T cells. Due to the inherent complexities of live cell culture and patient-to-patient variability, current techniques for generating therapeutically administered T cells, including engineered T cells, remain limited by cumbersome T cell manufacturing processes. Existing T cell manufacturing processes are neither easily scalable, repeatable, reliable, nor efficient, and often produce inferior T cell products that may be prone to depletion and loss of effector immune cell function. To date, engineered T cell adoptive immunotherapy has achieved only very limited success and routinely exhibits variable clinical activity. Therefore, such therapies are not suitable for broad clinical use. Therefore, there is still a need to develop compositions and methods for increasing and inducing antigen-specific T cells with desirable phenotypes and functions. [Overview of the project] [Means for solving the problem]

[0004] This disclosure provides novel and improved T-cell therapeutic agents for clinical development and use. While autologous T-cell therapeutic agents are safe to use, several dramatic improvements are needed to meet therapeutic standards, and development in this art has been rapid and challenging. The applicant's previously disclosed application provides a hallmark development in compositions and methods for T-cell therapy for cancer (WO2019 / 094642). This application stems from the remarkable discovery that depletion of certain cells expressing specific markers at different stages of ex vivo immune cell preparation provides a highly immunogenic cell composition. This disclosure also stems, in part, from the discovery of novel and improved methods for antigenic stimulation, thereby resulting in improved cell compositions for therapeutic development. Novel methods and compositions are provided herein, in part, that ex vivo stimulation and selective depletion of certain immune cells from a cell-proliferating environment provide novel therapeutic compositions and improved methods.

[0005] An improved ex vivo method for preparing tumor antigen-specific T cells, comprising the steps of: depleting CD14+ cells and / or CD25+ cells from a population of immune cells including antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14 and / or CD25-depleted immune cells including a first population of APCs and T cells, wherein the population of immune cells is derived from a biological sample of a human subject; and, over a first period of time, the population of CD14 and / or CD25-depleted immune cells including the first population of APCs and T cells is subjected to a polypeptide comprising (A) at least one tumor antigen epitope sequence expressed by cancer cells of a human subject having cancer or (B) The steps of: incubating in the presence of a polynucleotide encoding a polypeptide to form a population of cells including stimulated T cells; increasing the population of cells including stimulated T cells to form an enlarged population of cells including tumor antigen-specific T cells, wherein the tumor antigen-specific T cells include T cells that are specific to a complex comprising (i) at least one tumor antigen epitope sequence and (ii)(b)(ii) an MHC protein expressed by cancer cells or APCs of a human target; and administering the enlarged population of cells including tumor antigen-specific T cells to a human target, wherein the enlarged population of cells including tumor antigen-specific T cells is 1 × 10⁻¹⁶ 8 ~1 × 10 11 A method comprising the steps of including a total number of cells is provided herein.

[0006] An improved ex vivo method for preparing tumor antigen-specific T cells, comprising the steps of: depleting CD14+ cells and / or CD25+ cells from a population of immune cells including antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14 and / or CD25-depleted immune cells including a first population of APCs and T cells, wherein the population of immune cells is derived from a biological sample of a human subject; and incubating the population of CD14 and / or CD25-depleted immune cells including the first population of APCs and T cells for a first 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 having cancer, or (B) a polynucleotide encoding a polypeptide, thereby forming a population of cells including stimulated T cells; and A method is provided herein that includes the steps of: increasing a population of cells containing T cells to form an enlarged population of cells containing tumor antigen-specific T cells, wherein the tumor antigen-specific T cells include T cells that are specific to a complex containing (i) at least one tumor antigen epitope sequence and (ii)(b)(ii) an MHC protein expressed by cancer cells or APCs of a human subject; and administering the enlarged population of cells containing tumor antigen-specific T cells to a human subject, wherein the human subject has unresectable melanoma, or has previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor and has disease progression, or has received or is currently receiving a PD-1 inhibitor or a PD-L1 inhibitor for at least three months and has a stable disease or an asymptomatic progressive disease.

[0007] An improved ex vivo method for preparing tumor antigen-specific T cells, comprising the steps of: depleting CD14+ cells and / or CD25+ cells from a population of immune cells including antigen-presenting cells (APCs) and T cells, thereby forming a population of CD14 and / or CD25-depleted immune cells including a first population of APCs and T cells, wherein the population of immune cells is derived from a biological sample of a human subject; and expressing the population of CD14 and / or CD25-depleted immune cells including the first population of APCs and T cells over a first period of time, with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) and cancer cells of a human subject having cancer. A method is provided herein that comprises the steps of: incubating in the presence of mRNA encoding a polypeptide containing at least two different tumor antigen epitope sequences to form a population of cells containing stimulated T cells; and increasing the population of cells containing stimulated T cells to form an increased population of cells containing tumor antigen-specific T cells, wherein the tumor antigen-specific T cells include T cells that are specific to a complex comprising (i) at least one tumor antigen epitope sequence and (ii)(b)(ii) an MHC protein expressed by human target cancer cells or APCs.

[0008] An improved ex vivo method for preparing tumor antigen-specific T cells, comprising: (i) directly from a washed and / or cryopreserved peripheral blood mononuclear cell (PBMC) sample derived from a human subject; (ii) from a human subject-derived PBMC sample containing a percentage of immature dendritic cells (DCs) approximately the same as the percentage of immature DCs in the peripheral blood of a human subject; (iii) from a human subject-derived PBMC sample containing a percentage of mature DCs approximately the same as the percentage of mature DCs in the peripheral blood of a human subject; and (iv) from a human subject-derived PBMC sample containing a percentage of immature DCs approximately the same as the ratio of immature DCs to mature DCs in the peripheral blood of a human subject. (v) From human-derived PBMC samples containing the ratio of mature DCs to mature DCs, (vi) From human-derived PBMC samples that have not undergone the step of maturing immature DCs into mature DCs, (vii) From human-derived PBMC samples containing the percentage of APCs in the total cell population which is approximately the same as the percentage of APCs in the total cell population in the peripheral blood of human subjects, (viii) From human-derived PBMC samples containing the percentage of DCs in the total cell population which is approximately the same as the percentage of DCs in the total cell population in the peripheral blood of human subjects, From a human-derived PBMC sample containing a percentage of CD303+ cells in the total cell population that is approximately the same as the percentage of CD303+ cells in the total cell population in the peripheral blood of the subject, (ix) From a human-derived PBMC sample containing a percentage of CD141+ cells in the total cell population that is approximately the same as the percentage of CD141+ cells in the total cell population in the peripheral blood of the subject, (x) From a human-derived PBMC sample containing a percentage of macrophages in the total cell population that is approximately the same as the percentage of macrophages in the total cell population in the peripheral blood of the subject (i) CD14+ cells and / or CD25+ cells are depleted from an MC sample, or from a human subject-derived PBMC sample containing a percentage of CD19+ in the total cell population that is approximately the same as the percentage of CD19+ in the total cell population in peripheral blood of a human subject, thereby forming a population of CD14 and / or CD25 depleted PBMCs containing a first population of APCs and T cells; and (b) the population of CD14 and / or CD25 depleted immune cells containing a first population of APCs and T cells is subjected to a first period of time.A method is provided herein that comprises the steps of: incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) in the presence of (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by human target cancer cells having cancer, or (B) a polynucleotide encoding a polypeptide, thereby forming a population of cells comprising stimulated T cells; and increasing the population of cells comprising stimulated T cells, thereby forming an increased population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells that are specific to a complex comprising (i) at least one tumor antigen epitope sequence and (ii)(b)(ii) an MHC protein expressed by human target cancer cells or APCs.

[0009] In some embodiments, the method further includes the step of administering an enlarged population of cells, including tumor antigen-specific T cells, to a human subject.

[0010] In some embodiments, the incubation step includes incubating a population of CD14 and / or CD25-depleted immune cells, including a first population of APCs and T cells, for a first period in the presence of mRNA encoding a polypeptide comprising (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L) and (ii) at least two different tumor antigen epitope sequences expressed by cancer cells of a human subject having cancer.

[0011] In some embodiments, introduction includes electroporation or nucleofecting. In some embodiments, electroporation or nucleofecting is performed without separating the T cells from the APCs of the first population of APCs in step (a).

[0012] In some embodiments, the method further includes the step of administering an enlarged population of cells, including tumor antigen-specific T cells, to a human subject. In some embodiments, the incubation step includes incubating a population of CD14 and / or CD25-depleted immune cells, including a first population of APCs and T cells, for a first period in the presence of mRNA encoding a polypeptide containing (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L) and (ii) at least two different tumor antigen epitope sequences expressed by cancer cells of a human subject having cancer.

[0013] In some embodiments, the mRNA includes a 5' cap. In some embodiments, the 5' cap is cap-1 (CAP-1). In some embodiments, the mRNA includes a 3' poly-A tail. In some embodiments, the poly-A tail is 120–135 nucleotides long. In some embodiments, a first tumor antigen epitope sequence of at least two different tumor antigen epitope sequences is linked via a linker sequence to a second tumor antigen epitope sequence of at least two different tumor antigen epitope sequences. In some embodiments, the 5' cap is operably linked via a linker sequence to a sequence encoding at least two different tumor antigen epitope sequences. In some embodiments, at least two different tumor antigen epitope sequences are expressed as a single polypeptide chain. In some embodiments, the incubation step includes incubating a population of CD14 and / or CD25-depleted immune cells, including a first population of APCs and T cells, in the presence of LPS and IFNγ.

[0014] In some embodiments, at least two different tumor antigen epitope sequences are each 8 to 12 amino acids long. In some embodiments, at least two different tumor antigen epitope sequences are each 15 to 25 amino acids long. In some embodiments, the polypeptide contains 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 having cancer.

[0015] In some embodiments, an enlarged population of cells including tumor antigen-specific T cells is 1 × 10⁶ 8 ~1 × 10 11 It contains a total of 1 × 10⁶ cells. In some embodiments, the enlarged population of cells, including tumor antigen-specific T cells, is 1 × 10⁶. 8 ~1 × 10 11 Contains individual CD3+ cells.

[0016] In some embodiments, the human subject has an unresectable melanoma. Unlike resectable melanoma, tumor-infiltrating lymphocytes (TILs) cannot be obtained from unresectable melanoma; therefore, TILs cannot be used for the treatment of unresectable melanoma. One advantage of the methods and compositions provided herein is that they can be used for the treatment of unresectable melanoma.

[0017] In some embodiments, the human subject has previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor, and has disease progression.

[0018] In some embodiments, human subjects have received or are currently receiving PD-1 inhibitors or PD-L1 inhibitors for at least 3 months and have a stable disease or an asymptomatic progressive disease.

[0019] In some embodiments, the percentage of CD3+ cells in an enlarged population of cells including tumor antigen-specific T cells is at least 40%, 50%, or 60% of the total cell population.

[0020] In some embodiments, the percentage of CD107a+ cells in an enlarged population of cells including tumor antigen-specific T cells is at least 10% of the tumor antigen-specific T cell population.

[0021] In some embodiments, the percentage of TNFα+ cells in an enlarged population of cells including tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population.

[0022] In some embodiments, the percentage of IFNγ+ cells in an expanded population of cells including tumor antigen-specific T cells is at least 15% of the tumor antigen-specific T cell population.

[0023] In some embodiments, the percentage of TNFα+ and IFNγ+ cells in an enlarged population of cells including tumor antigen-specific T cells is at least 2% of the tumor antigen-specific T cell population.

[0024] In some embodiments, the percentage of TNFα+ and CD107a+ cells in an enlarged population of cells including tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cell population.

[0025] In some embodiments, the percentage of IFNγ+ and CD107a+ cells in an enlarged population of cells including tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population.

[0026] In some embodiments, the percentage of TNFα+, IFNγ+, and CD107a+ cells in an enlarged population of cells including tumor antigen-specific T cells is at least 0.1% of the tumor antigen-specific T cell population.

[0027] In some embodiments, the percentage of CD4+ T cells in an enlarged population of cells, including tumor antigen-specific T cells (naive T cells, CD62L+ and CD45RA+), is at most 15%.

[0028] In some embodiments, the percentage of CD4+ T cells in an enlarged population of cells, including tumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-), is at least 60%.

[0029] In some embodiments, the percentage of CD4+ T cells in an enlarged population of cells, including tumor antigen-specific T cells (effector T cells, CD62L- and CD45RA+), is at most 5%.

[0030] In some embodiments, the percentage of CD4+ T cells in an enlarged population of cells, including tumor antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-), is at least 10%.

[0031] In some embodiments, the percentage of CD8+ T cells in an enlarged population of cells, including tumor antigen-specific T cells (naive T cells, CD62L+CD45RA+), is at most 25%.

[0032] In some embodiments, the percentage of CD8+ T cells in an enlarged population of cells, including tumor antigen-specific T cells that are effector memory T cells (CD62L-CD45RA-), is at least 60%.

[0033] In some embodiments, the percentage of CD8+ T cells in an enlarged population of cells, including tumor antigen-specific T cells (CD62L-CD45RA+), is at most 10%.

[0034] In some embodiments, the percentage of CD8+ T cells in an enlarged population of cells, including tumor antigen-specific T cells that are central memory T cells (CD62L+CD45RA-), is at least 15%.

[0035] In some embodiments, an enlarged population of cells, including tumor antigen-specific T cells, produces cytokines and induces degranulation after recognition of target cells.

[0036] In some embodiments, human subjects are refractory to anti-checkpoint inhibitor therapy.

[0037] In some embodiments, the human subjects are between 18 and 75 years of age.

[0038] In some embodiments, the human subjects have a mutation in the BRAF gene and have previously received B-raf inhibitors or B-raf / MEK combination therapy.

[0039] In some embodiments, the depletion step includes depleting CD14+ and CD25+ cells from a human subject-derived peripheral blood mononuclear cell (PBMC) sample that has not been subjected to a monocyte maturation step to mature dendritic cells (DCs).

[0040] In some embodiments, the depletion step further includes depleting CD11b+ cells from a human subject-derived peripheral blood mononuclear cell (PBMC) sample that has not been subjected to a monocyte maturation step to mature dendritic cells (DCs).

[0041] In some embodiments, steps (b) and (c) are performed in less than 28 days.

[0042] In some embodiments, the proportion of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in an enlarged population of cells including tumor antigen-specific T cells is at least twice as high as the proportion of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in the biological sample.

[0043] In some embodiments, the proportion of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in an enlarged population of cells including tumor antigen-specific T cells is at least twice as high as the proportion of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in the biological sample.

[0044] In some embodiments, at least 0.1% of CD8+ T cells in an enlarged population of cells including tumor antigen-specific T cells are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells.

[0045] In some embodiments, at least 0.1% of CD4+ T cells in an enlarged population of cells including tumor antigen-specific T cells are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

[0046] In some embodiments, the augmentation step includes (A) contacting a population of cells containing stimulated T cells with a second population of mature APCs, wherein the second population of mature APCs (i) is incubated with FLT3L and (ii) presents at least one tumor antigen epitope sequence, and (B) augmenting the population of cells containing stimulated T cells over a second period of time, thereby forming an augmented population of T cells.

[0047] In some embodiments, prior to the step of contacting a population of cells containing stimulated T cells with a second population of mature APCs, the second population of mature APCs was incubated with FLT3L for at least 1 day.

[0048] In some embodiments, the biological sample is a peripheral blood sample, a leukocyte apheresis sample, or an apheresis sample.

[0049] In some embodiments, the method further includes the steps of collecting an enlarged population of cells containing tumor antigen-specific T cells, cryopreserving the enlarged population of cells containing tumor antigen-specific T cells, or preparing a pharmaceutical composition containing an enlarged population of cells containing tumor antigen-specific T cells.

[0050] In some embodiments, the incubation step includes incubating a population of CD14 / CD25-depleted immune cells, including a first population of APCs and T cells, for a first period of time in the presence of FLT3L and RNA encoding polypeptides.

[0051] In some embodiments, the human subject with cancer is the human subject from which the biological sample was obtained.

[0052] In some embodiments, the polypeptide is 8 to 50 amino acids long.

[0053] In some embodiments, the polypeptide comprises at least two tumor antigen epitope sequences, each expressed by cancer cells of a human subject having cancer.

[0054] In some embodiments, the step of depleting CD14+ cells and / or CD25+ cells from a population of immune cells including a first population of APCs and T cells includes the step of bringing the population of immune cells including a first population of APCs and T cells into contact with a CD14 binder and / or CD25 binder.

[0055] In some embodiments, the depletion step further includes depleting CD19+ cells from a population of immune cells, including a first population of APCs and T cells.

[0056] An ex vivo method for preparing tumor antigen-specific T cells, comprising the steps of: depleting CD11b+ cells from a population of immune cells including antigen-presenting cells (APCs) and T cells, thereby forming a population of CD11b-depleted immune cells including a first population of APCs and T cells, wherein the population of immune cells is derived from a biological sample of a human subject; and providing the population of CD11b-depleted immune cells including the first population of APCs and T cells with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) and (A) at least one tumor antigen epitope sequence expressed by cancer cells of a human subject having cancer over a first period of time. A method is provided herein that comprises the steps of: incubating in the presence of a polypeptide or a polynucleotide encoding a polypeptide, thereby forming a population of cells including stimulated T cells; and increasing the population of cells including stimulated T cells, thereby forming an increased population of cells including tumor antigen-specific T cells, wherein the tumor antigen-specific T cells include T cells that are specific to a complex comprising (i) at least one tumor antigen epitope sequence and (ii)(b)(ii) an MHC protein expressed by human target cancer cells or APCs.

[0057] A pharmaceutical composition is provided herein, comprising an enlarged population of cells including tumor antigen-specific T cells produced by the method described herein, and a pharmaceutically acceptable carrier.

[0058] (a) a population of immune cells derived from a biological sample, wherein the population of immune cells includes T cells stimulated by antigen-presenting cells (APCs) containing a polypeptide epitope-specific T cell receptor (TCR), (i) the amount of CD11b-expressing immune cells in the population of immune cells is relatively less than the amount of CD11b-expressing immune cells in the biological sample, and / or (ii) the amount of CD11c-expressing immune cells in the population of immune cells is relatively more than the amount of CD11c-expressing immune cells in the biological sample; and (b) a pharmaceutically acceptable excipient is provided herein.

[0059] (a) a population of immune cells derived from a biological sample, wherein the population of immune cells includes T cells stimulated by antigen-presenting cells (APCs) containing T cell receptors (TCRs) specific to polypeptide epitopes, and the APC-stimulated T cells are incubated with cytokines; (b) cytokines; and (c) pharmaceutically acceptable excipients.

[0060] (a) a population of immune cells derived from a biological sample of a subject administered with fms-like tyrosine kinase 3 ligand (FLT3L), the population of immune cells including T cells stimulated by antigen-presenting cells (APCs) containing a polypeptide epitope-specific T cell receptor (TCR), and (b) a pharmaceutically acceptable excipient.

[0061] In some embodiments, the population of immune cells is derived from a biological sample from the subject.

[0062] In some embodiments, the population of immune cells is derived from a biological sample from a subject that has been administered fms-like tyrosine kinase 3 ligand (FLT3L).

[0063] In some embodiments, APC-stimulated T cells are incubated with cytokines, and the pharmaceutical composition further comprises cytokines.

[0064] In some embodiments, the amount of immune cells expressing CD11b in a population of immune cells is relatively less than the amount of immune cells expressing CD11b in a biological sample.

[0065] In some embodiments, the amount of CD11c-expressing immune cells in a population of immune cells is relatively greater than the amount of CD11c-expressing immune cells in a biological sample.

[0066] In some embodiments, the amount of CD14-expressing immune cells in a population is relatively less than the amount of CD14-expressing immune cells in a biological sample.

[0067] In some embodiments, the amount of CD25-expressing immune cells in a population is relatively less than the amount of CD25-expressing immune cells in a biological sample.

[0068] In some embodiments, the amount of CD19-expressing immune cells in a population is relatively less than the amount of CD19-expressing immune cells in a biological sample.

[0069] In some embodiments, the APC is an APC stimulated by FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

[0070] In some embodiments, APC-stimulated T cells are T cells stimulated by FLT3L-stimulated APCs.

[0071] In some embodiments, the cytokine is IL-7, IL-15, or IL-21.

[0072] In some embodiments, APC-stimulated T cells include T cells stimulated by APCs loaded with antigens that present epitopes in MHC class I or MHC class II molecules.

[0073] In some embodiments, the antigen-loaded APC includes plasmacytoid dendritic cells (pDCs), CD11c+DCs, CD1c+DCs, or CD141+DCs.

[0074] In some embodiments, CD11b cells include CD16+ mononuclear cells.

[0075] In some embodiments, the pharmaceutical composition further comprises agents that promote cell growth and maintenance ex vivo, including growth factors, cytokines, amino acids, supplements, or combinations thereof.

[0076] In some embodiments, the amount of CD1c-expressing immune cells in a population of immune cells is relatively greater than the amount of CD1c-expressing immune cells in a biological sample.

[0077] In some embodiments, the amount of CD141-expressing immune cells or APCs in a population of immune cells is relatively greater than the amount of CD141-expressing immune cells or APCs in a biological sample.

[0078] In some embodiments, the cell population containing antigen-loaded APCs includes 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.

[0079] In some embodiments, APC-stimulated T cells include 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.

[0080] In some embodiments, APC-stimulated T cells include T cells stimulated by a cell population containing more than 90% CD11c+ cells.

[0081] In some embodiments, the pharmaceutical compositions described herein include T cells stimulated by a cell population containing more than 70% neoantigenic peptide-expressing cells, which are CD11c+, CD1c+, or CD141+ cells.

[0082] In some embodiments, the pharmaceutical composition contains at least 60% of the T cells in the pharmaceutical composition, which are specific to the epitope.

[0083] In some embodiments, the pharmaceutical compositions described herein contain a greater proportion of naive T cells that are induced or converted into neoantigen-primed T cells compared to cell compositions obtained by contacting isolated T cells with antigen-loaded APCs without reducing or depleting CD11b+ and / or CD19+ cells.

[0084] In some embodiments, the pharmaceutical compositions described herein contain more than 35% naive T cells that are induced or converted into epitope-specific antigen-specific activated T cells.

[0085] 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 depleting CD11b+ cells and / or CD19+ cells.

[0086] In some embodiments, the pharmaceutical compositions described herein contain at least 30% CD8+ T cells.

[0087] 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 depleting CD11b+ cells and / or CD19+ cells.

[0088] A method for treating cancer in a subject requiring such treatment is provided herein, comprising the step of administering a pharmaceutical composition described herein to the subject.

[0089] A method for preparing T cells containing a polypeptide epitope-specific T cell receptor (TCR) is provided herein, comprising the steps of (a) depleting CD11b-expressing cells from a population of immune cells including antigen-presenting cells and T cells, thereby forming a population of CD11b-depleted immune cells including T cells, and (b) incubating or amplifying the population of CD11b-depleted immune cells including T cells, to amplify memory T cells containing an epitope-specific TCR or induce naive T cells containing an epitope-specific TCR.

[0090] A method for preparing T cells containing an epitope-specific T cell receptor (TCR) is provided herein, comprising the steps of (a) enriching a population of immune cells containing APCs and T cells with respect to cells expressing CD11c, thereby forming a population of CD11c-enriched immune cells containing T cells, and (b) incubating or amplifying the CD11c-enriched immune cell population containing T cells, thereby increasing memory T cells containing an epitope-specific TCR or inducing naive T cells containing an epitope-specific TCR. In some embodiments, the method for preparing APCs includes APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

[0091] In some embodiments, the method further includes the step of preparing an APC preparation.

[0092] In some embodiments, a method for preparing an APC preparation includes the step of incubating the APC with FLT3L.

[0093] In some embodiments, a method for preparing an APC preparation includes the step of incubating the APC with a polypeptide or a polynucleotide encoding a polypeptide.

