T cell preparation compositions and methods

Abstract

The generation of antigen-specific T cells by controlled ex vivo induction or expansion can provide highly specific and beneficial T cell therapies. The present disclosure provides T cell preparation methods and therapeutic T cell compositions that can be used to treat subjects with cancer and other conditions, diseases and disorders, and individual antigen-specific T cell therapies.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 845,251, filed May 8, 2019, which is incorporated herein by reference in its entirety. Background Art

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

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

[0005] The present disclosure provides new and improved T cell therapeutics for clinical development and application. Although autologous T cell therapeutics are safe to use, some substantial improvements are still needed to meet treatment standards, and the development of this field is both rapid and full of difficulties. The applicant's previously disclosed application provides a landmark progress in compositions and methods for cancer T cell therapy (WO2019 / 094642). The present application stems from a surprising discovery that the consumption of certain cells expressing specific markers at different stages of in vitro immune cell preparation provides a highly immunogenic cell composition. The present disclosure is also partly derived from the discovery of new and improved methods for antigen stimulation, resulting in improved cell compositions for therapeutic agent development. New methods and compositions are provided herein, wherein, at least in part, the selective consumption of certain immune cells from in vitro stimulation and cell expansion environments provides new therapeutic compositions and improved methods.

[0006] Provided herein is an improved ex vivo method for preparing tumor antigen-specific T cells, the method comprising: depleting CD14+ cells and / or CD25+ cells from an immune cell population comprising antigen presenting cells (APCs) and T cells, thereby forming a CD14- and / or CD25-depleted immune cell population comprising a first population of APCs and T cells, wherein the immune cell population is derived from a biological sample of a human subject; and incubating the CD14- and / or CD25-depleted immune cell population comprising the first population of APCs and T cells for a first period of time in the presence of: FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) comprising at least one protein expressed by cancer cells of a human subject having cancer. (b) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (c) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (d) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (e) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (e) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (f) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (g) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (h) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (h) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (h) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (i) wherein the at least one tumor antigen epitope sequence is expressed by a human subject, or (ii ... 8 Up to 1x10 11 Total cells.

[0007] Provided herein is an improved ex vivo method for preparing tumor antigen-specific T cells, the method comprising: depleting CD14+ cells and / or CD25+ cells from an immune cell population comprising antigen presenting cells (APCs) and T cells, thereby forming a CD14- and / or CD25-depleted immune cell population comprising a first population of APCs and T cells, wherein the immune cell population is derived from a biological sample of a human subject; and incubating the CD14- and / or CD25-depleted immune cell population comprising the first population of APCs and T cells for a first period of time 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 a cancer cell of a human subject having cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a cell line comprising stimulated T cells. expanding the cell population comprising stimulated T cells to form an expanded cell population comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising: (i) the at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii); and administering the expanded cell population comprising tumor antigen-specific T cells to the human subject, wherein the human subject: has unresectable melanoma and has previously received a PD-1 inhibitor or a PD-L1 inhibitor and a regimen comprising a CTLA-4 inhibitor and has had disease progression, or has previously received or is currently receiving a PD-1 inhibitor or a PD-L1 inhibitor for at least 3 months and has stable disease or asymptomatic disease progression.

[0008] Provided herein is an improved ex vivo method for preparing tumor antigen-specific T cells, the method comprising: depleting CD14+ cells and / or CD25+ cells from an immune cell population comprising antigen-presenting cells (APCs) and T cells, thereby forming a CD14- and / or CD25-depleted immune cell population comprising a first population of APCs and T cells, wherein the immune cell population is derived from a biological sample of a human subject; and incubating the CD14- and / or CD25-depleted immune cell population comprising the first population of APCs and T cells for a first period of time in the presence of: FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and mRNA encoding a polypeptide comprising at least two different tumor antigen epitope sequences expressed by cancer cells of a human subject having cancer; thereby forming a cell population comprising stimulated T cells; and expanding the cell population comprising stimulated T cells to form an expanded cell population comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising the following components: (i) the at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by the cancer cells or APCs of the human subject of (b)(ii).

[0009] Provided herein is an improved ex vivo method for preparing tumor antigen-specific T cells, the method comprising: depleting CD14+ cells and / or CD25+ cells from: (i) a washed and / or cryopreserved peripheral blood mononuclear cell (PBMC) sample directly from a human subject, (ii) a PBMC sample from a human subject containing approximately the same percentage of immature dendritic cells (DCs) as the percentage of immature DCs in the peripheral blood of the human subject, (iii) a PBMC sample from a human subject containing approximately the same percentage of mature DCs as the percentage of mature DCs in the peripheral blood of the human subject, (iv) a PBMC sample from a human subject containing approximately the same percentage of mature DCs as the percentage of mature DCs in the peripheral blood of the human subject, (v) a PBMC sample from a human subject, wherein the ratio of immature DCs to mature DCs is approximately the same as the ratio of immature DCs to mature DCs in the peripheral blood of the human subject, (vi) a PBMC sample from a human subject, wherein the percentage of APCs in the total cell population is approximately the same as the percentage of APCs in the total cell population in the peripheral blood of the human subject, (vii) a PBMC sample from a human subject, wherein the percentage of DCs in the total cell population is approximately the same as the percentage of DCs in the peripheral blood of the human subject, (viii) a PBMC sample from a human subject a PBMC sample from a human subject, wherein the percentage of CD303+ cells in the total cell population is approximately the same as the percentage of CD303+ cells in the peripheral blood of the human subject, (ix) a PBMC sample from a human subject, wherein the percentage of CD141+ cells in the total cell population is approximately the same as the percentage of CD141+ cells in the peripheral blood of the human subject, (x) a PBMC sample from a human subject, wherein the percentage of macrophages in the total cell population is approximately the same as the percentage of macrophages in the peripheral blood of the human subject, or (xi) a PBMC sample from a human subject, wherein the percentage of CD19+ cells in the total cell population is approximately the same as the percentage of CD19+ cells in the peripheral blood of the human subject. (a) incubating the CD14- and / or CD25-depleted immune cell population comprising the first population of APCs and T cells for a first period of time 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 a cancer cell of a human subject having cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a cell population comprising stimulated T cells;and expanding the cell population comprising stimulated T cells to form an expanded cell population comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising: (i) the at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by a cancer cell or APC of the human subject of (b)(ii). ;

[0010] In some embodiments, the method further comprises administering the expanded cell population comprising tumor antigen-specific T cells to the human subject.

[0011] In some embodiments, the incubation comprises incubating the CD14 and / or CD25 depleted immune cell population comprising the first population of APCs and T cells for a first period of time in the presence of: (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) mRNA encoding a polypeptide comprising at least two different tumor antigen epitope sequences expressed by cancer cells of a human subject having cancer.

[0012] In some embodiments, introducing comprises electroporation or nucleofection. In some embodiments, the electroporation or nucleofection is performed without separating the APCs and the T cells of the first population of T cells from the APCs in step (a).

[0013] In some embodiments, the method further comprises administering to the human subject the expanded cell population comprising tumor antigen-specific T cells. In some embodiments, incubating comprises incubating the CD14 and / or CD25-depleted immune cell population comprising a first population of APCs and T cells for a first period of time in the presence of: (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) mRNA encoding a polypeptide comprising at least two different tumor antigen epitope sequences expressed by cancer cells of a human subject having cancer.

[0014] In some embodiments, the mRNA comprises a 5' cap. In some embodiments, the 5' cap is CAP-1. In some embodiments, the mRNA comprises a 3' poly A tail. In some embodiments, the poly A tail is 120 to 135 nucleotides in length. In some embodiments, the first tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences is connected to the second tumor antigen epitope sequence of the at least two different tumor antigen epitope sequences via a linker sequence. In some embodiments, the 5' cap is operably connected to a sequence encoding the at least two different tumor antigen epitope sequences via a linker sequence. In some embodiments, the at least two different tumor antigen epitope sequences are expressed as a single polypeptide chain. In some embodiments, incubation comprises incubating a CD14 and / or CD25-depleted immune cell population comprising a first population of APCs and T cells in the presence of LPS and IFNγ.

[0015] In some embodiments, the at least two different tumor antigen epitope sequences are each 8 to 12 amino acids in length. In some embodiments, the at least two different tumor antigen epitope sequences are each 15 to 25 amino acids in length. In some embodiments, the polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or more different tumor antigen epitope sequences expressed by cancer cells of a human subject suffering from cancer.

[0016] In some embodiments, the expanded cell population comprising tumor antigen-specific T cells comprises 1×10 8 Up to 1x10 11 In some embodiments, the expanded cell population comprising tumor antigen-specific T cells comprises 1×10 8 Up to 1x10 11 CD3+ cells.

[0017] In some embodiments, the human subject has unresectable melanoma. Unlike resectable melanoma, tumor infiltrating lymphocytes (TILs) cannot be obtained from unresectable melanoma; therefore, TILs cannot be used to treat unresectable melanoma. An advantage of the methods and compositions provided herein is that they can be used to treat unresectable melanoma.

[0018] In some embodiments, the human subject has previously received a PD-1 inhibitor or a PD-L1 inhibitor and a regimen containing a CTLA-4 inhibitor and had disease progression.

[0019] In some embodiments, the human subject has received or is currently receiving a PD-1 inhibitor or a PD-L1 inhibitor for at least 3 months and has stable disease or no symptomatic disease progression.

[0020] In some embodiments, the percentage of CD3+ cells in the expanded cell population comprising tumor antigen-specific T cells is at least 40%, 50%, or 60% of the total cell population.

[0021] In some embodiments, the percentage of CD107a+ cells in the expanded cell population comprising tumor antigen-specific T cells is at least 10% of the tumor antigen-specific T cell population.

[0022] In some embodiments, the percentage of TNFα+ cells in the expanded cell population comprising tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population.

[0023] In some embodiments, the percentage of IFNγ+ cells in the expanded cell population comprising tumor antigen-specific T cells is at least 15% of the tumor antigen-specific T cell population.

[0024] In some embodiments, the percentage of TNFα+ and IFNγ+ cells in the expanded cell population comprising tumor antigen-specific T cells is at least 2% of the tumor antigen-specific T cell population.

[0025] In some embodiments, the percentage of TNFα+ and CD107a+ cells in the expanded cell population comprising tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cell population.

[0026] In some embodiments, the percentage of IFNγ+ and CD107a+ cells in the expanded cell population comprising tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population.

[0027] In some embodiments, the percentage of TNFα+, IFNγ+, and CD107a+ cells in the expanded cell population comprising tumor antigen-specific T cells is at least 0.1% of the tumor antigen-specific T cell population.

[0028] In some embodiments, the percentage of CD4+ T cells that are naive T cells (CD62L+ and CD45RA+) in the expanded cell population comprising tumor antigen-specific T cells is at most 15%.

[0029] In some embodiments, the percentage of CD4+ T cells that are effector memory T cells (CD62L- and CD45RA-) in the expanded cell population comprising tumor antigen-specific T cells is at least 60%.

[0030] In some embodiments, the percentage of CD4+ T cells that are effector T cells (CD62L- and CD45RA+) in the expanded cell population comprising tumor antigen-specific T cells is at most 5%.

[0031] In some embodiments, the percentage of CD4+ T cells in the expanded cell population comprising tumor antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-) is at least 10%.

[0032] In some embodiments, the percentage of CD8+ T cells that are naive T cells (CD62L+CD45RA+) in the expanded cell population comprising tumor antigen-specific T cells is at most 25%.

[0033] In some embodiments, the percentage of CD8+ T cells that are effector memory T cells (CD62L-CD45RA-) in the expanded cell population comprising tumor antigen-specific T cells is at least 60%.

[0034] In some embodiments, the percentage of CD8+ T cells that are effector T cells (CD62L-CD45RA+) in the expanded cell population comprising tumor antigen-specific T cells is at most 10%.

[0035] In some embodiments, the percentage of CD8+ T cells that are central memory T cells (CD62L+CD45RA-) in the expanded cell population comprising tumor antigen-specific T cells is at least 15%.

[0036] In some embodiments, the expanded cell population comprising tumor antigen-specific T cells produces cytokines and causes degranulation upon recognition of target cells.

[0037] In some embodiments, the human subject is refractory to anti-checkpoint inhibitor therapy.

[0038] In some embodiments, the human subject is between 18 and 75 years old.

[0039] In some embodiments, the human subject has a mutation in the BRAF gene and has previously received a B-raf inhibitor or a B-raf / MEK combination therapy.

[0040] In some embodiments, depletion comprises depleting CD14+ cells and CD25+ cells from a peripheral blood mononuclear cell (PBMC) sample from a human subject that has not undergone a monocyte maturation step into mature dendritic cells (DCs).

[0041] In some embodiments, the depletion further comprises depleting CD11b+ cells from a peripheral blood mononuclear cell (PBMC) sample from a human subject that has not undergone a step of maturation of monocytes into mature dendritic cells (DCs).

[0042] In some embodiments, steps (b) and (c) are performed in less than 28 days.

[0043] In some embodiments, the proportion of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in the expanded cell population comprising tumor antigen-specific T cells is at least twice the proportion of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in the biological sample.

[0044] In some embodiments, the proportion of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in the expanded cell population comprising tumor antigen-specific T cells is at least twice the proportion of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in the biological sample.

[0045] In some embodiments, at least 0.1% of the CD8+ T cells in the expanded cell population comprising tumor antigen-specific T cells are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells.

[0046] In some embodiments, at least 0.1% of the CD4+ T cells in the expanded cell population comprising tumor antigen-specific T cells are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

[0047] In some embodiments, expanding comprises (A) contacting the cell population comprising stimulated T cells with a second population of mature APCs, wherein the second population of mature APCs (i) has been incubated with FLT3L and (ii) presents at least one tumor antigen epitope sequence; and (B) expanding the cell population comprising stimulated T cells for a second period of time, thereby forming an expanded T cell population.

[0048] In some embodiments, prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs, the second population of mature APCs has been incubated with FLT3L for at least 1 day.

[0049] In some embodiments, the biological sample is a peripheral blood sample, a leukapheresis sample, or an apheresis sample.

[0050] In some embodiments, the method further comprises harvesting the expanded cell population comprising tumor antigen-specific T cells, cryopreserving the expanded cell population comprising tumor antigen-specific T cells, or preparing a pharmaceutical composition containing the expanded cell population comprising tumor antigen-specific T cells.

[0051] In some embodiments, incubating comprises incubating the CD14 / CD25-depleted immune cell population comprising the first population of APCs and T cells in the presence of FLT3L and RNA encoding the polypeptide for a first period of time.

[0052] In some embodiments, the human subject suffering from cancer is the human subject from which the biological sample was obtained.

[0053] In some embodiments, the polypeptide is 8 to 50 amino acids in length.

[0054] In some embodiments, the polypeptide comprises at least two tumor antigen epitope sequences, each sequence being expressed by a cancer cell in a human subject having cancer.

[0055] In some embodiments, depleting CD14+ cells and / or CD25+ cells from the immune cell population comprising the first population of APCs and T cells comprises contacting the immune cell population comprising the first population of APCs and T cells with a CD14 binding agent and / or a CD25 binding agent.

[0056] In some embodiments, depleting further comprises depleting CD19+ cells from the population of immune cells comprising the first population of APCs and T cells.

[0057] Provided herein is an ex vivo method for preparing tumor antigen-specific T cells, the method comprising: depleting CD11b+ cells from an immune cell population comprising antigen presenting cells (APCs) and T cells, thereby forming a CD11b-depleted immune cell population comprising a first population of APCs and T cells, wherein the immune cell population is derived from a biological sample of a human subject; and incubating the CD11b-depleted immune cell population comprising the first population of APCs and T cells for a first period of time in the presence of: FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) comprising (B) a polypeptide of at least one tumor antigen epitope sequence expressed by cancer cells of a human subject having cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a cell population comprising stimulated T cells; and expanding the cell population comprising stimulated T cells to form an expanded cell population comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising the following components: (i) the at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by the cancer cells or APCs of the human subject of (b)(ii).

[0058] Provided herein is a pharmaceutical composition comprising an expanded cell population comprising tumor antigen-specific T cells produced by the methods described herein; and a pharmaceutically acceptable carrier.

[0059] Provided herein is a pharmaceutical composition comprising: (a) an immune cell population from a biological sample, wherein the immune cell population comprises T cells stimulated by antigen-presenting cells (APCs), wherein the T cells comprise a T cell receptor (TCR) specific for an epitope of a polypeptide, wherein (i) the amount of immune cells expressing CD11b in the immune cell population is proportionally lower than the amount of immune cells expressing CD11b in the biological sample, and / or (ii) the amount of immune cells expressing CD11c in the immune cell population is proportionally higher than the amount of immune cells expressing CD11c in the biological sample; and (b) a pharmaceutically acceptable excipient.

[0060] Provided herein is a pharmaceutical composition comprising: (a) an immune cell population from a biological sample, wherein the immune cell population comprises T cells stimulated by antigen-presenting cells (APCs), wherein the T cells comprise a T cell receptor (TCR) specific for an epitope of a polypeptide, wherein the APC-stimulated T cells have been incubated with a cytokine; (b) the cytokine; and (c) a pharmaceutically acceptable excipient.

[0061] Provided herein is a pharmaceutical composition comprising: (a) an immune cell population from a biological sample of a subject to which fms-like tyrosine kinase 3 ligand (FLT3L) has been administered, wherein the immune cell population comprises T cells stimulated by antigen-presenting cells (APCs), the T cells comprising a T cell receptor (TCR) specific for an epitope of a polypeptide; and (b) a pharmaceutically acceptable excipient.

[0062] In some embodiments, the population of immune cells is from a biological sample of a subject.

[0063] In some embodiments, the population of immune cells is from a biological sample of a subject to whom fms-like tyrosine kinase 3 ligand (FLT3L) has been administered.

[0064] In some embodiments, the APC-stimulated T cells have been incubated with a cytokine, and wherein the pharmaceutical composition further comprises a cytokine.

[0065] In some embodiments, the amount of immune cells expressing CD11b in the population of immune cells is proportionally lower than the amount of immune cells expressing CD11b in the biological sample.

[0066] In some embodiments, the amount of immune cells expressing CD11c in the population of immune cells is proportionally higher than the amount of immune cells expressing CD11c in the biological sample.

[0067] In some embodiments, the amount of CD14-expressing immune cells in the population is proportionally lower than the amount of CD14-expressing immune cells in the biological sample.

[0068] In some embodiments, the amount of immune cells expressing CD25 in the population is proportionally lower than the amount of immune cells expressing CD25 in the biological sample.

[0069] In some embodiments, the amount of immune cells expressing CD19 in the population is proportionally lower than the amount of immune cells expressing CD19 in the biological sample.

[0070] In some embodiments, the APC is an FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APC.

[0071] In some embodiments, the APC-stimulated T cells are APC-stimulated T cells stimulated with FLT3L.

[0072] In some embodiments, the cytokine is IL-7 or IL-15 or IL-21.

[0073] In some embodiments, the APC-stimulated T cells include T cells stimulated by antigen-loaded APCs presenting epitopes on MHC class I or MHC class II molecules.

[0074] In some embodiments, the antigen-loaded APCs include plasmacytoid dendritic cells (pDCs), CD11c+ DCs, CD1c+ DCs, or CD141+ DCs.

[0075] In some embodiments, the CD11b cells include CD16+ mononuclear cells.

[0076] In some embodiments, the pharmaceutical composition further comprises an agent that promotes the growth and maintenance of cells ex vivo, including growth factors, cytokines, amino acids, supplements, or a combination thereof.

[0077] In some embodiments, the amount of immune cells expressing CD1c in the population of immune cells is proportionally greater than the amount of immune cells expressing CD1c in the biological sample.

[0078] In some embodiments, the amount of immune cells or APCs expressing CD141 in the immune cell population is proportionally higher than the amount of immune cells or APCs expressing CD141 in the biological sample.

[0079] In some embodiments, the cell population comprising antigen-loaded APCs comprises greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 60%, or greater than 70% CD11c+ cells.

[0080] In some embodiments, the 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.

[0081] In some embodiments, the APC-stimulated T cells include T cells stimulated by a cell population comprising greater than 90% CD11c+ cells.

[0082] In some embodiments, the pharmaceutical compositions described herein comprise T cells stimulated from a cell population that contains greater than 70% neoantigen peptide-expressing cells, i.e., CD11c+, CD1c+, or CD141+ cells.

[0083] In some embodiments, the pharmaceutical composition comprises at least 60% of T cells specific for the epitope in the pharmaceutical composition.

[0084] In some embodiments, the pharmaceutical compositions described herein comprise a higher proportion of naive T cells that are induced or converted into neoantigen-primed T cells compared to a cell composition obtained by contacting isolated T cells with antigen-loaded APCs without reducing or depleting CD11b+ and / or CD19+ cells.

[0085] In some embodiments, the pharmaceutical compositions described herein comprise greater than 35% naive T cells that are induced or converted into antigen-specific activated T cells specific for the epitope.

[0086] In some embodiments, the pharmaceutical compositions described herein comprise a higher proportion of cancer neoantigen-specific CD8+ T cells compared to a cell composition obtained by contacting isolated T cells with antigen-loaded APCs without reducing or depleting CD11b+ cells and / or CD19+ cells.

[0087] In some embodiments, the pharmaceutical compositions described herein comprise at least 30% CD8+ T cells.

[0088] In some embodiments, the pharmaceutical compositions described herein comprise a higher proportion of memory T cells compared to a cell composition obtained by contacting isolated T cells with antigen-loaded APCs without reducing or depleting CD11b+ cells and / or CD19+ cells.

[0089] Provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described herein.

[0090] Provided herein is a method for preparing T cells comprising a T cell receptor (TCR) specific for an epitope of a polypeptide, the method comprising (a) depleting cells expressing CD11b from an immune cell population comprising antigen-presenting cells and T cells, thereby forming a CD11b-depleted immune cell population comprising T cells; and (b) incubating or expanding the CD11b-depleted immune cell population comprising T cells; wherein memory T cells comprising a TCR specific for the epitope are expanded, or naive T cells comprising a TCR specific for the epitope are induced.

[0091] Provided herein is a method for preparing a T cell comprising a T cell receptor (TCR) specific for an epitope, the method comprising (a) enriching an immune cell population comprising APCs and T cells for cells expressing CD11c, thereby forming an immune cell population comprising CD11c-enriched T cells; and (b) incubating or expanding the immune cell population comprising CD11c-enriched T cells; wherein memory T cells comprising a TCR specific for the epitope are expanded, or naive T cells comprising a TCR specific for the epitope are induced. In some embodiments, the APC preparation method comprises APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

[0092] In some embodiments, the method further comprises preparing an APC preparation.

[0093] In some embodiments, the method of preparing an APC preparation comprises incubating the APC with FLT3L.

[0094] In some embodiments, the method of preparing an APC preparation comprises incubating APCs with the polypeptide or a polynucleotide encoding the polypeptide.

[0095] Provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an immune cell population from a biological sample, wherein the immune cell population comprises T cells stimulated by antigen-presenting cells (APCs), the T cells comprising a T cell receptor (TCR) specific for an antigenic peptide sequence, and wherein the subject has been administered fms-like tyrosine kinase 3 ligand (FLT3L).

[0096] Provided herein is a method of treating cancer in a subject in need thereof, comprising: (a) administering FMS-like tyrosine kinase 3 receptor ligand (FLT3L) to the subject; and (b) administering to the subject an immune cell population from a biological sample, wherein the immune cell population comprises T cells stimulated by antigen-presenting cells (APCs), the T cells comprising a T cell receptor (TCR) specific for an antigenic peptide sequence.

[0097] Provided herein is a method for treating cancer in a subject in need thereof, comprising: (a) administering to the subject an immune cell population from a biological sample, wherein the immune cell population comprises T cells stimulated by antigen-presenting cells (APCs), and the T cells comprise a T cell receptor (TCR) specific for an antigenic peptide sequence; and (b) administering to the subject a polypeptide comprising the antigenic peptide sequence or a polynucleotide encoding the antigenic peptide sequence.

[0098] In some embodiments, the method further comprises administering FMS-like tyrosine kinase 3 receptor ligand (FLT3L) to the subject prior to administering the population of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1A Schematic diagram depicting an example protocol for antigen-specific T cell production.

[0100] Figure 1B Schematic diagram depicting an example protocol for antigen-specific T cell production.

[0101] Figure 1C Schematic diagram depicting an example alternative protocol for antigen-specific T cell production.

[0102] Figure 2 Depicts example results showing antigen-specific CD8 induced by long or short peptides + Memory T cell fraction. "Bulk" indicates that the sample containing T cells used for induction is entirely peripheral blood mononuclear cells (PBMC). "Treg" indicates that the sample containing T cells used for induction is PBMC depleted of cells expressing CD25.

[0103] Figure 3 Depicts an example flow cytometric analysis showing antigen-specific CD8 + The fraction of naive T cells.

[0104] Figure 4 Depicted are example results showing antigen-specific CD8 + T cell responses to HIV short peptides, previously identified short neoantigens (PINs), or peptide pools of long PINs. "Complete PBMCs" indicates that the sample containing T cells used for induction was complete PBMCs. "CD25 PBMCs" indicates that the sample containing T cells used for induction was depleted of CD25+ cells. Short, short peptides or shortmers; long, long peptides or longmers.

[0105] Figure 5A Depicts the expression of antigen-specific CD8 + Example flow cytometric analysis of naive T cell responses to a single previously identified neoantigen (PIN).

[0106] Figure 5B Depicts antigen-specific CD8 + Example flow cytometric analysis of naive T cell responses to a single previously identified neoantigen (PIN).

[0107] Figure 6Depicted are example results showing that antigen-specific CD8 + T cell responses to the indicated peptides.

[0108] Figure 7 Depicts antigen-specific CD8 + Example flow cytometry plots of T cell responses to the indicated mutant epitopes.

[0109] Figure 8A Depicted is an exemplary bar graph showing antigen-specific memory CD8 + The results of T cell responses to viral antigens. After up to three rounds of stimulation, all CD8 + Approximately 50% of the T cells were specific for the indicated viral epitopes (CMV pp65, EBVYVL, EBV BMLF1 and Mart-1).

