Compositions and methods for producing T cells

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 methods for producing T cells and therapeutic T cell compositions that can be used to treat subjects with personalized antigen-specific T cell therapies for cancer and other conditions, diseases, and disorders.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 386,810, filed December 9, 2022; U.S. Provisional Application No. 63 / 499,870, filed May 3, 2023, which applications are incorporated by reference in their entireties into this specification. [Background technology]

[0002] Tumor vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., adjuvants, cytokines, or TLR ligands) that cooperate to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. Such vaccines contain either common tissue-restricted tumor antigens or a mixture of common patient-specific antigens in the form of whole tumor cell preparations. Common tissue-restricted tumor antigens are ideally immunogenic proteins selectively expressed in tumors across many individuals and are typically 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. Because whole tumor cells are isolated from autologous patients, the cells may contain common tumor antigens in addition to patient-specific tumor antigens. Finally, there is a third class of tumor antigens, neoantigens, consisting of proteins (which may be patient-specific or common) with tumor-specific mutations that result in altered amino acid sequences, but these have rarely been used in vaccines. Such mutant proteins (a) are unique to the tumor cell because the mutation and its corresponding protein are present only in the tumor; (b) are more likely to be immunogenic because they circumvent central tolerance; and (c) provide excellent targets for immune recognition, including by both humoral and cellular immunity.

[0003] Adoptive immunotherapy or adoptive cell therapy (ACT) is the transfer of lymphocytes into a subject for disease therapy. Adoptive immunotherapy has yet to realize its potential for treating a variety of diseases, including cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immune deficiencies. However, most, if not all, adoptive immunotherapy strategies require T cell activation and expansion steps to generate clinically effective therapeutic doses of T cells. Due to the inherent complexity of live cell culture and interpatient variability, current technologies for generating therapeutic doses of T cells, including engineered T cells, remain limited by cumbersome T cell manufacturing processes. Existing T cell manufacturing processes are not easily scalable, repeatable, reliable, or efficient, and often produce inferior T cell products that may be prone to exhaustion and loss of effector immune cell function. To date, engineered T cell adoptive immunotherapy has met with limited success and routinely exhibits variable clinical activity. Therefore, such therapies are not suitable for widespread clinical use. Thus, there remains a need to develop compositions and methods for the expansion and induction of antigen-specific T cells with favorable phenotype and function. Summary of the Invention [Means for solving the problem]

[0004]

[0004] Although autologous T cell therapeutics are safe to use, significant improvements are needed to meet therapeutic standards, and development in this field has been both rapid and challenging.

[0005]

[0005] In one aspect, provided herein is a method of treating cancer in a human subject in need of cancer treatment, comprising the steps of administering to the human subject an expanded population of cells comprising tumor antigen-specific T cells, the expanded population of cells being from a population of immune cells comprising APCs and a first population of T cells, the first population being depleted of CD14+ and / or CD25+ cells and incubated 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 cancer cells of the human subject, or (B) a polynucleotide encoding the polypeptide; the tumor antigen-specific T cells comprising T cells specific to a complex comprising (i) at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by cancer cells or APCs of the human subject; and administering a cytokine to the human subject.

[0006] In another aspect, a method of treating cancer in a human subject in need thereof comprises the steps of: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a population of immune cells comprising a first population of CD14- and / or CD25-depleted APCs and T cells; (b) treating the first population of APCs and T cells from step (a) for a first period of time with (i) an FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of the human subject having cancer, or (B) said polypeptide. Provided herein are methods comprising the steps of: (a) forming a population of cells comprising stimulated T cells by incubating the stimulated T cells in the presence of a polynucleotide encoding the peptide; (c) forming an expanded population of cells comprising tumor antigen-specific T cells by expanding the population of cells comprising stimulated T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by cancer cells or APCs of the human subject of (b)(ii); (d) administering the expanded population of cells from (c) to the human subject; and (e) administering a cytokine to the human subject.

[0007] In some embodiments, the cytokine is interleukin-2 (IL-2). In some embodiments, the cytokine is administered on the same day as or after administering the expanded population of cells to the human subject.

[0008] In some embodiments, IL-2 is not administered to the subject during the therapy.

[0009] In some embodiments, the cytokine is administered 6 to 24 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered about 12 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered at a dose of 200,000 IU / kg to 1,000,000 IU / kg. In some embodiments, the cytokine is administered at a dose of about 600,000 IU / kg. In some embodiments, the cytokine is administered every 8 to 12 hours after administration of the expanded population of cells to the human subject.

[0009]

[0010] In some embodiments, at least 2, 3, 4, 5, or 6 doses of cytokine are administered. In some embodiments, up to 6 doses of cytokine are administered. In some embodiments, 6 doses of cytokine are administered. In some embodiments, the cytokine is administered intravenously. In some embodiments, the cytokine is administered intravenously at a dose of 600,000 IU / kg every 8-12 hours after administration of the expanded population of cells to the human subject, up to a maximum of 6 doses if tolerated.