[0094] A method for treating cancer in a subject requiring such treatment is provided herein, comprising the step of administering to 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) containing T cell receptors (TCRs) specific to an antigen peptide sequence, and the subject is administered fms-like tyrosine kinase 3 ligand (FLT3L).

[0095] A method for treating cancer in a subject requiring such treatment is provided herein, comprising the steps of (a) administering FMS-like tyrosine kinase 3 receptor ligand (FLT3L) to the subject, and (b) administering to 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) having a T cell receptor (TCR) specific to an antigen peptide sequence.

[0096] A method for treating cancer in a subject requiring such treatment is provided herein, comprising the steps of (a) administering to 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) having T cell receptors (TCRs) specific to an antigen peptide sequence, and (b) administering to a polypeptide comprising an antigen peptide sequence or a polynucleotide encoding an antigen peptide sequence.

[0097] In some embodiments, the method further includes the step of targeting and administering FMS-like tyrosine kinase 3 receptor ligand (FLT3L) prior to administering a population of immune cells. [Brief explanation of the drawing]

[0098] [Figure 1-1] Figure 1A shows an illustrative schematic diagram of an antigen-specific T cell production protocol.

[0099] [Figure 1-2] Figure 1B shows an illustrative schematic diagram of an antigen-specific T cell production protocol.

[0100] Figure 1C shows an illustrative alternative schematic diagram of an antigen-specific T cell production protocol.

[0101] [Figure 2] Figure 2 shows illustrative results illustrating the percentage of antigen-specific CD8+ memory T cells induced by long or short peptides. “Bulk” indicates that the sample containing the T cells used for induction is a whole peripheral blood mononuclear cell (PBMC). “Treg-” indicates that the sample containing the T cells used for induction is a PBMC with depleted CD25-expressing cells.

[0102] [Figure 3] Figure 3 shows an exemplary flow cytometry analysis illustrating the percentage of antigen-specific CD8+ naive T cells induced by the GAS7 peptide.

[0103] [Figure 4] Figure 4 shows exemplary results illustrating antigen-specific CD8+ T cell responses to peptide pools of short HIV peptides, short previously identified neoantigens (PINs), or long PINs. “Whole PBMC” indicates that the sample containing the T cells used for induction is the entire PBMC. “CD25-PBMC” indicates that CD25+ cells have been depleted from the sample containing the T cells used for induction. Short, short peptide, or shortmer; long, long peptide, or longmer.

[0104] [Figure 5] Figure 5A shows an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to a single previously identified neoantigen (PIN) under the conditions shown.

[0105] Figure 5B shows antigen-specific CD8 against a single previously identified neoantigen (PIN) under the indicated conditions. + This shows an exemplary flow cytometry analysis of the naive T cell response.

[0106] [Figure 6] Figure 6 shows exemplary results illustrating the antigen-specific CD8+ T cell response to the indicated peptide using PBMC samples from two human donors.

[0107] [Figure 7] Figure 7 shows exemplary flow cytometry plots of antigen-specific CD8+ T cell responses to the identified mutated epitopes in healthy donors before stimulation and after up to three rounds of stimulation.

[0108] [Figure 8-1] Figure 8A shows an illustrative bar graph illustrating the results of antigen-specific memory CD8+ T cell responses to viral antigens. After up to three rounds of stimulation, approximately 50% of all CD8+ T cells were specific to the identified viral epitopes (CMV pp65, EBV YVL, EBV BMLF1, and Mart-1).

[0109] [Figure 8-2] Figure 8B shows exemplary results from a recall assay of antigen-specific memory CD8+ T cell responses, where peptide-loaded antigen-presenting cells were incubated with viral antigen-loaded and unloaded APCs. The percentage of CD8+ T cells releasing the indicated cytokines from the two time points are shown in the chart.

[0110] [Figure 9] Figure 9 shows exemplary results from a cytotoxic assay used to evaluate whether the induced T cell culture could kill the antigen-expressing tumor line. The percentage of live and dead caspase-3 positive tumor cells relative to the total tumor cells is shown. Live caspase-3 positive tumor cells indicate cells undergoing early cell death.

[0111] [Figure 10]Figure 10 shows exemplary flow cytometry analysis of antigen-specific CD4+ T cell responses to peptide-loaded antigen-presenting cells, followed by incubation with PIN-loaded and unloaded APCs. The percentage of CD4+ T cells releasing IFNγ is shown.

[0112] [Figure 11] Figure 11 shows exemplary results for the percentage of antigen-specific CD4+ T cells releasing IFNγ after restimulation with mutant peptides or wild-type peptides.

[0113] [Figure 12] Figure 12 shows exemplary flow cytometry analysis illustrating the antigen-specific CD8+ naive T cell response to a short HIV5 peptide. Both short-term and long-term inductions are shown.

[0114] [Figure 13] Figure 13 shows an exemplary flow cytometry analysis of the percentage of antigen-specific CD8+ naive T cell responses to short ME1 peptides using whole PBMC samples derived from human donors.

[0115] [Figure 14] Figure 14 shows an exemplary flow cytometry analysis of a whole PBMC sample derived from a human donor, illustrating the antigen-specific CD8+ naive T cell response to a short HIV3 peptide.

[0116] [Figure 15] Figure 15 shows an exemplary flow cytometry analysis of an antigen-specific CD8+ naive T cell response to a long CSNK1A1 peptide using a whole PBMC sample derived from a human donor.

[0117] [Figure 16]FIG. 16 shows an exemplary flow cytometry analysis showing an antigen-specific CD8+ naive T cell response to a long CSNK1A1 peptide using a PBMC sample from a human donor depleted of CD25+ cells.

[0118] [Figure 17] FIG. 17 shows an exemplary flow cytometry analysis showing an antigen-specific CD8+ naive T cell response to a short GAS7 peptide using a PBMC sample from a human donor depleted of CD25+ cells.

[0119] [Figure 18] FIG. 18 shows an exemplary flow cytometry analysis showing an antigen-specific CD8+ naive T cell response to a short ACTN4 peptide using a PBMC sample from a human donor depleted of CD25+ cells.

[0120] [Figure 19] FIG. 19A shows an exemplary flow cytometry analysis showing an antigen-specific CD8+ naive T cell response to a short ACTN4 peptide using a PBMC sample from a human donor depleted of CD25+ cells. Short-term induction is shown.

[0121] FIG. 19B + shows an exemplary flow cytometry analysis showing an antigen-specific CD8 + naive T cell response to a short HIV3 peptide using a PBMC sample from a human donor depleted of CD25 cells. Long-term induction is shown.

[0122] [Figure 20] FIG. 20 shows an exemplary flow cytometry analysis of an antigen-specific CD8+ naive T cell response to a short HIV5 peptide using an entire PBMC sample from a human donor. Both short-term induction and long-term induction are shown.

[0123] [Figure 21]Figure 21 shows an exemplary flow cytometry analysis of a whole PBMC sample derived from a human donor, illustrating the antigen-specific CD8+ naive T cell response to a short HIV-3 peptide. Short-term induction is shown.

[0124] [Figure 22] Figure 22 shows exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to a short PRDX5 peptide using PBMC samples from human donors with depleted CD25+ cells. Both very short-term and long-term inductions are shown.

[0125] [Figure 23] Figure 23 shows an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to short HIV5 peptides using PBMC samples from human donors with depleted CD25+ cell tides. Both short-term and long-term inductions are shown.

[0126] [Figure 24] Figure 24 shows a schematic diagram of an example of a method for generating a therapeutic T cell composition, including the enlargement of memory T cells and the induction of naive T cells.

[0127] [Figure 25] Figure 25 shows exemplary methods for testing T cell functionality, phenotype, and / or function and / or T cell response.

[0128] [Figure 26] Figure 26 shows an example of a recall assay for testing T cell functionality, phenotype, and / or function, as well as T cell response.

[0129] [Figure 27-1]Figure 27A shows an exemplary flow cytometry analysis demonstrating the ability to deconvolute samples multiplexed with labeled samples acquired separately or as a mixture in a recall assay. Uniquely labeled samples were decomposed with minimal to no cross-contamination to other barcodes.

[0130] [Figure 27-2] Figure 27B shows an exemplary flow cytometry analysis demonstrating the detection of antigen-specific CD8+ T cells by multimer staining of a mixture of nine uniquely labeled samples in a recall assay.

[0131] [Figure 28-1] Figure 28A shows an exemplary flow cytometry analysis of a recall assay using six uniquely barcoded samples recalled using unloaded DCs and DCs loaded with neoantigen.

[0132] [Figure 28-2] Figure 28B shows an illustrative bar graph of the percentage of CD4+ T cells with the number of functions incubated with DCs loaded with the indicated peptide concentrations in a recall response assay. Samples from two induced cultures containing de novo CD4+ T cell responses were analyzed either alone or mixed with unrelated samples, without barcoding. Barcoding did not alter the detectable functions. The number of functions and the magnitude of the response evoked from the cells did not change significantly with sample barcoding.

[0133] [Figure 29] Figure 29A shows an illustrative bar graph illustrating the results of antigen-specific memory CD8+ T cell responses to viral antigens. CD8+ memory responses to CMV pp65, MART-1, and EBV BRLF1 and BMLF1 epitopes can be increased from 0.23% of CD8+ T cells in healthy donor starting material to over 60%.

[0134] Figure 29B shows antigen-specific memory CD8s recalled against viral antigens, then using DCs loaded and unloaded with the viral antigen. + Exemplary results from a T cell response recall assay are shown. CD8 cells from two time points releasing the indicated cytokines. + The percentage of T cells is shown in the chart.

[0135] [Figure 30-1] Figure 30A shows exemplary results of hit identification by detection and functional characterization of multiple specific de novo-induced CD4+ responses in the same culture. In the example shown, induction was performed in four replicate cultures targeting 10 HIV-derived epitopes, which are naive targets in HIV-negative healthy donors. Antigen-specific responses were detected in 4 / 4 biological replicates, with responses of varying magnitude.

[0136] Figure 30B shows de novo-induced CD4 with multiple specificities in the same culture. + Exemplary results of pooled deconvolution by response detection and functional characterization are shown. Multiple responses were detected in each replicate tested, and the same two epitopes (HIV#5 and HIV#7) produced the largest response in each case.

[0137] [Figure 30-2] Figure 30C shows exemplary results of susceptibility determination by detection and functional characterization of multiple de novo-induced CD4+ responses with multiple specificities in the same culture. Similar magnitudes were observed for each response in the pooled deconvolution assay. Responses to HIV#5, HIV#6, and HIV#4 demonstrated EC50 values ​​of 0.45 μM, 0.43 μM, and 9.1 μM, respectively.

[0138] [Figure 31] Figure 31 shows an illustrative schematic diagram of an antigen-specific T cell production protocol.

[0139] [Figure 32] Figure 32 shows an exemplary schematic diagram of a T cell induction protocol.

[0140] [Figure 33] Figure 33 shows an exemplary schematic diagram of a dendritic cell generation protocol.

[0141] [Figure 34] Figure 34 shows an exemplary pMHC multimers plot showing CD8+ T cell responses induced in leukapheresis materials from melanoma patients targeting patient-specific epitopes: SRSF1E>K, ARAP1Y>H and PKDREJG>R, and leukapheresis materials from melanoma patients targeting patient-specific epitopes (AASDH neoORF and seven model neoantigens: ACTN4K>N, CSNK1A1S>L, DHX40neoORF, GLI3P>LQARSR>W, FAM178BP>L and RPS26P>L). The first panel plots in the first and second columns show memory responses, and the remaining plots show de novo responses.

[0142] [Figure 35] Figure 35 shows exemplary data (left panel) of pMHC multimers plots of SRSF1E>K and ARAP1Y>H before and after peptide stimulation, and the pie chart shows the functionality of neoantigen-specific T cells upon re-challenge with neoantigen-loaded DCs; gated on pMHC multimer + CD8+ or CD4+ T cells. The multifunctional profiles of CD8+ memory, CD8+ de novo and CD4+ de novo responses induced in patients with melanoma are shown by combinations of 1, 2 or 3 functions (e.g., one or more functions are the production of one or more factors selected from IFNγ, TNFα, CD107a and 4-1BB).

[0143] [Figure 36]Figure 36 shows the specificity of memory and de novo responses induced in patients with melanoma against mutant and wild-type peptides. SRSF1E>K and ARAP1Y>H specific T cell responses were challenged with DCs loaded with mutant or wild-type neoantigen peptides at different concentrations (X-axis: 0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM), and IFN-γ+ and / or TNFα+ and / or CD107a+ (Y-axis) of total CD8+ T cells in the samples were measured; both responses showed a significant difference against the 0 μM concentration and were not responsive to the wild-type neoantigen peptide. Statistical analysis: FDR for adjusted p-values, P-values: *≤0.05, ***≤0.001, ****≤0.0001.

[0144] [Figure 37-1] Figure 37A shows the cytotoxic profile of the memory response induced in patients with melanoma, quantified by the frequency of CD8+CD107a+ T cells. This also shows target cell killing by these T cell responses, quantified by the frequency of aCAS3+ tumor cells. The cytotoxic ability of the induced CD8+ T cell response was evaluated by rechallenging with tumor cells transduced with mutant or wild-type neoantigens. Non-transduced tumor cells (parental A375 line) or tumor cells transduced with 200aa constructs were used. The constructs contained either the mutant or wild-type sequence with a central mutation. Upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells was measured during co-culture. Target ratio: 3.3:1 (SRSF1E>K).

[0145] [Figure 37-2]Figure 37B shows another example of the cytotoxicity profile of the memory response induced in patients with melanoma, quantified by the frequency of CD8+CD107a+ T cells. This also shows target cell death by these T cell responses, quantified by the frequency of aCAS3+ tumor cells. The cytotoxicity of the induced CD8+ T cell response was assessed by rechallenging tumor cells transduced with mutant or wild-type neoantigen. Untransduced tumor cells (parent A375 lineage) or tumor cells transduced with the 200aa construct were used. The construct contained either a mutant or wild-type sequence, or a central mutation. Upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells was measured during co-culture. Red circles highlight the pMHC+ percentage. Effector:Target ratio: 5:1 (SRSF1E>K). Statistical analysis: Unpaired t-test, P-values ​​**≦0.01, ****≦0.0001.

[0146] [Figure 37-3] Figure 37C shows the cytotoxicity profile of the de novo response induced in patients with melanoma, quantified by the frequency of CD8+CD107a+ T cells. This also shows target cell death by these T cell responses, quantified by the frequency of aCAS3+ tumor cells. The cytotoxicity of the induced CD8+ T cell response was evaluated by rechallenging tumor cells transduced with mutant or wild-type neoantigen. Untransduced tumor cells (parent A375 lineage) or tumor cells transduced with the 200aa construct were used. The construct contained either a mutant or wild-type sequence, or a central mutation. Upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells was measured during co-culture. Circles highlight pMHC+ percentage. Effector: Target ratio: 0.66:1 (ARAP1Y>H). Statistical analysis: Unpaired t-test, P-value** ≤ 0.01, **** ≤ 0.0001.

[0147] [Figure 38]Figure 38A shows the identification of neoantigen-specific CD4+ T cell responses in melanoma patients. Responses are identified based on IFN-γ and TNFα production (Y-axis) when rechallenged with DCs (0.8 μM) loaded with mutant neoantigen peptides. MKRN1S>L, CREBBPS>L, and TPCN1K>E were identified as positive responses.

[0148] Figure 38B shows the specificity of the CD4+ T cell response shown in Figure 38A to the mutated peptide and wild-type peptide shown. In the confirmation study, the CD4 T cell response shown in Figure 38A was challenged with mutant and wild-type neoantigen peptides at different concentrations (X-axis - 0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM), and IFNγ+ and / or TNFα+ (Y-axis) of total CD4+ in the samples were measured. Two of the CD4+ T cell responses (MKRN1) were identified. S>L and CREEBP S>L ) showed a significant difference compared to the 0 μM concentration, and was not responsive 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 for adjusted p-value, p-value < 0.05.

[0149] Figure 38C shows the multifunctionality profiles of these CD4+ T cell responses, indicated by combinations of 1, 2, 3, or 4 functions (e.g., one or more functions are the production of one or more factors selected from IFNγ, TNFα, CD107a, and 4-1BB). The multifunctionality of the identified CD4+ T cell responses was assessed by rechallenge with mutant neoantigen peptide-loaded DCs (0.8 μm). Percentages in the pie charts indicate the percentage of functional CD4+ T cells (1, 2, and / or 3 functions). Representative data shown were generated from post-stimulation CD4+ T cell responses induced in patients.

[0150] [Figure 39]Figure 39 shows the functionality of memory responses induced in two healthy donors with and without epacadostat administration, indicated by a combination of one, two, or three functions (for example, one or more functions being the production of one or more factors selected from IFNγ, TNFα, and CD107a).

[0151] [Figure 40] Figure 40 shows the percentage of induced de novo CD8+ T cell response ("hit rate," averaged across four healthy donors) in six replication inductions, with or without the addition of epacadostat.

[0152] [Figure 41] Figure 41A shows the absolute number of antigen-specific cells derived from healthy donors after induction by the T cell production protocol provided herein, with or without the addition of PD-1 blocking antibody.

[0153] Figure 41B shows the absolute number of antigen-specific cells derived from healthy donors after induction by the T cell production protocol provided herein, with or without the addition of PD-1 blocking antibody.

[0154] [Figure 42] Figure 42A shows the multimer positivity frequency as the percentage of CD8+ T cells from the de novo CD8+ T cell compartment, with or without IL-12 supplementation.

[0155] Figure 42B shows an illustrative graph of the percentage of CD8+ T cells from the de novo CD8+ T cell compartment with and without IL-12 supplementation.

[0156] [Figure 43]Figure 43 shows an exemplary graphical representation of the percent hit rates for highly immunogenic and low immunogenic antigens to which naive CD8 cells are responsive after performing different antigen presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Also shown is an exemplary graphical representation of the absolute numbers of antigen-specific cells after performing different antigen presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors using Mart-1 peptide or highly immunogenic and low immunogenic antigens.

[0157] [Figure 44-1] Figure 44A shows exemplary flow cytometry results for CD123 positive cells after performing the indicated antigen presenting cell enrichment and antigen loading protocols using PBMCs from three different healthy donors.

[0158] [Figure 44-2] Figure 44B shows an exemplary graphical representation of the absolute numbers of the indicated CD11c+ cell subsets after performing three antigen presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Treatments are as follows: base Flt3L, Flt3L treatment alone; CD11b, Flt3L treatment and depletion of CD11b-expressing cells; CD11b− / CD19−, Flt3L treatment, and depletion of CD11b-expressing cells and CD19-expressing cells.

[0159] [Figure 45] Figure 45 shows an exemplary graphical representation of the ratio of the total number of CD8 T cells to the indicated cells after performing three antigen presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Treatments are as follows: base Flt3L, Flt3L treatment alone; CD11b, Flt3L treatment and depletion of CD11b-expressing cells; CD11b− / CD19−, Flt3L treatment, and depletion of CD11b-expressing cells and CD19-expressing cells.

[0160] [Figure 46] Figure 46 shows exemplary flow cytometry results for CD11b-positive cells after performing the demonstrated antigen-presenting cell enrichment and antigen-loading protocols using PBMCs derived from three different healthy donors.

[0161] [Figure 47] Figure 47 shows exemplary flow cytometry results of CD19-positive cells after performing the demonstrated antigen-presenting cell enrichment and antigen-loading protocols using PBMCs derived from three different healthy donors.

[0162] [Figure 48] Figure 48 shows an exemplary graphical representation of the cell enlargement ratio after performing three antigen-presenting cell enrichment and antigen-loading protocols. The treatments are as follows: base FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment, and CD11b-expressing cell depletion; CD11b- / CD19-, FLT3L treatment, and CD11b-expressing cell and CD19-expressing cell depletion.

[0163] [Figure 49-1] Figure 49A shows exemplary data indicating the number of specific antigens to which naive CD8 T cells respond after performing three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs derived from healthy donors. Results were averaged across three healthy donors. Treatments were: base FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment, and CD11b-expressing cell depletion; CD11b- / CD19-, FLT3L treatment, and CD11b-expressing cell and CD19-expressing cell depletion. An exemplary graphical representation of the data is shown in the graph below.

[0164] [Figure 49-2]Figure 49B shows an exemplary graphical representation of the percentage hit rates for highly immunogenic (left) and low immunogenic (right) antigens to which naive CD8 cells responded after performing three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs derived from healthy donors. Results were averaged across three healthy donors. Treatments were: base FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment, and CD11b-expressing cell depletion; CD11b- / CD19-, FLT3L treatment, and CD11b-expressing and CD19-expressing cell depletion.

[0165] [Figure 50] Figure 50 shows an exemplary graphical representation of the number of antigen-specific cells in a population of cells activated by highly immunogenic and low immunogenic antigens to which T cells respond, after performing three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs derived from healthy donors. The treatments are as follows: base FLT3L, FLT3L treatment alone; CD11b, FLT3L treatment, and CD11b-expressing cell depletion; CD11b- / CD19-, FLT3L treatment, and CD11b-expressing cell and CD19-expressing cell depletion.

[0166] [Figure 51-1] Figure 51A shows an exemplary graph of the percentage of viable cells after performing three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs derived from healthy donors. The treatments were as follows: base, FLT3L treatment alone; base + CD11b- / CD19-, FLT3L treatment, and depletion of CD11b-expressing and CD19-expressing cells; +APC, base + CD11b- / CD19- with additional PBMC fractions, from which CD3, CD19, CD11b, CD25, and CD14-expressing cells were depleted.

[0167] Figure 51B shows an exemplary graphical representation of the percentage of viable cells after performing three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs derived from healthy donors. The treatments were as follows: base, FLT3L treatment alone; base + CD11b- / CD19-, FLT3L treatment, and depletion of CD11b-expressing and CD19-expressing cells; +APC, base + CD11b- / CD19- with additional PBMC fractions, from which CD3, CD19, CD11b, CD25, and CD14-expressing cells were depleted.

[0168] Figure 51C shows an exemplary graphical representation of the percentage of viable cells after performing three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs derived from healthy donors. The treatments were as follows: base, FLT3L treatment alone; base + CD11b- / CD19-, FLT3L treatment, and depletion of CD11b-expressing and CD19-expressing cells; +APC, base + CD11b- / CD19- with additional PBMC fractions, from which CD3, CD19, CD11b, CD25, and CD14-expressing cells were depleted.

[0169] [Figure 51-2] Figure 51D shows exemplary data for each donor, indicating the number of specific antigens to which CD8 cells respond, using an exemplary antigen-presenting cell enrichment protocol.

[0170] Figure 51E shows an exemplary graphical representation of the percentage hit rate for peptides to which CD8 cells were shown to be responsive, averaged across three healthy donors.

[0171] [Figure 52-1]Figure 52A shows exemplary flow cytometry analysis results from experiments in which populations of cells added to the culture process at different time points were labeled with a membrane-permeable amine-reactive dye (e.g., carboxyfluorescein succinimidyl ester or TagIT Violet®) before stimulation with antigen-loaded APCs. When applied to the second stimulation, a population of cells already cultured for 14 days was labeled with one dye, while another population of cells containing antigen-loaded APCs and a new preparation of T cells was labeled with the other dye. The two populations were then mixed together for restimulation or augmentation. The relative contribution of each of these populations to the overall antigen-specific T cell pool is indicated by the presence and dilution of each dye. In all examples, a population of cells cultured for 14 days (first stimulation), labeled with one dye, and then added to another population of cells labeled with the other dye, which had been stimulated with the antigen 1 day prior (standard protocol), 4 days prior (5-day early start), or 6 days prior (7-day early start).

[0172] [Figure 52-2] Figure 52B shows an exemplary schematic representation of three different T cell enlargement protocols, each having two stimuli, including an early initiation for antigen-loading APCs at 2, 5, or 7 days prior to contact with T cells.