[0110] Figure 8B Depicts example results of a recall assay involving antigen-specific memory CD8 + T cell responses to peptide-loaded antigen-presenting cells, followed by incubation with APCs loaded with and without viral antigens. The graph depicts the CD8 T cells releasing the indicated cytokines at two time points. + T cell fraction.

[0111] Figure 9 Depicted are example results of a cytotoxicity assay used to assess whether induced T cell cultures can kill tumor lines expressing an antigen. The fraction of live and dead caspase 3-positive tumor cells relative to total tumor cells is shown. Live caspase 3-positive tumor cells indicate that the cells are undergoing early cell death.

[0112] Figure 10 Depicts an example flow cytometric analysis involving antigen-specific CD4 + T cell responses to peptide-loaded antigen-presenting cells, followed by incubation with APCs loaded with and without PINs. IFNγ-releasing CD4 + The percentage of T cells.

[0113] Figure 11 Depicts antigen-specific CD4 IFNγ release after restimulation with mutant or wild-type peptides + Example results for the percentage of T cells.

[0114] Figure 12 Depicted is an example flow cytometric analysis showing antigen-specific CD8 +Naive T cell responses to short HTV5 peptides. Short-term and long-term induction are shown.

[0115] Figure 13 Depicted is an exemplary flow cytometric analysis showing antigen-specific CD8 + Fraction of naive T cells responding to short ME1 peptides.

[0116] Figure 14 Depicted is an example flow cytometric analysis showing antigen-specific CD8 + Naive T cell responses to short HTV3 peptides.

[0117] Figure 15 Depicted is an example flow cytometric analysis showing antigen-specific CD8 + Naive T cell responses to long CSNK1A1 peptides.

[0118] Figure 16 Depicted is an example flow cytometric analysis showing antigen-specific CD8 + Naive T cell responses to long CSNK1A1 peptides.

[0119] Figure 17 Depicted is an example flow cytometric analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short GAS7 peptides.

[0120] Figure 18 Depicted is an example flow cytometric analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short ACTN4 peptides.

[0121] Figure 19A Depicted is an example flow cytometric analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short ACTN4 peptides. Short-term induction is shown.

[0122] Figure 19B Depicted is an example flow cytometric analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8+ Naive T cell responses to short HIV3 peptides show long-term induction.

[0123] Figure 20 Describes the use of whole PBMC samples from human donors, antigen-specific CD8 + Example flow cytometric analysis of naive T cell responses to short HIV5 peptides. Short-term and long-term induction are shown.

[0124] Figure 21 Depicted is an example flow cytometric analysis showing antigen-specific CD8 + Naive T cell responses to short HIV3 peptides. Short-term induction is shown.

[0125] Figure 22 Depicted is an example flow cytometric analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short PRDX5 peptides. Very short-term and long-term induction are shown.

[0126] Figure 23 Depicted is an example flow cytometric analysis showing the use of depleted CD25 + Cells from PBMC samples of human donors, antigen-specific CD8 + Naive T cell responses to short HIV5 peptides. Short-term and long-term induction are shown.

[0127] Figure 24 A schematic diagram depicting an example of a method for producing a therapeutic T cell composition comprising expansion of memory T cells and induction of naive T cells.

[0128] Figure 25 Exemplary methods of testing the functionality, phenotype and / or function of T cells and / or T cell responses are depicted.

[0129] Figure 26 Depicted are examples of recall assays used to test T cell functionality, phenotype and / or function and / or T cell responses.

[0130] Figure 27A Depicted is an example flow cytometric analysis showing the ability to deconvolute multiplexed samples using labeled samples acquired individually or as a mixture in a recall assay. Uniquely labeled samples were resolved with no or minimal cross-contamination with other barcodes.

[0131] Figure 27BDepicted is an example flow cytometric analysis showing detection of antigen-specific CD8 in a recall assay by multimeric staining of a mixture of nine uniquely labeled samples. + T cells.

[0132] Figure 28A Depicted is an example flow cytometric analysis of a recall assay using six uniquely barcoded samples, where recall was performed with unloaded DCs and DCs loaded with neoantigens.

[0133] Figure 28B Depicts the functional number of CD4 T cells incubated with DCs loaded with the indicated concentrations of peptides in a recall response assay. + Example bar graphs of percentages of T cells. T cells containing de novo CD4 T cells were analyzed alone without barcoding or mixed with irrelevant samples. + Figure 2: Samples from two induced cultures of T cell responses. Barcoding did not alter detectable functionality. The number of functions elicited from the cells and the magnitude of the response did not change significantly with barcoding of the samples.

[0134] Figure 29A Depicted is an exemplary bar graph showing antigen-specific memory CD8 + Results of T cell responses to viral antigens. CD8 + Memory responses can be initiated from CD8 + T cells increased from 0.23% to >60%.

[0135] Figure 29B Depicts example results of a recall assay involving antigen-specific memory CD8 + T cell responses to viral antigens followed by recall using DCs loaded and unloaded with viral antigens. The figure depicts the CD8 T cells at two time points releasing the indicated cytokines. + T cell fraction.

[0136] Figure 30A Describes the de novo induction of CD4 + Example results of hit identification using response detection and functional characterization. In the example shown, four replicate cultures were induced against 10 HIV-derived epitopes that were primary targets in HIV-negative healthy donors. Antigen-specific responses were detected in 4 / 4 biological replicates, with varying magnitudes of response.

[0137] Figure 30BDescribes the de novo induction of CD4 + Example results of pooled deconvolution using detection and functional characterization of responses. Multiple responses were detected in each test replicate, with the same two epitopes (HIV#5 and HIV#7) generating the highest magnitude responses in each case.

[0138] Figure 30C Describes the de novo induction of CD4 + Responses were tested and functionally characterized to determine exemplary results for sensitivity. In pooled deconvolution analysis, similar magnitudes were observed for each response. Responses to HIV#5, HIV#6, and HIV#4 showed EC 50 0.45 μM, 0.43 μM and 9.1 μM respectively.

[0139] Figure 31 Schematic diagram depicting an example protocol for antigen-specific T cell production.

[0140] Figure 32 A schematic diagram depicting an example of a T cell induction protocol.

[0141] Figure 33 Schematic diagram depicting an example of a dendritic cell generation protocol.

[0142] Figure 34 Depicted are exemplary pMHC multimer maps showing pMHC from a patient-specific epitope SRSF1. E>K 、ARAP1 Y>H and PKDREJ G>R melanoma patients and targeting patient-specific epitopes (AASDH neoORF and seven model neoantigens: ACTN4 K>N 、CSNK1A1 S>L 、DHX40neoORF、GLI3 P>L QARSR >W 、FAM178B P>L and RPS26 P>L ) The first graph in the first and second rows indicates memory responses, and the remaining graphs indicate de novo responses.

[0143] Figure 35 Describing SRSF1 E>K and ARAP1 Y>H Example data of pMHC multimer plots before and after peptide stimulation (left panel), and pie charts depicting the functionality of neoantigen-specific T cells after re-challenge with DC loaded with neoantigen; pMHC multimer + CD8+ or CD4 + T cells were gated. The multifunctional profile of CD8+ memory, CD8+ de novo, and CD4+ de novo responses induced in melanoma patients was shown by a combination 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).

[0144] Figure 36 Delineating the specificity of memory and de novo responses induced by mutant and wild-type peptides in melanoma patients. E>K and ARAP1 Y>H Specific T cell responses were assessed using DCs loaded with mutant or wild-type neoantigen peptides at varying concentrations (X-axis: 0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM), and the expression of IFN-γ and / or TNFα and / or CD107a in total CD8+ T cells in the samples was measured (Y-axis); both responses showed significant differences from the 0 μM concentration, while there was no reactivity to the wild-type neoantigen peptide. Statistical analysis: FDR-adjusted p-values, P values: *≤0.05, ***≤0.001, ****≤0.0001.

[0145] Figure 37A Depicts the CD8 + CD107a + The cytotoxicity profile of the memory responses induced in melanoma patients was quantified by the frequency of T cells. It also depicts the target cell killing of these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The cytotoxic capacity of the induced CD8+ T cell responses was assessed by re-challenging tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The construct contained mutant or wild-type sequences, with a mutation in the center. Upregulation of CD107a on CD8+ T cells and upregulation of active caspase 3 on tumor cells were measured after co-culture. Target ratio: 3.3:1 (SRSF1 E>K ).

[0146] Figure 37B Depicts the CD8 + CD107a +Another example of the cytotoxicity profile of the memory response induced in a melanoma patient, quantified by the frequency of T cells. It also depicts the target cell killing of these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The cytotoxic capacity of the induced CD8+ T cell response was assessed by re-challenging tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The construct contained mutant or wild-type sequences, with a mutation in the center. Upregulation of CD107a on CD8+ T cells and upregulation of active caspase 3 on tumor cells were measured after co-culture. The red circle highlights the pMHC+ portion. Effector:target ratio: 5:1 (SRSF1 E>K Statistical analysis: Unpaired T test, **≤0.01, ****≤0.0001.

[0147] Figure 37C Depicts the CD8 + CD107a + Figure 3. Cytotoxicity profile of de novo responses induced in melanoma patients, quantified by the frequency of T cells. It also depicts the target cell killing of these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The cytotoxic capacity of the induced CD8+ T cell responses was assessed by re-challenging tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The construct contained mutant or wild-type sequences, with a mutation in the center. Upregulation of CD107a on CD8+ T cells and upregulation of active caspase 3 on tumor cells were measured after co-culture. Circles highlight the pMHC+ portion. Effector: target ratio: 0.66:1 (ARAP1Y>H). Statistical analysis: Unpaired T-test, **≤0.01, ****≤0.0001.

[0148] Figure 38A Depicted are the identification of neoantigen-specific CD4+ T cell responses in melanoma patients. Responses were identified based on the production of IFN-γ and TNFα (Y axis) when DCs were re-challenged with mutant neoantigen peptides (0.8 μM). MKRN1 S>L CREBBP S>L and TPCN1K>E were identified as positive responses.

[0149] Figure 38B Depicted are the mutant and wild-type peptides shown. Figure 38A The specificity of the CD4+ T cell response was depicted in the validation study. Figure 38AThe CD4 T cell responses shown in Figure 1 were challenged with mutant and wild-type neoantigen peptides at different concentrations (X-axis - 0 μM, 0.05 μM, 0.2 μM, 0.8 μM and 3.2 μM) and the total CD4+ IFNγ+ and / or TNFα+ in the samples were measured (Y-axis). Two of the CD4+ T cell responses (MKRN1 S>L and CREEBP S>L ) showed significant differences to 10 μM concentration and had no reactivity to wild-type neoantigenic peptide, but TPCN1 K>E The responses were reactive to both mutant and wild-type neoantigen peptides.Statistical analysis: FDR-adjusted p-value, P value < 0.05;

[0150] Figure 38C Depict the multifunctional spectrum of these CD4+T cell responses, as shown in the combination of 1, 2, 3 or 4 functions (for example, one or more functions are the generation of one or more factors selected from IFNγ, TNFα, CD107a and 4-1BB). The multifunctionality of the CD4+T cell responses identified was assessed by attacking DC (0.8 μm) loaded with mutant neoantigen peptides again. The percentages in the pie chart represent the percentages of functional CD4+T cells (1, 2 and / or 3 functions). The representative data depicted are generated by CD4+T cell responses after stimulation induced in the patient.

[0151] Figure 39 The functionality of the memory responses induced in two healthy donors with or without the addition of Epacadostat is depicted, as indicated by a combination of 1, 2, or 3 functions (e.g., one or more functions is the production of one or more factors selected from IFNγ, TNFα, and CD107α).

[0152] Figure 40 Depicts the de novo CD8 induction in six repeated inductions with or without the addition of Epacadostat. + Percent T cell response ("hit rate," averaged across four healthy donors).

[0153] Figure 41A Depicted are the absolute numbers of antigen-specific cells from healthy donors following induction using the T cell preparation protocol provided herein with or without the addition of PD-1 blocking antibodies.

[0154] Figure 41B Depicted are the absolute numbers of antigen-specific cells from healthy donors following induction using the T cell preparation protocol provided herein with or without the addition of PD-1 blocking antibodies.

[0155] Figure 42ADepicts the CD8 T cell compartment as a function of the de novo CD8+ T cell compartment with or without the addition of IL-12. + Frequency of multimer-positive T cells in percentage.

[0156] Figure 42B Depicted are exemplary graphical representations of the percentage of CD8+ T cells from the de novo CD8+ T cell compartment with or without the addition of IL-12.

[0157] Figure 43 Depicted are exemplary graphical representations of the percentage of hits to which naive CD8 cells responded to highly and poorly immunogenic antigens following different antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors. Also depicted are exemplary graphical representations of the absolute number of antigen-specific cells following different antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors using Mart-1 peptide or highly and poorly immunogenic antigens.

[0158] Figure 44A Depicted are exemplary flow cytometry results of CD123-positive cells following the indicated antigen-presenting cell enrichment and antigen loading protocols using PBMCs from three different healthy donors.

[0159] Figure 44B Depicted are exemplary graphical representations of the absolute numbers of the indicated CD11c+ cell subsets following three antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Treatments are: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of CD11b-expressing cells and CD19-expressing cells.

[0160] Figure 45 Depicted are exemplary graphical representations of the total number of CD8 T cells and the indicated cell ratios following three antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Treatments are: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of both CD11b-expressing and CD19-expressing cells.

[0161] Figure 46 Depicted are exemplary flow cytometry results of CD11b-positive cells following the indicated antigen-presenting cell enrichment and antigen loading protocols using PBMCs from three different healthy donors.

[0162] Figure 47Depicted are exemplary flow cytometry results of CD19-positive cells following the indicated antigen-presenting cell enrichment and antigen loading protocols using PBMCs from three different healthy donors.

[0163] Figure 48 Depicted are exemplary graphical representations of cell expansion folds following three antigen-presenting cell enrichment and antigen loading protocols: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; and CD11b- / CD19-, FLT3L treatment and depletion of both CD11b-expressing and CD19-expressing cells.

[0164] Figure 49A Depicted are exemplary data demonstrating the number of specific antigens to which naive CD8 T cells responded following three antigen-presenting cell enrichment and antigen loading protocols using PBMCs derived from healthy donors. Results were averaged across three healthy donors. Treatments were: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; and CD11b- / CD19-, FLT3L treatment and depletion of both CD11b-expressing and CD19-expressing cells. An exemplary graphical representation of the data is shown in the figure below.

[0165] Figure 49B Depicted are exemplary graphical representations of the percentage hits of naive CD8 cells to highly immunogenic (left) and poorly immunogenic (right) antigens following three antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Results were averaged across three healthy donors. Treatments were: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of both CD11b-expressing and CD19-expressing cells.

[0166] Figure 50 Depicted are exemplary graphical representations of the number of antigen-specific cells in populations activated by T cells in response to highly and poorly immunogenic antigens following three antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Treatments are: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of both CD11b-expressing and CD19-expressing cells.

[0167] Figure 51ADepicted are exemplary graphical representations of the percentage of viable cells following three antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Treatments are: basal, FLT3L treatment alone; basal+CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells; +APC, an additional PBMC fraction was added to the basal+CD11b- / CD19-, where the additional fraction was depleted of cells expressing CD3, CD19, CD11b, CD25, and CD14.

[0168] Figure 51B Depicted are exemplary graphical representations of the percentage of viable cells following three antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Treatments are: basal, FLT3L treatment alone; basal+CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells; +APC, an additional PBMC fraction was added to the basal+CD11b- / CD19-, where the additional fraction was depleted of cells expressing CD3, CD19, CD11b, CD25, and CD14.

[0169] Figure 51C Depicted are exemplary graphical representations of the percentage of viable cells following three antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Treatments are: basal, FLT3L treatment alone; basal+CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells; +APC, an additional PBMC fraction was added to the basal+CD11b- / CD19-, where the additional fraction was depleted of cells expressing CD3, CD19, CD11b, CD25, and CD14.

[0170] Figure 51D Depicted are exemplary data demonstrating the number of specific antigens to which CD8 cells from each donor responded using an exemplary antigen presenting cell enrichment protocol.

[0171] Figure 51E Depicted are exemplary graphical representations of the percentage hits to which CD8 cells responded to the indicated peptides, averaged across three healthy donors.

[0172] Figure 52A Depicted are exemplary flow cytometric analysis results from experiments in which cell populations added to the culture process at various times were stained with a membrane-permeable amine-reactive dye (e.g., carboxyfluorescein succinimidyl ester or TagIT Violet TM) mark, and then stimulate with the APC of load antigen.When being applied to second stimulation, the cell colony that has cultivated 14 days uses a kind of dye mark, and another cell colony of the new product containing the APC of load antigen and T cell uses another kind of dye mark, and then two cell colonies are mixed together and are stimulated again or expanded.Record the relative contribution to total antigen-specific T cell pool by the existence of every kind of dye and dilution rate of each in these colonies.In all cases, cell colony is cultivated 14 days (the 1st stimulation), uses a kind of dye mark, and then adds in another cell colony with another kind of dye mark that has carried out antigen stimulation 1 day in advance (standard protocol), 4 days (5 days leading start (head start)) or 6 days (7 days leading start) in advance.

[0173] Figure 52B Shown are exemplary schematics of three different T cell expansion protocols, each with two stimulations including a lead initiation of antigen loading of APCs 2, 5, or 7 days prior to contact with T cells.

[0174] Figure 52C Shows the use Figure 52B Figure 1 is an exemplary graphical representation of the time course of antigen-specific T cell numbers for three different T cell expansion protocols depicted in Figure 1. 1, standard protocol; 2, 5-day lead start; 3, 7-day lead start.

[0175] Figure 53 An exemplary graphical representation of the expansion fold of cultures treated with the indicated neoantigen peptides (pep) or neoantigen RNA is shown. PBMCs depleted of CD14 / CD25 were stimulated with antigen (peptide or mRNA encoding the antigen) after isolation or removal of CD3 lymphocytes. CD3 lymphocytes were reintroduced and stimulated for 14 days.

[0176] Figure 54 Shown are exemplary graphs of the number of multimer-positive antigen-specific cells in cultures nucleofected with the indicated neoantigen peptides (pep) or neoantigen RNA. Cultures were nucleofected in the presence or absence of T cells (-CD3). Irr, irradiated.

[0177] Figure 55 Depicts exemplary flow cytometric analysis showing antigen-specific CD8+ memory responses using viral peptides or RNA encoding the peptides and naive responses using neoantigen-encoding peptides or RNA in a short-term induction regimen. answer.

[0178] Figure 56A A schematic diagram depicts an exemplary process for generating RNA containing sequences encoding neoantigens and using them to load PBMCs and activate T cells.

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

[0180] Figure 57A A schematic diagram of an exemplary RNA concatemer construct encoding a neoantigen string is depicted.

[0181] Figure 57B Depicted in Figure 57A Schematic diagram of an exemplary arrangement of neoantigen strings in a 5'-3' direction within the construct shown.

[0182] Figure 58A A schematic diagram depicts an exemplary mRNA sequence for incorporating a 5'-cap structure into an mRNA encoding a concatenated neoantigen string for expression in PBMCs. The addition of an "A" nucleotide to the mRNA string is for Technology compatible.

[0183] Figure 58B Depicted are exemplary graphical representations of the percentage of viable cells 24 hours after expression of mRNA encoding tandem neoantigen strings with different 5'-cap structures in PBMCs.

[0184] Figure 58C Depicted are exemplary graphical representations of the total number of GFP-positive cells 24 hours after expression of mRNA encoding tandem neoantigen strings with different 5'-cap structures in PBMCs.

[0185] Figure 59A Depicted are exemplary results illustrating the use of modified nucleotides to prepare mRNA. mRNA was modified by replacing all (Full) or some (Part) uridine (U) and cytidine (C) residues within the mRNA. For example, 30% of the C residues in the Part C group were replaced with methylcytidine. The results show the effect on the expression of mRNA-encoded peptides in transfected PBMCs over time.

[0186] Figure 59B Depicted are exemplary data comparing the effects of commercial and in-house preparations of mRNA containing substituted uridine and / or cytidine on the generation of multimer-specific T cells stimulated with PBMCs loaded with mRNA.

[0187] Figure 59C Depicts exemplary data comparing Figure 59B Expansion of the generated stimulated T cells.

[0188] Figure 60ADepicted are exemplary schematics of mRNA constructs using short (9-10 amino acids, upper panel) and long (25 amino acids, lower panel) polymers for expression in cells.

[0189] Figure 60B An exemplary graph of multimer-specific CD8+ cells as a percentage of total CD8+ cells is depicted. Antigens used for multimer assays are indicated.

[0190] Figure 60C Depicted are exemplary flow cytometric analyses for detecting multimer-positive CD8+ T cells comparing APCs stimulated with short (9-10 amino acids) and long (25 amino acids) peptides and APCs containing proteins encoding the same short (9-10 amino acids) and long (25 amino acids) peptides.

[0191] Figure 61A Transfection is depicted Figures 61B-61D Schematic representation of exemplary RNA constructs for cells from the indicated experiments.

[0192] Figure 61B Depicted are exemplary graphical representations of the results from the multimer assay. Under all three conditions of PBMC treatment, RNA-transfected PBMCs were superior to peptide-loaded PBMCs in generating antigen-specific T cells. For the Gli3 antigen, a greater than 10-fold increase in multimer-positive cells was noted compared to peptide-loaded PBMCs.

[0193] Figure 61C Depicted are exemplary flow cytometry data showing the detection of Gli3 multimer-positive T cells in each of the indicated groups with and without CD3 cell depletion. Direct transfection of CD25+ PBMCs yielded more multimer-positive cells compared to PBMCs depleted of CD14 and CD25 cells or PBMCs thawed from frozen stocks.

[0194] Figure 61D Depicted are exemplary graphical representations of the results from the multimer assay. PBMCs treated overnight with FTL3L cells or CD25-depleted PBMCs were electroporated with RNA encoding a 25 amino acid length neoantigen sequence (long polymer) or an epitope length neoantigen sequence (short polymer). The percentage of neoantigen-positive cells in the culture was determined using the multimer technique.

[0195] Figure 61E Depicts from Figure 61DExemplary graphical representation of the expansion fold results of the experiment. PBMCs treated with FTL3L cells overnight or CD25-depleted PBMCs were electroporated with RNA encoding a 25 amino acid long neoantigen sequence (long polymer) or an epitope-length neoantigen sequence (short polymer). The expansion fold of cells after 26 days of culture and two stimulations is depicted.

[0196] Figure 62A Transfection is depicted Figures 62B-62D Schematic representation of exemplary RNA constructs for cells from the indicated experiments.

[0197] Figure 62B Depicted are exemplary graphical representations of the number of ACTN4- and Gli3-responsive live T cells from two donors at day 26 after maturation with the combinations indicated on the X-axis.

[0198] Figure 62C Depicted are exemplary data for the percentage of Gli3-responsive T cells from viable cells grown in the presence of the indicated maturation cocktails.

[0199] Figure 62D Depicted are exemplary flow cytometry data showing detection of Gli3 multimer-positive T cells grown in the presence of the indicated maturation cocktails.

[0200] Figure 63A Depicted are representative mass spectrometry data showing presentation of the indicated Gli3 epitopes by PBMCs using radioisotope incorporation. PBMCs were transfected with mRNA encoding multiple epitopes, including the Gli3 epitope, and peptide expression was detected using a reference peptide labeled with a heavier isotope.

[0201] Figure 63B Depicted is an exemplary graphical representation of the percentage of maximum presentation of the indicated epitopes by HLA-A02:01 over time following 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.

[0202] Figure 64A Depicted are exemplary graphical representations from recall assays showing the percent change in TNFα and / or IFNγ production (left) or the percentage of CD107a-positive cells (right) for neoantigen-specific-CD8 T cells challenged with increasing concentrations of the indicated peptides used to load APCs.

[0203] Figure 64BDepicted are exemplary graphical representations from multimeric assays showing the percent change in TNFα and / or IFNγ production (left) or the percentage of CD107a-positive cells (right) for neoantigen-specific-CD8 T cells challenged with increasing concentrations of the indicated peptides used to load APCs.

[0204] Figure 65 An exemplary Venn diagram depicting criteria considered to generate an optimal product personal T cell therapeutic using mRNA as an immunogen.

[0205] Figure 66 An exemplary flow chart showing the steps for selecting peptide sequences to prepare patient-specific T cell products is depicted.

[0206] Figure 67 An example is given for using Figure 1A The method for preparing T cells shown is advantageous in several ways for clinical approaches.

[0207] Figure 68 Depicted are exemplary representative flow cytometry data showing characterization of patient-specific T cell products produced by multiple engineering runs. Depicted are the proportion of CD3+ living cells (upper graph) and the proportion of CD8+ and CD4+ living CD3+ T cells (lower graph).

[0208] Figure 69A Depicted are exemplary graphical representations of data showing characterization of patient-specific T cell products produced by multiplex engineering runs. The percentage of multimer-positive CD8-positive cells is shown.

[0209] Figure 69B Depicted are exemplary representative flow cytometry data showing characterization of patient-specific T cell products produced by multiplex engineering runs. The percentages of multimer A-positive and multimer B-positive CD8 cells for the indicated epitopes are shown.

[0210] Figure 69C Depicted is an exemplary pie chart showing pMHC identified after re-challenge with DCs loaded with mutant neoantigens compared to unloaded DCs + CD8 + Multifunctionality of T cells.

[0211] Figure 70 Depicted are representative data indicating the CD4 + Also depicted are exemplary representative data showing changes in IFNγ and / or TNFα production in patient-specific T cell products produced by multiple engineering runs. + and / or TNFα+ and / or CD107a + CD4 + Characterization of cells.

[0212] Figure 71 Depicted is an exemplary graphical representation showing central memory T cells (T cm ), effector memory T cells (T em ), effector T cells (T eff ) and naive T cells The fraction of central memory T cells (T cm ):CD62L + CD45RA - , effector memory T cells (T em ):CD62L - CD45RA - , effector T cells (T eff ):CD62L - CD45RA + , naive T cells :CD62L+CD45RA.