[0010]

[0011] In some embodiments, a polynucleotide encoding IL-2 is administered to the subject. In some embodiments, the polynucleotide encoding IL-2 is RNA. In some embodiments, IL-2 is administered as a ribocytokine.

[0011]

[0012] In some embodiments, the method further comprises administering an immune checkpoint inhibitor to the human subject.

[0013] In some embodiments, the expanded population of cells administered is 0.75 x 10 8 ~1.25×10 10 Contains total cells.

[0012]

[0014] In some embodiments, the expanded population of cells administered is greater than 1.5 x 10 9 ~1.25×1010 In some embodiments, the expanded population of cells administered comprises 5 x 10 total cells. 8 ~1×10 10 In some embodiments, the expanded population of cells administered comprises 0.75 x 10 total cells. 8 ~1.25×10 9 Contains total cells.

[0013]

[0015] In some embodiments, the expanded population of cells administered comprises: a.0.75×10 8 ~1×10 9 total cells, b.0.75x10 8 ~0.75x10 9 of total cells, c.1x10 8 ~1.25x10 9 of total cells, d.1x10 8 ~1x10 9 of total cells, e.1x10 8 ~0.75x10 9 of total cells, f.1.25x10 8 ~1.25x10 9 of total cells, g.1.25×10 8 ~1×10 9 total cells, or h.1.25×10 8 ~0.75×10 9 total cells Includes.

[0014]

[0016] In some embodiments, the expanded population of cells administered comprises: a.1.5×10 9 ~1×10 10 total cells, b.1.5x10 9 ~0.75x10 10 of total cells, c.2x10 9 ~1.25x10 10 of total cells, d.2x10 9 ~1x10 10 of total cells, e.2x10 9 ~0.75x10 10 of total cells, f.2.5x10 9 ~1.25x10 10 of total cells, g.2.5×10 9 ~1×10 10 total cells, or h.2.5×10 9 ~0.75×10 10 total cells Includes.

[0015]

[0017] In some embodiments, the immune checkpoint inhibitor comprises an anti-PD1 agent.

[0018] In some embodiments, the immune checkpoint inhibitor comprises an anti-PD1 antibody.

[0016]

[0019] In some embodiments, the immune checkpoint inhibitor comprises pembrolizumab or nivolumab.

[0020] In some embodiments, the immune checkpoint inhibitor is administered after the expanded population of cells is administered. In some embodiments, the immune checkpoint inhibitor is administered before the expanded population of cells is administered. In some embodiments, the immune checkpoint inhibitor is administered at a dose of 200-400 mg, 2 mg / kg-4 mg / kg, 200 mg, 2 mg / kg, 400 mg, or 4 mg / kg. In some embodiments, the immune checkpoint inhibitor further comprises an anti-CTLA4 agent.

[0017]

[0021] In some embodiments, the anti-CTLA4 agent is an anti-CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody comprises ipilimumab.

[0022] In some embodiments, the immune checkpoint inhibitor is administered Q3W or Q6W.

[0018]

[0023] In some embodiments, the immune checkpoint inhibitor is administered Q6W.

[0024] In some embodiments, the immune checkpoint inhibitor is not administered for up to one week after administration of the expanded population of cells. In some embodiments, the immune checkpoint inhibitor is administered one to two weeks after administration of the expanded population of cells. In some embodiments, the immune checkpoint inhibitor is administered Q6W for up to 36 or 52 weeks after administration of the expanded population of cells. In some embodiments, the immune checkpoint inhibitor is not administered 36 or 52 weeks after administration of the expanded population of cells.

[0019]

[0025] In some embodiments, the method further comprises administering filgrastim to the human subject, wherein the filgrastim is administered after the expanded population of cells is administered. In some embodiments, the filgrastim is administered to the human subject after the subject's neutrophil count is greater than or equal to 1.0 x 10 for 3 days. 9 / L or >5.0 × 10 9 daily until a level of / L is reached.

[0020]

[0026] The method of any one of the above embodiments, wherein the human subject (i) has unresectable melanoma, (ii) has previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor and has disease progression, (iii) has received or is currently receiving a PD-1 inhibitor or a PD-L1 inhibitor for at least three months and has stable disease or asymptomatic progressive disease, or (iv) has discontinued a PD-1 inhibitor, PD-L1 inhibitor or CTLA-4 inhibitor due to toxicity, or (v) is not considered suitable to receive a CTLA-4 inhibitor.

[0021]

[0027] In some embodiments, the cancer is melanoma.

[0028] In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0022]

[0029] In some embodiments, the polynucleotide encoding the polypeptide is mRNA. In some embodiments, the polypeptide encoded by the mRNA comprises at least two tumor antigen epitope sequences. In some embodiments, the method includes incubating the APCs and a first population of T cells in the presence of IL-21 for a first period of time.