[0173] Figure 52C shows an illustrative graph of the number of antigen-specific T cells over time using three different T cell enlargement protocols shown in Figure 52B: 1) Standard protocol; 2) 5-day early start; 3) 7-day early start.

[0174] [Figure 53] Figure 53 shows an exemplary graph of the growth rate of cultures treated with the indicated neoantigen peptide (pep) or neoantigen RNA. PBMC cells, depleted of CD14 / CD25 after isolation or removal of CD3 lymphocytes, were stimulated with the antigen (peptide or antigen-encoding mRNA). CD3 lymphocyte cells were reintroduced and stimulated for 14 days.

[0175] [Figure 54] Figure 54 shows an exemplary graph of the number of multimer-positive antigen-specific cells in cultures treated with the indicated neoantigen peptide (pep) or neoantigen RNA nucleofection. Cultures were treated with nucleofection in the presence or absence of T cells (-CD3). Irr, irradiation was performed.

[0176] [Figure 55] Figure 55 shows exemplary flow cytometry analysis illustrating antigen-specific CD8+ memory responses using viral peptides or RNA encoding those peptides, and naive responses using neoantigen-coding peptides or RNA, in a short-term induction protocol.

[0177] [Figure 56-1] Figure 56A shows a schematic diagram of an exemplary process for generating RNA containing a neoantigen-coding sequence, loading it into PBMCs, and using them to activate T cells.

[0178] [Figure 56-2] Figure 56B shows a schematic diagram of an exemplary process for generating RNA containing a neoantigen sequence, loading it into PBMCs, and using them to activate T cells.

[0179] [Figure 57] Figure 57A shows a schematic diagram of an exemplary RNA concatemer construct encoding a neoantigen string.

[0180] Figure 57B shows a schematic diagram of an exemplary arrangement of the neoantigen string in 5'-3' orientation within the construct shown in Figure 57A.

[0181] [Figure 58]Figure 58A shows a schematic diagram of an exemplary mRNA sequence for incorporating a 5'-CAP structure into mRNA encoding a chained neoantigen string for expression in PBMCs. The addition of an "A" nucleotide to the mRNA string was used for compatibility with CleanCap® Technology.

[0182] Figure 58B shows an exemplary graph of the percentage of viable cells 24 hours after expression of mRNA encoding a chained neoantigen string with a different 5'-CAP structure in PBMCs.

[0183] Figure 58C shows an exemplary graph of the total number of GFP-positive cells 24 hours after expression of mRNA encoding a chained neoantigen string with a different 5'-CAP structure in PBMCs.

[0184] [Figure 59-1] Figure 59A shows exemplary results demonstrating the use of modified nucleotides to produce mRNA. mRNA was modified by substituting either all (complete) or some (partial) uridine (U) and cytidine (C) residues within the mRNA. For example, the partial C set contains 30% of the C residues replaced by methylcytidine. The results show the effect over time on the expression of the mRNA-encoded peptide in transfected PBMCs.

[0185] [Figure 59-2] Figure 59B shows illustrative data comparing the effects of commercially prepared and in-house prepared mRNAs containing substituted uridine and / or cytidine on generating multimer-specific T cells stimulated in mRNA-loaded PBMCs.

[0186] Figure 59C shows exemplary data comparing the increase in stimulated T cells generated as described in Figure 59B.

[0187] [Figure 60]Figure 60A shows a schematic diagram of exemplary mRNA constructs using short (9-10 amino acids, top) and long (25 amino acids, bottom) mRNAs used for expression in cells.

[0188] Figure 60B shows an exemplary graph of multimer-specific CD8+ cells as a percentage of total CD8+ cells. The antigens used in the multimer assay are shown.

[0189] Figure 60C shows an exemplary flow cytometry analysis for the detection of multimer-positive CD8+ T cells, comparing APCs stimulated with short-mer (9-10 amino acid) and long-mer (25 amino acid) peptides with APCs containing the same short-mer (9-10 amino acid) and long-mer (25 amino acid) peptides.

[0190] [Figure 61-1] Figure 61A shows a schematic diagram of an exemplary RNA construct to which the experimental cells shown in Figures 61B–61D are transfected.

[0191] Figure 61B shows an exemplary graphical representation of the results from the multimer assay. Under all three PBMC handling conditions, 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 was observed in multimer-positive cells compared to peptide-loaded PBMCs.

[0192] Figure 61C shows exemplary flow cytometry data illustrating the detection of Gli3 multimer-positive T cells in each shown set, with and without CD3 cell depletion. Transfection of CD25+ PBMCs directly results in increased multimer-positive cells compared to PBMCs depleted of CD14 and CD25 cells or PBMCs thawed from frozen stocks.

[0193] [Figure 61-2]Figure 61D shows an exemplary graphical representation of the results from the multimer assay. PBMCs treated overnight with FTL3L cells, or PBMCs depleted of CD25, were electroporated with RNA encoding either a 25-amino acid-length neoantigen sequence (longmer) or an epitope-length neoantigen sequence (shortmer). Percentage of neoantigen-positive cells in the culture were assayed using the multimer technique.

[0194] Figure 61E shows an illustrative graphical representation of the enlargement ratio results from the experiment described in Figure 61D. PBMCs treated overnight with FTL3L cells, or PBMCs with depleted CD25, were electroporated with RNA encoding either a 25-amino acid-length neoantigen sequence (longmer) or an epitope-length neoantigen sequence (shortmer). The enlargement ratios of the cells in culture and 26 days after two stimulations are shown.

[0195] [Figure 62-1] Figure 62A shows a schematic diagram of an exemplary RNA construct to which the experimental cells shown in Figures 62B–62D are transfected.

[0196] Figure 62B shows an exemplary graphical representation of the number of ACTN4 and Gli3-responsive live T cells from two donors at 26 days post-maturation, based on the given combinations on the X-axis.

[0197] Figure 62C shows exemplary data of the percentage of Gli3-responsive T cells from living cells grown in the presence of the matured mix shown.

[0198] [Figure 62-2] Figure 62D shows exemplary flow cytometry data illustrating detected Gli3 multimer-positive T cells grown in the presence of the matured mix shown.

[0199] [Figure 63-1]Figure 63A shows representative mass spectrometry data illustrating the detection of the presentation of the Gli3 epitope by PBMCs using radioisotope incorporation. PBMCs transfected with mRNA encoding multiple epitopes (including the Gli3 epitope), and peptide expression, are detected using a reference peptide labeled with a heavier isotope.

[0200] [Figure 63-2] Figure 63B shows an exemplary graph of the percentage of maximum surface presentation of epitopes indicated by HLA-A02:01 over time, after transfection of PBMCs with mRNA encoding each epitope. Each isotope-labeled epitope was detected by mass spectrometry. Maximum surface presentation was observed 6 hours after transfection.

[0201] [Figure 64-1] Figure 64A shows an exemplary graphical representation from a recall assay of percentage changes in TNFα and / or IFNγ production (left) or percentage of CD107a-positive cells (right) from neoantigen-specific CD8 T cells challenged with increasing concentrations of the indicated peptide used to load into APCs.

[0202] [Figure 64-2] Figure 64B shows an exemplary graphical representation from a multimer assay of percentage changes in TNFα and / or IFNγ production (left) or percentage of CD107a-positive cells (right) from neoantigen-specific CD8 T cells challenged with increasing concentrations of the indicated peptide used to load into APC.

[0203] [Figure 65] Figure 65 shows an illustrative Venn diagram of the criteria considered for generating a personalized T-cell therapeutic, which is the optimal product, using mRNA as an immunogen.

[0204] [Figure 66] Figure 66 shows an exemplary flow diagram illustrating the steps for selecting peptide sequences to prepare patient-specific T cell products.

[0205] [Figure 67] Figure 67 illustrates several embodiments that are advantageous for clinical approaches using T cells produced by the process shown in Figure 1A.

[0206] [Figure 68] Figure 68 shows exemplary representative flow cytometry data illustrating the characterization of patient-specific T cell products prepared by multiple engineering runs. CD3+ (upper panel) is shown as the percentage of viable cells, and CD8+ and CD4+ (lower panel) are shown as the percentage of viable CD3+ T cells.

[0207] [Figure 69] Figure 69A shows an exemplary graphical representation of data characterizing patient-specific T cell products prepared by multiple engineering runs. The percentage of multimer-positive CD8-positive cells is shown.

[0208] Figure 69B shows exemplary representative flow cytometry data illustrating the characterization of patient-specific T cell products prepared by multiple engineering runs. For the indicated epitopes, the percentage of multimer A-positive and multimer B-positive CD8 cells is shown.

[0209] Figure 69C shows the identified pMHCs during rechallenge in DCs loaded with mutant neoantigens compared to DCs that were not loaded. + CD8 + An exemplary pie chart illustrating the multifunctionality of T cells is shown.

[0210] [Figure 70]Figure 70 shows representative data illustrating changes in IFNγ and / or TNFα production by CD4+ cells in patient-specific T cell products prepared by multiple engineering runs. Exemplary representative data illustrating the characterization of IFNγ+ and / or TNFα+ and / or CD107a+CD4+ cells in patient-specific T cell products prepared by multiple engineering runs are also shown.

[0211] [Figure 71] Figure 71 shows an exemplary graph displaying the proportions of central memory T cells (Tcm), effector memory T cells (Tem), effector T cells (Teff), and naive T cells (Tnaive) in patient-specific T cell products prepared by multiple engineering runs. Central memory T cells (Tcm): CD62L+CD45RA-, effector memory T cells (Tem): CD62L-CD45RA-, effector T cells (Teff): CD62L-CD45RA+, naive T cells (Tnaive): CD62L+CD45RA+.

[0212] [Figure 72] Figure 72 shows an exemplary graphical representation of data from a multimer assay, showing the percentage of IFN-γ+ and / or TNFα+ and / or CD107a+ cells in total CD8+ cells (upper panel) or total CD4+ T cells (lower panel) measured during a challenge in DCs loaded with various concentrations of peptides in the sample. The peptide used is indicated for each graph.

[0213] [Figure 73] Figure 73 shows an exemplary graphical representation of data demonstrating the upregulation of CD107a on CD8+ T cells (top row) and active caspase 3 on tumor cells (bottom row). Measurements were obtained after co-culturing the A375 tumor cell line or the A375 tumor cell line with or without the peptide, with or without transduction.

[0214] [Figure 74] Figure 74 shows an exemplary graphical representation of data demonstrating that induced T cells can kill antigen-expressing cells. Neoantigen-specific T cells were tested via a recall response assay to determine whether they recognized autologous tumors or peptide-loaded autologous tumors. Readout: IFN-γ+ and / or TNFα+ and / or CD107a+ (Y-axis) of pMHC+(CD8+) and pMHC-(CD8+)% T cells. Significance was assigned using one-way ANOVA, P<0.05.

[0215] [Figure 75] Figure 75 shows an exemplary schematic diagram of cohorts and doses for use in the clinical study (NEO-PTC-01). [Modes for carrying out the invention]

[0216] T-cell therapeutic agents are expected to be relatively safe and well-tolerated adoptive T-cell products. However, based on the assessment of the risks associated with the product, there are three common classes of potential toxicity associated with T-cell therapeutic agents: (a) treatment-related toxicity due to lymphodepletion, cell injection, or cytokine release syndrome; (b) off-tumor off-target toxicity due to the proliferation of autoreactive clones or cross-reactivity of neoantigen-specific T cells; and (c) off-tumor on-target toxicity due to the presentation of neoantigens in non-tumor tissues. Novel immunotherapeutic agents and their use based on the discovery of neoantigens arising from mutational events specific to the tumor of an individual are described herein. Accordingly, the disclosures described herein provide methods and protocols for generating antigen-specific immune cells, such as T cells, for use in the treatment of diseases.

[0217] Compositions of neoantigen-responsive T cells for cancer immunotherapy are presented herein. Adoptive T cell therapy is a promising new approach for cancer treatment, but it requires several improvements. In general, T cells need to be appropriately cytotoxic to cancer cells, preserve non-cancerous cells in the body, not lose immunogenicity in the tumor environment, and provide long-term protection. Moreover, the use of virally transduced cells itself presents challenges. Therefore, striking the right balance to achieve a therapeutically effective composition that specifically targets cancer cells while preserving healthy cells requires several improvements at almost every step of the complex process, in order to halt disease progression, induce remission or at least substantial tumor regression, prevent cancer recurrence.

[0218] To aid in understanding this disclosure, several terms and phrases are defined below.

[0219] Antigens are foreign substances to the body that induce an immune response. "Neoantigens" refer to a class of tumor antigens arising from tumor-specific alterations in proteins. Neoantigens include, but are not limited to, tumor antigens arising from substitutions, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, and the expression of endogenous retroviral polypeptides in protein sequences.

[0220] A "neoepitope" refers to an epitope that is not present in reference cells such as non-infected cells, e.g., non-cancerous cells or germline cells, but is found in affected cells, e.g., cancer cells. This includes situations where the corresponding epitope is found in normal, non-infected cells or germline cells, but one or more mutations alter the epitope's sequence in affected cells, e.g., cancer cells, resulting in a neoepitope.

[0221] 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). A "somatic mutation" can occur in any cell of the body except embryonic cells (sperm and egg) and is not transmitted to offspring. Such changes can (but not necessarily) cause cancer or other diseases. In some embodiments, a mutation is a non-synonymous mutation. A "non-synonymous mutation" refers to a mutation that results in an amino acid change, such as an amino acid substitution in the translation product (e.g., a nucleotide substitution). A "frameshift" occurs when a mutation disrupts the normal phase of the gene's codon periodicity (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. Different mutations in a gene can achieve the same altered reading frame.

[0222] "Antigen processing" or "processing" refers to the breakdown of a polypeptide or antigen into processing products, which are fragments of the polypeptide or antigen (e.g., breakdown from polypeptide to peptide), and the association (e.g., by binding) of an MHC molecule with one or more of these fragments for presentation by a cell, such as an antigen-presenting cell, directed toward a specific T cell.

[0223] 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).

[0224] The term "affinity" refers to a measure of the strength of binding between two members of a binding pair (e.g., human leukocyte antigen (HLA) binding peptide and class I or II HLA, or peptide-HLA complex and T cell receptor (TCR)). D K refers to the dissociation constant between two members of a bond pair and has units of molar concentration. AK refers to the affinity constant between the two members of a bond pair and is the reciprocal of the dissociation constant. Affinity can be determined experimentally, for example, by surface plasmon resonance (SPR) using a commercially available Biacore SPR unit. off This refers to the off-rate constants of the two members of the binding pair (for example, the off-rate constant of the HLA-binding peptide and class I or II HLA, or the off-rate constant of the peptide-HLA complex and TCR). on This refers to the on-rate constants of the two members of the binding pair (for example, the on-rate constants of an HLA-binding peptide and a class I or II HLA, or a peptide-HLA complex and a TCR).

[0225] Through this disclosure, the “combined data” results are “IC 50 It can be expressed in units of ". Affinity is expressed as inhibitory concentration 50 (IC). 50 ), or it can also be expressed as the concentration at which 50% of the first member of the bond pair (e.g., the peptide) is replaced. Similarly, ln(IC) 50 ) is IC 50 This refers to the natural logarithm of IC. For example, IC 50 This could be the concentration of the test peptide in the binding assay at which 50% inhibition of binding to the labeled reference peptide is observed. Considering the conditions under which the assay is performed (e.g., limiting the HLA protein concentration and / or the labeled reference peptide concentration), these values ​​are K DThe values ​​can be approximated. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO94 / 20127 and WO94 / 03205, as well as in other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed in comparison to binding with a reference standard peptide. Binding was established in living cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del 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 system (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high-flux soluble phase assay (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurement of Class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell The determination can also be made using other assay systems, including the assay system using 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992).

[0226] The term “derived” is synonymous with “prepared” when used to describe an epitope. A derived epitope can be isolated from a natural source or synthesized according to standard protocols in the art. Synthetic epitopes may include artificial amino acid residues “amino acid mimetic,” such as D isomers of naturally occurring L amino acid residues, such as cyclohexylalanine, or unnatural amino acid residues. A derived or prepared epitope may be an analog of a native epitope. The term “derived from” refers to the origin or source and may include naturally occurring, recombinant, unpurified, purified, or differentiated molecules or cells. For example, augmented or induced antigen-specific T cells may be derived from T cells. For example, augmented 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 + They can originate from monocytes. For example, dendritic cells can originate from monocytes (e.g., CD14). + They can originate from monocytes. For example, APCs can originate from bone marrow cells.

[0227] An “epitope” is a collective feature of molecules that together form a site recognized by another molecule (e.g., an immunoglobulin, a T cell receptor, an HLA molecule, or a chimeric antigen receptor) (e.g., the charge of a peptide, as well as its primary, secondary, and tertiary structures). For example, an epitope may be a specific immunoglobulin; a set of amino acid residues involved in recognition by a major histocompatibility complex (MHC) receptor; or, in the context of T cells, residues recognized by a T cell receptor protein and / or a chimeric antigen receptor. Epitopes can be prepared by isolation from natural sources or synthesized according to standard protocols in the art. Synthetic epitopes may include artificial amino acid residues, amino acid mimetics (such as D isomers of naturally occurring L amino acid residues or amino acid residues that do not exist naturally). Throughout this disclosure, an epitope may, in some cases, be referred to as a peptide or a peptide epitope. In certain embodiments, the length of the peptides in this disclosure is limited. Embodiments with limited length occur when a protein or peptide containing an epitope described herein includes a region (i.e., a series of adjacent amino acid residues) that is 100% identical to the native sequence. For example, to avoid defining an epitope as being read across the entire native molecule, there is a limitation on the length of any region that is 100% identical to the native peptide sequence. Thus, for peptides containing an epitope described herein and a region that is 100% identical to the native peptide sequence, the region that is 100% identical to the native sequence generally has a length of less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues.In certain embodiments, the “epitope” described herein is comprised of a peptide having a region having less than 51 amino acid residues that are 100% identical to the native peptide sequence, in increments of up to 5 amino acid residues; for example, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue.

[0228] A "T cell epitope" refers to a peptide sequence that is bound by an MHC molecule in the form of a peptide-MHC (pMHC) complex. The peptide-MHC complex can be recognized and bound by the TCR of T cells (e.g., cytotoxic T lymphocytes or T helper cells).

[0229] "T cells" are CD4 + T cells and CD8 +This includes T cells. The term T cells also includes both T helper 1 T cells and T helper 2 T cells. T cells can be generated for clinical use by the methods described in this application. T cells or adoptive T cells referenced here for clinical use are cells isolated from a biological source, manipulated and cultured ex vivo, and prepared into drug candidates for specific therapies for cancer, such as melanoma. If the drug candidate cells pass specific qualitative and quantitative criteria for suitability for clinical use, the drug candidate may be named a drug product. In some cases, a drug product is selected from a number of drug candidates. In the context of this application, a drug product is T cells, more specifically a population of T cells, or more specifically a population of T cells having heterogeneous characteristics and subtypes. For example, a drug product disclosed herein may have a population of T cells including CD8+ T cells, CD4+ T cells, and at least a certain percentage of cells exhibit antigen specificity, and a certain percentage of each exhibit, among other things, a memory phenotype.

[0230] "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 granule cells.

[0231] An "immunogenic" peptide or "immunogenic" epitope or "immunogenic" peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated or humoral response, such as a cytotoxic T lymphocyte (CTL) response, a helper T lymphocyte (HTL) response, and / or a B lymphocyte response. The immunogenic peptides described herein can bind to an HLA molecule and subsequently induce a cell-mediated or humoral response to the peptide (e.g., a CTL (cytotoxic) response or an HTL response).

[0232] A "protective immune response" or "therapeutic immune response" refers to a CTL and / or HTL response to an antigen derived from a pathogenic antigen (e.g., a tumor antigen) that in some way prevents or at least partially halts disease symptoms, side effects, or progression. The immune response may also include an antibody response triggered by the stimulation of helper T cells.

[0233] A "T cell receptor" ("TCR") refers to a molecule found on the surface of T lymphocytes (T cells) that recognize antigens bound to major histocompatibility complex (MHC) molecules, whether naturally occurring or partially or entirely synthetically produced. The ability of T cells to recognize antigens associated with various diseases (e.g., cancer) or infectious organisms is conferred by their TCR, which consists of both alpha (α) and beta (β) chains or both gamma (γ) and delta (δ) chains. The proteins that make up these chains are encoded by DNA, using unique mechanisms to generate the vast diversity of TCRs. This multi-subunit immune recognition receptor associates 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 the APC surface is a central event in T cell activation.

[0234] As used herein, “chimeric antigen receptor” or “CAR” refers to an antigen-binding protein comprising an immunoglobulin antigen-binding domain (e.g., an immunoglobulin variable domain) and a T cell receptor (TCR) constant domain. As used herein, the “constant domain” of a TCR polypeptide comprises a membrane-proximal TCR constant domain, a TCR transmembrane domain and / or a TCR cytoplasmic domain, or fragments thereof. For example, in some embodiments, a CAR is a monomer comprising a polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCRβ constant domain. In some embodiments, a CAR is a dimer comprising a first polypeptide comprising an immunoglobulin heavy chain or light chain variable domain linked to a TCRα or TCRβ constant domain, and a second polypeptide comprising 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.

[0235] The "major histocompatibility complex" or "MHC" is a cluster of genes that plays a role in regulating cell interactions that result in physiological immune responses. The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes that occurs in all vertebrates and can include any class of MHC molecules, such as MHC class I and MHC class II molecules. 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 (e.g., Stites, et al., Immunology, 8 TH See Ed., Lange Publishing, Los Altos, Calif. (1994). For a detailed description of the MHC and HLA complexes, see Paul, Fundamental Immunology, 3 rd Ed., See Raven Press, New York (1993).

[0236] The major histocompatibility complex in the genome contains genetic regions whose gene products, expressed on the cell surface, are crucial for regulating immunological processes by binding to and presenting endogenous and / or exogenous antigens. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or affected cells in immune responses. MHC proteins or molecules bind to peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the cell surface and can display both self-antigens (peptide fragments derived from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. MHC-binding peptides result from the proteolytic cleavage of protein antigens and can represent potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes). MHC transports peptides to the cell surface, where they can be presented to specific cells such as cytotoxic T lymphocytes, T helper cells, or B cells. MHC regions can be divided into three subgroups: Class I, Class II, and Class III. MHC class I proteins can contain α chains and β2 microglobulin (which is not part of the MHC encoded by chromosome 15). These can present antigenic fragments to cytotoxic T cells. MHC class II proteins can contain α and β chains and can present antigenic fragments to T helper cells. MHC class III regions can encode other immune components, such as complement components and cytokines. MHC can be both polygenic (several MHC class I and MHC class II genes exist) and polymorphic (multiple alleles exist for each gene).

[0237] A "receptor" refers to a biomolecule or molecular group that can bind to a ligand. Receptors can function to transmit information in cells, cell formation, or organisms. A receptor comprises at least one receptor unit, for example, each receptor unit may consist of a protein molecule. A receptor has a structure that complements the structure of the ligand and can form a complex with the ligand as a binding partner. Information is transmitted, in particular, by a conformational change of the receptor after complex formation with the ligand on the cell surface. In some embodiments, the receptor should be understood to mean MHC class I and II proteins that can form a receptor / ligand complex with the ligand, in particular a peptide or peptide fragment of a suitable length. A "ligand" refers to a molecule that has a structure complementary to the structure of the receptor and can form a complex with this receptor. In some embodiments, the ligand should be understood to mean a peptide or peptide fragment that has a suitable length and a suitable binding motif in its amino acid sequence so that the peptide or peptide fragment can form a complex with an MHC protein, such as an MHC class I or MHC class II protein. In some embodiments, the term "receptor / ligand complex" should also be understood to mean a "receptor / peptide complex" or "receptor / peptide fragment complex" that includes a peptide or peptide fragment-presenting MHC molecule, such as an MHC class I or MHC class II molecule.