[0213] Figure 72 Depicted are exemplary graphical representations of data from a multimer assay showing IFN-γ measured in samples challenged with varying concentrations of peptide-loaded DCs. + and / or TNFα + and / or CD107a + cells as a percentage of total CD8 + cells (top panel) or total CD4 + Percentage of T cells (lower panel). Peptides used for each panel are indicated.

[0214] Figure 73 Depicted are exemplary graphical representations of data indicating that CD8 + Upregulation of CD107a on T cells (top row) and active caspase 3 on tumor cells (bottom row) were measured after co-culture of A375 tumor cell lines that were not transduced or transduced with the 200 amino acid construct or loaded or not with the peptide.

[0215] Figure 74 Depicted is an exemplary graphical representation of data indicating that induced T cells are able to kill cells expressing the antigen. Neoantigen-specific T cells were tested for recognition of autologous tumors or peptide-loaded autologous tumors by a recall response assay. Readout: pMHC + (CD8 + % of ) and pMHC - (CD8+ % of IFN-γ in T cells + and / or TNFα + and / or CD107a + (Y axis). Significance was assigned using one-way ANOVA, P < 0.05.

[0216] Figure 75 An exemplary schematic diagram of the cohorts and doses used in the clinical study (NEO-PTC-01) is depicted. DETAILED DESCRIPTION

[0217] T cell therapeutics are expected to be relatively safe and well-tolerated adoptive T cell products. However, based on an assessment of the risks associated with the product, there are generally three categories of potential toxicities associated with T cell therapeutics: (a) treatment-related toxicities due to lymphodepletion, cell infusion, or cytokine release syndrome; (b) off-tumor, off-target toxicities due to the expansion of autoreactive clones or cross-reactivity of neoantigen-specific T cells; and (c) off-tumor, on-target toxicities due to presentation of neoantigens on non-tumor tissues. Novel immunotherapeutics and their uses are described herein, which are based on the discovery of neoantigens caused by mutational events unique to individual tumors. Therefore, the present disclosure described herein provides methods and protocols for creating antigen-specific immune cells, such as T cells, for treating disease.

[0218] Here we present a composition of neoantigen-reactive T cells for use in cancer immunotherapy. Although adoptive T cell therapy is a promising new approach for cancer treatment, it still requires several improvements. In general, T cells must be sufficiently cytotoxic to cancer cells, must not affect non-cancerous cells in the body, should not lose immunogenicity in the tumor environment, and should provide long-term protection. In addition, the use of virally transduced cells presents its own challenges. Therefore, achieving the right balance to obtain a therapeutically effective composition that specifically targets cancer cells, spares healthy cells, delays disease progression, leads to improvement or at least significant tumor regression, and prevents cancer recurrence requires some improvement at almost all steps of the complex process.

[0219] To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0220] Antigens are foreign substances in the body that induce an immune response. "Neoantigens" refer to a class of tumor antigens that are generated by tumor-specific alterations in proteins. Neoantigens include, but are not limited to, tumor antigens generated by, for example, substitutions in protein sequences, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, and expression of endogenous retroviral polypeptides.

[0221] A "neo-epitope" is an epitope that is not present in a reference, such as a non-diseased cell, e.g., a non-cancerous cell, or a germline cell, but is found in a diseased cell, e.g., a cancer cell. This includes situations where the corresponding epitope is found in a normal, non-diseased cell or a germline cell, but due to one or more mutations in the diseased cell, e.g., a cancer cell, the sequence of the epitope is altered, thereby generating a neo-epitope.

[0222] "Mutation" refers to a change or difference (e.g., nucleotide substitution, addition, or deletion) in a nucleic acid sequence compared to a reference nucleic acid. "Somatic mutation" can occur in any cell of the body except germ cells (sperm and eggs) and is not passed on to children. These changes can (but not always) lead to cancer or other diseases. In some embodiments, the mutation is a non-synonymous mutation. "Non-synonymous mutation" refers to a mutation (e.g., nucleotide substitution) that results in an amino acid change in the translation product, such as an amino acid substitution. When a mutation disrupts the normal phase (also referred to as "reading frame") of the gene codon periodicity, a "frameshift" occurs, resulting in the translation of a non-natural protein sequence. Different mutations in a gene can achieve the same altered reading frame.

[0223] "Antigen processing" or "processing" refers to the degradation of a polypeptide or antigen into processing products, which are fragments of the polypeptide or antigen (e.g., degradation of a polypeptide into peptides) and the association (e.g., via binding) of one or more of these fragments with MHC molecules for presentation by a cell (e.g., an antigen-presenting cell) to a specific T cell.

[0224] "Antigen presenting cell" (APC) refers to a cell that presents peptide fragments of protein antigens associated with MHC molecules on its cell surface. The term includes professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).

[0225] The term "affinity" refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA) binding peptide and class I or class II HLA, or a peptide-HLA complex and a T cell receptor (TCR)). D Refers to the dissociation constant between two members of a binding pair and has units of molar concentration. K A K is the affinity constant between two members of a binding pair and is the reciprocal of the dissociation constant. Affinity can be determined experimentally, for example by surface plasmon resonance (SPR) using a commercially available Biacore SPR unit. off K refers to the dissociation rate constant of two members of a binding pair (e.g., the dissociation rate constant of an HLA-binding peptide from class I or II HLA or a peptide-HLA complex from a TCR).on It refers to the association rate constant of two members of a binding pair (e.g., the association rate constant of an HLA-binding peptide with class I or II HLA or a peptide-HLA complex with a TCR).

[0226] Throughout this disclosure, “combined data” results may be referred to as “IC 50 Affinity can also be expressed as inhibitory concentration 50 (IC 50 ), or the concentration at which 50% of the first member of the binding pair (e.g., peptide) is displaced. Similarly, ln(IC 50 ) refers to IC 50 For example, IC 50 It can be the concentration of the test peptide at which 50% inhibition of the binding of the labeled reference peptide is observed in the binding assay. Taking into account the conditions under which the assay is run (e.g., limiting HLA protein concentration and / or labeled reference peptide concentration), these values ​​can be close to K DValues. Assays for determining binding are well known in the art and are described in detail, for example, in PCT Publications WO 94 / 20127 and WO 94 / 03205, and other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney et al., J. Immunol. 154:247 (1995); and Sette et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to binding of a reference standard peptide. Binding can also be determined using other assay systems, including those using living cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)). , ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high-throughput soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurements of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

[0227] When used to discuss epitopes, the term "derived" is a synonym for "prepared." Derived epitopes can be isolated from natural sources or can be synthesized according to standard protocols in the art. Synthetic epitopes can contain artificial amino acid residues "amino acid mimetics," such as the D isomers of naturally occurring L amino acid residues or non-natural amino acid residues such as cyclohexylalanine. Derived or prepared epitopes can be analogs of natural epitopes. The term "derived from" refers to origin or source and can include naturally occurring, recombinant, unpurified, purified or differentiated molecules or cells. For example, expanded or induced antigen-specific T cells can be derived from T cells. For example, expanded or induced antigen-specific T cells can be derived from antigen-specific T cells in a biological sample. For example, mature APCs (e.g., professional APCs) can be derived from immature APCs (e.g., immature APCs). For example, APCs can be derived from monocytes (e.g., CD14 + Monocytes). For example, dendritic cells can be derived from monocytes (e.g., CD14 + For example, APCs can be derived from bone marrow cells.

[0228] An "epitope" is a set of features of a molecule (e.g., the charge of a peptide and the primary, secondary, and tertiary peptide structures) that together form a site recognized by another molecule (e.g., an immunoglobulin, a T cell receptor, an HLA molecule, or a chimeric antigen receptor). For example, an epitope can be a group of amino acid residues that participate in recognition by a specific immunoglobulin; a major histocompatibility complex (MHC) receptor; or, in the case of T cells, those residues recognized by a T cell receptor protein and / or a chimeric antigen receptor. Epitopes can be prepared by isolation from natural sources, or they can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues—amino acid mimetics (e.g., D isomers of naturally occurring L amino acid residues or non-naturally occurring amino acid residues). Throughout the disclosure, epitopes may in some cases be referred to as peptides or peptide epitopes. In certain embodiments, there are limits on the length of the peptides disclosed herein. Length-restricted embodiments occur when a protein or peptide comprising an epitope described herein comprises a region that is 100% identical to a native sequence (i.e., a continuous series of amino acid residues). In order to avoid that the definition of an epitope is read across the entire native molecule, for example, a limit is placed on the length of any region with 100% identity to the native peptide sequence. Thus, for peptides comprising an epitope as described herein and a region with 100% identity to a native peptide sequence, the region with 100% identity to the native sequence typically has a length of less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues. In certain embodiments, an “epitope” as described herein is contained in a peptide having a region of less than 51 amino acid residues in any increment down to 5 amino acid residues that is 100% identical to the native peptide sequence; for example, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue.

[0229] "T cell epitope" refers to a peptide sequence that is bound by an MHC molecule in the form of a peptide-MHC (pMHC) complex. The peptide-MHC complex can be recognized and bound by the TCR of a T cell (e.g., a cytotoxic T lymphocyte or a T helper cell).

[0230] "T cells" include CD4 +T cells and CD8 + T cell. The term T cell also includes T helper type 1 T cells and T helper type 2 T cells. T cells can be produced by the method described in the present application for clinical application. The T cells or adoptive T cells mentioned herein, such as those used in clinical application, are cells separated from biological sources, operated in vitro and cultured and prepared into candidate drugs for specific therapies (such as cancer, such as melanoma). When the candidate drug cell passes through specific qualitative and quantitative standards applicable to clinical application, the candidate drug can be designated as a drug product. In some cases, the drug product is selected from many candidate drugs. In the context of the present application, the drug product is a T cell, more specifically, a T cell colony, or more specifically, a T cell colony with heterogeneous characteristics and subtypes. For example, as disclosed herein, a drug product can have a T cell colony comprising CD8+T cells, CD4+T cells, at least a certain amount of cells showing antigen specificity, a certain percentage of each cell showing a memory phenotype, etc.

[0231] "Immune cells" refer to cells that play a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0232] An "immunogenic" peptide or "immunogenic" epitope or "immunogenic" peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated response or a humoral response, e.g., a cytotoxic T lymphocyte (CTL) response, a helper T lymphocyte (HTL) response, and / or a B lymphocyte response. The immunogenic peptides described herein are capable of binding to an HLA molecule and then inducing a cell-mediated response or a humoral response (e.g., a CTL (cytotoxic) response or an HTL response) to the peptide.

[0233] A "protective immune response" or "therapeutic immune response" refers to a CTL and / or HTL response directed against an antigen derived from a pathogenic antigen (e.g., a tumor antigen) that in some way prevents or at least partially prevents disease symptoms, side effects, or progression. The immune response may also include antibody responses promoted by stimulating helper T cells.

[0234] "T cell receptor" ("TCR") refers to a molecule, either natural or partially or completely synthetically produced, found on the surface of T lymphocytes (T cells) that recognize antigens bound to major histocompatibility complex (MHC) molecules. The ability of a T cell to recognize antigens associated with a variety of diseases (e.g., cancer) or infectious organisms is conferred by its TCR, which is composed of either an alpha (α) chain and a beta (β) chain or a gamma (γ) and delta (δ) chain. The proteins that make up these chains are encoded by DNA, which uses a unique mechanism to generate the enormous 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). Binding of the TCR to the peptide on the APC is a central event in T cell activation.

[0235] As used herein, "chimeric antigen receptor" or "CAR" refers to an antigen binding protein, which includes an immunoglobulin antigen binding domain (e.g., immunoglobulin variable domain) and a T cell receptor (TCR) constant domain. As used herein, the "constant domain" of a TCR polypeptide includes a membrane proximal TCR constant domain, a TCR transmembrane domain, and / or a TCR cytoplasmic domain or a fragment thereof. For example, in some embodiments, CAR is a monomer comprising a polypeptide comprising an immunoglobulin heavy chain variable domain connected to a TCR β constant domain. In some embodiments, CAR is a dimer comprising: a first polypeptide comprising an immunoglobulin heavy chain or light chain variable domain connected to a TCR α or TCR β constant domain, and a second polypeptide comprising an immunoglobulin heavy chain or light chain variable domain (e.g., κ or λ variable domain) connected to a TCR β or TCR α constant domain.

[0236] "Major histocompatibility complex" or "MHC" is a gene cluster that plays a role in controlling the cellular interactions that lead to physiological immune responses. The term "major histocompatibility complex" and the abbreviation "MHC" can include any class of MHC molecules, such as MHC class I and MHC class II molecules, and refers to a complex of genes present in all vertebrates. In humans, the MHC complex is also referred to as the human leukocyte antigen (HLA) complex. Therefore, "human leukocyte antigen" or "HLA" refers to the human major histocompatibility complex (MHC) protein (see, e.g., Stites et al., Immunology, 8th ed., Lange Publishing, Los Altos, Calif. (1994)). For a detailed description of MHC and HLA complexes, see Paul, Fundamental Immunology, 3rd ed., Raven Press, New York (1993).

[0237] The major histocompatibility complex (MHC) in the genome contains genetic regions whose gene products, expressed on the cell surface, are crucial for binding and presenting endogenous and / or exogenous antigens and, therefore, for regulating immune processes. MHC proteins or molecules are crucial for signaling between lymphocytes and antigen-presenting cells or diseased cells during immune responses. MHC proteins or molecules bind peptides and present them for recognition by T-cell receptors. Proteins encoded by MHC can be expressed on the cell surface and present self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. MHC-bound peptides can be generated by proteolytic cleavage of protein antigens and represent potential lymphocyte epitopes (e.g., T-cell epitopes and B-cell epitopes). MHC can transport peptides to the cell surface, where they are 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 include α chains and β2-microglobulin (which is not part of the MHC encoded by chromosome 15). They present antigen fragments to cytotoxic T cells. MHC class II proteins contain alpha and beta chains, which present antigen fragments to helper T cells. The MHC class III region encodes other immune components, such as complement components and cytokines. MHC can be either polygenic (with several MHC class I and class II genes) or polymorphic (with multiple alleles per gene).

[0238] "Receptor" refers to a biological molecule or group of molecules that can bind to a ligand. Receptors can be used to transmit information in cells, cell formations, or organisms. A receptor comprises at least one receptor unit, for example, wherein each receptor unit can be composed of a protein molecule. The receptor has a structure that is complementary to the structure of the ligand and can be complexed with the ligand as a binding partner. Information is transmitted specifically by conformational changes in the receptor after the ligand is complexed on the cell surface. In some embodiments, a receptor should be understood to refer in particular to proteins of MHC class I and class II that can form a receptor / ligand complex with a ligand (particularly a peptide or peptide fragment of suitable length). A "ligand" refers to a molecule that has a structure that is complementary to the structure of a receptor and can form a complex with the receptor. In some embodiments, a ligand should be understood to mean a peptide or peptide fragment having a suitable length and a suitable binding motif in its amino acid sequence so that the peptide or peptide fragment can form a complex with an MHC protein such as an MHC class I or MHC class II protein. In some embodiments, "receptor / ligand complex" should also be understood to mean a "receptor / peptide complex" or a "receptor / peptide fragment complex," which includes an MHC molecule, such as an MHC class I or class II molecule, presenting the peptide or peptide fragment.

[0239] A "native" or "wild-type" sequence refers to a sequence found in nature. As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and that can be isolated from a source in nature and has not been intentionally modified by man in the laboratory is naturally occurring.

[0240] The terms "peptide" and "peptide epitope" are used interchangeably with "oligopeptide" in this specification and refer to a series of residues typically linked to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues. A "synthetic peptide" refers to a peptide obtained from a non-natural source, e.g., an artificial peptide. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. "Synthetic peptides" include "fusion proteins."

[0241] The term "motif" refers to a pattern of residues in an amino acid sequence of a defined length, for example, a peptide of less than about 15 amino acid residues in length or less than about 13 amino acid residues in length, for example, for an HLA class I motif, having about 8 to about 13 (e.g., 8, 9, 10, 11, 12, or 13) amino acid residues, and for an HLA class II motif, having about 6 to about 25 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acid residues, which is recognized by a specific HLA molecule. The motif is generally different for each HLA protein encoded by a given human HLA allele. The pattern of primary and secondary anchor residues of these motifs is different. In some embodiments, an MHC class I motif recognizes peptides of 7, 8, 9, 10, 11, 12, or 13 amino acid residues in length. In some embodiments, the MHC class II motif recognizes a peptide that is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acid residues in length. A "cross-reactive binding" peptide is one that binds to more than one member of a binding pair member class (e.g., a peptide that is bound by both a class I HLA molecule and a class II HLA molecule).

[0242] The term "residue" refers to an amino acid residue or amino acid mimetic residue that is incorporated into a peptide or protein or that is encoded by a nucleic acid (DNA or RNA) through an amide bond or amide bond mimetic. The nomenclature used to describe a peptide or protein follows conventional practice. The amino group is present on the left side (amino terminus or N-terminus) of each amino acid residue and the carboxyl group is present on the right side (carboxyl terminus or C-terminus). When referring to amino acid residue positions in a peptide epitope, the amino acid residues are numbered in the amino to carboxyl direction, with the first position being the residue at the amino terminus of the peptide or protein of which the epitope or epitope may be a part. In the general formula representing the specific embodiment selected by the present invention, unless otherwise stated, the amino terminus and carboxyl terminus groups (although not specifically shown) are the forms they present at physiological pH values. In the amino acid structural formula, each residue is typically represented by standard three-letter or single-letter nomenclature. The L- form of an amino acid residue is represented by a capital single letter or a three-letter symbol with a capital initial, while the D- form of those amino acid residues with a D- form is represented by a lowercase single letter or a lowercase three-letter symbol. However, when the three-letter symbols or full names are used without capital letters, they may also refer to L amino acid residues. Glycine has no asymmetric carbon atom and is abbreviated as "Gly" or "G". The amino acid sequences of the peptides described herein are generally represented using standard single-letter symbols. (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine).

[0243] "Conservative amino acid substitution" is an amino acid substitution in which one of the amino acid residues is replaced by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, replacing tyrosine with phenylalanine is a conservative substitution. Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate peptide function are well known in the art.

[0244] "Pharmaceutically acceptable" refers to compositions or components of compositions that are generally non-toxic, inert, and / or physiologically compatible. "Pharmaceutically acceptable excipients" or "excipients" include materials such as adjuvants, carriers, pH adjusters and buffers, tonicity adjusters, wetting agents, preservatives, and the like. A "pharmaceutically acceptable excipient" is an excipient that is pharmaceutically acceptable.

[0245] According to the present disclosure, the term "vaccine" refers to a pharmaceutical preparation (pharmaceutical composition) or product that induces an immune response (e.g., a cellular or humoral immune response) upon administration that recognizes and attacks pathogens or diseased cells, such as cancer cells. Vaccines can be used to prevent or treat disease. The terms "personalized cancer vaccine" or "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 special circumstances of the individual cancer patient.

[0246] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to nucleotide polymers of any length, including DNA and RNA (e.g., mRNA). Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, polynucleotides and nucleic acids can be in vitro transcribed mRNA. In some embodiments, the polynucleotides administered using the methods of the present invention are mRNA.

[0247] The term "isolated" or "biologically pure" refers to a material that is substantially or essentially free of components that normally accompany the material when found in its native state. Thus, an isolated peptide as described herein does not contain some or all of the substances that are normally associated with the peptide in its native environment. For example, an "isolated" epitope can be an epitope that does not include the full sequence of the protein from which the epitope is derived. For example, a naturally occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide separated from some or all of the coexisting substances in the natural system is isolated. Such a polynucleotide can be part of a vector and / or such a polynucleotide or peptide can be part of a composition and still be "isolated" because such a vector or composition is not part of its natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and also include such molecules produced synthetically. In some embodiments, an isolated polypeptide, antibody, polynucleotide, vector, cell or composition is substantially pure. As used herein, the term "substantially pure" refers to a substance that is at least 50% pure (ie, free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0248] In the context of two or more nucleic acids or polypeptides, the term "identical" or percentage "identity" refers to that two or more sequences or subsequences are identical or have a specific percentage of identical nucleotides or amino acid residues when compared and aligned (if necessary, introducing gaps) for maximum correspondence, without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and variations thereof. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaning that when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. In some embodiments, identity exists over a region of the sequence that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the full length of the compared sequences, such as the amino acid sequence of a peptide or the coding region of a nucleotide sequence.

[0249] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein when referring to a human subject.

[0250] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of a therapeutic agent that is effective for "treating" a disease or condition in a subject or mammal. A therapeutically effective amount of an agent has a therapeutic effect, thereby preventing the development of the disease or condition; slowing the development of the disease or condition; slowing the progression of the disease or condition; alleviating to some extent one or more symptoms associated with the disease or condition; reducing morbidity and mortality; improving quality of life; or a combination of these effects.

[0251] The terms "treat," ...

[0252] When used to describe a cell sample (e.g., a peripheral blood mononuclear cell (PBMC) sample), the term "depleted" refers to a cell sample in which a subpopulation of cells has been removed or depleted. For example, an immune cell sample depleted of cells expressing CD25 refers to an immune cell sample in which cells expressing CD25 have been removed or depleted. For example, one or more binding agents can be used to remove or deplete one or more cells or cell types from a sample. For example, CD14 can be depleted or removed from a PBMC sample, for example, by using an antibody that binds to CD14. + cell.

[0253] "Stimulation" refers to a response induced by the binding of a stimulatory molecule to its cognate ligand, thereby mediating a signal transduction event. For example, stimulation of a T cell may refer to the binding of the TCR of a T cell to a peptide-MHC complex. For example, stimulation of a T cell may refer to the step in Protocol 1 or Protocol 2, where PBMCs are cultured with peptide-loaded APCs.

[0254] The term "enriched" refers to a composition or fraction in which a target species has been partially purified such that the concentration of the target species is substantially greater than the level of the species naturally present in the unenriched final product. The term "induced cells" refers to cells that have been treated with an inducing compound, cell, or cell population that affects the cell's protein expression, gene expression, differentiation state, shape, morphology, viability, etc.

[0255] A "reference" can be used to correlate and / or compare the results obtained in the methods of the present disclosure with disease specimens. Typically, a "reference" can be obtained based on one or more normal specimens, particularly specimens not affected by the disease, obtained from the individual or one or more different individuals (e.g., healthy individuals), such as individuals of the same species. A "reference" can be determined empirically by testing a sufficiently large number of normal specimens.

[0256] As used herein, unless otherwise specified, a tumor is a cancerous tumor, and the terms cancer and tumor are used interchangeably throughout the document.While tumors are cancers of solid tissues, several compositions and methods described herein are in principle applicable to blood cancers, leukemias.

[0257] Overview of T-cell therapy

[0258] The generation of antigen-specific T cells by controlled ex vivo induction or expansion of T cells (e.g., autologous T cells) can provide highly specific and beneficial T cell therapies (e.g., adoptive T cell therapy). The present disclosure provides T cell preparation methods and therapeutic T cell compositions that can be used to treat subjects with cancer and other conditions, diseases, and disorders. The purpose is to expand and induce antigen-specific T cells with good phenotype and function. The present disclosure provides compositions and methods for preparing T cells that can be used for antigen-specific T cell therapy (e.g., personal or personalized T cell therapy). The T cell composition provided herein can be a personal antigen-specific T cell therapy. Figure 1 graphically illustrates an overview of the process associated with T cell therapy: on the one hand, it includes identifying cancer and cancer-specific antigens in subjects with cancer, resulting in the production of new antigenic peptides; on the other hand, preparing activated antigen-specific cells for immunotherapy and administering cell products.

[0259] Neoantigens for T cell-based therapies

[0260] Traditional antigen-targeted immunotherapy focuses on tumor-associated antigens (TAAs), antigens including cancer testis antigens (typically germline-restricted gene products that are aberrantly expressed in tumors), or antigens derived from genes that show tissue-specific expression. However, tumors also display protein products of mutated genes known as neoantigens. The number and type of mutations can be easily determined using next-generation sequencing methods, and include single amino acid missense mutations, fusion proteins, and novel open reading frames (neoORFs) ranging in length from 1 to 100 or more amino acids. Neoantigens are antigens that contain non-silent mutations in the epitope, and the same antigen is not expressed in non-cancerous cells in the same person. Mutation-based antigens are particularly valuable because they bypass central tolerance (a process that occurs during normal thymic development that removes self-reactive T cells) and exhibit exquisite tumor specificity. Each non-synonymous (i.e., protein-coding) mutation has the ability to generate a new antigen that can be recognized by the patient's T cells. T cells that recognize these neoantigens can either directly kill tumor cells or catalyze a broader immune response against the tumor. The methods described herein aim to induce and expand such neoantigen-reactive T cells in a patient-specific manner and to use these cells for adoptive cell therapy.

[0261] In some embodiments, the neoantigens used herein comprise a point mutation.

[0262] In some embodiments, the neoantigens used herein comprise a frameshift mutation.

[0263] In some embodiments, the neoantigens used herein comprise crossover mutations.

[0264] In some embodiments, the neoantigens used herein comprise an insertion mutation caused by the insertion of one or more than one nucleotide.

[0265] In some embodiments, the neoantigens used herein comprise a deletion mutation resulting from the deletion of one or more than one nucleotide.

[0266] In some embodiments, the neoantigen may be caused by an insertion-deletion (in-del) mutation.

[0267] In some embodiments, the antigen or neoantigenic peptide binds to an HLA protein (e.g., HLA class I or HLA class II). In specific embodiments, the antigen or neoantigenic peptide binds to an HLA protein with greater affinity than the corresponding wild-type peptide. In specific embodiments, the IC of the antigen or neoantigenic peptide is 50 or K D At least less than 5000 nM, at least less than 500 nM, at least less than 100 nM, at least less than 50 nM or less.

[0268] In some embodiments, the length of the antigen or neoantigenic peptide can be about 8 to about 50 amino acid residues, or about 8 to about 30, about 8 to about 20, about 8 to about 18, about 8 to about 15, or about 8 to about 12 amino acid residues. In some embodiments, the length of the antigen or neoantigenic peptide can be about 8 to about 500 amino acid residues, or about 8 to about 450, about 8 to about 400, about 8 to about 350, about 8 to about 300, about 8 to about 250, about 8 to about 200, about 8 to about 150, about 8 to about 100, about 8 to about 50, or about 8 to about 30 amino acid residues.