[0238] A “native” or “wild-type” sequence refers to a sequence found in nature. The term “naturally occurring,” as used herein, refers to the fact that something can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0239] The terms “peptide” and “peptide epitope” are used herein interchangeably with “oligopeptide” and typically refer to a series of residues 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 an artificial peptide. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. “Synthetic peptide” includes “fusion protein.”

[0240] The term “motif” refers to a pattern of amino acid sequences of a defined length recognized by a particular HLA molecule, e.g., less than approximately 15 amino acid residues or less than approximately 13 amino acid residues, e.g., approximately 8 to approximately 13 amino acid residues for class I HLA motifs (e.g., 8, 9, 10, 11, 12, or 13), and approximately 6 to approximately 25 amino acid residues for class II HLA motifs (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25). Motifs are typically unique for each HLA protein encoded by a given human HLA allele. These motifs differ in their primary and secondary anchor residue patterns. In some embodiments, MHC class I motifs identify peptides with a length of 7, 8, 9, 10, 11, 12, or 13 amino acid residues. In some embodiments, the MHC class II motif identifies peptides with a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acid residues. A “cross-reactive” peptide refers to a peptide that binds to two or more members of the same class as the binding pair member (e.g., a peptide bound by both class I and class II HLA molecules).

[0241] The term "residue" refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or encoded by a nucleic acid (DNA or RNA). The nomenclature used to represent peptides or proteins follows conventional convention. The amino group is presented to the left (amino or N-terminus) of each amino acid residue, and the carboxyl group is presented to the right (carboxy or C-terminus). When amino acid residue positions are referenced in a peptide epitope, they are numbered from amino to carboxyl, with the first position being the residue located on the amino-terminal side of the epitope or the peptide or protein in which the epitope may be part. In formulas representing selected specific embodiments of the present invention, the amino and carboxyl terminal groups are of the type assumed at physiological pH values ​​unless otherwise specified. In amino acid structural formulas, each residue is generally represented by a standard three-letter or one-letter nomenclature. The L-type of an amino acid residue is represented by a single uppercase letter or by a three-letter symbol with the first letter uppercase, and the D-type of an amino acid residue is represented by a single lowercase letter or a three-letter symbol. However, when the three-letter symbol or full name is used without uppercase letters, it may refer to an L-type amino acid residue. Glycine does not have a chiral carbon atom and is simply referred to as "Gly" or "G". The amino acid sequences of peptides described herein are generally named using standard single-letter symbols (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine).

[0242] A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine with phenylalanine is a conservative substitution. Methods for identifying nucleotide and amino acid conservative substitutions that do not exclude peptide function are well known in the art.

[0243] "Pharmacologically acceptable" generally refers to a composition or component of a composition that is non-toxic, inert, and / or physiologically compatible. "Pharmaceutical excipients" or "excipients" include materials such as adjuvants, carriers, pH adjusters and buffers, osmotic regulators, wetting agents, preservatives, and others. "Pharmaceutical excipients" are pharmaceutically acceptable excipients.

[0244] In this disclosure, the term "vaccine" refers to a pharmaceutical preparation (pharmaceutical composition) or product that, after administration, induces an immune response, such as a cellular or humoral immune response, that recognizes and attacks diseased cells, such as pathogens or cancer cells. Vaccines can be used for the prevention or treatment of disease. The terms "personalized cancer vaccine," "personalized cancer vaccine," or "personal cancer vaccine" refer to a specific cancer patient and mean that the cancer vaccine is adapted to the needs or specific circumstances of that individual cancer patient.

[0245] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length, including DNA and RNA, e.g., mRNA. Nucleotides may 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, polynucleotides and nucleic acids may be in vitro transcribed mRNA. In some embodiments, the polynucleotide administered using the method of the present invention is mRNA.

[0246] The terms “isolated” or “biologically pure” refer to material that substantially or essentially contains no components that would normally be associated with the material when found in its native state. Therefore, isolated peptides described herein do not contain some or all of the material that would normally associate with the peptide in its in situ environment. For example, an “isolated” epitope may be an epitope that does not contain the entire sequence of the protein from which the epitope is derived. For example, a naturally occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide separated from some or all of the coexisting material in its natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or peptide may be part of a composition, and such a vector or composition can still be considered “isolated” in that it is not part of its natural environment. An isolated RNA molecule is the in vivo or in DNA molecule described herein. The material comprises a vitro RNA transcript and further comprises such molecules produced by synthesis. In some embodiments, the isolated polypeptide, antibody, polynucleotide, vector, cell, or composition is substantially pure. The term “substantially pure” as used herein means material that is at least 50% pure (i.e., free of impurities), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0247] In the context of two or more nucleic acids or polypeptides, the terms “identical” or “percent “identical” refer to two or more sequences or subsequences that are identical, or have a specified percentage of identical nucleotide or amino acid residues, when compared and aligned for maximum match (with gaps introduced if necessary) without considering any conserved amino acid substitutions as part of sequence identity. Percential identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. Such as BLAST, ALIGN, Megalign, BestFit, and GCG. This includes, but is not limited to, the Wisconsin Package and its variants. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaning that when they are compared and aligned for the greatest match as measured by a sequence comparison algorithm or by visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, and 99% nucleotide or amino acid residue identity. In some embodiments, the identity exists over a region of the sequence that is at least about 10, at least about 20, at least about 40–60 residues, at least about 60–80 residues, or any integer value in between. In some embodiments, the identity exists over a region longer than 60–80 residues, such as at least about 80–100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the coding region of an amino acid sequence or nucleotide sequence of a peptide.

[0248] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, dogs, cats, rodents, and others, that is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein to refer to human subjects.

[0249] The term “effective dose,” “therapeutic effective dose,” or “therapeutic effect” refers to the amount of a therapeutic agent that is effective in “treating” a disease or disorder in a subject or mammal. A therapeutic effective dose of a drug has a therapeutic effect and therefore can prevent the onset of a disease or disorder; slow the onset of a disease or disorder; slow the progression of a disease or disorder; reduce one or more of the symptoms associated with a disease or disorder to some extent; reduce morbidity and mortality; improve quality of life; or produce a combination of such effects.

[0250] The terms “to treat,” “treatment,” “to treat,” “to alleviate,” and “to alleviate” refer to both (1) therapeutic measures that cure, slow, reduce, and / or halt the progression of symptoms of a diagnosed pathological condition or disability, and (2) preventive or prophylactic measures that prevent or delay the onset of a targeted pathological condition or disability. Therefore, those who require treatment include those who already have a disability; those who are prone to developing a disability; and those for whom disability should be prevented.

[0251] The term "depleted," when used to describe a cell sample (e.g., a peripheral blood mononuclear cell (PBMC) sample), refers to a cell sample from which a subpopulation of cells has been removed or depleted. For example, an immune cell sample depleted of CD25-expressing cells refers to an immune cell sample from which CD25-expressing cells have been removed or depleted. For example, one or more binders can be used to remove or deplete one or more types of cells or cell groups from a sample. For example, CD14 + Cells can be depleted or removed from PBMC samples by using antibodies that bind to CD14, among other methods.

[0252] "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, T cell stimulation could refer to the binding of the T cell's TCR to a peptide-MHC complex. For example, T cell stimulation could refer to a step in protocol 1 or protocol 2 in which PBMCs are cultured with peptide-loaded APCs.

[0253] The term "concentrated" refers to a composition or fraction in which a target species has been partially purified so that the concentration of the target species is substantially higher than the naturally occurring level of that species in the finished product without concentration. The term "induced cells" refers to cells treated with an inducer compound, cells, or population of cells that affects cellular protein expression, gene expression, differentiation state, shape, morphology, viability, or other properties.

[0254] A “reference” can be used to correlate and / or compare results obtained in the methods of this disclosure from diseased specimens. Typically, a “reference” can be obtained based on one or more normal specimens, in particular specimens that are not affected by the disease, obtained from individuals of the same species or from one or more different individuals (e.g., healthy individuals). A “reference” can be determined empirically by examining a sufficiently large number of normal specimens.

[0255] As used herein, tumor refers to a cancerous tumor unless otherwise specified, and the terms cancer and tumor are used interchangeably throughout this document. A tumor is a cancer of solid tissue, but some of the compositions and methods described herein are applicable, in principle, to leukemia, which is a cancer of the blood. Overview of T-cell therapy

[0256] The generation of antigen-specific T cells by controlled ex vivo induction or amplification of T cells (e.g., autologous T cells) can provide highly specific and beneficial T cell therapies (e.g., adoptive T cell therapy). This disclosure provides methods for producing T cells and therapeutic T cell compositions that can be used to treat subjects with cancer and other conditions, diseases, and disorders. The goal is to amplify and induce antigen-specific T cells with favorable phenotypes and functions. This disclosure provides compositions and methods for producing T cells that can be used in antigen-specific T cell therapies (e.g., personal or individualized T cell therapies). The T cell compositions provided herein may be personal antigen-specific T cell therapies. Figure 1 graphically represents an overview of the process relating to T cell therapy: this includes, on the one hand, the identification of cancer and cancer-specific antigens in subjects with cancer, resulting in the production of neoantigenic peptides; on the other hand, the preparation of activated antigen-specific cells for immunotherapy and the administration of cell products. Neoantigens for T-cell based therapies

[0257] Traditional antigen-targeted immunotherapy has focused on antigens including tumor-associated antigens (TAAs), cancer-testicular antigens (typically germline-restricted gene products ectopically expressed in tumors), or antigens derived from genes exhibiting tissue-specific expression. However, tumors also display protein products of mutated genes called neoantigens. The number and type of mutations can be readily defined using next-generation sequencing approaches and include single-amino acid missense mutations, fusion proteins, and novel open reading frames (neoORFs) whose length varies from one to up to 100 or more amino acids. Neoantigens are antigens that contain non-silent mutations in their epitopes, and the same antigen is not expressed in non-cancer cells within the same human body. Mutation-based antigens are particularly useful because they possess bypassed central tolerance (a process that occurs in normal thymic development to eliminate autoreactive T cells) and demonstrate sophisticated tumor specificity. Each non-synonymous (i.e., protein-coding) mutation has the potential to generate a neoantigen that can be recognized by the patient's T cells. T cells that recognize such neoantigens can directly kill tumor cells and also function to catalyze a broader immune response against tumors. The methods described herein aim to induce and increase such neoantigen-reactive T cells in a patient-specific manner and to utilize such cells for adoptive cell therapy.

[0258] In some embodiments, the neoantigens used herein include point mutations.

[0259] In some embodiments, the neoantigens used herein include frameshift mutations.

[0260] In some embodiments, the neoantigens used herein include crossover mutations.

[0261] In some embodiments, the neoantigens used herein include insertion mutations resulting from the insertion of one or more nucleotides.

[0262] In some embodiments, the neoantigens used herein include deletion mutations resulting from the deletion of one or more nucleotides.

[0263] In some embodiments, the neoantigen may result from an insertion-deletion (indel) mutation.

[0264] 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 better affinity than the corresponding wild-type peptide. In specific embodiments, the antigen or neoantigen peptide has an IC of less than 5000 nM, less than 500 nM, less than 100 nM, less than 50 nM, or less than that IC. 50 or K D It holds.

[0265] In some embodiments, the antigen or neoantigen peptide may be about 8 to about 50 amino acid residues in length, or about 8 to about 30, about 8 to about 20, about 8 to about 18, about 8 to about 15, or about 8 to about 12 amino acid residues in length. In some embodiments, the antigen or neoantigen peptide may be about 8 to about 500 amino acid residues in length, or about 8 to about 450, about 8 to about 400, about 8 to about 350, about 8 to about 300, about 8 to about 250, about 8 to about 200, about 8 to about 150, about 8 to about 100, about 8 to about 50, or about 8 to about 30 amino acid residues in length.

[0266] In some embodiments, the antigen or neoantigen peptide may have 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, 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 neoantigen peptide may have 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, 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 embodiments, the antigen or neoantigen peptide may have a length of at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or fewer amino acid residues. In some embodiments, the antigen or neoantigen peptide may have a length of at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or fewer amino acid residues.

[0267] In some embodiments, the antigen or neoantigen peptide has a total length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids.

[0268] In some embodiments, the antigen or neoantigen peptide has a total length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids.

[0269] 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 more. 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 less.

[0270] 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 more. In some embodiments, the antigen or neoantigen peptide may have an HLA binding affinity of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, or 900 μM.

[0271] In some embodiments, the antigens or neoantigen peptides described herein may include carriers known in the art, such as thyroglobulin, albumins such as human serum albumin, tetanus toxoid, poly-L-lysine, poly-L-glutamic acid, and other polyamino acid residues, influenza virus proteins, hepatitis B virus core proteins, and others.

[0272] In some embodiments, the antigens or neoantigen peptides described herein are modified by terminal NH2 acylation, for example, by alkanoyl (C1-C1). 20 ) or can be modified by thioglycolylacetylation, terminal carboxylamidate, for example, ammonia, methylamine, etc. In some embodiments, these modifications can provide sites for linking to a support or other molecules.

[0273] In some embodiments, the antigens or neoantigen peptides described herein may include, but are not limited to, modifications such as glycosylation, side-chain oxidation, biotinylation, phosphorylation, and the addition of surface-active materials, such as lipids, or may be chemically modified, such as by acetylation. Furthermore, the bonds in the peptide may be other than peptide bonds, such as covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, or ionic bonds.

[0274] In some embodiments, the antigens or neoantigen peptides described herein may contain substitutions that alter the physical properties of the resulting peptide (e.g., stability or solubility). For example, an antigen or neoantigen peptide can be modified by substituting cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical properties, cysteine ​​tends to structurally alter the peptide sufficiently to form disulfide crosslinks and reduce its binding ability. Substitution of C with α-aminobutyric acid not only mitigates this problem but, in certain examples, actually improves binding and cross-binding ability. The substitution of cysteine ​​with α-aminobutyric acid may occur at any residue of the antigen or neoantigen peptide, for example, at an epitope or at an anchored or unanchored position of the analog within the peptide, or at any other position in the peptide.

[0275] In some embodiments, the antigen peptides or neoantigen peptides described herein are amino acid mimetic or non-natural amino acid residues, for example, D- or L-naphthyl alanine; D- or L-phenylglycine; D- or L-2-thieneyl alanine; D- or L-1,2,3 or 4-pyreneyl alanine; D- or L-3-thieneyl alanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyradinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine Lysine; D-(trifluoromethyl)-phenylglycine; D-(trifluoro-methyl)-phenylalanine; D-ρ-fluorophenylalanine; D-or L-ρ-biphenyl-phenylalanine; D-or L-ρ-methoxybiphenylphenylalanine; D-or L-2-indole(allyl)alanine; and D-or L-alkylalanine, where the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, isopentyl, or non-acidic amino acid residues. Non-natural amino acid aromatic rings include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides having a variety of amino acid mimetic or non-natural amino acid residues are particularly useful because they tend to exhibit increased stability in vivo. Such peptides may also possess improved shelf life or manufacturing characteristics.

[0276] In some embodiments, the peptide is brought into contact with immune cells to activate them and make them antigen-responsive.

[0277] In some embodiments, the peptide is brought into contact with immune cells ex vivo.

[0278] In some embodiments, the peptide is brought into contact with a biological system, such as immune cells in humans.

[0279] In some embodiments, immune cells are antigen-presenting cells.

[0280] In some embodiments, the immune cells are T cells.

[0281] This disclosure relates to a method for producing T cells that are specific to an immunogenic antigen.

[0282] This disclosure also relates to compositions comprising antigen-specific T cells stimulated by APCs. In some embodiments, one or more antigen peptides are loaded onto APCs, and the peptide-loaded APCs are then used to stimulate T cells to produce antigen-specific T cells. In some embodiments, the antigen is a neoantigen. In some embodiments, the APCs used for peptide loading are dendritic cells.

[0283] In some embodiments, the peptide sequence includes mutations not present in the non-cancer cells of interest. In some embodiments, the peptide is encoded by a gene or expressed gene in the cancer cell of interest. In some embodiments, the peptide sequence has a length of at least 8;9;10;11;12;13;14;15;16;17;18;19;20;21;22;23;24;25;26;27;28;29;30;40;50;60;70;80;90;100;150;200;250;300;350;400;450;500;600;700;800;900;1,000;1,500;2,000;2,500;3,000;4,000;5,000;7,500; or 10,000 or more naturally occurring amino acids.

[0284] In some embodiments, the peptide sequence binds to a protein encoded by a class I HLA allele and has a length of 8 to 12 naturally occurring amino acids. In some embodiments, the peptide sequence binds to a protein encoded by a class II HLA allele and has a length of 16 to 25 naturally occurring amino acids. In some embodiments, the peptide sequence comprises multiple antigen peptide sequences. In some embodiments, the multiple antigen 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 antigen peptide sequences.

[0285] In some embodiments, the antigens described herein are neoantigens. Candidate immunogenic neoantigen sequences can be identified by any suitable method known in the art. The methods of this disclosure may be useful, for example, for the production of therapies specific to a disease of interest or for the production of vaccines against a disease. Candidate immunogenic neoantigens may be previously identified neoantigens. In some embodiments, candidate immunogenic neoantigens do not need to be previously identified. Candidate immunogenic neoantigens for use in the methods and compositions described herein may be specific to a particular subject. In some embodiments, candidate neoantigens for use in the methods and compositions described herein may be specific to multiple subjects.

[0286] In both animals and humans, mutated epitopes may be potentially effective in inducing an immune response or activating T cells. In one embodiment, potentially immunogenic epitopes of infectious pathogens in a subject, such as viruses, can be determined. In one embodiment, potentially immunogenic mutated epitopes can be determined in subjects with diseases such as cancer. In some embodiments, a potentially immunogenic antigen or neoantigen for use in the methods described herein may be a differentiated antigen expressed in tumors and cells of the type of tissue from which it was produced. In some embodiments, a potentially immunogenic antigen or neoantigen for use in the methods described herein may be a cancer / germline antigen not expressed in another differentiated tissue. In some embodiments, a potentially immunogenic antigen or neoantigen for use in the methods described herein may be a mutated antigen. For example, a candidate immunogenic antigen or neoantigen peptide for use in the methods described herein may include an antigen or neoantigen of a fusion protein produced by a missense point mutation or a tumor-specific translocation of a gene segment. In some embodiments, a potentially immunogenic antigen or neoantigen for use in the methods described herein may be an overexpressed antigen. In some embodiments, potentially immunogenic antigens or neoantigens may be found in tumors. For example, potentially immunogenic antigens or neoantigens for use in the methods described herein may include proteins whose expression is tightly regulated in differentiated normal tissue cells.

[0287] Potentially immunogenic mutated epitopes can be determined by genome or exome sequencing of tumor tissue from cancer patients and healthy tissue using next-generation sequencing technology. For example, genes selected based on their mutation frequency and ability to act as antigens or neoantigens can be sequenced using next-generation sequencing technology. In one embodiment, sequencing data can be analyzed to identify potentially immunogenic mutated peptides that can bind to the target HLA molecule. In one embodiment, the data can be analyzed using a computer. In another embodiment, the sequence data can be analyzed for the presence of antigens or neoantigen peptides. In one embodiment, potentially immunogenic antigens or neoantigen peptides can be determined by their affinity for MHC molecules.

[0288] Potentially immunogenic antigens or neoantigen peptides can be determined by direct protein sequencing. For example, potentially immunogenic antigens or neoantigen peptides for use in the methods described herein can be identified using enzymatic protein sequencing of protein digests with multidimensional mass spectrometry techniques (e.g., tandem mass spectrometry (MS / MS)).

[0289] High-throughput methods for de novo sequencing of unknown proteins can be used to identify potentially immunogenic antigens or neoantigen 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 target tumor to identify potentially immunogenic neoantigens.

[0290] Potentially immunogenic antigens or neoantigen peptides can also be identified using MHC multimers to identify antigen-specific T cell responses. For example, high-throughput analysis of antigen-specific T cell responses in patient samples can be performed using MHC tetramer-based screening techniques. Tetramer-based screening techniques can be used as a secondary screening protocol for the initial identification of potentially immunogenic tumor-specific antigens or to assess whether a patient may have already been exposed to any potentially immunogenic antigens, thereby facilitating the selection of potentially immunogenic antigens for use in the methods described herein.

[0291] In some embodiments, specific neoantigens are targeted for immunotherapy. In some embodiments, neoantigenic peptides are synthesized. The neoantigenic peptides used herein are designed so that each peptide is specific to an HLA antigen and can bind to the HLA antigen with high binding affinity and specificity. In some embodiments, the peptides used herein are designed based on high-performance HLA binding prediction models created by the inventors, for example, the following patent applications / publications: WO2011143656, WO2017184590 and U.S. Patent Provisional Applications 62 / 783,914 and 62 / 826,827, all of which are incorporated herein by reference. NetMHCIIpan, even if it is the current prediction standard, cannot be considered error-free. Of the three class II loci (DR, DP, and DQ), data may only be available for certain common alleles of HLA-DR. In short, the newly created predictive model can be used to identify immunogenic antigen peptides, develop personalized drugs and other pharmaceuticals, and isolate and characterize antigen-specific T cells. The machine learning HLA-peptide presentation predictive model includes multiple predictor variables identified based on training data, the training data including sequence information of peptide sequences presented by HLA proteins expressed in cells and identified by mass spectrometry; training peptide sequence information including amino acid position information, and training peptide sequence information related to HLA proteins expressed in cells; and a function representing the relationship between the amino acid position information received as input and the presentation likelihood created as output, based on the amino acid position information and predictor variables. CD4+ T cell responses can have antitumor activity. In existing predictive methods, high rates of CD4+ T cell responses can be shown without using class II prediction (e.g., 60% of SLP epitopes in the NeoVax study (49% in NT-001) and 48% of mRNA epitopes in the BioNTech study). It is not always clear whether these epitopes are typically presented natively (by tumors or by phagocytic DCs).Therefore, it was desirable to bridge high CD4+T response rates to therapeutic efficacy by improving the identification of naturally presented class II epitopes. The roles of gene expression, enzymatic cleavage, and pathway / localization bias may not be robustly quantified. While the majority of existing MS data can be presumed to originate from autophagy, it may be unclear whether autophagy (class II presentation by tumor cells) or phagocytosis (class II presentation of tumor epitopes by APCs) is the more relevant pathway. Different data generation approaches may exist for learning the rules of class II presentation, including field standards and proposed approaches. Field standards may include affinity measurements that could be the basis for a NetMHCIIpan predictor, which offers low throughput and requires radioactive reagents, thus failing to capture the role of processing. The new approach involves mass spectrometry, where data from cell lines / tissues / tumors can help determine the process rules for autophagy (much of this data has already been published), and single-allele MS can enable the determination of allele-specific binding rules (multiple-allele MS data are presumed to be overly complex for efficient learning). The newly created prediction method includes a step of training a machine learning HLA-peptide presentation prediction model, the training step of inputting amino acid position sequences of HLA peptides isolated from one or more HLA-peptide complexes derived from cells expressing HLA class II alleles into the HLA-peptide presentation prediction model using a computer processor, the machine learning HLA-peptide presentation prediction model includes several predictor variables identified based on training data, the training data including sequence information of peptide sequences presented by HLA proteins expressed in cells and identified by mass spectrometry; training peptide sequence information including amino acid position information of the training peptides, and training peptide sequence information related to HLA proteins expressed in cells; and a function representing the relationship between the amino acid position information received as input and the presentation probability created as output, based on the amino acid position information and predictor variables.In some embodiments, the presented model has a recall of 0.1% to 10% and a positive predictive value of at least 0.25. In some embodiments, the presented model has a recall of 0.1% to 10% and a positive predictive value of at least 0.4. In some embodiments, the presented model has a recall of 0.1% to 10% and a positive predictive value of at least 0.6. In some embodiments, the mass spectrometry is single-allele mass spectrometry. In some embodiments, the peptide is presented by HLA proteins expressed in cells by autophagy. In some embodiments, the peptide is presented by HLA proteins expressed in cells by phagocytosis. In some embodiments, the quality of the training data is increased by using multiple quality metrics. In some embodiments, the multiple quality metrics include common contaminant peptide removal, high scoring peak intensity, high score, and high mass precision. In some embodiments, the scoring peak intensity is at least 50%. In some embodiments, the scoring peak intensity is at least 70%. In some embodiments, the peptide presented by the HLA protein expressed in the cell is the peptide presented by a single immunoprecipitated HLA protein expressed in the cell. In some embodiments, the multiple predictors include a peptide-HLA affinity predictor. In some embodiments, the multiple predictors include a source protein expression level predictor. In some embodiments, the multiple predictors include a peptide cleavage potential predictor. In some embodiments, the peptide presented by the HLA protein includes a peptide identified by searching a peptide database using a reverse database search strategy. In some embodiments, the HLA protein is an HLA-DR protein, and an HLA-DP or HLA-DQ protein. In some embodiments, the HLA protein is an HLA-DR protein selected from the group consisting of HLA-DR, and an HLA-DP or HLA-DQ protein.In some embodiments, the HLA proteins are HLA-DPB1*01:01 / HLA-DPA1*01:03, HLA-DPB1*02:01 / HLA-DPA1*01:03, HLA-DPB1*03:01 / HLA-DPA1*01:03, HLA-DPB1*04:01 / HLA-DPA1*01:03, HLA-DPB1*04:02 / HLA-DPA1*01:03, HLA-DPB1*06:01 / HLA-DPA1*01:03, HLA-DQB1*02:01 / HLA -DQA1*05:01, HLA-DQB1*02:02 / HLA-DQA1*02:01, HLA-DQB1*06:02 / HLA-DQA1*01:02, HLA-DQB1*06:04 / HLA-DQA1*01:02, HLA-DRB 1*01:01, HLA-DRB1*01:02, HLA-DRB1*03:01, HLA-DRB1*03:02, HLA-DRB1*04:01, HLA-DRB1*04:02, HLA-DRB1*04:03, HLA-DRB1*04 :04, HLA-DRB1*04:05, HLA-DRB1*04:07, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*08:02, HLA-DRB1*08:03, HLA-DRB1*08:04, HLA-DRB1*09:01, HLA-DRB1*10:01, HLA-DRB1*11:01, HLA-DRB1*11:02, HLA-DRB1*11:04, HLA-DRB1*12:01, HLA-DRB1*12:02, HLA- The HLA-DR protein is selected from the group consisting of 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 peptide presented by the HLA protein includes peptides identified by comparing the MS / MS spectrum of the HLA peptide with the MS / MS spectrum of one or more HLA peptides in a peptide database.

[0292] In some embodiments, the mutation is selected from the group consisting of point mutations, splice site mutations, frameshift mutations, readthrough mutations, and gene fusion mutations.

[0293] In some embodiments, the peptide presented by the HLA protein has a length of 15 to 40 amino acids. In some embodiments, the peptide presented by the HLA protein includes peptides identified by (a) isolating one or more HLA complexes from a cell line expressing a single HLA class II allele; (b) isolating one or more HLA-peptides from one or more isolated HLA complexes; (c) obtaining MS / MS spectra for one or more isolated HLA-peptides; and (d) obtaining peptide sequences corresponding to the MS / MS spectra of one or more isolated HLA-peptides from a peptide database, wherein one or more sequences obtained from step (d) identify the sequences of one or more isolated HLA-peptides.

[0294] Various antigenic peptides can be used to induce or enlarge T cells. Various antigenic peptides can be used to activate antigen-presenting cells (APCs), and then, by bringing the antigen-loaded APCs into contact with T cells, the APCs can activate the T cells.

[0295] In some embodiments, the peptide includes mutations selected from (A) point mutations, (B) splice site mutations, (C) frameshift mutations, (D) readthrough mutations, (E) gene fusion mutations, and combinations thereof. In some embodiments, the peptide includes a point mutation that binds to the target HLA protein with better affinity than the corresponding wild-type peptide.

[0296] In some embodiments, the peptide has an IC50 of 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or less than 10 nM.50 Then, it binds to the target HLA protein. In some embodiments, the peptide has an IC50 of 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or less than 10 nM. 50 or K D Then, it binds to the target HLA protein. In some embodiments, each peptide binds to the protein encoded by the HLA allele expressed by the target. In some embodiments, the TCR of induced or augmented antigen-specific T cells is 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or less than 10 nM IC 50 or K D Then, it binds to the peptide-HLA complex. In some embodiments, the TCR has an IC50 of 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or less than 10 nM. 50 or K D The peptide then binds to the HLA complex. In some embodiments, each of the at least one antigen peptide sequences contains a mutation not present in the non-cancer cells of interest. In some embodiments, each of the at least one antigen peptide sequences is encoded by a gene or expressed gene in the cancer cells of interest.

[0297] In some embodiments, the peptide has a length of at least 8;9;10;11;12;13;14;15;16;17;18;19;20;21;22;23;24;25;26;27;28;29;30;40;50;60;70;80;90;100;150;200;250;300;350;400;450;500;600;700;800;900;1,000;1,500;2,000;2,500;3,000;4,000;5,000;7,500; or 10,000 or more naturally occurring amino acids. In some embodiments, the peptide binds to a protein encoded by a class I HLA allele and has a length of 8 to 12 naturally occurring amino acids. In some embodiments, the peptide is bound to a protein encoded by a class II HLA allele and has a length of 16 to 25 naturally occurring amino acids. In some embodiments, the peptide comprises 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.

[0298] In some embodiments, the Disclosure provides peptides or polynucleotides encoding peptides identified using the methods briefly described above herein (e.g., peptides with tumor-specific mutations, viral peptides, or peptides associated with non-cancerous diseases).

[0299] In some embodiments, optical methods are used for the selection or identification of immunogenic antigens. In some embodiments, barcoded probes are used for the selection or identification of immunogenic antigens. In some embodiments, barcoded probes including a target-specific region and a barcoded region are used for the selection or identification of immunogenic antigens. In some embodiments, the target-specific region includes a nucleic acid sequence that hybridizes to or has at least about 90%, 95%, or 100% sequence complementarity to the nucleic acid sequence of a target polynucleotide. Preparation of activated antigen-specific T cells

[0300] Methods for stimulating T cells are provided herein. For example, antigen-specific T cells can be stimulated using the methods provided herein. T cells can be induced or activated using the methods provided herein. For example, activated T cells can be increased using the methods provided herein. For example, naive T cells can be induced using the methods provided herein. For example, antigen-specific CD8 can be stimulated using the methods provided herein. + T cells can be enlarged. For example, using the method provided herein, antigen-specific CD4 + T cells can be enlarged. For example, using the method provided herein, antigen-specific CD8 having a memory phenotype can be increased. + T cells can be enlarged. For example, the therapeutic composition may contain antigen-specific CD8+ T cells. For example, the therapeutic composition may contain antigen-specific memory T cells.

[0301] T cells can be activated ex vivo by a composition containing a neoantigenic peptide or a polynucleotide encoding a neoantigenic peptide.

[0302] T cells can be activated ex vivo by a composition containing antigen-loaded antigen-presenting cells.

[0303] In some embodiments, APCs and / or T cells are derived from a biological sample obtained from the subject.

[0304] In some embodiments, APCs and / or T cells are derived from a biological sample that consists of peripheral blood mononuclear cells (PBMCs).

[0305] In some embodiments, subjects are administered FLT3L prior to the step of obtaining a biological sample for preparing APCs and / or T cells.

[0306] In some embodiments, APCs and / or T cells are derived from a biological sample that is a leukocyte apheresis sample.

[0307] In some embodiments, antigen-presenting cells are first loaded with neoantigenic peptides ex vivo and used to prepare neoantigen-activated T cells. In some embodiments, the compositions provided herein include APC-stimulated T cells, such as APCs preloaded with antigen peptides. The compositions may include a population of immune cells, including T cells derived from a sample (e.g., a biological sample), where the T cells include APC-stimulated T cells. In some embodiments, mRNA encoding one or more neoantigenic peptides is introduced into the APC for neoantigenic peptide expression. Such APCs are used to stimulate or activate T cells.

[0308] In some embodiments, the biological sample comprises a percentage of at least one antigen-specific T cells in a composition that is at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5%. In some embodiments, the biological sample comprises antigen-activated T cells representing 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, less than 5%, or less than 10% of the total cell count in a biological sample derived from peripheral blood or leukocyte apheresis. In some embodiments, the biological sample contains antigen-activated T cells representing 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 the biological sample derived from peripheral blood or leukocyte apheresis. In some embodiments, the biological sample includes antigen-naive T cells. In some embodiments, the biological sample contains antigen-naive cells representing 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%, 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 more than 95% of the total cell count in the biological sample derived from peripheral blood or leukocyte apheresis. 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%, 0.05%, 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 in biological samples derived from peripheral blood or leukocyte apheresis 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%, and 10%. In some embodiments, the percentage of at least one antigen-specific T cells in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of total immune cells. In some embodiments, at least one antigen-specific CD8 in the biological sample + 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, at least one antigen-specific CD4 is present in the biological sample. + 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, neoantigen-specific CD8 in the biological sample + The percentage of T cells is at most about 0.5%. In some embodiments, antigen-specific CD4 in biological samples + The percentage of T cells in biological samples is at most about 0.5%. Preparation of APCs loaded with neoantigen

[0309] In some embodiments, the composition comprises a population of immune cells incubated with one or more cytokines, growth factors, or ligands, such as ligands that bind to the 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, IFN-γ, R848, LPS, ss-rna40, and poly(I:C). In some embodiments, the composition comprises a population of immune cells incubated with one or more APCs or APC preparations. For example, the composition may include a population of immune cells incubated with APCs stimulated with one or more cytokines, growth factors, and / or ligands, or APC preparations stimulated with cytokines, growth factors, and / or ligands. For example, the composition may include a population of immune cells incubated with APCs stimulated with one or more cytokines, or APC preparations stimulated with cytokines. For example, the composition may include a population of immune cells incubated with APCs stimulated with one or more growth factors or APC preparations stimulated with growth factors.

[0310] In some embodiments, APC is autologous APC, homogeneous inter-plant APC, or artificial APC.

[0311] Immune cells are characterized by cell surface molecules. In some embodiments, immune cells are selected based on cell surface markers, preferably by using antibodies that can bind to cell surface receptors, for example, from a biological sample. In some embodiments, some cells are negatively selected to enrich one or more cell types that do not express the negatively selected cell surface molecules.

[0312] In some embodiments, antigen-presenting cells (APCs) are prepared from a biological sample by selecting APCs or precursor cells, and the APCs or precursor cells can be cultured in the presence of a neoantigenic peptide to produce neoantigen-loaded APCs, which are used to activate T cells. Some of the relevant cell surface markers for selecting and / or enriching sets of cells are described later.

[0313] CD1 (Surface Antigen Class 1) is a family of glycoproteins expressed on the surface of various human antigen-presenting cells. It is associated with class I MHC molecules and is involved in the presentation of lipid antigens to T cells.

[0314] CD11b or integrin alpha-M (ITGAM) is also known as macrophage-1 antigen (Mac-1) or complement receptor 3 (CR3), and is a heterodimer integrin alpha-M beta-2 (α M β2) It is a single protein subunit that forms a molecule. ITGAM is also known as CR3A and surface antigen classification molecule 11b (CD11b). α M The second strand of β2 is the common integrin β2 subunit known as CD18, and therefore integrin α M β2 belongs to the β2 subfamily (or leukocyte) integrins. α M β2 is expressed on the surface of many leukocytes involved in the innate immune system, including monocytes, granule cells, macrophages, and natural killer cells. It mediates inflammation by regulating leukocyte adhesion and migration and has been linked to several immune processes, including phagocytosis, cell-mediated cytotoxicity, chemotaxis, and cell activation. It is involved in the complement system through its ability to bind to inactivated complement component 3b (iC3b). M The β2 ITGAM (alpha) subunit is directly involved in cell adhesion and propagation, but it cannot mediate cell migration without the presence of the β2 (CD18) subunit.

[0315] CD11c, also known as integrin alpha-X (complement component 3 receptor 4 subunit) (ITGAX), is the gene that encodes CD11c. CD11c is an integrin alpha-X chain protein. Integrins are heterodimeric membrane endogenous proteins composed of alpha and beta chains. This protein combines with the beta-2 chain (ITGB2) to form a leukocyte-specific integrin called the inactivated C3b (iC3b) receptor 4 (CR4). The alpha-X beta-2 complex appears to overlap with the properties of alpha-M beta-2 integrin in the adhesion of neutrophils and monocytes to stimulated endothelial cells, as well as in the phagocytosis of complement-coated particles. CD11c is a type I transmembrane protein found at high levels on the surface of most human dendritic cells, but also on the surface of monocytes, macrophages, neutrophils, and some B cells. It induces cell activation and helps trigger neutrophil respiratory bursts; it is expressed in hairy cell leukemia, acute non-lymphocytic leukemia, and some B-cell chronic lymphocytic leukemias.

[0316] CD14 is a surface antigen preferentially expressed on the surface of monocytes / macrophages. It works in cooperation with other proteins to mediate the innate immune response to bacterial lipopolysaccharides. Alternative splicing results in multiple transcript variants encoding the same protein. CD14 exists in two forms: one tethered to the membrane by a glycosylphosphatidylinositol tail (mCD14), and the other in a soluble form (sCD14). Soluble CD14 appears after shedding of mCD14 (48 kDa) or is secreted directly from intracellular vesicles (56 kDa). CD14 acts as a co-receptor for the detection of bacterial lipopolysaccharide (LPS) (together with Toll-like receptors TLR4 and MD-2). CD14 can bind to LPS only in the presence of lipopolysaccharide-binding proteins (LBPs). While LPS is considered its primary ligand, CD14 also recognizes other pathogen-associated molecular patterns, such as lipoteichoic acid.

[0317] CD25 is expressed by normal T cells after stimulation, and in human peripheral blood, CD4 + CD25 hi It was shown that only T cells act as "suppressors."

[0318] In some embodiments, the APC includes dendritic cells (DCs). In some embodiments, the APC is CD14 + Derived from monocytes. In some embodiments, APCs can be obtained from skin, spleen, bone marrow, thymus, lymph nodes, peripheral blood, or umbilical cord blood. In some embodiments, CD14 + Monocytes are derived from biological samples of the subject, including PBMCs. For example, CD14 + Monocytes can be isolated, concentrated, or purified from a biological sample derived from the subject, including PBMCs. In some embodiments, CD14 + Monocytes are stimulated by one or more cytokines or growth factors. 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, IL-15, IFN-γ, IFN-α, R848, LPS, ss-rna40, poly(I:C) or a combination thereof. In some embodiments, CD14 + The monocytes were derived from a second biological sample containing PBMCs.

[0319] In some embodiments, the isolated population of APCs can be concentrated or substantially concentrated. In some embodiments, the isolated population of APCs is at least 30%, at least 50%, at least 75%, or at least 90% homogeneous. In some embodiments, the isolated population of APCs is at least 60%, at least 75%, or at least 90% homogeneous. APCs such as APCs may include, for example, APCs derived from monocytic dendritic cell precursors in culture, as well as endogenously derived APCs present in tissues such as peripheral blood, umbilical cord blood, skin, spleen, bone marrow, thymus, and lymph nodes.

[0320] APCs and cell populations substantially enriched with APCs can be isolated by a method similarly provided by the present invention. This method generally includes the steps of obtaining a population of cells containing APC precursors and differentiating the APC precursors into immature or mature APCs, and may also include the isolation of APCs from the differentiated population of immature or mature APCs.

[0321] APC precursor cells can be obtained by methods known in the art. APC precursors can be isolated, for example, by density gradient separation, fluorescence-activated cell sorting (FACS), immunological cell separation techniques such as panning, complement lysis, rosetting, magnetic cell separation techniques, nylon wool separation, and combinations of such methods. Methods for immunoselecting APCs include the step of using antibodies against cell surface markers associated with APC precursors, such as anti-CD34 and / or anti-CD14 antibodies coupled to a substrate.

[0322] A concentrated population of APC precursors can also be obtained. Methods for obtaining such a concentrated precursor population are known in the art. For example, a concentrated population of APC precursors can be isolated from a tissue source by selective removal of cells adhering to a substrate. For example, using a tissue source such as bone marrow or peripheral blood, a concentrated population of non-adherent APC precursors can be obtained by removing adherent monocytes from a cell preparation using a commercially available plastic substrate (e.g., beads or magnetic beads).

[0323] Monocyte APC precursors can also be obtained from tissue sources by using an APC precursor adhesive substrate. For example, peripheral blood leukocytes isolated by leukocyte apheresis are brought into contact with a monocytic APC precursor adhesive substrate having a high surface area-to-volume ratio, and adhesive monocytic APC precursors are separated. In additional embodiments, the substrate to be coupled may be a granular or fibrous substrate having a high surface area-to-volume ratio, such as microbeads, microcarrier beads, pellets, granules, powders, capillaries, microvilli membranes, etc. Furthermore, the granular or fibrous substrate may be glass, polystyrene, plastic, glass-coated polystyrene microbeads, etc.

[0324] APC precursors can also be cultured in vitro for differentiation and / or proliferation. Methods for the differentiation / proliferation of APC precursors are known in the art. Generally, proliferation can be achieved by culturing the precursors in the presence of at least one cytokine that induces APC (e.g., dendritic cells) differentiation / proliferation. Typically, such cytokines are granule cell colony-stimulating factor (G-CSF) or granule cell / macrophage colony-stimulating factor (GM-CSF). In addition, other agents can be used to inhibit the proliferation and / or maturation of non-APC cell types in culture, thereby further enriching the population of APC precursors. Typically, such agents include cytokines such as IL-13, IL-4, or IL-15, etc.

[0325] Isolated populations of APC precursors are cultured and differentiated to obtain immature or mature APCs. Suitable tissue culture media include, but are not limited to, AIM-V®, RPMI 1640, DMEM, X-VIVO, and others. The tissue culture media are typically supplemented with amino acids, vitamins, divalent cations, and cytokines to promote the differentiation of precursors toward the APC phenotype. Typically, the pro-differentiation cytokines are GM-CSF and / or IL-4.

[0326] Furthermore, cultures of APC precursors during enlargement, differentiation, and maturation into the APC phenotype may contain plasma to promote APC development. A typical plasma concentration is approximately 5%. In addition, for example, if the APC precursor is isolated by adhesion to a substrate, CD14 may be present in the early stages of culture. + To enhance the phenotype, the culture medium during the adhesion step may contain plasma. A typical plasma concentration during adhesion is approximately 1% or more.

[0327] Monocytic APC precursors can be cultured for any suitable time. In certain embodiments, the suitable culture time for differentiation from the precursor to immature APCs may be about 1 to 10 days, for example, about 4 to 7 days. Differentiation from the precursor to immature APCs is indicated by cell surface markers (e.g., CD11c + , CD83 low CD86 - / low HLA-DR + Immature APCs can be monitored by methods known to those skilled in the art, such as by the presence or absence of ) ). Immature APCs can also be cultured in appropriate tissue culture media to maintain them in a state for further differentiation or antigen uptake, processing, and presentation. For example, immature APCs can be maintained in the presence of GM-CSF and IL-4.

[0328] In some embodiments, APC precursors can be isolated prior to differentiation. In some embodiments, the isolated population can be enriched or substantially enriched for APC precursors. In some embodiments, APC precursors are isolated by a CD14-specific probe. In one exemplary embodiment, CD14-expressing cells are detected by FACS using a CD14-specific probe directly conjugated to a fluorescent molecule (e.g., FITC or PE), or by an unlabeled antibody specific to CD14 and a second antibody labeled specifically for this first antibody. + Cells are sorted by FACS to determine CD14 low and CD14 -It can also be isolated from cells. CD14 high Gating for positivity can be determined, for example, by referring to CD14 staining in PBMC-derived monocytes. Typically, the CD14-specific conjugate is, for example, an anti-CD14 antibody (e.g., a monoclonal or its antigen-binding fragment). Numerous anti-CD14 antibodies suitable for use in this invention are well known to those skilled in the art, and many of them are commercially available. Differentiation into immature APCs (CD14-negative) can be performed after isolation.

[0329] In another embodiment, a CD14-specific probe is coupled to a substrate, and CD14 + Cells are isolated by affinity selection. CD14 + A population of cells, including CD14 cells, was exposed to a coupled substrate. + The cells are specifically adhered. Next, non-adherent CD14 - The cells are washed away from the substrate, and then adherent cells are eluted to obtain an isolated cell population substantially enriched with the APC precursor. 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 for affinity isolation of cell populations using substrate-coupled antibodies specific to surface markers are generally known.

[0330] During culture, immature APCs can be exposed to a predetermined antigen as needed. A suitable predetermined antigen may include any antigen for which T cell modulation is desired. In one embodiment, immature APCs are cultured in the presence of prostate-specific membrane antigen (PSMA) for cancer immunotherapy and / or tumor growth inhibition. Other antigens may include, for example, bacterial cells, viruses, partially purified or purified bacterial or viral antigens, tumor cells, tumor-specific or tumor-associated antigens (e.g., tumor cell lysates, tumor cell membrane preparations, antigens isolated from tumors, fusion proteins, liposomes, etc.), recombinant cells expressing antigens on their surface, autoantigens, and any other antigens. Any of the antigens may also be presented as peptides or recombinantly produced proteins or portions thereof. After contact with the antigen, the cells can be cultured for any suitable time to allow antigen uptake and processing, thereby increasing the population of antigen-specific APCs, etc.

[0331] For example, in one embodiment, immature APCs can be cultured after antigen uptake to promote maturation from immature APCs to mature APCs that present the antigen in the context of MHC molecules. Methods for APC maturation are known. Such maturation can be carried out by culturing in the presence of known maturation factors, such as cytokines (e.g., TNF-α, IL-1β, or CD40 ligand), bacterial products (e.g., LPS or BCG), and others. Maturation from immature APCs to mature APCs can be monitored by methods known in the art, such as by measuring the presence or absence of cell surface markers (e.g., upregulation of CD83, CD86, and MHC molecules), or by examining the expression of mature APC-specific mRNA or protein using an oligonucleotide array.

[0332] If necessary, immature APCs can be cultured in appropriate tissue culture media to increase the cell population and / or maintain them in a state for further differentiation or antigen uptake. For example, immature APCs can be maintained and / or increased in the presence of GM-CSF and IL-4. Alternatively, immature APCs can be cultured in the presence of anti-inflammatory molecules, such as anti-inflammatory cytokines (e.g., IL-10 and TGF-β), to inhibit their maturation.

[0333] In another embodiment, an isolated population of APCs is enriched for mature APCs. An isolated population of mature APCs can be obtained by culturing a differentiated population of immature APCs in the presence of the aforementioned maturation factors (e.g., bacterial products and / or pro-inflammatory cytokines), thereby inducing maturation. Immature APCs can be isolated by removing CD14+ cells.

[0334] In yet another embodiment of the present invention, APCs can be preserved, for example, by cryopreservation, either 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, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate, and inorganic salts. A controlled, slow cooling rate can be of significant importance. Different cryoprotective agents and different cell types typically have different optimal cooling rates. The heat of the fusion phase, in which water turns to ice, should typically be minimized. For example, the cooling procedure can be performed using a programmable freezing device or a methanol bath procedure. Programmable freezing devices allow for the determination of the optimal cooling rate and facilitate standard, reproducible cooling. Programmable speed-controlled freezers, such as Cryomed or Planar, allow for adjustment of the freezing regime to a desired cooling rate curve.