[0269] In some embodiments, the antigen or neoantigenic peptide can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues in length. In some embodiments, the neoantigenic peptide can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more amino acid residues in length. In some embodiments, the antigenic or neoantigenic peptide may be up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or fewer amino acid residues in length. In some embodiments, the antigenic or neoantigenic peptide may be up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or fewer amino acid residues in length.

[0270] In some embodiments, the total length of the antigen or neoantigenic peptide is at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

[0271] In some embodiments, the total length of the antigenic or neoantigenic peptide is 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.

[0272] In some embodiments, the neoantigenic peptides may have a pi value of about 0.5 to about 12, about 2 to about 10, or about 4 to about 8. In some embodiments, the neoantigenic peptides may have a pi value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the neoantigenic peptides may have a pi value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

[0273] In some embodiments, the antigen or neoantigenic peptide may have an HLA binding affinity of about 1 pM to about 1 mM, about 100 pM to about 500 μM, about 500 pM to about 10 μM, about 1 nM to about 1 μM, or about 10 nM to about 1 μM. In some embodiments, the antigen or neoantigenic peptide may have an HLA binding affinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 μM or more. In some embodiments, the antigen or neoantigenic peptide may have an HLA binding affinity of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 μΜ.

[0274] In some embodiments, the antigens or neoantigenic peptides described herein may include carriers such as those known in the art, for example, thyroglobulin, albumin such as human serum albumin, tetanus toxoid, polyamino acid residues (such as poly-L-lysine, poly-L-glutamic acid), influenza virus proteins, hepatitis B virus core protein, etc.

[0275] In some embodiments, the antigens or neoantigenic peptides described herein may be acylated by terminal -NH2 (e.g., by an alkanoyl (C1-C 20 ) or thioacetylation), terminal carboxyl amidation (e.g., ammonia, methylamine, etc.). In some embodiments, these modifications can provide sites for attachment to supports or other molecules.

[0276] In some embodiments, the antigens or neoantigen peptides described herein may include modifications such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surfactants (e.g., lipids), or may be chemically modified, such as acetylation, etc. In addition, the bonds in the peptides may be bonds other than peptide bonds, such as covalent bonds, ester bonds or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0277] In some embodiments, the antigens or neoantigenic peptides described herein may comprise substitutions to change the physical properties (e.g., stability or solubility) of the resulting peptide. For example, an antigen or neoantigenic peptide can be modified by replacing cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical properties, cysteine ​​has a tendency to form disulfide bonds and structurally alters the peptide sufficiently to reduce binding ability. Replacing C with α-aminobutyric acid not only alleviates this problem, but actually improves binding and cross-binding ability in some cases. The replacement of cysteine ​​with α-aminobutyric acid can occur on any residue of the antigen or neoantigenic peptide, such as at an anchored or non-anchored position of an epitope or analog within the peptide or at other positions of the peptide.

[0278] In some embodiments, the antigenic peptides or neoantigenic peptides described herein may comprise amino acid mimetics or non-natural amino acid residues, such as D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2-thienylalanine; D- or L-1, 2, 3 or 4-pyrenylalanine; D- or L-3-thienylalanine; D- or L-(2-pyridyl)-alanine; D- or L-(3-pyridyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenyl Glycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoro-methyl)-phenylalanine; D-ρ-fluorophenylalanine; D- or L-ρ-biphenyl-phenylalanine; D- or L-ρ-methoxybiphenylphenylalanine; D- or L-2-indole(allyl)alanine; and D- or L-alkylalanine, wherein the alkyl group can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-butyl (sec-isotyl), isopentyl or a non-acidic amino acid residue. The aromatic rings of non-natural amino acids include, for example, thiazolyl, thienyl, pyrazolyl, benzimidazolyl, naphthyl, furyl, pyrrolyl and pyridyl aromatic rings. Modified peptides with various amino acid mimetics or non-natural amino acid residues are particularly useful because they tend to show increased in vivo stability. Such peptides can also have improved shelf life or preparation properties.

[0279] In some embodiments, the peptide is contacted with immune cells to activate the cells and render them antigen-responsive.

[0280] In some embodiments, the peptide is contacted with the immune cell ex vivo.

[0281] In some embodiments, the peptide is contacted with an immune cell in a living system, such as a human.

[0282] In some embodiments, the immune cell is an antigen presenting cell.

[0283] In some embodiments, the immune cell is a T cell.

[0284] The present disclosure relates to methods for preparing T cells specific for immunogenic antigens.

[0285] The present disclosure also relates to compositions comprising antigen-specific T cells stimulated with APCs. In some embodiments, one or more antigenic peptides are loaded onto APCs, where the peptide-loaded APCs are then used to stimulate T cells to generate antigen-specific T cells. In some embodiments, the antigen is a neoantigen. In some embodiments, the APCs used for peptide loading are dendritic cells.

[0286] In some embodiments, the peptide sequence comprises a mutation that is not present in the non-cancerous cell of the subject. In some embodiments, the peptide is encoded by a gene or an expressed gene of a cancer cell of the subject. In some embodiments, the length of the peptide sequence is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500 or 10,000 or more naturally occurring amino acids.

[0287] In some embodiments, the peptide sequence is in conjunction with the protein encoded by class I HLA allele and has a length of 8-12 natural amino acids. In some embodiments, the peptide sequence is in conjunction with the protein encoded by class II HLA allele and has a length of 16-25 naturally occurring amino acids. In some embodiments, the peptide sequence comprises a plurality of antigenic peptide sequences. In some embodiments, a plurality of antigenic peptide sequences comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 antigenic peptide sequences.

[0288] In some embodiments, the antigens described herein are neoantigens. Candidate immunogenic neoantigen sequences can be identified by any suitable method known in the art. The methods of the present disclosure can be used, for example, to produce specific therapies for a subject's disease or to produce vaccines for a disease. Candidate immunogenic neoantigens can be previously identified neoantigens. In some embodiments, candidate immunogenic neoantigens may not have been previously identified. Candidate immunogenic neoantigens used in the methods and compositions described herein can be specific to a subject. In some embodiments, candidate neoantigens used in the methods and compositions described herein can be specific to multiple subjects.

[0289] In both animals and humans, mutated epitopes may be effective in inducing an immune response or activating T cells. In one embodiment, potentially immunogenic epitopes of infectious agents such as viruses can be determined in a subject. In one embodiment, potentially immunogenic mutated epitopes can be determined in a subject with a disease such as cancer. In some embodiments, potential immunogenic antigens or neoantigens for use with the methods described herein may be differentiation antigens expressed in cells of tumors and tissue types that produce them. In some embodiments, potential immunogenic antigens or neoantigens for use with the methods described herein may be cancer / germline antigens that are not expressed in another differentiated tissue. In some embodiments, potential immunogenic antigens or neoantigens for use with the methods described herein may be mutated antigens. For example, candidate immunogenic antigens or neoantigenic peptides for use with the methods described herein may comprise missense point mutations or antigens or neoantigens of fusion proteins produced by tumor-specific translocation of gene segments. In some embodiments, potential immunogenic antigens or neoantigens for use with the methods described herein may be overexpressed antigens. In some embodiments, potential immunogenic antigens or neoantigens can be found in tumors. For example, potentially immunogenic antigens or neoantigens for use with the methods described herein can include proteins whose expression is tightly regulated in cells of differentiated normal tissues.

[0290] Next generation sequencing technology can be used to determine potential immunogenic mutation epitopes by sequencing the genome or exome of tumor tissue and healthy tissue of cancer patients. For example, next generation sequencing technology can be used to sequence genes selected based on their mutation frequency and ability to act as antigens or new antigens. In one embodiment, sequencing data can be analyzed to identify potential immunogenic mutation peptides that can bind to the subject's HLA molecules. In one embodiment, a computer can be used to analyze the data. In another embodiment, the presence of antigens or new antigenic peptides in the sequence data can be analyzed. In one embodiment, it can be determined by the affinity of the potential immunogenic antigen or new antigenic peptide to the MHC molecule.

[0291] Potential immunogenic antigens or neoantigenic peptides can be determined by direct protein sequencing. For example, protein sequencing of enzymatic protein digests using multidimensional mass spectrometry techniques (e.g., tandem mass spectrometry (MS / MS)) can be used to identify potential immunogenic antigens or neoantigenic peptides for use in the methods described herein.

[0292] High-throughput methods for de novo sequencing of unknown proteins can be used to identify potentially immunogenic antigens or neoantigenic peptides. For example, high-throughput methods for de novo sequencing of unknown proteins, such as meta-shotgun protein sequencing, can be used to analyze the proteome of a subject's tumor to identify potentially immunogenic expressed neoantigens.

[0293] MHC multimers can also be used to identify potential immunogenic antigens or new antigenic peptides to identify antigen-specific T cell responses. For example, high-throughput analysis of antigen-specific T cell responses in patient samples can be performed using MHC tetramer-based screening techniques. Tetramer-based screening techniques can be used for the preliminary identification of potential immunogenic tumor-specific antigens, or alternatively can be used as a secondary screening scheme to assess which potential immunogenic antigens a patient may have been exposed to, thereby facilitating the selection of potential immunogenic antigens for use in the methods described herein.

[0294] In some embodiments, specific neoantigens are targeted for immunotherapy. In some embodiments, neoantigen peptides are synthesized. The neoantigen peptides used herein are designed so that each peptide is specific for an HLA antigen and can bind to an HLA antigen with high binding affinity and specificity. In some embodiments, the peptides used herein are designed based on a high-performance HLA binding prediction model generated by the inventors and have been described in, for example, the following patent applications / publications: WO2011143656, WO2017184590, and U.S. provisional applications 62 / 783,914 and 62 / 826,827; all of which are incorporated herein by reference. NetMHCIIpan may be the current prediction standard, but it may be considered inaccurate. Among the three class II loci (DR, DP, and DQ), data may only exist for certain common alleles of HLA-DR. Briefly, the newly generated prediction model facilitates the identification of immunogenic antigenic peptides and can be used for drug development, such as personalized medicine, and the isolation and characterization of antigen-specific T cells. The machine-learning HLA-peptide presentation prediction model includes: a plurality of predictor variables determined based on at least training data, wherein the training data comprises: sequence information of peptides presented by HLA proteins expressed in cells and identified by mass spectrometry; training peptide sequence information comprising amino acid position information, wherein the training peptide sequence information is associated with HLA proteins expressed in cells; and a function representing the relationship between the amino acid position information received as input and the likelihood of presentation generated as output based on the amino acid position information and the predictor variables. CD4+ T cell responses can have anti-tumor activity. Existing prediction methods that do not use 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) can demonstrate high CD4+ T cell response rates. It may be unclear whether these epitopes are normally presented naturally (by tumors or phagocytic DCs). Therefore, it is hoped that high CD4+ T cell responses can be translated into therapeutic efficacy by improving the identification of naturally presented class II epitopes. The role of gene expression, enzymatic cleavage, and pathway / localization preferences may not yet be robustly quantified. It may not be clear whether autophagy (class II presentation by tumor cells) or phagocytosis (class II presentation of tumor epitopes by APCs) is the more relevant pathway, although most existing MS data may be presumed to be derived from autophagy. There may be different data generation methods to learn the rules of class II presentation, including field standards and the proposed method. Field standards could include affinity measurements, which may be the basis of the NetMHCIIpan predictor, provide low throughput and require radioactive reagents, and it omits the role of processing.The new approach includes mass spectrometry, where data from cell lines / tissues / tumors can help determine the processing rules of autophagy (most of which have already been published), while monoallelic MS can allow for the determination of allele-specific binding rules (assuming that multi-allelic MS data is too complex for effective learning). The newly generated prediction method includes training a machine learning HLA-peptide presentation prediction model, where the training includes inputting into the HLA-peptide presentation prediction model, using a computer processor, amino acid position information sequences of HLA-peptides isolated from one or more HLA-peptide complexes from cells expressing an HLA class II allele; the machine learning HLA-peptide presentation prediction model includes: a plurality of predictor variables determined based on at least training data, the training data comprising: sequence information of peptides presented by HLA proteins expressed in the cells and identified by mass spectrometry; training peptide sequence information comprising amino acid position information of the training peptides, wherein the training peptide sequence information is associated with the HLA proteins expressed in the cells; and amino acid sequences representing the amino acid positions received as input. A function of the relationship between position information and a presentation probability generated as an output based on the amino acid position information and the predictor variables. In some embodiments, the presentation model has a positive predictive value of at least 0.25 at a recall of 0.1%-10%. In some embodiments, the presentation model has a positive predictive value of at least 0.4 at a recall of 0.1%-10%. In some embodiments, the presentation model has a positive predictive value of at least 0.6 at a recall of 0.1%-10%. In some embodiments, the mass spectrometry method is monoallelic mass spectrometry. In some embodiments In some embodiments, the peptides are presented by HLA proteins expressed in cells via autophagy. In some embodiments, the peptides are presented by HLA proteins expressed in cells via phagocytosis. In some embodiments, the quality of the training data is improved by using multiple quality metrics. In some embodiments, the multiple quality metrics include removal of common contaminant peptides, high-scoring peak intensity, high score, and high mass accuracy. In some embodiments, the scored peak intensity is at least 50%. In some embodiments, the scored peak intensity is at least 70%. In some embodiments, the peptides presented by HLA proteins expressed in cells are peptides presented by a single immunoprecipitated HLA protein expressed in the cells. In some embodiments, the multiple predictor variables include a peptide-HLA affinity predictor variable. In some embodiments, the multiple predictor variables include a source protein expression level predictor variable. In some embodiments, the multiple predictor variables include a peptide cleavage predictor variable. In some embodiments, the peptides presented by HLA proteins include peptides identified by searching a peptide database using an inverse database search strategy. In some embodiments, the HLA proteins are HLA-DR and HLA-DP or HLA-DQ proteins.In some embodiments, the HLA protein is an HLA-DR protein selected from HLA-DR and HLA-DP or HLA-DQ proteins. In some embodiments, the HLA protein is an HLA-DR protein selected from the group consisting of HLA-DPB1*01:01 / HLA-DPA1*01:03, HLA-DPB1*02:01 / HLA-DPA1*01:03, HLA-DPB1*03:01 / HLA-DPA1*01:03, HLA-DPB1*04:01 / HLA-DPA1*01:03, HLA-DPB1*04:02 / HLA-DPA1*01:03, HLA-DPB1*06:01 / HLA-DPA1*01:03, HLA -DQB1*02:01 / HLA-DQA1*05:01,HLA-DQB1*02:02 / HLA-DQA1*02:01, HLA-DQB1*06:02 / HLA-DQA1*01:02,HLA-DQB1*06:04 / HLA-D QA1*01:02, HLA-DRB1*01:01, HLA-DRB1*01:02, HLA-DRB1*03:01, HLA-DRB1*03:02, HLA-DRB1*04:01, HLA-DRB1*04:02, HLA-DRB 1*04:03, HLA-DRB1*04:04, HLA-DRB1*04:05, HLA-DRB1*04:07, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*08:02, HLA-DRB1* 08:03, HLA-DRB1*08:04, HLA-DRB1*09:01, HLA-DRB1*10:01, HLA-DRB1*11:01, HLA-DRB1*11:02, HLA-DRB1*11:04, HLA-DRB1*12 :01, HLA-DRB1*12:02, HLA-DRB1*13:01, HLA-DRB1*13:02, HLA-DRB1*13:03, HLA-DRB1*14:01, HLA-DRB1*15:01, HLA-DRB1*15:02, HLA-DRB1*15:03, HLA-DRB1*16:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB3*03:01, HLA-DRB4*01:01, and HLA-DRB5*01:01). In some embodiments, the peptides presented by the HLA protein include peptides identified by comparing the MS / MS spectrum of the HLA-peptide to the MS / MS spectra of one or more HLA-peptides in a peptide database.

[0295] In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a readthrough mutation, and a gene fusion mutation.

[0296] In some embodiments, the peptides presented by the HLA protein have a length of 15-40 amino acids. In some embodiments, the peptides presented by the HLA protein include peptides identified by: (a) isolating one or more HLA complexes from a cell line expressing a single HLA class II allele; (b) isolating one or more HLA-peptides from the one or more isolated HLA complexes; (c) obtaining MS / MS spectra of the one or more isolated HLA-peptides; and (d) obtaining peptide sequences corresponding to the MS / MS spectra of the one or more isolated HLA-peptides from a peptide database; wherein the sequences of the one or more isolated HLA-peptides are identified from the one or more sequences obtained in step (d).

[0297] Various antigenic peptides can be used to induce or expand T cells. Various antigenic peptides can be used to activate antigen-presenting cells (APCs), which in turn activate T cells by contacting them with antigen-loaded APCs.

[0298] In some embodiments, the peptide comprises a mutation selected from the group consisting of: (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a read-through mutation, (E) a gene fusion mutation, and combinations thereof. In some embodiments, the peptide comprises a point mutation and binds to an HLA protein of the subject with greater affinity than the corresponding wild-type peptide.

[0299] In some embodiments, the peptides are expressed at an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 In some embodiments, the peptide binds to an HLA protein of the subject with an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or 10 nM. 50 or K D In some embodiments, each peptide binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, the TCR of the induced or expanded antigen-specific T cells has an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K D In some embodiments, the TCR binds to the peptide-HLA complex with an IC of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. 50 or K DIn some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in a non-cancerous cell of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or an expressed gene of a cancer cell of the subject.

[0300] In some embodiments, the peptide is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 naturally occurring amino acids in length. In some embodiments, the peptide binds to a protein encoded by a class I HLA allele and has a length of 8-12 naturally occurring amino acids. In some embodiments, the peptide binds to a protein encoded by a class II HLA allele and has a length of 16-25 naturally occurring amino acids. In some embodiments, the peptide comprises a plurality of peptides. In some embodiments, the plurality of peptides comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 or more antigenic peptides.

[0301] In some aspects, the present disclosure provides peptides (eg, peptides having tumor-specific mutations, viral peptides, or peptides associated with non-cancerous diseases) identified using the methods briefly described above, or polynucleotides encoding the peptides.

[0302] In some embodiments, optical methods are used to select or identify immunogenic antigens. In some embodiments, barcoded probes are used to select or identify immunogenic antigens. In some embodiments, barcoded probes comprising a target-specific region and a barcoded region are used to select or identify immunogenic antigens. In some embodiments, the target-specific region comprises a nucleic acid sequence that hybridizes to or has at least about 90%, 95%, or 100% sequence complementarity with a nucleic acid sequence of a target polynucleotide.

[0303] Preparation of activated antigen-specific T cells

[0304] Methods for stimulating T cells are provided herein. For example, the methods provided herein can be used to stimulate antigen-specific T cells. The methods provided herein can be used to induce or activate T cells. For example, the methods provided herein can be used to expand activated T cells. For example, the methods provided herein can be used to induce naive T cells. For example, the methods provided herein can be used to expand antigen-specific CD8 + For example, the methods provided herein can be used to expand antigen-specific CD4 + For example, the methods provided herein can be used to expand antigen-specific CD8 T cells with a memory phenotype. + T cells. For example, the therapeutic composition may comprise antigen-specific CD8+ T cells. For example, the therapeutic composition may comprise antigen-specific memory T cells.

[0305] T cells can be activated ex vivo using a composition comprising a neoantigenic peptide or a polynucleotide encoding a neoantigenic peptide.

[0306] T cells can be activated ex vivo using a composition comprising antigen-loaded antigen-presenting cells.

[0307] In some embodiments, the APCs and / or T cells are derived from a biological sample obtained from a subject.

[0308] In some embodiments, the APCs and / or T cells are derived from a biological sample as peripheral blood mononuclear cells (PBMCs).

[0309] In some embodiments, FLT3L is administered to the subject prior to obtaining the biological sample for preparing APCs and / or T cells.

[0310] In some embodiments, the APCs and / or T cells are derived from a biological sample as a leukapheresis sample.

[0311] In some embodiments, antigen presenting cells are first loaded with neoantigen peptides in vitro and used to prepare neoantigen activated T cells. In some embodiments, provided herein are compositions comprising T cells stimulated by APCs such as preloaded antigen peptides. The composition may comprise an immune cell colony comprising T cells from a sample (e.g., a biological sample), wherein the T cells include T cells stimulated by APCs. In some embodiments, mRNA encoding one or more neoantigen peptides is introduced into APCs for expression of neoantigen peptides. Such APCs are used to stimulate or activate T cells.

[0312] In some embodiments, the biological sample comprises at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5% of at least one antigen-specific T cell in the composition. In some embodiments, the biological sample comprises less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or less than 10% of the total cell count in a biological sample derived from peripheral blood or leukapheresis. In some embodiments, the biological sample comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% of the total cell count in the biological sample derived from peripheral blood or leukapheresis.

[0313] In some embodiments, the biological sample comprises antigen-naive T cells. In some embodiments, the biological sample comprises greater than about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total cell count in the biological sample derived from peripheral blood or leukapheresis.

[0314] In some embodiments, at least one antigen-specific CD8 + The percentage of T cells is less than about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% in a biological sample derived from peripheral blood or leukapheresis. In some embodiments, at least one antigen-specific CD4 +The percentage of T cells is at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% of a biological sample derived from peripheral blood or leukapheresis.

[0315] In some embodiments, the percentage of the at least one antigen-specific T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total immune cells. In some embodiments, the at least one antigen-specific CD8 T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total immune cells. + The percentage of T cells is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of the total immune cells. In some embodiments, at least one antigen-specific CD4 + In some embodiments, the percentage of T cells in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total immune cells. In some embodiments, the percentage of antigen-specific T cells in the biological sample is at most about 0.5%. In some embodiments, the percentage of neoantigen-specific CD8 T cells in the biological sample is at most about 0.5%. + The percentage of T cells is at most about 0.5%. In some embodiments, the antigen-specific CD4 + The percentage of T cells is at most about 0.5% of the biological sample.

[0316] Preparation of neoantigen-loaded APCs

[0317] In some embodiments, the composition includes an immune cell colony that has been incubated with one or more cytokines, growth factors or ligands such as ligands bound to the cell surface receptors of APC 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-α, R848, LPS, ss-rna40 and poly-IC. In some embodiments, the composition includes an immune cell colony that has been incubated with one or more APCs or APC products. For example, the composition can include an immune cell colony that has been incubated with APCs or cytokines, growth factors and / or ligand-stimulated APCs or cytokines, growth factors and / or ligand-stimulated APC products. For example, the composition can include an immune cell colony that has been incubated with APCs or cytokine-stimulated APC products stimulated by one or more cytokines. For example, a composition may comprise a population of immune cells that has been incubated with one or more growth factor-stimulated APCs or a preparation of growth factor-stimulated APCs. For example, a composition may comprise a population of immune cells that has been incubated with one or more ligand-stimulated APCs or a preparation of ligand-stimulated APCs.

[0318] In some embodiments, the APC is an autologous APC, an allogeneic APC, or an artificial APC.

[0319] Immune cells are characterized by cell surface molecules. In some embodiments, immune cells are preferably selected based on cell surface markers, for example, by selecting from a biological sample using antibodies that can bind to cell surface receptors. In some embodiments, some cells are negatively selected to enrich for one or more cell types that do not express the cell surface molecules for which they were negatively selected.

[0320] In some embodiments, antigen presenting cells (APCs) are prepared from a biological sample by selecting from APCs or precursor cells, which can be cultured in the presence of neoantigenic peptides to generate neoantigen-loaded APCs for activating T cells. Some relevant cell surface markers for selecting and / or enriching a group of cells are described below.

[0321] CD1 (Cluster of Differentiation 1) is a family of glycoproteins expressed on the surface of various human antigen-presenting cells. They are associated with class I MHC molecules and are involved in presenting lipid antigens to T cells.

[0322] CD11b or integrin αM (ITGAM) is a heterodimeric integrin α-Mβ-2 (α MITGAM is also known as CR3A and cluster of differentiation molecule 11b (CD11b). M The second chain of β2 is the common integrin β2 subunit called CD18, so integrin α M β2 belongs to the β2 subfamily (or leukocyte) integrin. α M β2 is expressed on the surface of many leukocytes involved in the innate immune system, including monocytes, granulocytes, macrophages, and natural killer cells. It mediates inflammation by regulating leukocyte adhesion and migration and participates in various immune processes such as phagocytosis, cell-mediated cytotoxicity, chemotaxis, and cell activation. It participates in the complement system due to its ability to bind inactivated complement component 3b (iC3b). Integrin α M The ITGAM (α) subunit of β2 is directly involved in inducing cell adhesion and spreading, but cannot mediate cell migration in the absence of the β2 (CD18) subunit.

[0323] CD11c, also known as integrin αX (complement component 3 receptor subunit 4) (ITGAX), is the gene encoding CD11c. CD11c is the integrin αX chain protein. Integrins are heterodimeric integral membrane proteins composed of α and β chains. This protein combines with the β2 chain (ITGB2) to form the leukocyte-specific integrin known as inactivated C3b (iC3b) receptor 4 (CR4). The αXβ2 complex appears to overlap with the properties of the αMβ2 integrin in terms of neutrophil and monocyte adhesion to stimulated endothelial cells and phagocytosis of complement-coated particles. CD11c is a type I transmembrane protein found at high levels on most human dendritic cells, but also on monocytes, macrophages, neutrophils, and some B cells, where it induces cell activation and helps trigger the neutrophil respiratory burst. It is expressed in hairy cell leukemia, acute nonlymphocytic leukemia, and some B-cell chronic lymphocytic leukemias.

[0324] CD14 is a surface antigen preferentially expressed on monocytes / macrophages. It collaborates with other proteins to mediate the innate immune response to bacterial lipopolysaccharide (LPS). Alternative splicing results in multiple transcript variants encoding the same protein. CD14 exists in two forms: one anchored to the membrane by a glycosylphosphatidylinositol tail (mCD14) and the other a soluble form (sCD14). Soluble CD14 is either produced after shedding of mCD14 (48 kDa) or secreted directly from intracellular vesicles (56 kDa). CD14 acts as a co-receptor (along with Toll-like receptors TLR4 and MD-2) for detecting bacterial lipopolysaccharide (LPS). CD14 can only bind LPS in the presence of lipopolysaccharide binding protein (LBP). Although LPS is considered its primary ligand, CD14 also recognizes other pathogen-associated molecular patterns, such as lipoteichoic acid.