[0335] After complete freezing, APCs can be rapidly transferred to long-term cryogenic storage containers. In a typical embodiment, the sample can be stored at low temperatures in liquid nitrogen (-196°C) or its vapor (-165°C). Considerations and procedures for handling, cryopreservation, and long-term storage of hematopoietic stem cells, particularly those derived from bone marrow or peripheral blood, are largely applicable to the APCs of the present invention.

[0336] Frozen cells are preferably thawed rapidly (e.g., in a water bath maintained at 37-41°C) and immediately cooled after thawing. It may be desirable to treat the cells to prevent cell clumping after thawing. Various procedures can be used to prevent clumping, including but not limited to the addition of DNAse, low molecular weight dextran and citrate, hydroxyethyl starch, and other pre- and / or post-freezing additives. If cryoprotective agents are toxic in humans, they should be removed prior to the therapeutic use of thawed APCs. One way to remove cryoprotective agents is by dilution to an insignificantly low concentration. Once frozen APCs have been thawed and recovered, they can be used for T cell activation as described herein with respect to unfrozen APCs.

[0337] In one embodiment, the composition for T cell activation includes a population of immune cells depleted of one or more types of immune cells. For example, the composition may include a population of immune cells depleted of one or more types of immune cells expressing one or more proteins, such as one or more cell surface receptors. In some embodiments, the composition includes a population of immune cells derived from a biological sample, including at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigen peptide sequence, and the amount of CD14 and / or CD25-expressing immune cells in the population is relatively different from the amount of CD14 and / or CD25-expressing immune cells in the biological sample. For example, the composition may include a population of immune cells derived from a biological sample, including at least one antigen-specific T cell containing a T cell receptor (TCR) specific to at least one antigen peptide sequence, and the amount of CD14-expressing immune cells in the population is relatively different from the amount of CD14-expressing immune cells in the biological sample. For example, the composition may include a population of immune cells derived from a biological sample, each containing at least one antigen-specific T cell with a T cell receptor (TCR) specific to at least one antigen peptide sequence, and the amount of CD25-expressing immune cells in the population is relatively different from the amount of CD25-expressing immune cells in the biological sample. For example, the composition may include a population of immune cells derived from a biological sample, each containing at least one antigen-specific T cell with a T cell receptor (TCR) specific to at least one antigen peptide sequence, and the amount of CD14 and CD25-expressing immune cells in the population is relatively different from the amount of CD14 and CD25-expressing immune cells in the biological sample. For example, the composition may include a population of immune cells derived from a biological sample, and the amount of CD14 and CD25-expressing immune cells in the population is relatively less than the amount of CD14 and CD25-expressing immune cells in the biological sample.

[0338] A method for preparing a cell composition for cancer immunotherapy, comprising: I. the step of preparing antigen-loaded antigen-presenting cells (APCs), (a) obtaining peripheral blood mononuclear cells (PBMCs) from a subject pretreated with fms-like tyrosine kinase 3 ligand (FLT3L); (b) (i) a plurality of cancer neoantigen peptides, or one or more polynucleotides encoding a plurality of cancer neoantigen peptides, each of which a portion of the cancer neoantigen peptide or 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 obtaining a cell population that promotes cell growth and maintenance ex vivo; and (iv) CD11b low Alternatively, a step comprising ex vivo contact of PBMCs with a drug for reducing or depleting CD11b+ cells from a cell population in order to obtain an APC loaded with a CD11b-depleted antigen; II. CD11b low A method is provided herein that comprises the steps of: III. ex vivo contacting an APC loaded with a CD11b-depleted antigen with isolated T cells; and III. preparing antigen-primed T cells for a cell composition for cancer immunotherapy.

[0339] An improved method for preparing tumor antigen-specific T cells ex vivo, comprising the steps of (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells including antigen-presenting cells (APCs) and T cells, thereby forming a population of immune cells depleted of CD14 and / or CD25, wherein the population of immune cells is derived from a biological sample of a human subject, and (b) subjecting the first population of APCs and T cells from step (a) over a first period of time to (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L) and (ii) at least one tumor antigen expressed by cancer cells of a human subject having cancer. A method is provided herein comprising the steps of: (b) incubating in the presence of a polypeptide containing a proto-epitope sequence or a polynucleotide encoding a polypeptide, thereby forming a population of cells including stimulated T cells; (c) augmenting the stimulated T cells of step (b), thereby forming an augmented population of cells including tumor antigen-specific T cells, wherein the tumor antigen-specific T cells include T cells that are specific to a complex comprising (i) at least one tumor antigen epitope sequence of step (b)(ii) and (ii)(b)(ii) an MHC protein expressed by cancer cells or APCs of a human target; and (c) administering the augmented population of cells of step (c) to a human target, wherein the augmented population of cells of step (c) is 1 × 10⁻¹⁶ 8 ~1 × 10 11 A method comprising steps, including a total number of cells, is provided herein.

[0340] In some embodiments, subjects are pre-treated with FLT3L 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 before PBMC isolation or leukocyte apheresis. In some embodiments, subjects are pre-treated with FLT3L at least approximately 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks before PBMC isolation or leukocyte apheresis.

[0341] In some embodiments, the cell population is enriched with CD11c+ cells. In some embodiments, the antigen-loaded APCs include dendritic cells (DCs). In some embodiments, the antigen-loaded APCs include plasmacytoid dendritic cells (pDCs). In some embodiments, the antigen-loaded APCs include CD1c+DCs. In some embodiments, the antigen-loaded APCs include CD141+DCs. In some embodiments, the cell population includes macrophages. In some embodiments, the method further includes the step of reducing or depleting CD19+ cells from the cell population in order to activate or enrich neoantigen-activated T cells. In some embodiments, the method further includes the step of reducing or depleting both CD11b+ and CD19+ cells from the cell population in order to activate or enrich neoantigen-activated T cells.

[0342] In some embodiments, the method further includes a step of reducing or depleting CD14+ cells from a cell population in order to prepare and concentrate antigen-activated T cells. In some embodiments, the method further includes a step of reducing or depleting CD25+ cells from a cell population in order to prepare and concentrate antigen-activated T cells. In some embodiments, the method further includes a step of reducing or depleting one or more of CD19+, CD14+, CD25+, or CD11b+ cells from a cell population in order to activate or concentrate neoantigen-activated T cells.

[0343] In some embodiments, the stimulant for activating cells includes FL3TL.

[0344] In some embodiments, agents that promote cell growth and maintenance ex vivo include growth factors, cytokines, amino acids, supplements, or combinations thereof.

[0345] In some embodiments, an antigen-loaded APC can stimulate T cells for 2, 3, 4, 5, 6, or 7 days.

[0346] In some embodiments, each of the multiple cancer neoantigen peptides is 8 to 30 amino acids long.

[0347] In some embodiments, each of the multiple neoantigenic peptides contains a neoantigenic epitope. In some embodiments, the multiple cancer neoantigenic peptides comprise 2, 3, 4, 5, 6, 7, or 8 neoantigenic peptides; each of the multiple neoantigenic peptides has the neoantigenic peptide characteristics described in a previous section.

[0348] In some embodiments, the neoantigenic peptide used in the preparation of antigen-loaded APCs is a long peptide containing at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, or any number in between. In some embodiments, the neoantigenic peptide used in the preparation of antigen-loaded APCs contains amino acids adjacent to either side of the mutation to facilitate endogenous processing of the neoantigenic peptide for increased presentation rate to T cells.

[0349] Longer immunogenic peptides can be designed in several ways. In some embodiments, if the HLA-binding peptide is predicted or known, the longer immunogenic peptide may consist of (1) individual binding peptides having 2-5 amino acid extensions toward the N and C-terminuses of each corresponding gene product; or (2) concatenations of some or all of the binding peptides having extension sequences toward each. In other embodiments, if sequencing reveals a long (>10 residue) epitope sequence present in the tumor, e.g., a neoepitope (e.g., by frameshift, read-through, or intron inclusion resulting in a novel peptide sequence), the longer neoantigen peptide may consist of the entire stretch of novel tumor-specific amino acids, either as a single longer peptide or several overlapping longer peptides. In some embodiments, the use of longer peptides is presumed to enable endogenous processing by patient cells, which may result in more effective antigen presentation and induction of T cell responses. In some embodiments, two or more peptides may be used, in which case these peptides overlap and tile across the longer neoantigen peptide.

[0350] In some embodiments, each of the multiple neoantigenic peptides contains the same neoantigenic epitope. In some embodiments, the multiple neoantigenic peptides contain two or more neoantigenic epitopes.

[0351] In some embodiments, one or more polynucleotides encoding multiple cancer neoantigen peptides are DNA.

[0352] In some embodiments, one or more polynucleotides encoding multiple cancer neoantigen peptides are inserted into one or more mammalian expression vectors.

[0353] In some embodiments, one or more polynucleotides encoding multiple cancer neoantigen peptides are messenger RNAs.

[0354] In some embodiments, the present invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules containing modified nucleosides.

[0355] In some embodiments, the present invention provides gene therapy vectors comprising RNA, oligoribonucleotides, and polyribonucleotides.

[0356] In some embodiments, the present invention provides gene therapy methods and gene transcription silencing methods, including those described above.

[0357] In some embodiments, the polynucleotide encodes a single neoantigenic peptide.

[0358] In some embodiments, one polynucleotide encodes two or more neoantigenic peptides.

[0359] In some embodiments, the polynucleotide is a messenger RNA. In some embodiments, each messenger RNA contains in tandem coding sequences of two or more neoantigenic peptides.

[0360] In some embodiments, each messenger RNA contains in tandem coding sequences for 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more neoantigenic peptides. Typically, mRNA contains a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In some embodiments, mRNA is self-amplifying mRNA. In the context of the present invention, mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methodologies are known to those skilled in the art. For example, various commercially available in vitro... In vitro transfer kits are available.

[0361] The stability and translation efficiency of RNA can be modified. For example, RNA can be stabilized and its translation increased by one or more modifications that have a stabilizing effect on RNA and / or increase its translation efficiency. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. In order to increase the expression of RNA used in accordance with the present invention, RNA can be modified within its coding region, i.e., the sequence encoding the expressed peptide or protein, so as to increase its GC content and thereby increase mRNA stability and perform codon optimization without altering the sequence of the expressed peptide or protein, thereby enhancing translation in cells.

[0362] In some embodiments, the mRNA may contain multiple neoantigenic epitopes. In some embodiments, a long polyribonucleotide sequence capable of encoding a neo-ORF, such as a mutated GATA3 sequence encoding a neo-ORF, may be used. In some embodiments, the mRNA of a large portion of a gene containing a sequence encoding a neoantigenic peptide, or even the entire coding region thereof, is delivered to immune cells for endogenous processing and presentation of the antigen.

[0363] In some embodiments, the coding sequence for each neoantigenic peptide is 24 to 120 nucleotides long.

[0364] In some embodiments, the mRNA is 50 to 10,000 nucleotides long. In some embodiments, the mRNA is 100 to 10,000 nucleotides long. In some embodiments, the mRNA is 200 to 10,000 nucleotides long. In some embodiments, the mRNA is 50 to 5,000 nucleotides long. In some embodiments, the mRNA is 100 to 5,000 nucleotides long. In some embodiments, the mRNA is 100 to 1,000 nucleotides long. In some embodiments, the mRNA is 300 to 800 nucleotides long. In some embodiments, the mRNA is 400 to 700 nucleotides long. In some embodiments, the mRNA is 450 to 600 nucleotides long. In some embodiments, the mRNA is at least 200 nucleotides long. In some embodiments, the mRNA has more than 250 nucleotides, more than 300 nucleotides, more than 350 nucleotides, more than 400 nucleotides, more than 450 nucleotides, more than 500 nucleotides, more than 550 nucleotides, more than 600 nucleotides, more than 650 nucleotides, more than 700 nucleotides, more than 750 nucleotides, more than 800 nucleotides, more than 850 nucleotide lengths, more than 900 nucleotide lengths, more than 950 nucleotide lengths, more than 1000 nucleotide lengths, more than 2000 nucleotide lengths, more than 3000 nucleotide lengths, more than 4000 nucleotide lengths, or more than 5000 nucleotide lengths.

[0365] In some embodiments, mRNA encoding one or more neoantigenic peptides is modified, and the modification relates to the 5'-UTR. In some embodiments, the modification relates to providing RNA having a 5'-cap or a 5'-cap analogue to the 5'-UTR. The term “5'-cap” refers to a cap structure found at the 5' end of an mRNA molecule, and generally consists of a guanosine nucleotide attached to mRNA via an unusual 5'-to-5' triphosphate linkage. In some embodiments, this guanosine is methylated at position 7. The term “conventional 5'-cap” refers to the naturally occurring RNA 5'-cap, 7-methylguanosine cap (mG). In the context of the present invention, the term “5'-cap” includes 5'-cap analogues that resemble the RNA cap structure and are modified to possess the ability to stabilize RNA and / or enhance RNA translation when attached to RNA in vivo and / or in cells. In some embodiments, mRNA is capped concurrently via transcription.

[0366] 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 long. 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.

[0367] In some embodiments, the poly-A tail contains 100 to 250 adenosine units. In some embodiments, the poly-A tail contains 120 to 130 adenine units. In some embodiments, the poly-A tail contains 120 adenine units. In some embodiments, the poly-A tail contains 121 adenine units. In some embodiments, the poly-A tail contains 122 adenine units. In some embodiments, the poly-A tail contains 123 adenine units. In some embodiments, the poly-A tail contains 124 adenine units. In some embodiments, the poly-A tail contains 125 adenine units. In some embodiments, the poly-A tail is 129 bases.

[0368] In some embodiments, the coding sequences of two consecutive neoantigenic peptides are separated by a spacer or linker.

[0369] 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 is a lysine-encoding sequence such as AAA or AAG. Another exemplary spacer sequence is CAACTGGGATTG.

[0370] In some embodiments, the mRNA includes one or more additional structures to enhance antigen epitope processing and presentation by the APC.

[0371] In some embodiments, the linker or spacer region may contain a cleavage site. The cleavage site ensures that the protein product containing the epitope sequence string is cleaved into separate epitope sequences for presentation. To avoid accidental cleavage of epitopes within the sequence, preferred cleavage sites are placed adjacent to certain epitopes. In some embodiments, the design of epitopes and cleavage regions in the mRNA encoding the epitope string is non-random.

[0372] In certain embodiments, the mRNA encoding the neoantigen peptide of the present invention is administered to a subject requiring it. In some embodiments, the mRNA to be administered contains at least one modified nucleoside phosphate.

[0373] In some embodiments, T cells are activated by neoantigenic peptides from artificial antigen-presenting cells. In some embodiments, an artificial scaffold is used to activate T cells with neoantigenic peptides, loading the artificial scaffold with neoantigenic peptides coupled to MHC antigens that can bind the neoantigenic peptides with high affinity.

[0374] In some embodiments, the additional structure includes a structure that encodes a specific domain derived from a protein selected from the group consisting of MITD, SP1, and the 10th fibronectin domain: 10FnIII.

[0375] In some embodiments, antigen-loaded APCs are prepared by contacting cells derived from peripheral blood or leukocyte apheresis with multiple cancer neoantigen peptides, or one or more polynucleotides encoding multiple cancer neoantigen peptides, one or more times.

[0376] In some embodiments, the method includes the step of incubating one or more APCs or APC preparations with a first medium containing at least one cytokine or growth factor for a first period of time.

[0377] In some embodiments, the method includes the step of incubating one or more APC preparations together with at least one peptide for a second period of time.

[0378] In some embodiments, the enriched cells further comprise CD1c+ cells.

[0379] In some embodiments, the cell population is enriched for CD11c+ and CD141+ cells.

[0380] In some embodiments, the cell population containing antigen-loaded APCs includes 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 more than 95% CD11c+ cells.

[0381] In some embodiments, the cell population containing antigen-loaded APCs includes 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, and less than 4% CD11b+ expressing cells.

[0382] In some embodiments, the cell population containing antigen-loaded APCs includes 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 more than 95% CD11c+ neoantigenic peptide-expressing cells.

[0383] In some embodiments, the cell population containing antigen-loaded APCs includes neoantigenic peptide-expressing cells that are CD11c+CD1c+ or CD141+ cells, comprising 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 more than 95%.

[0384] In some embodiments, the neoantigen-loaded APC includes a mature APC.

[0385] In some embodiments, the method includes the step of obtaining a biological sample derived from the subject, which contains at least one APC and at least one PBMC or at least one (on)T cell.

[0386] In some embodiments, the method includes the step of depleting cells expressing CD14 and / or CD25 and / or CD19 from a biological sample, thereby obtaining a CD14 and / or CD25 and / or CD19 cell-depleted sample.

[0387] In some embodiments, the method includes the step of incubating a CD14 and / or CD25 and / or CD19 cell-depleted sample with FLT3L for a first period of time.

[0388] In some embodiments, the method includes the step of incubating at least one peptide with a CD14 and / or CD25 and / or CD19 cell-depleted sample for a second period, thereby obtaining a first mature APC peptide-loaded sample. Preparation of neoantigen-activated T cells using neoantigen-loaded APCs.

[0389] In some embodiments, APCs loaded with neoantigens prepared by the method described above are incubated with T cells to obtain antigen-activated T cells. This method may include a step of generating at least one antigen-specific T cell, where the antigen is a neoantigen. In some embodiments, the step of generating at least one antigen-specific T cell includes a step of generating multiple antigen-specific T cells.

[0390] In some embodiments, T cells are obtained from a biological sample derived from the subject.

[0391] In some embodiments, T cells are obtained from a biological sample derived from the same subject from which the APCs originate. In some embodiments, T cells are obtained from a biological sample derived from a different subject from which the APCs originate.

[0392] In some embodiments, APCs and / or T cells are derived from a biological sample that is a peripheral blood mononuclear cell (PBMC). In some embodiments, APCs and / or T cells are derived from a biological sample that is a leukocyte apheresis sample.

[0393] In some embodiments, the APC includes dendritic cells (DCs).

[0394] In some embodiments, the APC is derived from CD14+ monocytes, or is CD14-enriched APC, or is CD141-enriched APC.

[0395] In some embodiments, CD14+ monocytes are enriched from a biological sample derived from the subject, including peripheral blood mononuclear cells (PBMCs).

[0396] In some embodiments, APC is PBMC. In some embodiments, PBMC is newly isolated PBMC. In some embodiments, PBMC is frozen PBMC. In some embodiments, PBMC is autologous PBMC isolated from the subject or patient.

[0397] In some embodiments, PBMCs are loaded with antigens, which may be peptides or polypeptides, or polynucleotides such as mRNA encoding peptides and polypeptides. PBMCs (monocytes, DCs, phagocytic cells) can take up and process antigens via phagocytosis and present them on their surface for T cell activation. Peptides or polypeptides loaded into PBMCs can 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, for example, RNA can act as a self-adjuvant, so mRNA antigen loading does not require adjuvant supplementation.

[0398] In some embodiments, PBMCs are directly isolated or thawed from frozen samples and incubated with a neoantigen, a composition containing a neoantigen, or one or more antigens such as one or more nucleic acids or polynucleotides encoding one or more antigens. In some embodiments, PBMC samples are not further cultured for differentiation of one or more cellular components within the PBMCs or subjected to further maturation (e.g., maturation of antigen-presenting cells, or differentiation from monocytes to dendritic cells) before the PBMCs are exposed to one or more antigens or nucleic acids encoding one or more antigens. In some embodiments, one or more cell types are depleted or removed from the newly isolated or newly thawed PBMC population before the cells are exposed to or incubated with one or more antigens or nucleic acids encoding one or more antigens. In some embodiments, CD14+ cells are depleted from the PBMCs. In some embodiments, CD25+ cells are depleted from the PBMCs. In some embodiments, CD11b+ cells are depleted from the PBMCs. In some embodiments, CD14+ and CD25+ cells are depeated from PBMCs before incubation with one or more antigens or one or more nucleic acids encoding one or more antigens. In some embodiments, CD11b+ and / or CD14+ and / or CD25+ cells are depeated from PBMCs. In some embodiments, the method provided herein includes the step of preparing tumor antigen-specific T cells by depleting CD14+ and / or CD25+ cells from a human subject-derived PBMC sample containing a percentage of immature dendritic cells (DCs) approximately the same as the percentage of immature DCs in the peripheral blood of a human subject. In some embodiments, the method provided herein includes the step of preparing tumor antigen-specific T cells by depleting CD14+ and / or CD25+ cells from a human subject-derived PBMC sample containing a percentage of mature DCs approximately the same as the percentage of mature DCs in the peripheral blood of a human subject.In some embodiments, the method provided herein includes the step of preparing tumor antigen-specific T cells by depleting CD14+ cells and / or CD25+ cells from a human subject-derived PBMC sample containing a ratio of immature DCs to mature DCs that is approximately the same as the ratio of immature DCs to mature DCs in the peripheral blood of a human subject. In some embodiments, the method provided herein includes the step of preparing tumor antigen-specific T cells by depleting CD14+ cells and / or CD25+ cells from a human subject-derived PBMC sample that has not been subjected to the step of maturing immature DCs into mature DCs.

[0399] In some embodiments, CD14+ monocytes are stimulated with one or more cytokines or growth factors.

[0400] 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, IL-15, IFN-γ, IFN-α, R848, LPS, ss-rna40, poly(I:C) or a combination thereof.

[0401] In some embodiments, CD14+ monocytes are derived from a second biological sample containing PBMCs.

[0402] In some embodiments, the second biological sample originates from the same subject.

[0403] In some embodiments, the biological sample includes peripheral blood mononuclear cells (PBMCs).

[0404] In some embodiments, at least one antigen-specific T cell is stimulated in a medium containing IL-7, IL-15, an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof.

[0405] In some embodiments, the IDO inhibitor is epacadostat, navoximod, 1-methyltryptophan, or a combination thereof.

[0406] In some embodiments, subjects are administered FLT3L prior to the step of obtaining a biological sample for preparing APCs and / or T cells.

[0407] In some embodiments, T cells are obtained from biological samples derived from subjects described in a previous section of this disclosure.

[0408] In some embodiments, the biological sample is a sample newly obtained from the subject or a frozen sample.

[0409] In some embodiments, the incubation step is performed 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, poly(I:C) or any combination thereof.

[0410] In some embodiments, the method includes the step of stimulating T cells with IL-7, IL-15, or a combination thereof. In some embodiments, the method includes the step of stimulating T cells with IL-7, IL-15, or a combination thereof in the presence of an IDO inhibitor, a PD-1 antibody, or IL-12. In some embodiments, the stimulated T cells are enlarged ex vivo under suitable T cell growth conditions in the presence of FLT3L, with one or more cytokines or growth factors including GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, R848, LPS, ss-rna40, poly(I:C), or a combination thereof. In some embodiments, the method further includes the step of administering antigen-specific T cells.

[0411] In some embodiments, the method includes the step of incubating APCs prepared as described in a previous section together with T cells in the presence of a medium containing at least one cytokine or growth factor to generate neoantigen-activated T cells.

[0412] In some embodiments, the incubation step includes incubating a first APC preparation with T cells for more than 7 days. In some embodiments, the incubated T cells are stimulated T cells that grow in vitro for more than 7 days in the presence of the APC preparation, cytokines, and growth factors.

[0413] In some embodiments, the incubation step includes incubating the first APC preparation with T cells for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or longer.

[0414] In some embodiments, the first period of one or more periods is approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 days.

[0415] In some embodiments, the total duration of the separate periods is less than 28 days. In some embodiments, the total duration of the separate periods is between 20 and 27 days. In some embodiments, the total duration of the separate periods is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 days.

[0416] In some embodiments, the method includes the step of incubating a first APC preparation with T cells for more than 7 days. In some embodiments, the method includes the step of incubating a first APC preparation 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 the step of incubating a first APC preparation with T cells for 7–20, 8–20, 9–20, 10–20, 11–20, or 12–20 days. In some embodiments, the method includes the step of incubating a first APC preparation with T cells for about 10–15 days.