[0325] CD25 is expressed by conventional T cells upon stimulation and has been shown to be expressed only by CD4 + CD25 hi T cells are "suppressors."

[0326] In some embodiments, the APC comprises a dendritic cell (DC). In some embodiments, the APC is derived from a CD14 + In some embodiments, APCs can be obtained from skin, spleen, bone marrow, thymus, lymph nodes, peripheral blood, or umbilical cord blood. In some embodiments, CD14 + The monocytes are derived from a biological sample from a subject comprising PBMCs. For example, CD14 can be isolated, enriched, or purified from a biological sample from a subject comprising PBMCs. + In some embodiments, CD14 is stimulated with one or more cytokines or growth factors. + In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, R848, LPS, ss-RNA40, poly I:C, or a combination thereof. In some embodiments, CD14 + The monocytes are derived from a second biological sample comprising PBMCs.

[0327] In some embodiments, the isolated APC population can be enriched or substantially enriched. In some embodiments, the isolated APC population is at least 30%, at least 50%, at least 75%, or at least 90% homogeneous. In some embodiments, the isolated APC population is at least 60%, at least 75%, or at least 90% homogeneous. APCs, such as APCs, can include, for example, APCs derived from mononuclear dendritic precursors in culture, as well as endogenously derived APCs present in tissues such as peripheral blood, umbilical cord blood, skin, spleen, bone marrow, thymus, and lymph nodes.

[0328] APCs and cell populations substantially enriched for APCs can be isolated by methods further provided herein. The methods generally include obtaining a cell population comprising APC precursors, differentiating the APC precursors into immature or mature APCs, and further comprising isolating APCs from the differentiated immature or mature APC populations.

[0329] APC precursor cells can be obtained by methods known in the art. APC precursors can be isolated by, for example, density gradient separation, fluorescence activated cell sorting (FACS), immune cell separation techniques such as panning, complement lysis, rosetting, magnetic cell separation techniques, nylon wool separation, and combinations of such methods. Methods for immunoselection of APCs include, for example, the use of antibodies directed against cell surface markers associated with APC precursors, such as anti-CD34 and / or anti-CD14 antibodies coupled to a substrate.

[0330] Enriched APC precursor populations can also be obtained. Methods for obtaining such enriched precursor populations are known in the art. For example, enriched APC precursor populations can be isolated from a tissue source by selectively removing cells adhering to a substrate. Using tissue sources such as bone marrow or peripheral blood, commercially processed plastic substrates (e.g., beads or magnetic beads) can be used to remove adherent mononuclear cells from cell preparations to obtain a non-adherent APC precursor enriched population.

[0331] Monocyte APC precursors can also be obtained from tissue sources by using an APC precursor adhesion substrate. For example, peripheral blood leukocytes separated by, for example, leukapheresis are contacted with a mononuclear APC precursor adhesion substrate having a high surface area: volume ratio, and the adhered mononuclear APC precursors are separated. In another embodiment, the coupled substrate can be a granular or fibrous substrate having a high surface: volume ratio, such as microbeads, microcarrier beads, pellets, particles, powders, capillaries, microporous membranes, etc. In addition, the granular or fibrous substrate can be glass, polystyrene, plastic, glass-coated polystyrene microbeads, etc.

[0332] APC precursors can also be cultured in vitro for differentiation and / or expansion. Methods for differentiation / expansion of APC precursors are known in the art. Typically, expansion can be achieved by culturing the precursors in the presence of at least one cytokine that induces APC (e.g., dendritic cells) differentiation / proliferation. Typically, these cytokines are granulocyte colony-stimulating factor (G-CSF) or granulocyte / macrophage colony-stimulating factor (GM-CSF). In addition, other reagents can be used to inhibit the proliferation and / or maturation of non-APC cell types in the culture, thereby further enriching the APC precursor population. Typically, such reagents include cytokines, for example, IL-13, IL-4, or IL-15, etc.

[0333] The isolated APC precursor population is cultured and differentiated to obtain immature or mature APCs. Suitable tissue culture media include, for example, but are not limited to RPMI 1640, DMEM, X-VIVO, etc. Tissue culture medium is usually supplemented with amino acids, vitamins, divalent cations and cytokines to promote the differentiation of precursors to APC phenotype. Typically, the cytokines that promote differentiation are GM-CSF and / or IL-4.

[0334] In addition, the culture of APC precursors can include plasma to promote the development of APCs during the expansion, differentiation, and maturation of APC phenotypes. Typical plasma concentrations are approximately 5%. In addition, for example, in the case of isolating APC precursors by adhesion to a substrate, plasma can be included in the culture medium during the adhesion step to promote the early development of CD14 + Typical plasma concentrations during adhesion are approximately 1% or higher.

[0335] The mononuclear cell APC precursors can be cultured for any suitable time. In certain embodiments, the suitable culture time for differentiating the precursors into immature APCs can be from about 1 day to about 10 days, such as from about 4 days to about 7 days. The differentiation of the precursors into immature APCs can be determined by methods known to those skilled in the art, such as by the presence or absence of cell surface markers (e.g., CD11c + , CD83 low, CD86 - / Low, HLA-DR + ) to monitor the differentiation of immature APCs from precursors. Immature APCs can also be cultured in appropriate tissue culture media to maintain the immature APCs in a state of further differentiation or antigen uptake, processing, and presentation. For example, immature APCs can be maintained in the presence of GM-CSF and IL-4.

[0336] In some embodiments, APC precursors can be isolated before differentiation. In some embodiments, the isolated population can be enriched or substantially enriched for APC precursors. In some embodiments, APC precursors are isolated using a CD14-specific probe. In an exemplary embodiment, cells expressing CD14 are detected by FACS using a CD14-specific probe directly conjugated to a fluorescent molecule (e.g., FITC or PE), or using an unlabeled antibody specific for CD14 and a labeled second antibody specific for the first antibody. CD14 can also be isolated by FACS sorting. + cells and CD14 低 and CD14 - Cell separation. CD14 can be determined by, for example, CD14 staining on PBMC-derived monocytes. 高 Positive gating. Typically, a CD14-specific binding agent is, for example, an anti-CD14 antibody (e.g., a monoclonal or antigen-binding fragment thereof). Many anti-CD14 antibodies suitable for use in the present invention are well known to those skilled in the art, and many are commercially available. After separation, differentiation into immature APCs (CD14 negative) can occur.

[0337] In another embodiment, a CD14-specific probe is coupled to a substrate and CD14 is isolated by affinity selection. + cells. This will include CD14 + The cell population is exposed to the coupled substrate and CD14 + Cells adhere specifically. Non-adherent CD14 - The cells are then eluted to obtain a substantially isolated cell population enriched for APC precursors. The CD14-specific probe can be, for example, an anti-CD14 antibody. The substrate can be, for example, a commercially available tissue culture plate or beads (e.g., glass or magnetic beads). Methods for affinity separation of cell populations using substrate-coupled surface marker-specific antibodies are well known.

[0338] During the culture process, immature APCs can be optionally exposed to predetermined antigens. Suitable predetermined antigens may include any antigen for which T cell regulation is desired. In one embodiment, immature APCs are cultured in the presence of prostate-specific membrane antigen (PSMA) for cancer immunotherapy and / or tumor growth inhibition. Other antigens may include, for example, bacterial cells, viruses, partially purified or purified bacterial or viral antigens, tumor cells, tumor-specific or tumor-associated antigens (e.g., tumor cell lysates, tumor cell membrane preparations, antigens isolated from tumors, fusion proteins, liposomes, etc.), recombinant cells expressing antigens on their surfaces, autoantigens, and any other antigens. Any antigen can also be presented as a peptide or recombinantly produced protein or portion thereof. After contact with the antigen, the cells can be cultured for any suitable time to allow antigen uptake and processing to expand the antigen-specific APC population, etc.

[0339] For example, in one embodiment, immature APCs can be cultured after antigen uptake to promote the maturation of immature APCs into mature APCs that present antigens in the context of MHC molecules. Methods for APC maturation are known. For example, such maturation can be performed by culturing in the presence of known maturation factors such as cytokines (e.g., TNF-α, IL-1β, or CD40 ligands), bacterial products (e.g., LPS or BCG), etc. The maturation of immature APCs into mature APCs can be monitored by methods known in the art, for example, by measuring the presence or absence of cell surface markers (e.g., upregulation of CD83, CD86, and MHC molecules) or using, for example, oligonucleotide arrays to detect the expression of mature APC-specific mRNA or protein.

[0340] Optionally, immature APCs can be cultured in a suitable tissue culture medium to expand the cell population and / or maintain the immature APCs in a state for further differentiation or antigen uptake. For example, immature APCs can be maintained and / or expanded in the presence of GM-CSF and IL-4. Immature APCs can also be cultured in the presence of anti-inflammatory molecules such as anti-inflammatory cytokines (e.g., IL-10 and TGF-β) to inhibit the maturation of immature APCs.

[0341] In another aspect, the isolated APC population is enriched for mature APCs. An isolated mature APC population can be obtained by culturing a differentiated immature APC population in the presence of maturation factors (e.g., bacterial products and / or proinflammatory cytokines) as described above, thereby inducing maturation. Immature APCs can be isolated by removing CD14+ cells.

[0342] According to another aspect of the present invention, for example, APC can be preserved by freezing before or after exposure to a suitable antigen. Useful cryopreservatives include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, polyvinyl pyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, isoerythritol, D-ribitol, D-mannitol, D-sorbitol, inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, monoacetin, and inorganic salts. A controlled slow cooling rate may be critical. Different cryoprotectants and different cell types typically have different optimal cooling rates. The heat of the melting stage, in which water becomes ice, should generally be minimal. The cooling process can be carried out using, for example, a programmable freezing device or a methanol bath program. Programmable freezing equipment allows determination of the optimal cooling rate and contributes to standard, repeatable cooling. Programmable rate-controlled freezers, such as Cryomed or Planar, allow the freezing protocol to be adjusted to a desired cooling rate curve.

[0343] After complete freezing, the APCs can be quickly transferred to a long-term cryogenic storage container. In a typical embodiment, the sample can be cryogenically stored in liquid nitrogen (-196°C) or its vapor (-165°C). The considerations and procedures for the handling, cryopreservation, and long-term storage of hematopoietic stem cells, particularly those from bone marrow or peripheral blood, are largely applicable to the APCs of the present invention.

[0344] Frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37-41° C.) and cooled immediately after thawing. In order to prevent the cells from clumping when thawing, it may be necessary to treat the cells. In order to prevent clumping, various procedures can be used, including but not limited to adding DNA enzymes, low molecular weight dextran and citrate, hydroxyethyl starch, etc. before and / or after freezing. If the cryoprotectant is toxic to the human body, it should be removed before the thawed APC is used therapeutically. One method of removing the cryoprotectant is to dilute it to a very small concentration. Once the frozen APCs have been melted and recovered, they can be used to activate T cells as described herein for non-frozen APCs.

[0345] On the one hand, the composition for T cell activation includes an immune cell colony that has exhausted one or more types of immune cells.For example, the composition can include an immune cell colony that has exhausted expression of one or more proteins, such as one or more cell surface receptors.In some embodiments, the composition includes an immune cell colony from a biological sample, and the biological sample includes at least one antigen-specific T cell, and the antigen-specific T cell includes a T cell receptor (TCR) with specificity for at least one antigen peptide sequence, wherein the amount of the immune cell expressing CD14 and / or CD25 in the colony is proportionally different from the amount of the immune cell expressing CD14 and / or CD25 in the biological sample.For example, the composition can include an immune cell colony from a biological sample, and the biological sample includes at least one antigen-specific T cell, and the antigen-specific T cell includes a T cell receptor (TCR) with specificity for at least one antigen peptide sequence, wherein the amount of the immune cell expressing CD14 in the colony is proportionally different from the amount of the immune cell expressing CD14 in the biological sample. For example, a composition may comprise a population of immune cells from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the amount of immune cells expressing CD25 in the population is proportionally different from the amount of immune cells expressing CD25 in the biological sample. For example, a composition may comprise a population of immune cells from a biological sample comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the amount of immune cells expressing CD14 and CD25 in the population is proportionally different from the amount of immune cells expressing CD14 and CD25 in the biological sample. For example, a composition may comprise a population of immune cells from a biological sample, wherein the amount of immune cells expressing CD14 and CD25 in the population is proportionally less than the amount of immune cells expressing CD14 and CD25 in the biological sample.

[0346] The present invention provides a method for preparing a cell composition for cancer immunotherapy, which comprises: I. preparing antigen-loaded antigen-presenting cells (APCs), which comprises: (a) obtaining peripheral blood mononuclear cells (PBMCs) from a subject pretreated with fms-like tyrosine kinase 3 ligand (FLT3L); (b) contacting the PBMCs with the following substances ex vivo: (i) a plurality of cancer neoantigen peptides, or one or more polynucleotides encoding the plurality of cancer neoantigen peptides, and wherein each cancer neoantigen peptide or a portion thereof is bound to a protein encoded by an HLA allele expressed in the subject, (ii) a stimulator for activating cells, (iii) an agent for promoting cell growth and maintenance ex vivo, thereby obtaining a cell population, and (iv) an agent for reducing or depleting CD11b+ cells from the cell population, so as to obtain CD11b 低 or CD11b-depleted antigen-loaded APC; II. separating T cells from the CD11b 低 or CD11b-depleted APCs loaded with antigens ex vivo; III. preparing antigen-primed T cells for use in cell compositions for cancer immunotherapy.

[0347] Provided herein is an improved method for preparing tumor antigen-specific T cells ex vivo, the method comprising: (a) depleting CD14+ cells and / or CD25+ cells from an immune cell population comprising antigen presenting cells (APCs) and T cells, thereby forming a CD14 and / or CD25-depleted immune cell population comprising a first population of APCs and T cells, wherein the immune cell population is derived from a biological sample of a human subject; (b) incubating the APCs and the first population of T cells from step (a) for a first period of time in the presence of: FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) comprising at least 1% to 2% of the immune cell population. (b) a polypeptide encoding at least one tumor antigen epitope sequence expressed by a cancer cell of a human subject having cancer, or (b) a polynucleotide encoding the polypeptide; thereby forming a cell population comprising stimulated T cells; (c) expanding the stimulated T cells from step (b) to form an expanded cell population comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising the following components: (i) the at least one tumor antigen epitope sequence from step (b)(ii), and (ii) an MHC protein expressed by a cancer cell or APC of a human subject from (b)(ii). Provided herein is a method comprising administering the expanded cell population from (c) to the human subject, wherein the expanded cell population from step (c) comprises 1x10 8 Up to 1x10 11 Total cells.

[0348] In some embodiments, the subject is pretreated with FLT3L at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week prior to isolation of PBMCs or leukapheresis. In some embodiments, the subject is pretreated with FLT3L at least about 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks prior to isolation of PBMCs or leukapheresis.

[0349] In some embodiments, the cell colony is enriched for CD11c+ cells. In some embodiments, the APC loaded with antigens comprises dendritic cells (DC). In some embodiments, the APC loaded with antigens comprises plasmacytoid dendritic cells (pDC). In some embodiments, the APC loaded with antigens comprises CD1c+DC. In some embodiments, the APC loaded with antigens comprises CD141+DC. In some embodiments, the cell colony comprises macrophages. In some embodiments, the method further comprises reducing or consuming CD19+ cells from the cell colony for activating or enriching neoantigen-activated T cells. In some embodiments, the method further comprises reducing or consuming CD11b+ and CD19+ cells from the cell colony for activating or enriching neoantigen-activated T cells.

[0350] In some embodiments, the method further comprises reducing or depleting CD14+ cells from the cell population for the preparation and enrichment of antigen-activated T cells. In some embodiments, the method further comprises reducing or depleting CD25+ cells from the cell population for the preparation and enrichment of antigen-activated T cells. In some embodiments, the method further comprises reducing or depleting one or more of CD19+, CD14+, CD25+, or CD11b+ cells from the cell population for the activation or enrichment of neoantigen-activated T cells.

[0351] In some embodiments, the stimulus used to activate the cells comprises FL3TL.

[0352] In some embodiments, agents that promote the growth and maintenance of cells ex vivo include growth factors, cytokines, amino acids, supplements, or combinations thereof.

[0353] In some embodiments, antigen-loaded APCs can stimulate T cells for 2, 3, 4, 5, 6, or 7 days.

[0354] In some embodiments, each of the plurality of cancer neoantigenic peptides is 8-30 amino acids in length.

[0355] In some embodiments, each of the plurality of neoantigenic peptides comprises a neoantigenic epitope. In some embodiments, the plurality of cancer neoantigenic peptides comprises 2, 3, 4, 5, 6, 7, or 8 neoantigenic peptides; and each of the plurality of neoantigenic peptides has the neoantigenic peptide properties described in the previous section.

[0356] In some embodiments, the neoantigenic peptide used to prepare antigen-loaded APCs is a long peptide comprising at least 20 amino acids, or at least 30 amino acids, or at least 40 amino acids, or at least 50 amino acids, or any number of amino acids therebetween. In some embodiments, the neoantigenic peptide used to prepare antigen-loaded APCs comprises flanking amino acids on either side of the mutation that promote endogenous processing of the neoantigenic peptide to increase the rate of presentation to T cells.

[0357] Longer immunogenic peptides can be designed in several ways. In some embodiments, when HLA binding peptides are predicted or known, the longer immunogenic peptides can be composed of: (1) a single binding peptide extending 2-5 amino acids to the N-terminus and C-terminus of each corresponding gene product; or (2) a concatenation of some or all binding peptides with the extension sequence of each binding peptide. In other embodiments, when sequencing reveals the presence of long (>10 residues) epitope sequences, such as neoepitopes, in tumors (e.g., due to frameshifting, read-through, or intron inclusion resulting in new peptide sequences), the longer neoantigenic peptides can be composed of the entire new tumor-specific amino acid stretch as a single longer peptide or several overlapping longer peptides. In some embodiments, it is speculated that the use of longer peptides allows for endogenous processing by patient cells and can result in more efficient antigen presentation and T cell response induction. In some embodiments, two or more peptides can be used, where the peptides overlap and are layered on the long neoantigenic peptide.

[0358] In some embodiments, each of the plurality of neoantigenic peptides comprises the same neoantigenic epitope. In some embodiments, the plurality of neoantigenic peptides comprises more than one neoantigenic epitope.

[0359] In some embodiments, the one or more polynucleotides encoding the plurality of cancer neoantigenic peptides are DNA.

[0360] In some embodiments, one or more polynucleotides encoding the plurality of cancer neoantigenic peptides are inserted into one or more mammalian expression vectors.

[0361] In some embodiments, the one or more polynucleotides encoding the plurality of cancer neoantigenic peptides are messenger RNAs.

[0362] In some embodiments, the present invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising modified nucleosides.

[0363] In some embodiments, the present invention provides gene therapy vectors comprising the RNA, oligoribonucleotides and polyribonucleotides.

[0364] In some embodiments, the present invention provides gene therapy methods and gene transcription silencing methods comprising the above.

[0365] In some embodiments, the polynucleotide encodes a neoantigenic peptide.

[0366] In some embodiments, the one polynucleotide encodes more than one neoantigenic peptide.

[0367] In some embodiments, the polynucleotide is a messenger RNA. In some embodiments, each messenger RNA comprises two or more coding sequences for tandem neoantigenic peptides.

[0368] In some embodiments, each messenger RNA comprises two, three, four, five, six, seven, eight, nine or ten or more tandem coding sequences for neoantigenic peptides. Typically, mRNA comprises 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, the mRNA is a self-amplifying mRNA. In the context of the present invention, mRNA can be generated by in vitro transcription of a DNA template. In vitro transcription methods are known to those skilled in the art. For example, a variety of in vitro transcription kits are commercially available.

[0369] Can change the stability and the translation efficiency of RNA.For example, RNA can be stabilized, and its translation increases by one or more modifications with stabilization and / or improve RNA translation efficiency.For example, in PCT / EP2006 / 009448, such modification is described, and the document is incorporated herein by reference.In order to increase the expression of the RNA used according to the present invention, this RNA can be modified in the coding region (i.e. the peptide or protein sequence of encoding expression), and the sequence of the peptide or protein of expression is not changed, thereby increase GC content to increase mRNA stability and carry out codon optimization, thereby strengthen the translation in cell.

[0370] In some embodiments, mRNA can include multiple neoantigen epitopes. In some embodiments, long polyribonucleotide sequences can be used that can encode neo-ORFs, for example, mutant GATA3 sequences that encode neo-ORFs. In some cases, mRNA containing a large portion or even the entire coding region of a gene encoding a sequence for a neoantigenic peptide is delivered to immune cells for endogenous processing and presentation of the antigen.

[0371] In some embodiments, the coding sequence for each neoantigenic peptide is 24-120 nucleotides in length.

[0372] In some embodiments, the mRNA is 50-10,000 nucleotides long. In some embodiments, the mRNA is 100-10,000 nucleotides long. In some embodiments, the mRNA is 200-10,000 nucleotides long. In some embodiments, the mRNA is 50-5,000 nucleotides long. In some embodiments, the mRNA is 100-5,000 nucleotides long. In some embodiments, the mRNA is 100-1,000 nucleotides long. In some embodiments, the mRNA is 300-800 nucleotides long. In some embodiments, the mRNA is 400-700 nucleotides long. In some embodiments, the mRNA is 450-600 nucleotides long. In some embodiments, the mRNA is at least 200 nucleotides long. In some embodiments, the mRNA is greater than 250 nucleotides, greater than 300 nucleotides, greater than 350 nucleotides, greater than 400 nucleotides, greater than 450 nucleotides, greater than 500 nucleotides, greater than 550 nucleotides, greater than 600 nucleotides, greater than 650 nucleotides, greater than 700 nucleotides, greater than 750 nucleotides, greater than 800 nucleotides, greater than 850 nucleotides, greater than 900 nucleotides, greater than 950 nucleotides, greater than 1000 nucleotides, greater than 2000 nucleotides, greater than 3000 nucleotides, greater than 4000 nucleotides, or greater than 5000 nucleotides in length.

[0373] In some embodiments, the mRNA encoding one or more neoantigenic peptides is modified, wherein the modification involves the 5'-UTR. In some embodiments, the modification involves providing an RNA having a 5'-cap or a 5'-cap analog in the 5'-UTR. The term "5'-cap" refers to a cap structure found on the 5' end of an mRNA molecule, and is typically composed of a guanosine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate bond. In some embodiments, this guanosine is methylated at position 7. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, a 7-methylguanosine cap (mG). In the context of the present invention, the term "5'-cap" includes 5'-cap analogs similar to RNA cap structures, and is modified to have the ability to stabilize RNA and / or enhance RNA translation in vivo and / or within cells (if attached to RNA). In some embodiments, mRNA is co-transcriptionally capped.

[0374] In some embodiments, the mRNA encoding one or more neoantigenic peptides comprises a 3'-UTR comprising a poly A tail. In some embodiments, the poly A tail is 100-200 bp in length. In some embodiments, the poly A tail is longer than 20 nucleotides. In some embodiments, the poly A tail is longer than 50 nucleotides. In some embodiments, the poly A tail is longer than 60 nucleotides. In some embodiments, the poly A tail is longer than 70 nucleotides. In some embodiments, the poly A tail is longer than 80 nucleotides. In some embodiments, the poly A tail is longer than 90 nucleotides. In some embodiments, the poly A tail is longer than 100 nucleotides. In some embodiments, the poly A tail is longer than 110 nucleotides. In some embodiments, the poly A tail is longer than 120 nucleotides. In some embodiments, the poly A tail is longer than 130 nucleotides. In some embodiments, the poly A tail is longer than 140 nucleotides. In some embodiments, the poly A tail is longer than 150 nucleotides. In some embodiments, the poly A tail is longer than 160 nucleotides. In some embodiments, the poly A tail is longer than 170 nucleotides. In some embodiments, the poly A tail is longer than 180 nucleotides. In some embodiments, the poly A tail is longer than 190 nucleotides. In some embodiments, the poly A tail is longer than 200 nucleotides. In some embodiments, the poly A tail is longer than 210 nucleotides. In some embodiments, the poly A tail is longer than 220 nucleotides. In some embodiments, the poly A tail is longer than 230 nucleotides. In some embodiments, the poly A tail is longer than 100 nucleotides. In some embodiments, the poly A tail is longer than 240 nucleotides. In some embodiments, the poly A tail is longer than 100 nucleotides. In some embodiments, the poly A tail is about 250 nucleotides.

[0375] In some embodiments, the poly A tail comprises 100-250 adenosine units. In some embodiments, the poly A tail comprises 120-130 adenine units. In some embodiments, the poly A tail comprises 120 adenine units. In some embodiments, the poly A tail comprises 121 adenine units. In some embodiments, the poly A tail comprises 122 adenine units. In some embodiments, the poly A tail comprises 123 adenine units. In some embodiments, the poly A tail comprises 124 adenine units. In some embodiments, the poly A tail comprises 125 adenine units. In some embodiments, the poly A tail is 129 bases.

[0376] In some embodiments, the coding sequences for two consecutive neoantigenic peptides are separated by a spacer or linker.

[0377] In some embodiments, the spacer or linker comprises up to 5000 nucleotide residues. An exemplary spacer sequence is GGCGGCAGCGGCGGCGGCGGCAGCGGCGGC. Another exemplary spacer sequence is GGCGGCAGCCTGGGCGGCGGCGGCAGCGGC. Another exemplary spacer sequence is GGCGTCGGCACC. Another exemplary spacer sequence is CAGCTGGGCCTG. Another exemplary spacer sequence is a sequence encoding lysine, such as AAA or AAG. Another exemplary spacer sequence is CAACTGGGATTG.

[0378] In some embodiments, the mRNA comprises one or more additional structures to enhance processing and presentation of the antigenic epitope by APCs.

[0379] In some embodiments, the linker or spacer 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. Preferred cleavage sites are placed adjacent to certain epitopes to avoid inadvertently cleaving epitopes within the sequence. In some embodiments, the design of the epitope and cleavage region on the mRNA encoding the epitope string is non-random.

[0380] In certain embodiments, mRNA encoding a neoantigenic peptide of the present invention is administered to a subject in need thereof. In some embodiments, the mRNA to be administered comprises at least one modified nucleoside-phosphate.

[0381] In some embodiments, T cells are activated with neoantigen peptides by artificial antigen presenting cells. In some embodiments, T cells are activated with neoantigen peptides using artificial scaffolds loaded with neoantigen peptides coupled to MHC antigens to which they can bind with high affinity.

[0382] In some embodiments, the additional structure comprises encoding a specific domain from a protein selected from MITD, SP1, and the tenth fibronectin domain: 10FnIII.

[0383] In some embodiments, cells derived from peripheral blood or leukapheresis are contacted with a plurality of cancer neoantigen peptides or one or more polynucleotides encoding the plurality of cancer neoantigen peptides once or more to prepare antigen-loaded APCs.

[0384] In some embodiments, the method comprises incubating the APCs in one or more APC preparations with a first medium comprising at least one cytokine or growth factor for a first period of time.

[0385] In some embodiments, the method comprises incubating one or more APC preparations with at least one peptide for a second period of time.

[0386] In some embodiments, the enriched cells further include CD1c+ cells.

[0387] In some embodiments, the cell population is enriched for CD11c+ and CD141+ cells.

[0388] In some embodiments, the cell population comprising antigen-loaded APCs comprises greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more CD11c+ cells.

[0389] In some embodiments, the cell population comprising antigen-loaded APCs comprises less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, 4% or less of cells expressing CD11b+.

[0390] In some embodiments, the cell population comprising antigen-loaded APCs comprises greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of neoantigen peptide-expressing cells that are CD11c+.

[0391] In some embodiments, the cell population comprising antigen-loaded APCs comprises greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the neoantigen peptide expressing cells that are CD11c+CD1c+ or CD141+ cells.

[0392] In some embodiments, the neoantigen-loaded APCs comprise mature APCs.

[0393] In some embodiments, the method comprises obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC or at least one T cell.

[0394] In some embodiments, the method comprises 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.

[0395] In some embodiments, the method comprises incubating the CD14 and / or CD25 and / or CD19 cell-depleted sample with FLT3L for a first period of time.

[0396] In some embodiments, the method comprises incubating at least one peptide with the CD14 and / or CD25 and / or CD19 cell-depleted sample for a second period of time, thereby obtaining a first mature APC peptide-loaded sample.

[0397] Preparation of neoantigen-activated T cells using neoantigen-loaded APCs

[0398] In some embodiments, the APCs (APCs) loaded with neoantigens prepared by the above method are incubated with T cells to obtain antigen-activated T cells. The method may include generating at least one antigen-specific T cell, wherein the antigen is a neoantigen. In some embodiments, generating at least one antigen-specific T cell includes generating multiple antigen-specific T cells.

[0399] In some embodiments, T cells are obtained from a biological sample from a subject.

[0400] In some embodiments, the T cells are obtained from a biological sample from the same subject from which the APCs were derived. In some embodiments, the T cells are obtained from a biological sample from a different subject than the subject from which the APCs were derived.

[0401] In some embodiments, the APCs and / or T cells are derived from a biological sample that is a peripheral blood mononuclear cell (PBMC).In some embodiments, the APCs and / or T cells are derived from a biological sample that is a leukapheresis sample.

[0402] In some embodiments, the APCs comprise dendritic cells (DCs).

[0403] In some embodiments, the APCs are derived from CD14+ monocytes, or are CD14-enriched APCs, or are CD141-enriched APCs.

[0404] In some embodiments, the CD14+ monocytes are enriched from a biological sample from a subject comprising peripheral blood mononuclear cells (PBMCs).

[0405] In some embodiments, the APCs are PBMCs. In some embodiments, the PBMCs are freshly isolated PBMCs. In some embodiments, the PBMCs are frozen PBMCs. In some embodiments, the PBMCs are autologous PBMCs isolated from a subject or patient.

[0406] In some embodiments, PBMC is loaded with antigen, wherein the antigen can be a peptide or polypeptide or a polynucleotide encoding the peptide and polypeptide, such as mRNA. PBMC (monocyte, DC, phagocyte) can absorb antigen by phagocytosis, process and present it on the surface for T cell activation. The peptide or polypeptide loaded on PBMC can be supplemented with an adjuvant to increase immunogenicity. In some embodiments, PBMC is loaded with nucleic acid antigen. Nucleic acid antigen can be in the form of mRNA, comprising a sequence encoding one or more antigens. In some embodiments, mRNA antigen loading does not require a supplementary adjuvant, because, for example, RNA can serve as an adjuvant on its own.

[0407] In some embodiments, PBMC is directly separated or thawed from frozen sample, and is subjected to incubation with one or more antigens such as neoantigens or compositions comprising neoantigens or one or more nucleic acids or polynucleotides encoding the one or more antigens. In some embodiments, before PBMC is exposed to one or more antigens or nucleic acids encoding the one or more antigens, PBMC samples are not further cultured for differentiation or subjected to further maturation of one or more cell components in PBMC (for example, maturation of antigen presenting cells, or differentiation of monocytes to dendritic cells). In some embodiments, before cells are exposed to one or more antigens or nucleic acids encoding the one or more antigens or incubated therewith, one or more cell types are consumed or removed from freshly separated PBMC cell colonies or freshly thawed PBMC colonies. In some embodiments, CD14+ cells are consumed from PBMC. In some embodiments, CD25+ cells are consumed from PBMC. In some embodiments, CD11b+ cells are consumed from PBMC. In some embodiments, before incubation with one or more antigens or one or more nucleic acids encoding the one or more antigens, CD14+ and CD25+ cells are consumed from PBMC. In some embodiments, CD11b+ and / or CD14+ and / or CD25+ cells are consumed from PBMC. In some embodiments, provided herein are methods including preparing tumor antigen-specific T cells by consuming CD14+ cells and / or CD25+ cells from PBMC samples from human subjects, the percentage of immature dendritic cells (DC) included in the sample is about the same as the percentage of immature DC in the peripheral blood of the human subjects. In some embodiments, provided herein are methods including preparing tumor antigen-specific T cells by consuming CD14+ cells and / or CD25+ cells from PBMC samples from human subjects, the percentage of mature DC included in the sample is about the same as the percentage of mature DC in the peripheral blood of the human subjects. In some embodiments, provided herein are methods including preparing tumor antigen-specific T cells by consuming CD14+ cells and / or CD25+ cells from PBMC samples from human subjects, the ratio of immature DC to mature DC in the sample is about the same as the ratio of immature DC to mature DC in the peripheral blood of the human subjects. In some embodiments, the methods provided herein comprise preparing tumor antigen-specific T cells by depleting CD14+ cells and / or CD25+ cells from a PBMC sample from a human subject that has not undergone a step of maturation of immature DCs to mature DCs.

[0408] In some embodiments, CD14+ monocytes are stimulated with one or more cytokines or growth factors.

[0409] In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848, LPS, ss-RNA40, poly I:C, or a combination thereof.

[0410] In some embodiments, the CD14+ monocytes are from a second biological sample comprising PBMCs.

[0411] In some embodiments, the second biological sample is from the same subject.

[0412] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0413] In some embodiments, the at least one antigen-specific T cell is stimulated in a medium comprising IL-7, IL-15, an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof.

[0414] In some embodiments, the IDO inhibitor is epacadostat, navoximod, 1-methyltryptophan, or a combination thereof.

[0415] In some embodiments, FLT3L is administered to the subject prior to obtaining the biological sample for preparing APCs and / or T cells.

[0416] In some embodiments, T cells are obtained from a biological sample from a subject, as described in the previous section of this disclosure.

[0417] In some embodiments, the biological sample is freshly obtained from a subject or is a frozen sample.

[0418] In some embodiments, the incubation is performed in the presence of at least one cytokine or growth factor, which includes 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.

[0419] In some embodiments, the method includes stimulating T cells with IL-7, IL-15 or a combination thereof. In some embodiments, the method includes stimulating T cells with IL-7, IL-15 or a combination thereof in the presence of an IDO inhibitor, a PD-1 antibody or IL-12. In some embodiments, the one or more tumor antigen epitope sequences or the APC loaded with the one or more tumor antigen epitope sequences or the APC loaded with (for example, expressing) the nucleic acid sequence (such as mRNA sequence) encoding the one or more tumor antigen epitope sequences, one or more cytokines or growth factors (including GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, R848, LPS, ss-rna40, poly I: C, FLT3L or a combination thereof) in the presence of suitable ex vivo T cell growth conditions, expanding stimulated T cells. In some embodiments, the method further includes administering antigen-specific T cells to the subject.

[0420] In some embodiments, the method comprises incubating APCs prepared as described in the previous section with T cells in the presence of a medium comprising at least one cytokine or growth factor to generate neoantigen-activated T cells.

[0421] In some embodiments, the incubation comprises incubating the first APC preparation of the APC preparation with the T cells for more than 7 days. In some embodiments, the incubated T cells are stimulated T cells that have been expanded in vitro for more than 7 days in the presence of APC preparations, cytokines, and growth factors.

[0422] In some embodiments, the incubating comprises incubating the first of the APC preparations with the T cells for more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0423] In some embodiments, the first of the one or more time periods is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 days.

[0424] In some embodiments, the total time period of the separate time periods is less than 28 days. In some embodiments, the total time period of the separate time periods is 20-27 days. In some embodiments, the total time period of the separate time periods is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 days.

[0425] In some embodiments, the method comprises incubating the first APC preparation of the APC preparation with the T cells for more than 7 days. In some embodiments, the method comprises incubating the first APC preparation of the APC preparation with the T cells for more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the method comprises incubating the first APC preparation of the APC preparation with the T cells for 7-20, 8-20, 9-20, 10-20, 11-20, or 12-20 days. In some embodiments, the method comprises incubating the first APC preparation of the APC preparation with the T cells for about 10-15 days.

[0426] In some embodiments, the method comprises incubating the second APC preparation of the APC preparation with the T cells for 5-9 days. In some embodiments, the method comprises incubating the second APC preparation of the APC preparation with the T cells for 5, 6, 7, 8, or 9 days. In some embodiments, the method further comprises removing the one or more cytokines or growth factors from the second medium after the third time period and before starting the fourth time period.

[0427] In some embodiments, the method comprises incubating a third APC preparation of the APC preparation with T cells for 5-9 days. In some embodiments, the method comprises incubating a third APC preparation of the APC preparation with T cells for 5, 6, 7, 8, or 9 days.

[0428] In some embodiments, the method comprises incubating a first preparation of APCs with T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days, incubating a second preparation of APCs with T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days, and incubating a third preparation of APCs with T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days.

[0429] In some embodiments, the method is carried out in vitro. In some embodiments, T cells are cultured in a culture medium containing cytokines. In some embodiments, the example of a cytokine includes IL-7. In some embodiments, the example of a cytokine includes IL-15. In some embodiments, the example of a cytokine includes IL-7 and IL-15. In some embodiments, T cells are cultured in a culture medium comprising IL-7 and / or IL-15. In some embodiments, the final concentration of the cytokine in the T cell culture or culture medium is at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL or 20 ng / mL. In some embodiments, the final concentration of IL-7 in the T cell culture or culture medium is at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the final concentration of IL-15 in the T cell culture or culture medium is at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the T cells are cultured in a culture medium further comprising FLT3L. In some embodiments, the final concentration of FLT3L in the T cell culture or culture medium is at least 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, or 200 ng / mL.In some embodiments, T cells are incubated, induced, or stimulated in a medium containing FLT3L for a first time period. In some embodiments, T cells are incubated, induced, or stimulated in a medium containing additional FLT3L for a second time period. In some embodiments, T cells are incubated, induced, or stimulated in a medium containing additional FLT3L for a third time period. In some embodiments, T cells are incubated, induced, or stimulated in a medium containing additional FLT3L for a fourth, fifth, or sixth time period, with freshly added FLT3L in each time period.

[0430] In some embodiments, T cells are cultured in the presence of neoantigens, such as neoantigens presented by APCs, wherein the culture medium comprises high potassium [K]. + In some embodiments, during incubation with APCs or T cells, a high [K] + In some embodiments, during incubation with APCs or T cells, the [K] + In some embodiments, the content of the culture medium is kept constant during the in vitro culture of the T cells. In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] +In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + In some embodiments, the [K] + The content is about 40mM.

[0431] In some embodiments, at least for a period of time during incubation of T cells with the neoantigen, the [K] + The content is about 40mM. In some embodiments, the new antigen can be presented by the APC loaded with the new antigen. In some embodiments, in [K] + T cells are tested for T effector function, CD8+ cytotoxicity, cytokine production, and memory phenotype in the presence of [K]. In some embodiments, at high [K] + T cells grown in the presence of [K] express an effector T cell phenotype. In some embodiments, at high [K] + In some embodiments, at high [K] + T cells grown in the presence of do not express T cell exhaustion markers.

[0432] In some embodiments, the stimulated T cells are immune cell populations comprising activated T cells stimulated with APCs comprising a neoantigen peptide-MHC complex. In some embodiments, the method may include incubating an immune cell population from a biological sample with an APC comprising a peptide-MHC complex to obtain a stimulated immune cell sample; determining the expression of one or more cell markers of at least one immune cell in the stimulated immune cell sample; and determining the binding of at least one immune cell in the stimulated immune cell sample to the peptide-MHC complex; wherein determining the expression of certain cell surface markers or other deterministic markers such as intracellular factors or released substances such as cytokines and determining the binding to the neoantigen peptide-MHC complex is performed simultaneously. In some embodiments, the one or more cell markers include TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, enzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3 or any combination thereof. In some embodiments, the one or more cell markers include cytokines. In some embodiments, the one or more cell markers include degranulation markers. In some embodiments, the one or more cell markers include cell surface markers. In some embodiments, the one or more cell markers include proteins. In some embodiments, determining the combination of at least one immune cell of the immune cell sample that stimulates and the peptide-MHC complex includes determining the combination of at least one immune cell of the immune cell sample that stimulates and the MHC tetramer of the MHC that comprises the peptide and the peptide-MHC complex. In some embodiments, the MHC is class I MHC or class II MHC. In some embodiments, the peptide-MHC complex comprises one or more markers.

[0433] In some embodiments, the activation of T cells is verified by detecting the release of cytokines by activated T cells. In some embodiments, the cytokines are one or more of the following: TNF-α, IFN-γ, or IL-2. In some embodiments, the activation of T cells is verified by their specific antigen binding and cytokine release. In some embodiments, the activation of T cells is verified by their ability to kill tumor cells in vitro. Samples of activated T cells can be used to verify the activation state of T cells. In some embodiments, samples from T cells are removed from T cell cultures to determine the cell composition and activation state by flow cytometry.

[0434] In some embodiments, the percentage of at least one antigen-specific T cell in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total T cells or total immune cells. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 5%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 7%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 10%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 12%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 15%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 20%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 25%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 30%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 40%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 50%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 60%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 70%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 80%. In some embodiments, the percentage of at least one antigen-specific T cell in the composition is about 90%.

[0435] In some embodiments, the percentage of at least one antigen-specific CD8+ T cell in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of total 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 cell in the composition is about 5%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cell in the composition is about 7%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cell in the composition is about 10%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 12%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 15%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 20%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 25%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 30%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 40%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 50%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 60%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 70% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0436] 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.

[0437] In some embodiments, the percentage of the at least one antigen-specific T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells.

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

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

[0440] In some embodiments, the antigen is a neoantigen, a tumor-associated antigen, an overexpressed antigen, a viral antigen, a minor histocompatibility antigen, or a combination thereof.

[0441] In some embodiments, the number of at least one antigen-specific CD8+ T cell in the composition is at least about 1x10^6, 2x10^6, 5x10^6, 1x10^7, 2x10^7, 5x10^7, 1x10^8, 2x10^8, or 5x10^8 antigen-specific CD8+ T cells.

[0442] In some embodiments, the number of at least one antigen-specific CD4+ T cell in the composition is at least about 1x10^6, 2x10^6, 5x10^6, 1x10^7, 2x10^7, 5x10^7, 1x10^8, 2x10^8, or 5x10^8 antigen-specific CD4+ T cells.

[0443] Pharmaceutical composition

[0444] Provided herein are compositions (e.g., pharmaceutical compositions) comprising an immune cell colony. The composition can include at least one antigen-specific T cell comprising a T cell receptor (TCR). The composition can include at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence.

[0445] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, including excipients and adjuvants, which aid in processing the active agent into a pharmaceutically acceptable product. Suitable formulations may depend on the selected route of administration. Any known techniques, carriers, and excipients may be used as suitable techniques, carriers, and excipients as understood in the art.

[0446] In some cases, the pharmaceutical composition is formulated as a cell-based therapeutic, such as a T cell therapeutic. In some embodiments, the pharmaceutical composition comprises a peptide-based therapy, a nucleic acid-based therapy, an antibody-based therapy, and / or a cell-based therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic or a nucleic acid-based therapeutic, wherein the nucleic acid encodes a polypeptide. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic or a nucleic acid-based therapeutic, wherein the nucleic acid encodes a polypeptide; wherein the peptide-based therapeutic or nucleic acid-based therapeutic is contained in a cell, wherein the cell is a T cell. In some embodiments, the pharmaceutical composition comprises an antibody-based therapeutic. The composition may comprise T cells specific for two or more immunogenic antigens or neoantigenic peptides.

[0447] In one aspect, provided herein is a pharmaceutical composition comprising (a) an immune cell population comprising T cells from a biological sample, wherein the T cells comprise at least one antigen-specific T cell, the antigen-specific T cell being an APC-stimulated T cell and comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the APC is a FLT3L-stimulated APC; and (b) a pharmaceutically acceptable excipient.

[0448] On the one hand, there is provided herein a pharmaceutical composition comprising: (a) an immune cell colony from a biological sample, the biological sample comprising at least one antigen-specific T cell, the antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, and (b) a pharmaceutically acceptable excipient; wherein the amount of immune cells expressing CD14 and / or CD25 in the colony is proportionally different from the number of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the at least one antigen-specific T cell comprises T cells stimulated by at least one APC. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the colony is proportionally less than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the colony is proportionally greater than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the at least one antigen-specific T cell comprises at least one CD4+ T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD8+ T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD4-enriched T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD8-enriched T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory CD4+ T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory CD8+ T cell. In some embodiments, the percentage of the at least one antigen-specific T cell in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total T cells or total immune cells. In some embodiments, the percentage of at least one antigen-specific CD8+ T cell in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0449] In addition to the active ingredient, the pharmaceutical composition may also contain a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other substances known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance will depend on the route of administration.

[0450] Acceptable carriers, excipients, or stabilizers are those that are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; ); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as or polyethylene glycol (PEG).

[0451] An acceptable carrier is physiologically acceptable to the patient to whom it is administered and maintains the therapeutic properties of the compound to be administered. Acceptable carriers and their formulations are generally described in, for example, Remington's Pharmaceutical Sciences (18th edition, A. Gennaro, Mack Publishing Co., Easton, PA 1990). An example of a carrier is normal saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is involved in carrying or transporting the subject compound from the site of administration of one organ or body part to another organ or body part, or in an in vitro assay system. An acceptable carrier is compatible with the other ingredients of the formulation and is harmless to the subject to which it is administered. An acceptable carrier should also not change the specific activity of the neoantigen.

[0452] In one aspect, provided herein are pharmaceutically acceptable or physiologically acceptable compositions comprising solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coating materials, isotonic agents, and absorption enhancers or retarder compatible with drug administration. Therefore, pharmaceutical compositions or pharmaceutical preparations refer to compositions suitable for use as medicines in subjects. Compositions can be formulated to be compatible with specific routes of administration (i.e., systemic or local). Therefore, compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0453] In some embodiments, the composition may further include an acceptable additive to improve the stability of the immune cells in the composition. Acceptable additives may not change the specific activity of the immune cells. Examples of acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. Acceptable additives may be combined with acceptable carriers and / or excipients such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 that are used to increase the stability of the peptide and reduce the gelation of the solution. The surfactant may be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition in storage.

[0454] Pharmaceutical compositions can be administered, for example, by injection. Compositions for injection include aqueous solutions (which are water-soluble) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, or phosphate-buffered saline (PBS). Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, by using coatings such as lecithin, in the case of dispersions, by maintaining the desired particle size, and by using surfactants to maintain fluidity. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride, can be included in the composition. The resulting solution can be packaged for use as is, or lyophilized; the lyophilized product can then be merged with a sterile solution before administration. For intravenous injection, or injection at the affected part, the active ingredient will be in the form of an acceptable aqueous solution for parenteral administration, which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art can use, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection to prepare suitable solutions. As needed, preservatives, stabilizers, buffers, antioxidants and / or other additives can be included. Sterile injectable solutions can be prepared by incorporating the required amount of the active ingredient into a suitable solvent together with a combination of the above-listed ingredients or ingredients (if necessary), followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing an alkaline dispersion medium and the required other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods can be vacuum drying and freeze drying, which obtain the active ingredient plus any additional required ingredient powder from its previously filtration-sterilized solution.

[0455] For example, the composition can be routinely administered intravenously, for example, by injecting a unit dose. For injection, the active ingredient can be in the form of a parenteral acceptable aqueous solution that is substantially pyrogen-free and has suitable pH, isotonicity, and stability. Suitable solutions can be prepared using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, and lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. Additionally, the composition can be administered via atomization.

[0456] When a composition is contemplated for use in a pharmaceutical or any of the methods provided herein, it is contemplated that the composition may be substantially free of pyrogens, such that the composition does not induce an inflammatory response or an unsafe allergic reaction when administered to a human patient. Testing a composition for pyrogens and preparing a composition that is substantially free of pyrogens is well known to those of ordinary skill in the art and can be accomplished using commercially available kits.

[0457] 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 clearance or hydrolysis of peptides; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can also be used to stabilize or increase or decrease the absorption of pharmaceutical compositions (including liposome carriers). To prevent digestion, the compound can be complexed with a composition to render it resistant to acid and enzymatic hydrolysis, or the compound can be complexed in an appropriately resistant carrier such as a liposome. Means for protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13: 1760-1764; Samanen (1996) J. Pharm. Pharmacol. 48: 119-135; and U.S. Pat. No. 5,391,377).

[0458] The composition can be used in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be used depends on the ability of the subject to be treated, the subject's immune system to utilize the active ingredient, and the degree of desired binding capacity. The precise amount of active ingredient that needs to be used depends on the practitioner's judgment and is specific for each individual. The appropriate regimen for initial administration and booster injection is also different, but typically, initial administration is first followed by repeated administration at intervals of one or more hours by subsequent injection or other administration. Alternatively, continuous intravenous infusion that is sufficient to maintain blood concentration is considered.

[0459] In some embodiments, the present invention relates to an immunogenic composition, such as a pharmaceutical composition capable of eliciting a neoantigen-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic composition comprises a neoantigen therapeutic agent described herein (e.g., a peptide, a polynucleotide, a TCR, a CAR, a cell containing a TCR or a CAR, a dendritic cell containing a polypeptide, a dendritic cell containing a polynucleotide, an antibody, etc.) corresponding to a tumor-specific antigen or a neoantigen.

[0460] In some embodiments, the pharmaceutical compositions described herein are capable of eliciting a specific cytotoxic T cell response, a specific helper T cell response, or a B cell response.

[0461] In some embodiments, the antigen polypeptide or polynucleotide can be provided as an antigen presenting cell (e.g., dendritic cell) containing such polypeptide or polynucleotide. In other embodiments, such antigen presenting cells are used to stimulate T cells for patients. In some embodiments, the antigen presenting cell is a dendritic cell. In related embodiments, the dendritic cell is an autologous dendritic cell pulsed with a neoantigen peptide or nucleic acid. The neoantigen peptide can be any suitable peptide that produces an appropriate T cell response. In some embodiments, the T cell is a CTL. In some embodiments, the T cell is an HTL. Therefore, one embodiment of the present disclosure is an immunogenic composition containing at least one antigen presenting cell (e.g., dendritic cell) pulsed or loaded with one or more 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 can be loaded with neoantigen peptides or polynucleotides in vitro. In related embodiments, such APCs or PBMCs are injected back into the patient's body. The polynucleotide can be any suitable polynucleotide capable of transducing dendritic cells, resulting in presentation of new antigenic peptides and induction of immunity. In some embodiments, such antigen presenting cells (APCs) (e.g., dendritic cells) or peripheral blood mononuclear cells (PBMCs) are used to stimulate T cells (e.g., autologous T cells). In related embodiments, the T cells are CTLs. In other related embodiments, the T cells are HTLs. In some embodiments, the T cells are CD8 + In some embodiments, the T cells are CD4 + These T cells are then injected into the patient.

[0462] 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. Administration of any therapeutic agent can be performed simultaneously or sequentially and in any order.

[0463] In some embodiments, the pharmaceutical compositions described herein for therapeutic treatment (e.g., immunogenic compositions) may be formulated for parenteral, topical, nasal, oral, or external administration. In some embodiments, the pharmaceutical compositions described herein are administered parenterally, such as intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the composition may be administered intratumorally. The composition may be administered at the site of surgical resection to induce a local immune response to the tumor. In some embodiments, a composition for parenteral administration is described herein, comprising a solution of a neoantigenic peptide, and the immunogenic composition is dissolved or suspended in an acceptable carrier such as an aqueous carrier. A variety of aqueous carriers may be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterilized by filtration. The resulting aqueous solution may be packaged for use as is, or lyophilized, and the lyophilized product may be combined with a sterile solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0464] The ability of an adjuvant to increase the immune response to an antigen is generally manifested as a significant increase in immune-mediated reactions or a decrease in disease symptoms. For example, an increase in humoral immunity can be manifested as a significant increase in antibody titers against the antigen, and an increase in T cell activity can be manifested as increased cell proliferation or cytotoxicity or cytokine secretion. Adjuvants can also alter the immune response, for example, by changing a response that is primarily humoral or type 2 T helper cells to one that is primarily cellular or type 1 T helper cells.