[0417] In some embodiments, the method includes the step of incubating a second APC preparation with T cells for 5 to 9 days. In some embodiments, the method includes the step of incubating a second APC preparation with T cells for 5, 6, 7, 8, or 9 days. In some embodiments, the method further includes the step of removing one or more cytokines or growth factors from the second medium after the third period and before the start of the fourth period.

[0418] In some embodiments, the method includes the step of incubating a third APC preparation with T cells for 5 to 9 days. In some embodiments, the method includes the step of incubating a third APC preparation with T cells for 5, 6, 7, 8, or 9 days.

[0419] In some embodiments, the method includes the steps of: incubating a first APC preparation of the APC preparation 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 preparation of the APC preparation 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 preparation of the APC preparation 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.

[0420] In some embodiments, the method is performed ex vivo. In some embodiments, T cells are cultured in a medium containing cytokines. In some embodiments, an example of cytokines includes IL-7. In some embodiments, an example of cytokines includes IL-15. In some embodiments, an example of cytokines includes 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 cytokines in the T cell culture or medium have a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the IL-7 in the T cell culture or medium has a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the IL-15 in the T cell culture or medium has a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the T cells are cultured in a medium further containing FLT3L.In some embodiments, the FLT3L in the T cell culture or medium has a final concentration of at least 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, or 200 ng / mL. In some embodiments, T cells are incubated, induced, or stimulated in a medium containing FLT3L for a first period of time. In some embodiments, T cells are incubated, induced, or stimulated in a medium containing additionally added FLT3L for a second period of time. In some embodiments, T cells are incubated, induced, or stimulated in a medium containing additionally added FLT3L for a third period of time. In some embodiments, T cells are incubated, induced, or stimulated over a fourth, fifth, or sixth period in a medium containing additionally added FLT3L, with newly added FLT3L during each period.

[0421] In some embodiments, T cells are cultured in the presence of neoantigens, such as neoantigens presented by APCs, and the culture medium is high in potassium [K]. + Contents include. In some embodiments, T cells have high [K] in the culture medium for at least a certain period during incubation with APC or T cells. + The culture is performed in the presence of [K] in the culture medium. In some embodiments, [K] + The content is altered over at least a certain period during incubation with APCs or T cells. In some embodiments, the content in the medium is kept constant throughout the period of T cell ex vivo culture. In some embodiments, the [K] content in the T cell culture medium is altered. + The content is ≥5 mM. In some embodiments, [K] in T cell culture medium + The content is ≥6 mM. In some embodiments, [K] in T cell culture medium+ The content is ≧ 7 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 8 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 9 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 10 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 11 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 12 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 13 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 14 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 15 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 16 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 17 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 18 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 19 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 20 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 22 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 25 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 30 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 35 mM. In some embodiments, [K] in the T cell culture medium + The content is ≧ 40 mM. In some embodiments, [K] in the T cell culture medium + The content is about 40 mM.

[0422] In some embodiments, [K] in T cell culture medium + The content is about 40 mM for at least a certain period during incubation of the neoantigen with T cells. In some embodiments, the neoantigen may be presented by an APC loaded with the neoantigen. In some embodiments, the T cells are [K] + In the presence of [K], T effector function, CD8+ cytotoxicity, cytokine production, and memory phenotype are examined. In some embodiments, high [K] + T cells grown in the presence of [K] express the effector T cell phenotype. In some embodiments, high [K] + T cells grown in the presence of [K] express memory cell markers. In some embodiments, high [K] + T cells grown in the presence of [specific substance] do not express T cell depletion markers.

[0423] In some embodiments, stimulated T cells are a population of immune cells, including activated T cells stimulated by an APC containing a neoantigenic peptide-MHC complex. In some embodiments, the method may include the steps of: incubating a population of immune cells derived 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 cellular markers of at least one immune cell in the stimulated immune cell sample; and determining the binding of at least one immune cell in the stimulated immune cell sample to the peptide-MHC complex, wherein the steps of determining the expression of a specific cell surface marker or other determinant marker, such as an intracellular factor or a released drug, such as a cytokine, and determining binding to the neoantigen-MHC complex are performed simultaneously. In some embodiments, one or more cell markers include TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, granzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3, or any combination thereof. In some embodiments, one or more cell markers include cytokines. In some embodiments, one or more cell markers include degranulation markers. In some embodiments, one or more cell markers include cell surface markers. In some embodiments, one or more cell markers include proteins. In some embodiments, the step of determining the binding of at least one immune cell of a stimulated immune cell sample to a peptide-MHC complex includes the binding of at least one immune cell of a stimulated immune cell sample to an MHC tetramer containing the peptide and MHC of the peptide-MHC complex. In some embodiments, the MHC is class I MHC or class II MHC. In some embodiments, the peptide-MHC complex includes one or more labels.

[0424] In some embodiments, T cell activation is verified by detecting cytokine release by activated T cells. In some embodiments, the cytokines are one or more of TNF-α, IFN-γ, or IL-2. In some embodiments, T cell activation is verified by its specific antigen binding and cytokine release. In some embodiments, T cell activation is verified by its ability to kill tumor cells in vitro. The activation state of T cells can be verified using a sample of activated T cells. In some embodiments, a T cell-derived sample is isolated from a T cell culture, and its cell composition and activation state are determined by flow cytometry.

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

[0426] 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 at least one antigen-specific CD8+ T cells in the composition is about 5%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 7%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 10%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 12%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 15%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 20%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 25%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 30%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 40%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 50%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 60%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is approximately 70% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0427] In some embodiments, the percentage of at least one antigen-specific CD4+ T cells in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

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

[0429] In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in a biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.05%, 0.1%, or 0.5% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0430] In some embodiments, the percentage of at least one antigen-specific CD4+ T cells in a biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.05%, 0.1%, or 0.5% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0431] In some embodiments, the antigen is a neoantigen, tumor-associated antigen, overexpressed antigen, viral antigen, minor histocompatibility antigen, or a combination thereof.

[0432] In some embodiments, the number of at least one antigen-specific CD8+ T cells in the composition is at least about 1 × 10^6, 2 × 10^6, 5 × 10^6, 1 × 10^7, 2 × 10^7, 5 × 10^7, 1 × 10^8, 2 × 10^8, or 5 × 10^8 antigen-specific CD8+ T cells. In some embodiments, the number of at least one antigen-specific CD4+ T cells in the composition is at least about 1 × 10^6, 2 × 10^6, 5 × 10^6, 1 × 10^7, 2 × 10^7, 5 × 10^7, 1 × 10^8, 2 × 10^8, or 5 × 10^8 antigen-specific CD4+ T cells. Pharmaceutical composition

[0433] A composition comprising a population of immune cells (e.g., a pharmaceutical composition) is provided herein. The composition may include at least one antigen-specific T cell comprising a T cell receptor (TCR). The composition may include at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence.

[0434] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, including excipients and auxiliary agents that facilitate the processing of the active agent into preparations that can be used as pharmaceuticals. The appropriate formulation may depend on the chosen route of administration. Any of the well-known techniques, carriers, and excipients can be used as appropriate and as understood in the art. In some cases, the pharmaceutical composition is formulated as a cell-based therapeutic agent, such as a T-cell therapeutic agent. In some embodiments, the pharmaceutical composition includes a peptide-based therapeutic agent, a nucleic acid-based therapeutic agent, an antibody-based therapeutic agent, and / or a cell-based therapeutic agent. In some embodiments, the pharmaceutical composition includes a peptide-based therapeutic agent or a nucleic acid-based therapeutic agent encoding a polypeptide. In some embodiments, the pharmaceutical composition includes a peptide-based therapeutic agent or a nucleic acid-based therapeutic agent encoding a polypeptide, and the peptide-based therapeutic agent or nucleic acid-based therapeutic agent is contained in cells, and the cells are T cells. In some embodiments, the pharmaceutical composition includes an antibody-based therapeutic agent. The composition may contain T cells specific to two or more immunogenic antigens or neoantigen peptides.

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

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

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

[0438] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; serum albumin This includes proteins such as cellulose, 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 TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

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

[0440] In one embodiment, pharmaceutically acceptable or physiologically acceptable compositions comprising solvents (aqueous or nonaqueous), solutions, emulsions, dispersions, coatings, isotonic and absorption enhancers or retarders, which are compatible with drug administration, are provided herein. Thus, a pharmaceutical composition or pharmaceutical formulation refers to a composition suitable for drug use in a subject. The composition can be formulated to be compatible with a specific route of administration (i.e., systemic or topical). Thus, the composition comprises carriers, diluents or excipients suitable for administration by various routes.

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

[0442] Pharmaceutical compositions can be administered, for example, by injection. Compositions for injection include aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride may be included in the composition. The resulting solution may be packaged for use as is or lyophilized; lyophilized preparations can later be combined with a sterile solution prior to administration. For intravenous or site-administered injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogenically free and has a suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare a suitable solution using, for example, an isotonic medium such as sodium chloride injection, Ringer's injection, or Ringer's lactate injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. A sterile injection solution can be prepared, as required, by incorporating the active ingredient in the required amount into a suitable solvent, along with one or a combination of the components listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile medium containing a base dispersion medium and other required components derived from the components listed above. For sterile powders for the preparation of sterile injection solutions, preferred preparation methods may be vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired components from a previously sterile filtered solution.

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

[0444] When a composition is considered for use in a pharmaceutical or by any method provided herein, it is intended that the composition be substantially free of pyrogens so as not to cause an inflammatory or dangerous allergic reaction when administered to a human patient. Testing a composition for pyrogens and preparing a composition substantially free of pyrogens are well understood by one or ordinary skill of the art and can be achieved using commercially available kits.

[0445] 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. Drugs that delay absorption include, for example, aluminum monostearate and gelatin. Detergents containing liposome carriers can also be used to stabilize, increase, or decrease the absorption of pharmaceutical compositions. To protect against digestion, compounds can be complexed with the composition to make it resistant to acidic and enzymatic hydrolysis, or compounds can be complexed in a suitably 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 No. 5,391,377).

[0446] The composition can be administered in a therapeutically effective dose in a manner compatible with the drug formulation. The amount to be administered depends on the target being treated, the capacity of the target's immune system to utilize the active ingredient, and the desired degree of binding affinity. The precise amount of active ingredient required to be administered is at the physician's discretion and is specific to each individual. Suitable regimens for initial administration and booster shots are also variable but are typically represented by repeated doses at one or more time intervals following the initial dose, or by subsequent injections or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain blood concentration may be intended.

[0447] In some embodiments, the present invention relates to immunogenic compositions, such as pharmaceutical compositions, that can induce a neoantigen-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic composition includes neoantigen therapeutic agents described herein (e.g., peptides, polynucleotides, TCRs, CARs, cells containing TCRs or CARs, dendritic cells containing polypeptides, dendritic cells containing polynucleotides, antibodies, etc.) that correspond to tumor-specific antigens or neoantigens.

[0448] In some embodiments, the pharmaceutical compositions described herein can induce a specific cytotoxic T cell response, a specific helper T cell response, or a B cell response.

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

[0450] In some embodiments, CTLs are injected into the patient. In some embodiments, HTLs are injected into the patient. In some embodiments, both CTLs and HTLs are injected into the patient. The administration of either therapeutic agent can be carried out simultaneously, sequentially, or in any order.

[0451] In some embodiments, the pharmaceutical compositions described herein for therapeutic treatment (e.g., immunogenic compositions) can be formulated for parenteral, topical, nasal, oral, or topical administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the compositions can be administered intratumorally. The compositions can be administered to the site of surgical excision to induce a local immune response against the tumor. In some embodiments, compositions for parenteral administration comprising a solution of neoantigen peptide are described herein, and the immunogenic composition is dissolved or suspended in an acceptable carrier, such as an aqueous carrier. Various aqueous carriers can be used, such as water, buffer water, 0.9% saline, 0.3% glycine, hyaluronic acid, and others. These compositions can be sterilized by conventional well-known sterilization techniques or by sterile filtration. The resulting aqueous solutions can be packaged for use as is or lyophilized, and the lyophilized preparations are combined with a sterile solution prior to administration. The composition may contain pH adjusters and buffers, osmotic pressure adjusters, wetting agents, and other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0452] The ability of adjuvants to enhance the immune response to an antigen is typically manifested by a significant increase in immune-mediated responses or a reduction in disease symptoms. For example, increased humoral immunity may manifest as a significant increase in the titer of antibodies produced against the antigen, and increased T cell activity may manifest as increased cell proliferation or cellular cytotoxicity or cytokine secretion. Adjuvants can also alter the immune response, for example, by shifting a primarily humoral or T helper 2 response to a primarily cellular or T helper 1 response.

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

[0454] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in therapeutic settings. While not theoretically constrained, CpG oligonucleotides act by activating the innate (maladaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, living or dead viruses, dendritic cell immunogenic compositions, autologous cell immunogenic compositions, and polysaccharide conjugates, in both prophylactic and therapeutic immunogenic pharmaceutical compositions. Importantly, this is CD4 +Even without the assistance of T cells, CpG enhances dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and strong cytotoxic T-lymphocyte (CTL) generation. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of adjuvants such as alum or Freund's incomplete adjuvants (IFAs), which normally promote TH2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar preparations, which are particularly useful for inducing strong responses when antigens are relatively weak. This can also accelerate the immune response, and in several experiments, it has been possible to reduce antigen doses with antibody responses comparable to full-dose immunogenic drug compositions without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484). U.S. Patent No. 6,406,705 describes the use of a combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce an antigen-specific immune response. A commercially available CpG TLR9 antagonist is a dSLIM (double stem-loop immunomodulator) by Mologen (Berlin, DE), which is an ingredient in the pharmaceutical compositions described herein. Other TLR-binding molecules, such as RNAs that bind to TLR7, TLR8, and / or TLR9, may also be used.

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

[0456] In some embodiments, the immunogenic composition according to this disclosure may contain two or more different adjuvants. Furthermore, the present invention encompasses pharmaceutical compositions comprising any adjuvant substance including any of the above or any combination thereof. In some embodiments, the immunogenic composition comprises a neoantigen therapeutic agent (e.g., peptides, polynucleotides, TCRs, CARs, cells containing TCRs or CARs, dendritic cells containing polypeptides, dendritic cells containing polynucleotides, antibodies, etc.), and the adjuvants may be administered separately in any appropriate order.

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

[0458] Fatty acids for generating lipid-modified peptides may include C2-C30 saturated, monounsaturated, or polyunsaturated fatty acid acyl groups. Exemplary fatty acids may include palmitoyl, myristoyl, stearoyl, and decanoyl groups. In some examples, a lipid moiety having adjuvant properties is attached to the polypeptide of interest to induce or enhance immunogenicity in the absence of an exogenous adjuvant. Lipid-modified peptides or lipopeptides may be referred to as self-adjuvant lipopeptides. Any of the fatty acids described above and elsewhere in this specification may induce or enhance the immunogenicity of the polypeptide of interest. Fatty acids that can induce or enhance immunogenicity may include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, and decanoyl groups.

[0459] Polypeptides, such as naked peptides or lipid-conjugated peptides, can be incorporated into liposomes. In some cases, lipid-conjugated peptides can be incorporated into liposomes. For example, the lipid portion of a lipid-conjugated peptide can spontaneously integrate into the lipid bilayer of the liposome. Therefore, lipopeptides can be presented on the "surface" of liposomes. Exemplary liposomes suitable for incorporation into formulations include, but are not limited to, multilayer vesicles (MLVs), oligolamellar vesicles (OLVs), monolayer vesicles (UVs), small monolayer vesicles (SUVs), intermediate-sized monolayer vesicles (MUVs), large monolayer vesicles (LUVs), giant monolayer vesicles (GUVs), multilayer vesicles (MVVs), single or oligolayer vesicles (REVs) prepared by reverse-phase evaporation, multilayer vesicles (MLV-REVs) prepared by reverse-phase evaporation, stable plurilamellar vesicles (SPLVs), freeze-thawed MLVs (FATMLVs), vesicles prepared by extrusion (VETs), vesicles prepared by French press (FPVs), vesicles prepared by fusion (FUVs), dehydration-rehydration vesicles (DRVs), and bubblesomes (BSVs).

[0460] Depending on the preparation method, liposomes may be monolayered or multilayered, and their size can vary, with diameters ranging from approximately 0.02 μM to over 10 μm. Liposomes can adsorb to many types of cells and release ingested drugs (e.g., peptides described herein). In some cases, liposomes fuse with target cells, thereby releasing the contents of the liposome into the target cells. Liposomes can be endocytized by phagocytic cells. Following endocytosis, intralysosomal degradation of liposomal lipids and release of encapsulated drugs may occur.

[0461] The liposomes provided herein may include carrier lipids. In some embodiments, the carrier lipid is a phospholipid. Carrier lipids that can form 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 distearoyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidyl glycerol (DPPG), distearoyl phosphatidylglycerol (DSPG), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidic acid (DPPA); dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidylserine (DPPS), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidylserine (DSPS), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), and others or combinations thereof. In some embodiments, the liposomes further include sterols (e.g., cholesterol) that modulate liposome formation. The carrier lipid may be any known non-phosphorylated polar lipid.

[0462] The pharmaceutical composition can be encapsulated in liposomes using known techniques. Biodegradable microspheres can also be used as carriers for the pharmaceutical composition of the present invention.

[0463] Pharmaceutical compositions can be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to patients are well known to those skilled in the art. Basically, the material is dissolved in an aqueous solution, appropriate phospholipids and lipids are added, along with surfactants if required, and the material is dialyzed or sonicated as needed.

[0464] Microspheres formed from polymers or proteins are well known to those skilled in the art and can be individually prepared for passage through the gastrointestinal tract and direct entry into the bloodstream. Alternatively, compounds can be incorporated, and microspheres or complexes of microspheres can be implanted for slow release over periods ranging from several days to several months.

[0465] Immunogenic pharmaceutical compositions based on cells can also be administered to subjects. For example, immunogenic pharmaceutical compositions based on antigen-presenting cells (APCs) can be formulated using any of the well-known techniques, carriers, and excipients as appropriate and as understood in the art. APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells. In some cases, an immunogenic pharmaceutical composition based on APCs may be an immunogenic pharmaceutical composition based on dendritic cells.

[0466] Immunogenic drug compositions based on dendritic cells can be prepared by any method known in the art. In some cases, immunogenic drug compositions based on dendritic cells are ex The preparation can be carried out by vivo or in vivo methods. Ex vivo methods may include the use of autologous DCs pulsed ex vivo with the polypeptides described herein to activate or load the DCs prior to administration to a patient. In vivo methods may include the step of targeting a specific DC receptor using an antibody coupled with the polypeptides described herein. The DC-based immunogenic pharmaceutical composition may further include DC activators such as TLR3, TLR-7-8, and CD40 agonists. The DC-based immunogenic pharmaceutical composition may further include adjuvants and pharmaceutically acceptable carriers.

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

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

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

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

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

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

[0473] Immunogenic pharmaceutical compositions may contain preservatives such as thiomersal or 2-phenoxyethanol. In some cases, immunogenic pharmaceutical compositions are substantially mercury-free (e.g., <10 μg / mL), for example, thiomersal-free. Alpha-tocopherol succinate can be used as a substitute for mercury compounds.

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

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

[0476] Immunogenic pharmaceutical compositions may contain one or more buffers, such as Tris buffers; borate buffers; succinate buffers; histidine buffers (especially with aluminum hydroxide adjuvants); or citrate buffers. In some cases, the buffers are present in concentrations ranging from 5 to 20 or 10 to 50 mM.

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

[0478] The immunogenic pharmaceutical composition may be sterilized. The immunogenic pharmaceutical composition may be pyrogenically free, for example, containing <1 EU (endotoxin units, standard scale) per dose, or <0.1 EU per dose. The composition may be gluten-free.

[0479] The immunogenic pharmaceutical composition may contain detergents, such as polyoxyethylene sorbitan ester surfactants (known as "Tween®" products) or octoxynol (e.g., octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol). Detergents may be present in trace amounts. The immunogenic pharmaceutical composition may contain less than 1 mg / mL of octoxynol-10 and polysorbate 80, respectively. Other trace residue components may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0480] Immunogenic pharmaceutical compositions can be formulated as sterile solutions or suspensions in suitable media known in the art. The pharmaceutical compositions can be sterilized by conventional sterilization techniques or by sterile filtration. The resulting aqueous solutions can be packaged for use as is or lyophilized, and the lyophilized preparations are combined with a sterile solution prior to administration.

[0481] For example, a pharmaceutical composition containing an active agent such as an immune cell as disclosed herein in combination with one or more adjuvants can be formulated to contain a specific molar ratio. For example, a molar ratio of about 99:1 to about 1:99 of the active agent such as an immune cell as described herein in combination with one or more adjuvants can be used. In some examples, the molar ratio range of the active agent such as an immune cell as described herein in combination with one or more adjuvants can be selected from about 80:20 to about 20:80; about 75:25 to about 25:75; about 70:30 to about 30:70; about 66:33 to about 33:66; about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90. The molar ratio of the active agent such as an immune cell as described herein in combination with one or more adjuvants may be about 1:9, and in some cases, it may be about 1:1. Combinations of the activating agents such as immune cells described herein with one or more adjuvants may be formulated together in the same dosing unit, e.g., one vial, suppository, tablet, capsule, or aerosol spray; or each agent, form, and / or compound may be formulated in separate units, e.g., two vials, suppositories, tablets, two capsules, tablets and vials, aerosol sprays, or the like.

[0482] In some cases, immunogenic pharmaceutical compositions may be administered with additional agents. The choice of additional agents may depend, at least in part, on the condition being treated. Additional 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 has a therapeutic effect against pathogen infection (e.g., viral infection), including drugs used to treat inflammatory conditions, such as NSAIDs, e.g., ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. For example, checkpoint inhibitors may be PD-1 / PD-L1 antagonists selected from the group consisting of nivolumab (ONO-4538 / BMS-936558, MDX1 106, OPDIVO), pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011), and MPDL328OA (ROCHE). As another example, the formulation may also contain one or more supplements such as vitamin C, E, or other antioxidants.

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

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

[0485] In some embodiments, the pharmaceutical composition includes a preservative or stabilizer. In some embodiments, the preservative or stabilizer is selected from cytokines, growth factors, adjuvants, or chemicals. In some embodiments, the composition includes at least one agent that helps preserve cell viability after at least one freeze-thaw cycle. In some embodiments, the composition includes at least one agent that helps preserve cell viability after at least two or more freeze-thaw cycles.

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

[0487] In some cases, pharmaceutical compositions are formulated according to routine procedures as pharmaceutical compositions adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in a sterile isotonic aqueous buffer. If necessary, the composition may include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the components are supplied separately or mixed together in a unit dosage form as dry lyophilized powder or water-free concentrate in sealed containers such as ampoules or sachets indicating the content of the active agent. If the composition is to be administered by infusion, it may be prepared in an infusion bottle containing sterile pharmaceutical-grade water or saline. If the composition is to be administered by injection, ampoules of sterile water or saline for injection may be provided so that the components can be mixed prior to administration. Manufacturing method:

[0488] Methods for producing antigen-specific T cells are provided herein. Methods for preparing T cell compositions, such as therapeutic T cell compositions, are provided herein. For example, the method may include the step of amplifying or inducing antigen-specific T cells. Preparing T cells (e.g., inducing or amplifying) may also mean producing T cells of any type (e.g., CD4). + T cells and CD8 +The method broadly encompasses procedures for isolating, stimulating, culturing, inducing, and / or increasing T cells. In one embodiment, a method is provided herein for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence, comprising the step of incubating the APC with a population of immune cells derived from a biological sample in which cells expressing CD14 and / or CD25 are depleted. In some embodiments, the method comprises the step of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence, and the method comprises the step of incubating the APC with a population of immune cells derived from a biological sample in which cells expressing CD11b and / or CD19 are depleted. In some embodiments, the method comprises the step of incubating the APC with a population of immune cells derived from a biological sample in which cells expressing any CD11b and / or CD19 and / or CD14 and / or CD25 or any combination thereof.