[0465] Suitable adjuvants are known in the art (see WO2015 / 095811) and include, but are not limited to, poly(I:C), poly-ICLC, STING agonists, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, Delivery systems, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulator derived from saponin (Aquila Biotech, Worcester, Mass., USA), mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. Several immunoadjuvants specific for dendritic cells have been described (Dupuis M et al., Cell Immunol. 1998; 186(1): 18-27; Allison AC; Dev Biol Stand. 1998; 92: 3-11) (Mosca et al., Frontiers in Bioscience, 2007; 12: 4050-4060) (Gamvrellis et al., Immunol & Cell Biol. 2004; 82: 506-516). Cytokines can also be used. Several cytokines have been directly implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into potent antigen-presenting cells of T lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1β, IL-4, IL-6, and CD40L) (U.S. Pat. No. 5,849,589, which is incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0466] It has also been reported that CpG immunostimulatory oligonucleotides enhance the effect of adjuvants in therapeutic settings. Without being bound by theory, CpG oligonucleotides work by activating innate (non-adaptive) immune systems via Toll-like receptors (TLRs) (mainly TLR9). The TLR9 activation of CpG triggers enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live viruses or killed viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cell immunogenic pharmaceutical compositions, and polysaccharide conjugates in preventive and therapeutic immunogenic pharmaceutical compositions. Importantly, it enhances the maturation and differentiation of dendritic cells, thereby leading to the activation of enhanced TH1 cells and the generation of strong cytotoxic T lymphocytes (CTL), even in the absence of CD4+ T cell help. Even in the presence of adjuvants that usually promote TH2 bias, such as alum or incomplete Freund's adjuvant (IFA), the TH1 bias induced by TLR9 stimulation is also maintained. When formulated with other adjuvants or co-administered or in formulations such as microparticles, nanoparticles, lipid emulsions or similar preparations, CpG oligonucleotides demonstrate even greater adjuvant activity, which is particularly useful for inducing strong responses when antigens are relatively weak. They can also accelerate immune responses and enable antigen dosages to be reduced, and in some experiments, have suitable antibody responses (Arthur M.Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484) with full-dose immunogenic pharmaceutical compositions that do not contain CpG. United States Patent (USP) 6,406,705 describes the use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens in combination to induce antigen-specific immune responses. A commercially available CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) from Mologen (Berlin, DE), a component of pharmaceutical compositions as described herein. Other TLR binding molecules can also be used, such as RNA in conjunction with TLR7, TLR8 and or TLR9.

[0467] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly (I and / or poly C) (e.g., poly I:CI2U), non-CpG bacterial DNA or RNA, ssRNA against TLR840, and immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which can act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives that can be used in the context of the present invention can be readily determined by a skilled artisan without undue experimentation. Additional adjuvants include colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0468] In some embodiments, the immunogenic composition according to the present disclosure may include more than one different adjuvant. In addition, the present invention includes a pharmaceutical composition comprising any adjuvant substance, including any of the above-mentioned adjuvants and combinations thereof. In some embodiments, the immunogenic composition comprises a neoantigen therapeutic agent (e.g., a peptide, a polynucleotide, a TCR, a CAR, a cell containing a TCR or a CAR, a dendritic cell containing a polypeptide, a dendritic cell containing a polynucleotide, an antibody, etc.), and the adjuvant can be administered separately in any suitable order.

[0469] Lipidation can be divided into several different types, such as N-myristoylation, palmitoylation, GPI-anchor addition, prenylation and several other types of modification. N-myristoylation is the covalent attachment of myristate (C14 saturated acid) to glycine residues. Palmitoylation is the thioester connection of long-chain fatty acids (C16) to cysteine ​​residues. GPI-anchor addition is a glycosyl-phosphatidylinositol (GPI) connection through an amide bond. Prenylation is the thioether connection of an isoprenoid lipid (e.g., farnesyl (C-15), geranylgeranyl (C-20)) to a cysteine ​​residue. Other types of modification may include attachment of S-diacylglycerols to the sulfur atom of cysteine, conjugation via the O-octanoyl group of a serine or threonine residue, conjugation to the S-archaeol of a cysteine ​​residue, and cholesterol attachment.

[0470] The fatty acid for generating the lipidated peptide can comprise C2 to C30 saturated, monounsaturated or polyunsaturated fatty acyl groups. Exemplary fatty acids can comprise palmitoyl, myristoyl, stearoyl and decanoyl. In some cases, the lipid moiety with adjuvant properties is attached to the target polypeptide to cause or enhance immunogenicity in the absence of an exogenous adjuvant. Lipidated peptides or lipopeptides can be referred to as self-adjuvant lipopeptides. Any fatty acid described above and elsewhere herein can cause or enhance the immunogenicity of the target polypeptide. The fatty acid that can cause or enhance immunogenicity can comprise palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl and decanoyl.

[0471] Polypeptides such as naked peptides or lipidated peptides can be incorporated into liposomes. Sometimes, lipidated peptides can be incorporated into liposomes. For example, the lipid portion of the lipidated peptide can spontaneously integrate into the lipid bilayer of the liposome. Thus, the lipopeptide can be presented on the "surface" of the liposome. Exemplary liposomes suitable for incorporation into the formulation include, but are not limited to, multilamellar vesicles (MLVs), oligolamellar vesicles (OLVs), unilamellar vesicles (UVs), small unilamellar vesicles (SUVs), medium-sized unilamellar vesicles (MUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), multivesicular vesicles (MVVs), unilamellar or oligolamellar vesicles prepared by reverse phase evaporation (REVs), multilamellar vesicles prepared by reverse phase evaporation (MLV-REVs), stable multilamellar vesicles (SPLVs), frozen and thawed MLVs (FATMLVs), vesicles prepared by extrusion (VETs), vesicles prepared by French press (FPVs), vesicles prepared by fusion (FUVs), dehydration-rehydration vesicles (DRVs), and foam bodies (BSVs).

[0472] Depending on the method of preparation, liposomes can be unilamellar or multilamellar and can vary in size, ranging from about 0.02 μm to greater than about 10 μm in diameter. Liposomes can adsorb many types of cells and then release the incorporated agent (e.g., a peptide as described herein). In some cases, the liposomes fuse with the target cells, whereupon the contents of the liposomes are subsequently emptied into the target cells. The liposomes can be endocytosed by phagocytes. Endocytosis can be followed by intralysosomal degradation of the liposome lipids and release of the encapsulating agent.

[0473] Liposomes provided herein may also include a carrier lipid. In some embodiments, the carrier lipid is a phospholipid. The carrier lipids capable of forming liposomes include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Other suitable phospholipids also include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic acid (DPPA); dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE) etc. or its combination. In some embodiments, liposomes also include sterols (such as cholesterol) that regulate liposome formation. Carrier lipids can be any known non-phosphate polar lipids.

[0474] The pharmaceutical composition can be encapsulated in liposomes using known techniques. Biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of the present invention.

[0475] The pharmaceutical composition can be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to patients are known to those skilled in the art. Basically, the material is dissolved in an aqueous solution, appropriate phospholipids and lipids and surfactants (if necessary) are added, and the material is dialyzed or sonicated as needed.

[0476] Microspheres formed from polymers or proteins are well known to those skilled in the art and can be adapted for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, a compound can be incorporated and implanted into the microspheres or microsphere complexes for slow release over a period of days to months.

[0477] Cell-based immunogenic pharmaceutical compositions can also be administered to a subject. For example, an immunogenic pharmaceutical composition based on antigen presenting cells (APCs) can be formulated using any known technology, carrier, and excipient as understood in the art. APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells. Sometimes, an APC-based immunogenic pharmaceutical composition can be an immunogenic pharmaceutical composition based on dendritic cells.

[0478] Dendritic cell-based immunogenic pharmaceutical compositions can be prepared by any method known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared by in vitro or in vivo methods. The in vitro method may include the use of autologous DC pulsed with the polypeptides described herein to activate or load the DCs prior to administration to the patient. The in vivo method may include the use of antibodies coupled to the polypeptides described herein to target specific DC receptors. The DC-based immunogenic pharmaceutical composition may further include a DC activator, such as a TLR3, TLR-7-8, and CD40 agonist. The DC-based immunogenic pharmaceutical composition may further include an adjuvant and a pharmaceutically acceptable carrier.

[0479] Adjuvants can be used to enhance the immune response (humoral and / or cellular response) elicited in a patient receiving an immunogenic pharmaceutical composition. Sometimes, an adjuvant can elicit a Th1 type response. Other times, an adjuvant can elicit a Th2 type response. A Th1 type response can be characterized by the production of cytokines such as IFN-γ, while a Th2 type response can be characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

[0480] In some aspects, lipid-based adjuvants, such as MPLA and MDP, can be used with the immunogenic pharmaceutical compositions disclosed herein. For example, monophosphoryl lipid A (MPLA) is an adjuvant that increases the presentation of liposomal antigens to specific T lymphocytes. In addition, muramyl dipeptide (MDP) can also be used as a suitable adjuvant with the immunogenic pharmaceutical formulations described herein.

[0481] Adjuvants can also include stimulatory molecules such as cytokines. Non-limiting examples of cytokines include: CCL20, alpha-interferon (IFNα), beta-interferon (IFNβ), gamma-interferon (IFNγ), platelet-derived growth factor (PDGF), TNFα, GM-CSF, epidermal growth factor (EGF), skin T cell-attracting chemokine (CTACK), epidermal thymus-expressed chemokine (TECK), mucosal-associated epithelial cell chemokine (MEC) IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1-, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA -1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, angiogenic factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI and TAP2.

[0482] Other adjuvants include: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic factor, fibroblast growth factor IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANKLIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[0483] In some aspects, the adjuvant can be a modulator of toll-like receptors. Examples of toll-like receptor modulators include TLR9 agonists and are not limited to small molecule modulators of toll-like receptors, such as imiquimod. Sometimes, the adjuvant is selected from bacterial toxins, polyoxypropylene-polyethylene glycol block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions or combinations thereof. Sometimes, the adjuvant is an oil-in-water emulsion. The oil-in-water emulsion may include at least one oil and at least one surfactant, wherein the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplet diameter in the emulsion may be less than 5 μm, and may even have a submicron diameter, and these small sizes are achieved by a microfluidizer to provide a stable emulsion. Droplets less than 220 nm in size can be sterilized by filtration.

[0484] In some cases, the immunogenic pharmaceutical composition may include carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, antibacterial agents, chelating agents, suspending agents, thickeners and / or preservatives), water, oil (including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.), saline solutions, dextrose and glycerol aqueous solutions, flavorings, coloring agents, anti-adhesives and other acceptable additives, adjuvants or adhesives, other pharmaceutically acceptable auxiliary substances required for approximate physiological conditions, such as pH buffers, tonicity regulators, emulsifiers, wetting agents, etc. The example of excipient includes starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. In another case, the pharmaceutical product is substantially free of preservatives. In other cases, the pharmaceutical product may contain at least one preservative. It will be appreciated that while any suitable carrier known to those of ordinary skill in the art may be used to administer the pharmaceutical compositions described herein, the type of carrier will vary depending upon the mode of administration.

[0485] The immunogenic pharmaceutical composition may include a preservative, such as thimerosal or 2-phenoxyethanol. In some cases, the immunogenic pharmaceutical composition is substantially free (e.g., <10 μg / mL) of mercury species, e.g., free of thimerosal. Alpha-tocopheryl succinate may be used as a substitute for mercury compounds.

[0486] To control tonicity, the immunogenic pharmaceutical composition may contain physiological salts, such as sodium salts. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride.

[0487] The immunogenic pharmaceutical composition may have an osmolality of 200 to 400 mOsm / kg, 240-360 mOsm / kg, or 290-310 mOsm / kg.

[0488] The immunogenic pharmaceutical composition may comprise one or more buffers, such as Tris buffer; borate buffer; succinate buffer; histidine buffer (particularly containing aluminum hydroxide adjuvant); or citrate buffer. In some cases, the buffer is included at 5-20 or 10-50 mM.

[0489] The pH of the immunogenic pharmaceutical composition can be about 5.0 to about 8.5, about 6.0 to about 8.0, about 6.5 to about 7.5, or about 7.0 to about 7.8.

[0490] The immunogenic pharmaceutical composition may be sterile. The immunogenic pharmaceutical composition may be pyrogen-free, for example, containing <1 EU (endotoxin unit, a standard measure) per dose, and may be <0.1 EU per dose. The composition may be gluten-free.

[0491] The immunogenic pharmaceutical composition may include a detergent, for example, a polyoxyethylene sorbitan ester surfactant (known as a "Tween") or an octoxynol (such as octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol). The detergent may be present only in trace amounts. The immunogenic pharmaceutical composition may include less than 1 mg / mL of each of octoxynol-10 and polysorbate 80. Other residual components in trace amounts may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0492] The immunogenic pharmaceutical composition can be formulated as a sterile solution or suspension in a suitable vehicle known in the art. The pharmaceutical composition can be sterilized by conventional, known sterilization techniques, or can be sterilized by filtration. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized product combined with the sterile solution before administration.

[0493] For example, a pharmaceutical composition comprising an active agent (such as an immune cell disclosed herein) can be formulated with one or more adjuvant combinations to have a certain mol ratio. For example, an active agent such as an immune cell as described herein and one or more adjuvant combinations can be used. The mol ratio of about 99:1 to about 1:99 can be used. In some cases, the mol ratio of an active agent such as an immune cell as described herein and one or more adjuvant combinations 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 mol ratio of an active agent such as an immune cell as described herein and one or more adjuvant combinations can be about 1:9, and in some cases can be about 1:1. Active agents, such as the immune cells described herein, in combination with one or more adjuvants, can be formulated together in the same dosage unit, e.g., one vial, suppository, tablet, capsule, aerosol spray; or each agent, form and / or compound can be formulated in a separate unit, e.g., two vials, two suppositories, two tablets, two capsules, one tablet and one vial, aerosol spray, etc.

[0494] In some cases, the immunogenic pharmaceutical composition may be administered with an additional agent. The selection 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 having a therapeutic effect on pathogen infection (e.g., viral infection), including, for example, drugs used to treat inflammatory conditions, such as NSAIDs, for example, ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. For example, the checkpoint inhibitor can be a PD-1 / PD-L1 agonist selected from the group consisting of: nivolumab (ONO-4538 / BMS-936558, MDX1106, OPDIVO), pembrolizumab (MK-3475, KEYTRUDA), pidilizumab (CT-011) and MPDL328OA (ROCHE). As another example, the formulation can additionally contain one or more supplements, such as vitamin C, E or other antioxidants.

[0495] The pharmaceutical composition comprising an active agent such as an immune cell as described herein and a combination of one or more adjuvants can be prepared in a conventional manner using one or more physiologically acceptable carriers, which include excipients, diluents and / or adjuvants, such as carriers that promote the processing of active agents into products that can be used. Suitable formulations can rely at least in part on the selected route of administration. Medicaments as described herein can be delivered to patients using a variety of routes of administration or modes (including oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous and intramuscular administration, and by inhalation).

[0496] The active agent can be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and can be presented in unit dose form in ampoules, prefilled syringes, small volume infusions, or in preservative-added multidose containers. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, for example, a solution in aqueous polyethylene glycol.

[0497] In some embodiments, the pharmaceutical composition comprises a preservative or stabilizer. In some embodiments, the preservative or stabilizer is selected from a cytokine, a growth factor, an adjuvant, or a chemical. In some embodiments, the composition comprises at least one agent that helps maintain cell viability through at least one freeze-thaw cycle. In some embodiments, the composition comprises at least one agent that helps maintain cell viability through at least one freeze-thaw cycle.

[0498] For injectable preparations, vehicle can be selected from those suitable known in the art, including aqueous solution or oily suspension or emulsion (with sesame oil, corn oil, cottonseed oil or peanut oil), and elixir, mannitol, dextrose or sterile aqueous solution, and similar pharmaceutical vehicles. Said preparation can also include biocompatible, biodegradable polymer composition, such as poly (lactic acid-co-glycolic acid). These materials can be made into microspheres or nanospheres, loaded drugs, and further coated or derivatized to provide excellent sustained release performance. The vehicle suitable for periocular or intraocular injection includes, for example, a suspension of therapeutic agent in injection-grade water, liposomes and a vehicle suitable for lipophilic substances. Other vehicles used for periocular or intraocular injection are well known in the art.

[0499] In some cases, pharmaceutical composition is formulated into a pharmaceutical composition suitable for intravenous administration to human body according to conventional procedures. Generally speaking, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. When needed, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to alleviate the pain at the injection site. Usually, the composition is supplied separately or mixed together with a unit dosage form supply, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or a sachet (sachette) indicating the amount of the active agent. When the composition is administered by infusion, it can be distributed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the composition can be mixed before administration.

[0500] Preparation method

[0501] Provided herein are methods for preparing antigen-specific T cells. Provided herein are methods for preparing T cell compositions, such as therapeutic T cell compositions. For example, the methods may include expanding or inducing antigen-specific T cells. Preparing (e.g., inducing or expanding) T cells may also refer to preparing T cells and broadly encompasses isolating, stimulating, culturing, inducing, and / or expanding any type of T cell (e.g., CD4 T cells). + T cells and CD8 +In one aspect, there is provided herein a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence, the method comprising incubating APC with an immune cell colony from a biological sample that is depleted of cells expressing CD14 and / or CD25. In some embodiments, the method includes preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence, the method comprising incubating APC with an immune cell colony from a biological sample that is depleted of cells expressing CD11b and / or CD19. In some embodiments, the method includes incubating APC with an immune cell colony from a biological sample that is depleted of cells expressing any CD11b and / or CD19 and / or CD14 and / or CD25 or any combination thereof.

[0502] In a second aspect, the present invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample.

[0503] In a third aspect, the present invention provides a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising: incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample for a first period of time; and then incubating at least one T cell from the biological sample with APCs.

[0504] In a fourth aspect, the present invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising incubating an immune cell population from a biological sample with one or more APC preparations for one or more separate time periods, the time period being less than 28 days from incubation of the immune cell population with the first APC preparation of the one or more APC preparations, wherein at least one antigen-specific memory T cell is expanded or at least one antigen-specific naive T cell is induced.

[0505] In a fifth aspect, the present invention provides a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, the method comprising incubating a population of immune cells from a biological sample with three or fewer APC preparations for three or fewer time periods, wherein at least one antigen-specific memory T cell is expanded, or at least one antigen-specific naive T cell is induced.

[0506] In some embodiments, a method of preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods, thereby stimulating the T cells to become antigen-specific T cells, wherein the percentage of antigen-specific T cells is 100% of the total CD4 + T cells, total CD8 + or at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of T cells, total T cells, or total immune cells. In some embodiments, the method of preparing antigen-specific T cells comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence comprises incubating an immune cell population from a biological sample with three or fewer APC preparations for three or fewer separate time periods, thereby stimulating the T cells to become antigen-specific T cells. In some embodiments, the method of preparing antigen-specific T cells comprising a T cell receptor (TCR) having specificity for at least one antigenic peptide sequence comprises incubating an immune cell population from a biological sample with two or fewer APC preparations for two or fewer separate time periods, thereby stimulating the T cells to become antigen-specific T cells.

[0507] In some embodiments, a method is provided herein, which includes incubating an immune cell population from a biological sample with one or more APC preparations for one or more separate time periods to stimulate T cells to become antigen-specific T cells, wherein the APC preparation is a PBMC cell population, and before the APC population is loaded with antigen, cells expressing one or more cell surface markers are consumed from the cell population. In some embodiments, CD14+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD25+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD19+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD3+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD25+ cells and CD14+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+ and CD25+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+ and CD14+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+, CD14+ and CD25+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+ and CD19+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+, CD19+ and CD25+ cells are consumed before the APC population is loaded with antigen. In some embodiments, CD11b+, CD14+, CD19+ and CD25+ cells are consumed before the APC population is loaded with antigen. In some embodiments, the method includes adding a cell PBMC-derived population of APC enriched with CD3+ cells depleted to any of the above-mentioned consumed APC populations. In some embodiments, the cell PBMC-derived population of the APC enrichment is depleted of CD3+, and the cells are depleted of any one or more of CD11b+, CD14+, CD19+ or CD25+.

[0508] 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 thereof to the PBMC sample, thereby loading the antigen onto the APCs within the PBMCs for presentation of the antigen to the T cells in the PBMCs.

[0509] In some embodiments, the method comprises: (a) obtaining a biological sample from a subject, the biological sample comprising at least one antigen presenting cell (APC); (b) enriching cells expressing CD11c from the biological sample, thereby obtaining CD11c + Cell-enriched samples; (c) CD11c+ The cell-enriched sample is incubated with at least one cytokine or growth factor for a first period of time; (d) at least one peptide is combined with the CD11c of (c) + (e) incubating the APC sample with one or more cytokines or growth factors for a third time period to obtain a mature APC sample; (f) incubating the APCs of the mature APC sample with a sample comprising PBMCs depleted of CD11b and / or CD14 and / or CD25 for a fourth time period; (g) incubating the PBMCs with the APCs of the mature APC sample for a fifth time period; (h) incubating the PBMCs with the APCs of the mature APC sample for a sixth time period; and (i) administering at least one T cell of the PBMCs to a subject in need thereof.

[0510] In some embodiments, the method comprises: (a) obtaining a biological sample from a subject, the biological sample comprising at least one antigen presenting cell (APC); (b) enriching cells expressing CD14 from the biological sample, thereby obtaining CD14 + Cell-enriched samples; (c) CD14 + The cell-enriched sample is incubated with at least one cytokine or growth factor for a first period of time; (d) at least one peptide is combined with the CD14 of (c) + (e) incubating the APC peptide-loaded sample with one or more cytokines or growth factors for a third time period to obtain a mature APC sample; (f) incubating the APCs of the mature APC sample with a CD14 and / or CD25-depleted sample comprising PBMCs for a fourth time period; (g) incubating the PBMCs with the APCs of the mature APC sample for a fifth time period; (h) incubating the PBMCs with the APCs of the mature APC sample for a sixth time period; and (i) administering at least one T cell of the PBMCs to a subject in need thereof.

[0511] In some embodiments, the method comprises: (a) obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD11b and / or CD19 from the biological sample, thereby obtaining a CD11b and / or CD19 cell-depleted sample; (c) incubating the CD11b and / or CD19 cell-depleted sample with FLT3L for a first period of time; (d) incubating at least one peptide with the CD11b and / or CD19 cell-depleted sample of (c) for a second period of time, thereby obtaining an APC-peptide-loaded sample. (e) incubating the APC peptide-loaded sample with at least one PBMC for a third time period, thereby obtaining a first stimulated PBMC sample; (f) incubating the PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth time period, thereby obtaining a second stimulated T cell sample; (g) incubating the PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth time period, thereby obtaining a third stimulated PBMC sample; and (h) administering at least one T cell from the third stimulated PBMC sample to a subject in need thereof.

[0512] In some embodiments, the method comprises: (a) obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD11b and / or CD19 and / or CD14 and / or CD25 from the biological sample, thereby obtaining a CD11b and / or CD19 cell-depleted sample; (c) incubating the CD11b and / or CD19 and / or CD14 and / or CD25 cell-depleted sample with FLT3L for a first period of time; (d) combining at least one peptide with the CD11b and / or CD19 and / or CD14 and / or CD25 cell-depleted sample of (c); (e) incubating the APC peptide-loaded sample with at least one PBMC for a third time period to obtain a first stimulated PBMC sample; (f) incubating the PBMCs of the first stimulated PBMC sample with APCs of the mature APC sample for a fourth time period to obtain a second stimulated T cell sample; (g) incubating the PBMCs of the second stimulated PBMC sample with APCs of the mature APC sample for a fifth time 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 in need thereof.

[0513] In some embodiments, the method comprises: (a) obtaining a biological sample from a subject, the biological sample comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD14 and / or CD25 from the biological sample, thereby obtaining a CD14 and / or CD25 cell-depleted sample; (c) incubating the CD14 and / or CD25 cell-depleted sample with FLT3L for a first period of time; (d) incubating at least one peptide with the CD14 and / or CD25 cell-depleted sample of (c) for a second period of time, thereby obtaining an APC peptide-loaded sample; (e) incubating the APC peptide-loaded sample with at least one PBMC for a third time period, thereby obtaining a first stimulated PBMC sample; (f) incubating the PBMCs from the first stimulated PBMC sample with APCs from the mature APC sample for a fourth time period, thereby obtaining a second stimulated T cell sample; (g) incubating the PBMCs from the second stimulated PBMC sample with APCs from the mature APC sample for a fifth time period, thereby obtaining a third stimulated PBMC sample; (h) administering at least one T cell from the third stimulated PBMC sample to a subject in need thereof.

[0514] In some embodiments, a method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating an APC with a population of immune cells from a biological sample depleted of cells expressing CD14 and / or CD25.

[0515] In some embodiments, provided herein is a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, the method comprising incubating an immune cell population from a biological sample with one or more APC preparations for one or more separate time periods, the time period being less than 28 days from the incubation of the immune cell population with the first APC preparation of the one or more APC preparations, wherein at least one antigen-specific memory T cell is expanded, or at least one antigen-specific naive T cell is induced. In some embodiments, provided herein is a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigen peptide sequence, the method comprising incubating an immune cell population from a biological sample with 3 or fewer APC preparations for 3 or fewer separate time periods, wherein at least one antigen-specific memory T cell is expanded, or at least one antigen-specific naive T cell is induced.

[0516] In some embodiments, the method for preparing an antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigen peptide sequence includes contacting an immune cell (e.g., PBMC) colony with an APC. In some embodiments, the method for preparing an antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigen peptide sequence includes incubating an immune cell (e.g., PBMC) colony with an APC for a period of time. In some embodiments, the immune cell colony is from a biological sample. In some embodiments, the immune cell colony is from a sample (e.g., a biological sample) of cells that have been depleted of expression CD14. In some embodiments, the immune cell colony is from a sample (e.g., a biological sample) of cells that have been depleted of expression CD25. In some embodiments, the immune cell colony is from a sample (e.g., a biological sample) of cells that have been depleted of expression CD14 and cells that have been expressed CD25.