[0489] In a second aspect, the following is provided: 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, comprising the step of incubating an APC stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample.

[0490] In a third aspect, the present invention provides a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence, comprising the steps of: incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells derived from a biological sample for a first period of time; and subsequently incubating at least one T cell from the biological sample with an APC.

[0491] A fourth aspect 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, comprising the steps of incubating a population of immune cells derived from a biological sample with one or more APC preparations for one or more separate periods of less than 28 days, wherein at least one antigen-specific memory T cell is enlarged or at least one antigen-specific naive T cell is induced.

[0492] A fifth aspect of the invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence, comprising the steps of incubating a population of immune cells derived from a biological sample with three or fewer APC preparations over three or fewer separate periods, thereby increasing at least one antigen-specific memory T cell or inducing at least one antigen-specific naive T cell.

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

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

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

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

[0497] In some embodiments, the method includes (a) obtaining a biological sample derived from a subject, comprising at least one antigen-presenting cell (APC), and (b) concentrating cells expressing CD14 from the biological sample, thereby enabling the expression of CD14 + The steps include obtaining a sample in which cells are concentrated, and (c)CD14 +(d) Incubating the cell-enriched sample with at least one cytokine or growth factor over a first period, and (c) incubating at least one peptide with the CD14 of (d) over a second period. + (i) The procedure includes the steps of (a) incubating a concentrated sample with a peptide to obtain an APC peptide-loaded sample, (e) incubating the APC peptide-loaded sample with one or more cytokines or growth factors for a third period to obtain a mature APC sample, (f) incubating the APC of the mature APC sample with a CD14 and / or CD25-depleted sample containing PBMCs for a fourth period, (g) incubating the PBMCs with the APC of the mature APC sample for a fifth period, (h) incubating the PBMCs with the APC of the mature APC sample for a sixth period, and (i) administering at least one T cell of the PBMCs to a subject in need.

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

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

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

[0501] 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 antigen peptide sequence includes the step of incubating the APC with a population of immune cells derived from a biological sample in which cells expressing CD14 and / or CD25 are depleted.

[0502] In some embodiments, a method is provided herein for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence, comprising the steps of incubating a population of immune cells derived from a biological sample with one or more APC preparations for one or more separate periods of less than 28 days, thereby increasing the number of at least one antigen-specific memory T cells or inducing at least one antigen-specific naive T cell. In some embodiments, a method is provided herein for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence, comprising the steps of incubating a population of immune cells derived from a biological sample with three or fewer APC preparations for three or fewer separate periods, thereby increasing the number of at least one antigen-specific memory T cell or inducing at least one antigen-specific naive T cell.

[0503] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence includes the step of 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 antigen peptide sequence includes the step of incubating a population of immune cells (e.g., PBMCs) with an APC for a certain period of time. In some embodiments, the population of immune cells is derived from a biological sample. In some embodiments, the population of immune cells is derived from a sample (e.g., a biological sample) in which CD14-expressing cells are depleted. In some embodiments, the population of immune cells is derived from a sample (e.g., a biological sample) in which CD25-expressing cells are depleted. In some embodiments, the population of immune cells is derived from a sample (e.g., a biological sample) in which both CD14-expressing cells and CD25-expressing cells are depleted.

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

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

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

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

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

[0509] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence includes the step of incubating a population of immune cells derived from a biological sample with one or more APC preparations for one or more separate periods, thereby inducing or increasing antigen-specific T cells, wherein one or more separate periods are less than 28 days calculated from the incubation of the immune cell population with the first APC preparation of the one or more APC preparations. In some embodiments, the step of incubating a population of immune cells derived from a biological sample with one or more APC preparations for one or more separate 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 is antigen-specific CD8 + The number of cells can be increased. In some embodiments, the IDO inhibitor is used in memory CD8 + The functional profile of the T cell response can be maintained. PD-1 antibodies can increase the absolute number of antigen-specific memory CD8+ T cell responses. PD-1 antibodies can increase the proliferation rate of cells treated with such antibodies. The addition of IL-12 increases antigen-specific cells and / or CD8 + This can lead to an increase in the frequency of T cells.

[0510] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence includes the steps of incubating a population of immune cells derived from a biological sample with one or more APC preparations over one or more separate periods, thereby increasing or inducing antigen-specific T cells, antigen-specific CD4 + T cells or antigen-specific CD8 +The percentage of T cells is the total T cells and the total CD4. + T cells, total CD8 + The step includes T cells, total immune cells, or total cells, which constitute 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%.

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

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

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

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

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

[0516] In some embodiments, the method includes the step of depleting CD14 and / or CD25-expressing cells from a biological sample. + The step of depleting the cells includes contacting the APC with a CD14 binder. In some embodiments, the APC is CD14 + It is derived from monocytes. In some embodiments, APCs are concentrated from biological samples. For example, APCs may be isolated, concentrated, or purified from a biological sample of the subject, including PBMCs.

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

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

[0519] In some embodiments, the percentage of antigen-specific T cells in this 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 this method is approximately 0.1% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, or 65% to 70%. In some embodiments, antigen-specific CD8 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 naive CD8 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 memory CD8 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 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 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% of total T cells or total immune cells. In some embodiments, antigen-specific CD8 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%. In some embodiments, antigen-specific naive CD8 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%. In some embodiments, antigen-specific memory CD8 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%. 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%.

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

[0521] In some embodiments, the method includes the step of incubating one or more APC preparations with a first medium containing at least one cytokine or growth factor for a first period. In some embodiments, the first 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 period is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days or less. In some embodiments, the first period is at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 days. In some embodiments, the first period is 3, 4, 5, 6, 7, 8, 9, or 10 days or less. In some embodiments, 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.

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

[0523] In some embodiments, the method includes the step of incubating one or more APC preparations with a second medium containing one or more cytokines or growth factors for a third period, thereby obtaining mature APCs. In some embodiments, the one or more cytokines or growth factors include GM-CSF (granule cell macrophage colony-stimulating factor), IL-4, FLT3L, IFN-γ, LPS, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848 (reximod), LPS, ss-rna40, poly(I:C), CpG, or a combination thereof. In some embodiments, the third period is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days or less. In some embodiments, the third period is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days. In some embodiments, the third period is 2, 3, 4, or 5 days or less. In some embodiments, the third period is at least 1, 2, 3, or 4 days.

[0524] In some embodiments, the method further includes the step of removing one or more cytokines or growth factors from the second medium after the third period and before the start of the fourth period. Antigen-loaded PBMCs for in vitro T cell induction

[0525] In some embodiments, the method provided herein includes the steps of isolating PBMCs from a human blood sample and directly loading an antigen onto the PBMCs. PBMCs in direct contact with the antigen can readily take up the antigen by phagocytosis and present the antigen to T cells that may be present in or added to a culture. In some embodiments, the method provided herein includes the steps of isolating PBMCs from a human blood sample and nucleofection or electroporation of polynucleotides, e.g., mRNA encoding one or more antigens, into the PBMCs. In some embodiments, the antigen delivered to the PBMCs offers significant advantages in terms of time and production efficiency compared to antigen-presenting cells that mature into DCs. One or more cell types may be further depleted from the PBMCs. In some embodiments, CD3+ cells may be depleted from the PBMCs during the initial period of antigen loading, and the CD3+ cells may be returned to the culture so that the PBMCs can stimulate CD3+ T cells. In some embodiments, CD25+ cells may be depleted from the PBMCs. In some embodiments, CD14+ cells may be depleted from the PBMCs. In some embodiments, CD19+ cells may be depleted from PBMCs. In some embodiments, both CD14-expressing and CD25-expressing cells may be depleted from PBMCs. In some embodiments, CD11b+ cells are depleted from the PBMC sample before antigen loading. In some embodiments, both CD11b+ and CD25+ cells are depleted from the PBMC sample before antigen loading.

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

[0527] In some embodiments, PBMCs are homogeneous for the target of the treatment. In some embodiments, PBMCs are homogeneous for adoptive cell therapy using antigen-specific T cells.

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

[0529] In some embodiments, mRNA is used as an immunogen for uptake and antigen presentation. One advantage of using mRNA to load PBMCs over peptide antigens is that RNA is self-adjuvant and does not require further adjuvants. Another advantage of using mRNA is that the peptide is endogenously processed and presented. In some embodiments, the mRNA includes a short-mer construct encoding a 9-10 amino acid peptide that constitutes an epitope. In some embodiments, the mRNA includes a long-mer construct encoding a peptide of about 25 amino acids. In some embodiments, the mRNA includes concatenation of multiple epitopes. In some embodiments, the concatemer may include one or more epitopes derived from the same antigenic protein. In some embodiments, the concatemer may include one or more epitopes derived from several different antigenic proteins. Several embodiments are described in the Examples section. Antigen loading of PBMCs by antigen loading may include various mechanisms for the delivery and incorporation of nucleic acids into PBMCs. In some embodiments, the delivery or integration mechanism includes transfection, electroporation, nucleofection, chemical delivery, such as lipid-encapsulated or liposome-mediated delivery.

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

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

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

[0533] In some embodiments, the method further includes the step of administering one or more of at least one antigen-specific T cells to a target. 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 administration is by injection, the active agent may be formulated in an aqueous solution, specifically in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain a formulation agent, such as a suspension agent, stabilizer, and / or dispersant. In another embodiment, the pharmaceutical composition does not contain any other substances, including adjuvants added to enhance the peptide-stimulated immune response. In some embodiments, the method further includes the step of administering one or more of at least one antigen-specific T cells to a target as part of the pharmaceutical composition described herein. 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 chemical substances. In some embodiments, at least one antigen-specific T cell is administered to the subject within 28 days of collecting a PBMC sample from the subject.

[0534] In addition to the formulations previously described, active agents may also be formulated as depot preparations. Such long-acting formulations may be administered by implantation or transdermal delivery (e.g., subcutaneous or intramuscular), intramuscular injection, or transdermal patch. Therefore, for example, the agent may be formulated using a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, for example, as a sparingly soluble salt.

[0535] Methods for treating subjects having a disease, disorder, or condition are also provided herein. These methods may include the step of administering a composition or pharmaceutical composition disclosed herein to a subject having a disease, disorder, or condition.

[0536] This disclosure provides methods for treatment including immunogenic therapy. Methods for treatment of diseases (e.g., cancer or viral infection) are provided. The method may include the step of administering an effective amount of a composition comprising immunogenic antigen-specific T cells to a subject according to the method provided herein. In some embodiments, the antigen includes a viral antigen. In some embodiments, the antigen includes a tumor antigen.

[0537] Non-limiting examples of therapeutic agents that can be prepared include peptide-based therapies, nucleic acid-based therapies, antibody-based therapies, T-cell-based therapies, and antigen-presenting cell-based therapies.

[0538] In some other embodiments, compositions for the manufacture of pharmaceuticals for use in therapeutics or the use of pharmaceutical compositions are provided herein. In some embodiments, the treatment method includes the step of administering a target effective amount of T cells that specifically recognize an immunogenic neoantigen peptide. In some embodiments, the treatment method includes the step of administering a target effective amount of a TCR that specifically recognizes an immunogenic neoantigen peptide, for example, a TCR expressed in T cells.

[0539] In some embodiments, cancer includes carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, colorectal cancer, rectal cancer, soft cell carcinoma. The group is selected from histiocytosis, Kaposi's sarcoma, B-cell lymphoma (including low-gra...

Claims

1. The use of an enlarged population of cells, including tumor antigen-specific T cells, in the manufacture of a pharmaceutical product for treating cancer in a subject, The tumor antigen-specific T cells include stimulated T cells that have been augmented in vitro from a peripheral blood mononuclear cell (PBMC) sample derived from the subject, and the PBMC sample includes a first population of antigen-presenting cells (APCs) and T cells, and the first population of antigen-presenting cells (APCs) and T cells is (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) A polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject having cancer, (B) Polynucleotide encoding the polypeptide In the presence of , they are incubated for a first period, thereby forming a population of cells including stimulated T cells. The first population of APCs and T cells is a population of immune cells derived from a sample containing APCs and T cells from which CD25+ cells and / or CD14+ cells have been depleted. The tumor antigen-specific T cells include T cells that are specific to a complex comprising the at least one tumor antigen epitope sequence and the MHC protein expressed by the human target cancer cells or APC. Here, (i) At least 0.1% of the CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells, are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells, or (ii) At least 0.1% of the CD4+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells, are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells, (a) The cancer is an unresectable melanoma, (b) The cancer is in a subject who has previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor and has disease progression, or (c) Use in which the cancer is in a subject who has received or is currently receiving a PD-1 inhibitor or PD-L1 inhibitor for at least three months and has a stable disease or an asymptomatic progressive disease.

2. The use according to claim 1, wherein the sample is washed and / or cryopreserved peripheral blood mononuclear cells (PBMCs).

3. An enlarged population of the aforementioned cells, including tumor antigen-specific T cells, is 1 × 10⁻⁶ ８ ~1 x 10 １１ The use according to claim 1 or 2, comprising a total of 1000 cells.

4. The population of cells, including stimulated T cells, in the first period, (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. The use according to any one of claims 1 to 3, comprising a first population of APCs and T cells incubated in the presence of .

5. The use according to any one of claims 2 to 4, wherein the washed and / or cryopreserved PBMC sample directly depletes CD14+ cells and CD25+ cells.

6. The use according to claim 4 or 5, wherein the mRNA comprises a 5' CAP and a poly-A tail.

7. The use according to claim 6, wherein the 5'CAP is CAP-1 and the polyA tail comprises 120 to 135 nucleotide residues.

8. The use according to any one of claims 4 to 7, wherein the polypeptide comprising at least two different tumor antigen epitope sequences expressed by cancer cells of the human subject having cancer comprises at least three, four, five, six, seven, eight, nine, ten or more different tumor antigen epitope sequences expressed by cancer cells of the human subject having cancer.

9. The use according to any one of claims 4 to 8, wherein each of the at least two different tumor antigen epitope sequences is 8 to 12 amino acid long or 15 to 25 amino acid long.

10. The use according to any one of claims 1 to 9, wherein the percentage of CD3+ cells in the enlarged population of cells, including tumor antigen-specific T cells, is at least 40%, at least 50%, or at least 60% of the total cells in the enlarged population of cells.

11. (a) The percentage of CD107a+ cells in the enlarged population of cells, including tumor antigen-specific T cells, is at least 10% of the tumor antigen-specific T cell population; (b) The percentage of TNFα+ cells in the enlarged population of cells, including tumor antigen-specific T cells, is at least 5% of the tumor antigen-specific T cell population; (c) The percentage of IFNγ+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 15% of the tumor antigen-specific T cell population; (d) The percentage of TNFα+ and IFNγ+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 2% of the tumor antigen-specific T cell population; (e) The percentage of TNFα+ and CD107a+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 0.5% of the tumor antigen-specific T cell population; (f) The percentage of IFNγ+ and CD107a+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 5% of the tumor antigen-specific T cell population; (g) The percentage of TNFα+, IFNγ+, and CD107a+ cells in the enlarged population of the cells, including tumor antigen-specific T cells, is at least 0.1% of the tumor antigen-specific T cell population. The use described in any one of claims 1 to 10.

12. (a) The percentage of CD4+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-), is at least 60%; (b) The percentage of CD4+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are effector T cells (CD62L- and CD45RA+), is at most 5%; (c) The percentage of CD4+ T cells in the enlarged population of cells, including tumor antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-), is at least 10%; (d) The percentage of CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+), is at most 25%; (e) The percentage of CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-), is at least 60%; (f) The percentage of CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are effector T cells (CD62L- and CD45RA+), is at most 10%; and / or (g) The percentage of CD8+ T cells in the enlarged population of the cells, including tumor antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-), is at least 15%. The use described in any one of claims 1 to 11.

13. The use according to any one of claims 1 to 12, wherein the first population of APCs and T cells is a population of immune cells derived from a biological sample in which CD11b+ cells and / or CD19+ cells have been further depleted.

14. The use according to any one of claims 4 to 13, wherein the at least two different tumor antigen epitope sequences are expressed as a single polypeptide chain, and the first tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences is linked to the second tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences via a linker sequence.

15. The aforementioned subject is, (i) refractory to anti-checkpoint inhibitor therapy; (ii) being between 18 and 75 years of age; and / or, (iii) Having a mutation in the BRAF gene and having previously received B-raf inhibitor or B-raf / MEK combination therapy, The use described in any one of claims 1 to 14.

16. The use according to any one of claims 1 to 15, wherein the incubation and growth period of the first population of APCs and T cells is less than 28 days.

17. An improved ex vivo method for preparing tumor antigen-specific T cells, (a) Deplete CD14+ cells and / or CD25+ cells directly from washed and / or cryopreserved peripheral blood mononuclear cell (PBMC) samples derived from human subjects, This involves the step of forming a CD14 and / or CD25 depleted population of PBMCs, which includes a first population of APCs and T cells, (b) The first population of APCs and T cells from step (a) is subjected to a first period of time. (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) A polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject having cancer, or (B) Polynucleotide encoding the polypeptide Incubate in the presence of This involves the step of forming a population of cells including stimulated T cells, (c) A step of increasing the number of stimulated T cells from step (b), thereby forming an enlarged population of cells including tumor antigen-specific T cells. Includes, The tumor antigen-specific T cells are (i) at least one tumor antigen epitope sequence from step (b), and (ii) MHC protein expressed by the human target cancer cells or APC from step (b). A method comprising T cells that are specific to a complex containing the above.

18. Step (b) above is: (i) The FLT3L and (ii) Polynucleotide encoding the polypeptide The method according to claim 17, comprising incubating the first population of APCs and T cells from step (a) in the presence of a first period.

19. A composition for treating cancer in human subjects, comprising an enlarged population of cells including tumor antigen-specific T cells, The tumor antigen-specific T cells are T cells that have been augmented in vitro from a human subject-derived peripheral blood mononuclear cell (PBMC) sample and stimulated with tumor antigens ex vivo, wherein the PBMC sample comprises a first population of antigen-presenting cells (APCs) and T cells, and the first population of antigen-presenting cells (APCs) and T cells is subjected to the depletion of CD25+ and / or CD14+ cells, (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) A polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject having cancer, (B) Polynucleotide encoding the polypeptide In the presence of [unspecified substance], a population of cells including stimulated T cells was formed during the first period of incubation. The T cells stimulated with tumor antigens in the aforementioned ex vivo are specific to a complex comprising the at least one tumor antigen epitope sequence and the MHC protein expressed by the human target cancer cells or APCs. Here, (i) At least 0.1% of the CD8+ T cells in the enlarged population of cells including the T cells stimulated with the tumor antigen in ex vivo are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells, or (ii) In the enlarged population of cells including the T cells stimulated with the tumor antigen in ex vivo, at least 0.1% of the CD4+ T cells are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells, (a) The cancer is an unresectable melanoma, (b) The cancer is in a subject who has previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor and has disease progression, or (c) A composition wherein the cancer is in a subject who has received or is currently receiving a PD-1 inhibitor or PD-L1 inhibitor for at least three months and has a stable disease or an asymptomatic progressive disease.

20. The composition according to claim 19, wherein the PBMC sample is washed and / or cryopreserved PBMC.

21. The enlarged population of cells, including T cells stimulated with tumor antigens in the aforementioned ex vivo, is 1 × 10 ８ ~1 x 10 １１ The composition according to claim 19 or 20, comprising a total of 1000 cells.

22. The composition according to any one of claims 19 to 21, wherein the first population of APCs and T cells is incubated for the first period in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

23. The population of cells, including T cells stimulated with tumor antigens in the aforementioned ex vivo, during the first period, (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 the cancer cells of the human subject having cancer. The composition according to any one of claims 19 to 22, comprising a first population of APCs and T cells incubated in the presence of .

24. The washed and / or cryopreserved PBMC sample directly depletes CD14+ and CD25+ cells, and then, (A) A polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, (B) Polynucleotide encoding the polypeptide The composition according to any one of claims 20 to 22, wherein the composition is incubated for a first period in the presence of the present, thereby forming a population of cells including T cells stimulated with the tumor antigen in ex vivo.

25. The composition according to claim 24, wherein the polynucleotide encoding the polypeptide is mRNA and comprises a 5'CAP and a polyA tail.

26. The composition according to claim 25, wherein the 5' CAP is CAP-1 and the poly A tail comprises 120 to 135 nucleotide residues.

27. The composition according to claim 23 or 24, wherein the polypeptide comprises at least two different tumor antigen epitope sequences expressed by cancer cells of the human subject having cancer, or further comprises at least three, four, five, six, seven, eight, nine, ten or more different tumor antigen epitope sequences expressed by cancer cells of the human subject having cancer.

28. The composition according to claim 23 or 27, wherein each of the at least two different tumor antigen epitope sequences is 8 to 12 amino acid long or 15 to 25 amino acid long.

29. The composition according to any one of claims 19 to 28, wherein the percentage of CD3+ cells in the enlarged population of cells, including T cells stimulated with tumor antigens in ex vivo, is at least 40%, at least 50%, or at least 60% of the total cell population.

30. (a) The percentage of CD107a+ cells in the enlarged population of cells is at least 10% of the tumor antigen-specific T cell population; (b) The percentage of TNFα+ cells in the enlarged population of cells is at least 5% of the tumor antigen-specific T cell population; (c) The percentage of IFNγ+ cells in the enlarged population of the cells is at least 15% of the tumor antigen-specific T cell population; (d) The percentage of TNFα+ and IFNγ+ cells in the enlarged population of the cells is at least 2% of the tumor antigen-specific T cell population; (e) The percentage of TNFα+ and CD107a+ cells in the enlarged population of the cells is at least 0.5% of the tumor antigen-specific T cell population; (f) The percentage of IFNγ+ and CD107a+ cells in the enlarged population of the cells is at least 5% of the tumor antigen-specific T cell population; (g) The percentage of TNFα+, IFNγ+, and CD107a+ cells in the enlarged population of the cells is at least 0.1% of the tumor antigen-specific T cell population. The composition according to any one of claims 19 to 29.

31. (a) The percentage of CD4+ T cells in the enlarged population of the cells, which are effector memory T cells (CD62L- and CD45RA-), is at least 60%; (b) The percentage of CD4+ T cells in the enlarged population of effector T cells (CD62L- and CD45RA+) is at most 5%; (c) The percentage of CD4+ T cells in the enlarged population of the cells, which are central memory T cells (CD62L+ and CD45RA-), is at least 10%; (d) The percentage of CD8+ T cells in the enlarged population of naive T cells (CD62L+ and CD45RA+) is at most 25%; (e) The percentage of CD8+ T cells in the enlarged population of the cells, which are effector memory T cells (CD62L- and CD45RA-), is at least 60%; (f) The percentage of CD8+ T cells in the enlarged population of effector T cells (CD62L- and CD45RA+) is at most 10%; and / or (g) The percentage of CD8+ T cells in the enlarged population of the cells, which are central memory T cells (CD62L+ and CD45RA-), is at least 15%. The composition according to any one of claims 19 to 30.

32. The composition according to any one of claims 19 to 31, wherein the first population of APCs and T cells is further subjected to the depletion of CD11b+ cells and / or CD19+ cells.

33. The composition according to any one of claims 23 to 32, wherein the at least two different tumor antigen epitope sequences are expressed as a single polypeptide chain, and the first tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences is linked to the second tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences via a linker sequence.

34. The aforementioned subject is, (i) refractory to anti-checkpoint inhibitor therapy; (ii) being between 18 and 75 years of age; and / or, (iii) Having a mutation in the BRAF gene and having previously received B-raf inhibitor or B-raf / MEK combination therapy, The composition according to any one of claims 19 to 33.

35. The composition according to any one of claims 19 to 34, wherein the incubation and growth period of the first population of APCs and T cells is less than 28 days.

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