[0517] In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating an APC stimulated by FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with an immune cell population from a biological sample. In some embodiments, provided herein is a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence, the method comprising: incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with an immune cell population from a biological sample for a first period of time; thereafter incubating at least one T cell from the biological sample with an APC.

[0518] In some embodiments, the method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigen peptide sequence comprises contacting an immune cell population from a sample (e.g., a biological sample) with an FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, the method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigen peptide sequence comprises contacting an immune cell population from a sample (e.g., a biological sample) with an APC stimulated by an FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, the method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) having specificity for at least one antigen peptide sequence comprises incubating an immune cell population from a sample (e.g., a biological sample) with an APC stimulated by an FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, a method for preparing a pharmaceutical composition comprising at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample (e.g., for a period of time); and then contacting the T cells of the biological sample with an APC. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises contacting a population of immune cells from a sample (e.g., a biological sample) with one or more APC preparations. In some embodiments, a method for preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a sample (e.g., a biological sample) with one or more APC preparations for one or more separate time periods. In some embodiments, the method of preparing at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a sample (e.g., a biological sample) with one or more APC preparations for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate time periods. In some embodiments, the one or more separate time periods are less than 28 days, calculated from the incubation of the immune cell population with the first of the one or more APC preparations.

[0519] In some embodiments, the method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating an immune cell population with APCs for a period of time, wherein the immune cell population is from a biological sample comprising PBMCs. In some embodiments, the method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating an immune cell population with APCs for a period of time, wherein the immune cell population is from a biological sample depleted of cells expressing CD14 and / or CD25.

[0520] In some embodiments, a method of preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with APCs stimulated with FMS-like tyrosine kinase 3 receptor ligand (FLT3L) for a period of time.

[0521] In some embodiments, a method of preparing a pharmaceutical composition comprising antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample; and then contacting the T cells from the biological sample with APCs.

[0522] In some embodiments, a method for preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating an immune cell population from a biological sample with one or more APC preparations for one or more separate time periods, thereby inducing or expanding antigen-specific T cells, wherein the one or more separate time periods are less than 28 days from the time the immune cell population is incubated with the first APC preparation of the one or more APC preparations. In some embodiments, the immune cell population from a biological sample is incubated with one or more APC preparations for one or more separate time periods in a medium containing IL-7, IL-15, or a combination thereof. In some embodiments, the medium further comprises an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof. The IDO inhibitor can be epacadostat, navoximod, 1-methyltryptophan, or a combination thereof. In some embodiments, an IDO inhibitor can increase antigen-specific CD8 + In some embodiments, IDO inhibitors can maintain memory CD8 +Functional spectrum of T cell responses. PD-1 antibodies can increase the absolute number of antigen-specific memory CD8+ T cell responses. PD-1 antibodies can increase the proliferation rate of cells treated with this antibody. Addition of IL-12 can lead to an increase in antigen-specific cells and / or CD8 + The frequency of T cells increases.

[0523] In some embodiments, a method of preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods, thereby expanding or inducing antigen-specific T cells, wherein the antigen-specific T cells, antigen-specific CD4 + T cells or antigen-specific CD8 + The percentage of T cells is total T cells, total CD4 + T cells, total CD8 + or at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of T cells, total immune cells, or total cells.

[0524] In some embodiments, a method of preparing antigen-specific T cells comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence comprises incubating a population of immune cells from a biological sample with three or fewer APC preparations for three or fewer separate time periods, thereby stimulating the T cells to become antigen-specific T cells.

[0525] In some embodiments, the immune cell colony is from a biological sample that is depleted of cells expressing CD14 and / or CD25. In some embodiments, the APC is an APC stimulated by FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, the APC comprises one or more APC products. In some embodiments, the APC product comprises 3 or fewer APC products. In some embodiments, the APC product is sequentially incubated with immune cells in one or more separate time periods.

[0526] In some embodiments, the biological sample is from a subject. In some embodiments, the subject is a human. For example, the subject can be a patient or a donor. In some embodiments, the subject has a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the antigen-specific T cells comprise CD4 + and / or CD8 + In some embodiments, the antigen-specific T cells comprise CD4-enriched T cells and / or CD8-enriched T cells. For example, CD4-enriched T cells can be isolated, enriched, or purified from a biological sample from a subject comprising PBMCs. + T cells and / or CD8 + In some embodiments, the antigen-specific T cells are naive CD4 + and / or naive CD8 + In some embodiments, the antigen-specific T cells are memory CD4 + and / or memory CD8 + T cells.

[0527] In some embodiments, the at least one antigenic peptide sequence comprises a mutation selected from the group consisting of: (A) a point mutation, and the cancer antigenic peptide is expressed with an IC of less than 500 nM. 50 and bind to the HLA protein of the subject with greater affinity than the corresponding wild-type peptide, (B) splice site mutation, (C) frameshift mutation, (D) read-through mutation, (E) gene fusion mutation, and combinations thereof. In some embodiments, each of the at least one antigenic peptide sequence binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in the non-cancerous cell of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by an expression gene of a cancer cell of the subject. In some embodiments, one or more of the at least one antigenic peptide sequence has a length of 8-50 naturally occurring amino acids. In some embodiments, the at least one antigenic peptide sequence comprises a plurality of antigenic peptide sequences. In some embodiments, the plurality of antigenic peptide sequences comprise 2-50, 3-50, 4-50, 5-50, 6-50, 7-50, 8-50, 9-50 or 10-50 antigenic peptide sequences.

[0528] In some embodiments, the APC comprises an APC loaded with one or more antigenic peptides comprising one or more of at least one antigenic peptide sequence. In some embodiments, the APC is an autologous APC or an allogeneic APC. In some embodiments, the APC comprises a dendritic cell (DC).

[0529] In some embodiments, the method comprises depleting cells expressing CD14 and / or CD25 from a biological sample. In some embodiments, depleting CD14 + The cell comprises contacting a CD14 binding agent with an APC. In some embodiments, the APC is derived from a CD14 + In some embodiments, APCs are enriched from a biological sample. For example, APCs can be isolated, enriched, or purified from a biological sample from a subject that contains PBMCs.

[0530] In some embodiments, APCs are stimulated with one or more cytokines or growth factors. In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, or a combination thereof. In some embodiments, the one or more cytokines or growth factors include IL-4, GM-CSF, TNF-α, IL-1β, PGE1, IL-6, IL-7, or a combination thereof.

[0531] In some embodiments, the APCs are from a second biological sample. In some embodiments, the second biological sample is from the same subject.

[0532] In some embodiments, the percentage of antigen-specific T cells in the methods is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells. In some embodiments, the percentage of antigen-specific T cells in the methods is about 0.1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to 65%, or about 65% to about 70% of total T cells or total immune cells. In some embodiments, the percentage of antigen-specific CD8 + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the total T cells or total immune cells. In some embodiments, the method comprises antigen-specific naive CD8 +The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the total T cells or total immune cells. In some embodiments, the method comprises antigen-specific memory CD8 + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the total T cells or total immune cells. In some embodiments, the method wherein the antigen-specific CD4 + The percentage of T cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the total T cells or total immune cells. In some embodiments, the method wherein the antigen-specific CD4 + In some embodiments, the percentage of antigen-specific T cells in a 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 the total T cells or total immune cells. In some embodiments, the percentage of antigen-specific T cells in a biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the percentage of antigen-specific CD8 T cells in a biological sample 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%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the percentage of T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the percentage of T cells in a biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. + The percentage of T cells is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the percentage of T cells in a biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. +The percentage of T cells is up to about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0533] In some embodiments, the biological sample is freshly obtained from a subject or is a frozen sample.

[0534] In some embodiments, the method comprises incubating one or more APC preparations with a first medium comprising at least one cytokine or growth factor for a first period of time. In some embodiments, the first period of time is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 or 18 days. In some embodiments, the first period of time is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days.

[0535] In some embodiments, the first period of time is at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 days.

[0536] In some embodiments, the first time period is no more than 3, 4, 5, 6, 7, 8, 9, or 10 days.In some embodiments, the at least one cytokine or growth factor comprises GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-γ, LPS, IFN-α, R848, LPS, ss-rna40, poly I:C, or any combination thereof.

[0537] In some embodiments, the method comprises incubating the one or more APC preparations with the at least one peptide for a second period of time. In some embodiments, the second period of time is no more than 1 hour.

[0538] In some embodiments, the method includes incubating one or more APC products with a second medium comprising one or more cytokines or growth factors for a third time period to obtain mature APCs. In some embodiments, the one or more cytokines or growth factors include GM-CSF (granulocyte macrophage colony stimulating factor), IL-4, FLT3L, IFN-γ, LPS, TNF-α, IL-1β, PGE1, IL-6, IL-7, IFN-α, R848 (Resiquimod), LPS, ss-rna40, poly I: C, CpG or a combination thereof. In some embodiments, the third time period is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 days. In some embodiments, the third time period is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 days. In some embodiments, the third period of time is no more than 2, 3, 4, or 5 days. In some embodiments, the third period of time is at least 1, 2, 3, or 4 days.

[0539] In some embodiments, the method further comprises removing the one or more cytokines or growth factors from the second medium after the third period of time and before initiating the fourth period of time.

[0540] Antigen-loaded PBMCs for in vitro T cell induction

[0541] In some embodiments, the methods provided herein include isolating PBMCs from a human blood sample and directly loading the PBMCs with antigens. PBMCs in direct contact with the antigen can easily absorb the antigen through phagocytosis and present the antigen to T cells that may be present in the culture or added to the culture. In some embodiments, the methods provided herein include isolating PBMCs from a human blood sample and nuclear transfection or electroporation of polynucleotides encoding one or more antigens, such as mRNA, into the PBMCs. In some embodiments, antigen delivery to PBMCs, rather than antigen-presenting cells that mature into DCs, provides great advantages in terms of time and preparation efficiency. PBMCs can further deplete one or more cell types. In some embodiments, in the initial stage of antigen loading, PBMCs can be depleted of CD3+ cells, and CD3+ cells are returned to the PBMC culture to stimulate CD3+ T cells. In some embodiments, PBMCs can be depleted of CD25+ cells. In some embodiments, PBMCs can be depleted of CD14+ cells. In some embodiments, PBMCs can be depleted of CD19+ cells. In some embodiments, PBMCs can be depleted of CD14 and CD25 expressing cells. In some embodiments, CD11b+ cells are depleted from the PBMC sample prior to antigen loading. In some embodiments, CD11b+ and CD25+ cells are depleted from the PBMC sample prior to antigen loading.

[0542] In some embodiments, PBMCs isolated from human blood samples can be minimally processed as possible prior to antigen loading. Increased processing of PBMCs, such as freezing and thawing cells, multiple cell depletion steps, etc., may compromise the health and viability of the cells.

[0543] In some embodiments, the PBMCs are allogeneic to the subject being treated. In some embodiments, the PBMCs are allogeneic to the subject being treated with adoptive cell therapy of antigen-specific T cells.

[0544] In some embodiments, PBMC is HLA matched for treating the subject. In some embodiments, PBMC is allogeneic and matched for the HLA subtype of the subject, while CD3+ T cells are autologous. PBMC is loaded with respective antigens (e.g., as determined by analysis of a peptide presentation analysis platform such as RECON), co-cultured with the subject's PBMC comprising T cells, to stimulate antigen-specific T cells.

[0545] In some embodiments, mRNA is used as an immunogen for uptake and antigen presentation. One advantage of using mRNA over peptide antigen loading of PBMCs is that RNA acts as an adjuvant on its own, without the need for additional adjuvants. Another advantage of using mRNA is that peptides are endogenously processed and presented. In some embodiments, mRNA comprises a shortmer construct that encodes a peptide of 9-10 amino acids comprising an epitope. In some embodiments, mRNA comprises a longmer construct that encodes a peptide of approximately 25 amino acids. In some embodiments, mRNA comprises a series of multiple epitopes. In some embodiments, a concatemer may comprise one or more epitopes from the same antigenic protein. In some embodiments, a concatemer may comprise one or more epitopes from several different antigenic proteins. Several embodiments are described in the Examples section. Loading antigens to PBMCs by antigen loading may include various mechanisms for delivering and incorporating nucleic acids into PBMCs. In some embodiments, delivery or incorporation mechanisms include transfection, electroporation, nuclear transfection, chemical delivery, such as lipid-encapsulated or liposome-mediated delivery.

[0546] Using PBMC loaded with antigen to stimulate T cells can save the maturation time required in the method of generating DC from PBMC sample before T cell stimulation. In some embodiments, using PBMC loaded with antigen, such as PBMC loaded with mRNA as APC, the total preparation time is shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. In some embodiments, using PBMC loaded with antigen as APC shortens the total preparation time by 3 days. In some embodiments, using PBMC loaded with antigen as APC shortens the total preparation time by 4 days. In some embodiments, using PBMC loaded with antigen as APC shortens the total preparation time by 5 days. In some embodiments, using PBMC loaded with antigen as APC shortens the total preparation time by 6 days. In some embodiments, using PBMC loaded with antigen as APC shortens the total preparation time by 7 days.

[0547] In some embodiments, using mRNA as antigen may be preferred because it is easy to design and prepare nucleic acid, and is easy to transfect PBMC.In some embodiments, the PBMC of load mRNA can stimulate T cell and generate more antigen-specific T cells.In some embodiments, the PBMC of load mRNA can stimulate T cell and generate the antigen-specific T cells of higher yield.In some embodiments, the PBMC of load mRNA can stimulate T cell and generate the higher presentation with input antigen, i.e., there is reactive antigen-specific T cell to a variety of antigens.In some embodiments, the PBMC of load mRNA can stimulate the T cell with at least 1,2,3,4,5,6,7,8,9,10 or more antigen-reactive in expansion cell pool.In some embodiments, the PBMC of load mRNA can stimulate the T cell with at least 1,2,3,4,5,6,7,8,9,10 or more antigen-reactive compared with the APC (such as the DC of load peptide) of conventional load antigen.

[0548] Treatment

[0549] Provided herein is a method for treating cancer in a subject, comprising: I. contacting cancer neoantigen-loaded antigen-presenting cells (APCs) with isolated T cells ex vivo, wherein the cancer neoantigen-loaded antigen-presenting cells (APCs) are CD11b-depleted; II. preparing cancer neoantigen-triggered T cells for use in a cell composition for ex vivo cancer immunotherapy; and III. administering the cell composition for cancer immunotherapy to the subject, wherein the administration alleviates or improves at least one or more conditions or symptoms associated with cancer, thereby treating the subject, wherein the cancer neoantigen-loaded APCs and cancer neoantigen-triggered T cells each express a protein encoded by an HLA allele expressed in the subject, and the neoantigen can specifically bind to the protein.

[0550] In some embodiments, the method further includes administering one or more of the at least one antigen-specific T cells to a subject. In some embodiments, the therapeutic composition comprising T cells is administered by injection. In some embodiments, the therapeutic composition comprising T cells is administered by infusion. When administered by injection, the active agent can be formulated in an aqueous solution, particularly in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or saline buffer. The solution may contain a formulation agent, such as a suspending agent, a stabilizer, and / or a dispersant. In another embodiment, the pharmaceutical composition does not include an adjuvant or any other substance added to enhance the immune response stimulated by the peptide. In some embodiments, the method further includes administering one or more of the at least one antigen-specific T cells as a pharmaceutical composition as described herein to a subject. In some embodiments, the pharmaceutical composition includes a preservative or stabilizer. In some embodiments, the preservative or stabilizer is selected from a cytokine, a growth factor, an adjuvant, or a chemical substance. 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.

[0551] In addition to the preparations described above, active agents can also be formulated into depot products. Such long-acting preparations can be administered by implantation or transdermal delivery (e.g., subcutaneous or intramuscular), intramuscular injection, or using a transdermal patch. Thus, for example, the medicament can be formulated with a suitable polymeric material or hydrophobic material (e.g., an emulsion in an acceptable oil) or an ion exchange resin, or formulated as a slightly soluble derivative, e.g., as a slightly soluble salt.

[0552] Also provided herein are methods of treating a subject having a disease, disorder, or condition. The methods of treatment may comprise administering a composition or pharmaceutical composition disclosed herein to a subject having a disease, disorder, or condition.

[0553] The present disclosure provides methods for treating diseases such as cancer or viral infections. Methods can include administering to a subject an effective amount of a composition comprising immunogenic antigen-specific T cells according to the methods provided herein. In some embodiments, the antigen comprises a viral antigen. In some embodiments, the antigen comprises a tumor antigen.

[0554] Non-limiting examples of therapeutics that can be prepared include peptide-based therapeutics, nucleic acid-based therapeutics, antibody-based therapeutics, T-cell-based therapeutics, and antigen-presenting cell-based therapeutics.

[0555] In some other aspects, provided herein is a use of a composition or pharmaceutical composition in the preparation of a medicament for treatment. In some embodiments, the treatment method comprises administering to a subject an effective amount of T cells that specifically recognize an immunogenic neoantigenic peptide. In some embodiments, the treatment method comprises administering to a subject an effective amount of TCRs that specifically recognize an immunogenic neoantigenic peptide, such as TCRs expressed in T cells.

[0556] In some embodiments, the cancer is selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, colorectal cancer, rectal cancer, soft tissue sarcoma, Kaposi's sarcoma, B cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma), NHL, small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunogenic NHL, high-grade lymphocytic NHL, high-grade small non-lytic cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloma, hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with nevus hamartomatosis, edema, Meigs syndrome, and combinations thereof.

[0557] The methods described herein are particularly useful in personalized medicine settings, where immunogenic neoantigenic peptides identified according to the methods described herein are used to develop therapeutics (such as vaccines or therapeutic antibodies) for the same individual. Thus, a method for treating a disease in a subject can include identifying an immunogenic neoantigenic peptide in a subject according to the methods described herein; synthesizing the peptide (or a precursor thereof, such as a polynucleotide encoding the peptide (e.g., mRNA)); and preparing T cells specific for the identified neoantigen; and administering the neoantigen-specific T cells to the subject. In some embodiments, a method for treating a disease in a subject can include identifying an immunogenic neoantigenic peptide in a subject according to the methods described herein; synthesizing a polynucleotide encoding the immunogenic neoantigenic peptide or a precursor thereof, such as mRNA; and preparing T cells specific for the identified neoantigen; and administering the neoantigen-specific T cells to the subject.

[0558] The medicaments and compositions provided herein can be used alone or in combination with conventional treatment regimens such as surgery, radiation, chemotherapy and / or bone marrow transplantation (autologous, isogenic, allogenic or unrelated).For example, a group of tumor antigens can be identified using the methods described herein, and it can be used, for example, in most cancer patients.

[0559] In some embodiments, in addition to the composition comprising the immunogenic therapeutic agent, at least one or more chemotherapeutic agents may be administered. In some embodiments, the one or more chemotherapeutic agents may belong to different classes of chemotherapeutic agents.

[0560] In practicing the treatments or methods of use provided herein, a therapeutically effective amount of a therapeutic agent can be administered to a subject suffering from a disease or condition. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors.

[0561] The subject can be, for example, a mammal, a human, a pregnant woman, an elderly person, an adult, a teenager, a prepubescent child, a child, a toddler, an infant, a neonate or a newborn baby. The subject can be a patient. In some cases, the subject can be a human. In some cases, the subject can be a child (i.e., a young person below puberty). In some cases, the subject can be an infant. In some cases, the subject can be a formula-fed infant. In some cases, the subject can be an individual participating in a clinical study. In some cases, the subject can be a laboratory animal, such as a mammal or a rodent. In some cases, the subject can be a mouse. In some cases, the subject can be an obese or overweight subject.

[0562] In some embodiments, the subject has been previously treated with one or more different cancer treatment modalities. In some embodiments, the subject has been previously treated with one or more of radiation therapy, chemotherapy, or immunotherapy. In some embodiments, the subject has been treated with one, two, three, four, or five lines of prior therapy. In some embodiments, the prior therapy was cytotoxic therapy.

[0563] In some embodiments, a disease or condition that can be treated with the methods disclosed herein is cancer. Cancer is the abnormal growth of cells that often proliferates in an uncontrolled manner and, in some cases, metastasizes (spreads). Tumors can be cancerous or benign. A benign tumor is one that can grow but not spread. A cancerous tumor is malignant, which means it can grow and spread to other parts of the body. If cancer spreads (metastasizes), the new tumor has the same name as the original (primary) tumor.

[0564] The methods of the present disclosure can be used to treat any type of cancer known in the art. Non-limiting examples of cancers treated by the methods of the present disclosure ...

Claims

1. 1x10 for treating cancer in a subject in need thereof 8 Up to 1x10 11 An expanded cell population of cells comprising tumor antigen-specific T cells is prepared by the following method, the method comprising: (a) depleting CD14+ cells and CD25+ cells from an immune cell population comprising antigen presenting cells (APCs) and T cells, thereby forming a CD14- and CD25-depleted immune cell population comprising a first population of APCs and T cells, wherein the immune cell population is derived from a peripheral blood sample of a human subject; (b) incubating the APCs and the first population of T cells from step (a) for a first period of time in the presence of: (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by a cancer cell of a human subject suffering from cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a cell population comprising stimulated T cells; and (c) expanding the stimulated T cells from step (b) to form an expanded cell population, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising: (i) the at least one tumor antigen epitope sequence from step (b)(ii), and (ii) an MHC protein expressed by a cancer cell or APC from the human subject of (b)(ii), and wherein at least 0.1% of the CD8+ T cells in the expanded cell population are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells.

2. The expanded cell population of claim 1, wherein the polypeptide comprises at least two tumor antigen epitope sequences expressed by cancer cells of the human subject, wherein the at least two tumor antigen epitope sequences are different.

3. The expanded cell population according to claim 2, wherein the at least two tumor antigen epitope sequences are each 8 to 12 amino acids in length. The cell population according to claim 2 , wherein the at least two tumor antigen epitope sequences are each 15 to 25 amino acids in length.

5. The expanded cell population of claim 1, wherein the polypeptide comprises at least three different tumor antigen epitope sequences expressed by cancer cells of a human subject suffering from cancer.

6. The expanded cell population of claim 1, wherein the expanded cell population comprises CD3+ cells, wherein the percentage of CD3+ cells in the expanded cell population is at least 40% of the total cell population.

7. The expanded cell population of claim 1, wherein the percentage of CD107a+ cells in the expanded cell population is at least 10% of the tumor antigen-specific T cell population.

8. The expanded cell population of claim 1, wherein the percentage of TNFα+ cells in the expanded cell population is at least 5% of the tumor antigen-specific T cell population.

9. The expanded cell population of claim 1, wherein the percentage of IFNγ+ cells in the expanded cell population is at least 15% of the tumor antigen-specific T cell population.

10. The expanded cell population of claim 1, wherein the percentage of TNFα+ and IFNγ+ cells in the expanded cell population is at least 2% of the tumor antigen-specific T cell population.

11. The expanded cell population of claim 1, wherein the percentage of TNFα+ and CD107a+ cells in the expanded cell population is at least 0.5% of the tumor antigen-specific T cell population.

12. The expanded cell population of claim 1, wherein the percentage of IFNγ+ and CD107a+ cells in the expanded cell population is at least 5% of the tumor antigen-specific T cell population.

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

14. The expanded cell population of claim 1, wherein the expanded cell population comprises CD4+ cells, and the percentage of CD4+ T cells in the expanded cell population that are naive T cells (CD62L+ and CD45RA+) is at most 15%.

15. The expanded cell population of claim 1, wherein the percentage of CD4+ T cells in the expanded cell population that are effector memory T cells (CD62L- and CD45RA-) is at least 60%.

16. The expanded cell population of claim 1, wherein the percentage of CD4+ T cells that are effector T cells (CD62L- and CD45RA+) in the expanded cell population is at most 5%.

17. The expanded cell population of claim 1, wherein the percentage of CD4+ T cells in the expanded cell population that are central memory T cells (CD62L+ and CD45RA-) is at least 10%.

18. The expanded cell population of claim 1, wherein the expanded cell population comprises CD8+ cells, wherein the percentage of CD8+ T cells in the expanded cell population that are naive T cells (CD62L+ and CD45RA+) is at most 25%.

19. The expanded cell population of claim 1, wherein the percentage of CD8+ T cells in the expanded cell population that are effector memory T cells (CD62L- and CD45RA-) is at least 60%.

20. The expanded cell population of claim 1, wherein the percentage of CD8+ T cells that are effector T cells (CD62L- and CD45RA+) in the expanded cell population is at most 10%.

21. The expanded cell population of claim 1, wherein the percentage of CD8+ T cells in the expanded cell population that are central memory T cells (CD62L+ and CD45RA-) is at least 15%.

22. The expanded cell population of claim 1, wherein the expanded cell population produces cytokines and induces degranulation upon recognition of target cells.

23. The expanded cell population of claim 1, wherein the human subject is refractory to anti-checkpoint inhibitor therapy.

24. The expanded cell population of claim 1, wherein the human subject has a mutation in the BRAF gene and has previously received a B-raf inhibitor or a B-raf / MEK combination therapy.

25. The expanded cell population of claim 1, wherein the depletion comprises depleting CD14+ cells and CD25+ cells from a peripheral blood mononuclear cell (PBMC) sample from a human subject that has not undergone a step of maturation of monocytes into mature dendritic cells (mature DCs).

26. The expanded cell population of claim 1, wherein the ratio of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in the expanded cell population is at least twice the ratio of CD8+ tumor antigen-specific T cells to the total number of CD8+ T cells in the biological sample.

27. The expanded cell population of claim 1, wherein the ratio of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in the expanded cell population is at least twice the ratio of CD4+ tumor antigen-specific T cells to the total number of CD4+ T cells in the biological sample.

28. The expanded cell population of claim 1, wherein at least 0.1% of the CD4+ T cells in the expanded cell population are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

29. The expanded cell population of claim 1, wherein: (I) the cancer is unresectable melanoma, (II) the subject has previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor and has had disease progression, or (III) The subject has received or is currently receiving a PD-1 inhibitor or PD-L1 inhibitor for at least 3 months and has stable disease or asymptomatic disease progression.

30. A pharmaceutical composition comprising the expanded cell population of claim 1; and a pharmaceutically acceptable carrier.

Images