[0023]

[0030] In some embodiments, the method includes expanding a population of cells comprising stimulated T cells in the presence of IL-21. In some embodiments, the population of immune cells is from a biological sample from a human subject.

[0024]

[0031] 1. A method of treating cancer in a human subject in need thereof, comprising: administering to the human subject an expanded population of cells comprising tumor antigen-specific T cells, wherein the expanded population of cells is from a population of immune cells comprising APCs and a first population of T cells, the first population having been depleted of CD14+ and / or CD25+ cells and incubated 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 cancer cells of the human subject, or (B) a polynucleotide encoding said polypeptide; the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by cancer cells or APCs of the human subject; and the expanded population of cells comprises 5×10 8 ~1×10 10 Methods are provided herein for detecting a total number of cells.

[0025]

[0032] In some embodiments, the human subject has discontinued a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor due to toxicity; or is deemed unsuitable to receive a CTLA-4 inhibitor.

[0026]

[0033]

[0010] In one embodiment, a method of treating cancer in a human subject in need thereof comprises the steps of: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a CD14- and / or CD25-depleted population of immune cells comprising a first population of APCs and T cells; (b) incubating the first population of APCs and 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 cancer cells of the human subject with cancer, or (B) a polynucleotide encoding said polypeptide. (b)(ii) to form a population of cells comprising stimulated T cells by (c) expanding the population of cells comprising stimulated T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence, and (ii) an MHC protein expressed by cancer cells or APCs of the human subject of (b)(ii); and (d) administering the expanded population of cells from (c) to a human subject, wherein the human subject has discontinued a PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor due to toxicity; or is deemed not suitable to receive a CTLA-4 inhibitor.

[0027]

[0034] In some embodiments, the expanded population of cells is 5×10 8 ~1×10 10 Contains total cells.

[0035] In some embodiments, the method further comprises administering a cytokine to the human subject. In some embodiments, the cytokine is interleukin-2 (IL-2).

[0028]

[0036] In some embodiments, depleting comprises depleting only CD25+ cells, hi some embodiments, depleting comprises depleting CD25+ cells and CD56+ cells.

[0029]

[0037] In some embodiments, the methods do not include administering IL2 to the human subject.

[0038] In one embodiment, an anti-cancer monotherapy comprising autologous T cells from a subject who has previously been treated with anti-PD1, anti-PDL1, or anti-CTLA4 therapy, comprising approximately 5×10 7 ~2×10 9 Provided herein are therapies comprising one or more doses of total cells.

[0030]

[0039] In some embodiments, the cancer is metastatic melanoma. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0031]

[0040] In some embodiments, no other therapeutic agents are administered to the subject during the period of T cell therapy. [Brief explanation of the drawings]

[0032] [Figure 1A]

[0041] FIG. 1A depicts an exemplary schematic of a protocol for producing antigen-specific T cells. [Figure 1B]

[0042] FIG. 1B depicts an exemplary schematic of a protocol for producing antigen-specific T cells. [Figure 1C]

[0043] FIG. 1C depicts an exemplary alternative schematic of a protocol for producing antigen-specific T cells. [Figure 2]

[0044] Figure 2 depicts exemplary results showing the percentage of antigen-specific CD8+ memory T cells induced by long or short peptides. "Bulk" indicates that the sample containing T cells used for induction is whole peripheral blood mononuclear cells (PBMCs). "Treg-" indicates that the sample containing T cells used for induction is PBMCs depleted of CD25-expressing cells. [Figure 3]

[0045] FIG. 3 shows an exemplary flow cytometry analysis showing the percentage of antigen-specific CD8+ naive T cells induced with GAS7 peptide. [Figure 4]

[0046] Figure 4 depicts exemplary results showing antigen-specific CD8+ T cell responses to peptide pools of HIV short peptides, short identified neoantigens (PINs), or long PINs. "Total PBMC" indicates that the sample containing the T cells used for induction was total PBMC. "CD25-PBMC" indicates that the sample containing the T cells used for induction was depleted of CD25+ cells. Short, short peptides, or shortmers; long, long peptides, or longmers. [Figure 5A]

[0047] FIG. 5A depicts an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to a previously identified single neoantigen (PIN) under the conditions indicated. [Figure 5B]

[0048] Figure 5B depicts an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to a previously identified single neoantigen (PIN) under the indicated conditions. [Figure 6]

[0049] FIG. 6 depicts exemplary results showing antigen-specific CD8+ T cell responses to the indicated peptides using PBMC samples from two human donors. [Figure 7]

[0050] FIG. 7 depicts exemplary flow cytometry plots of antigen-specific CD8+ T cell responses to the indicated mutant epitopes in healthy donors before and after up to three stimulations. [Figure 8A]

[0051] Figure 8A depicts an exemplary bar graph showing the results of antigen-specific memory CD8+ T cell responses to viral antigens. After up to three stimulations, approximately 50% of all CD8+ T cells were specific for the indicated viral epitopes (CMV pp65, EBV YVL, EBV BMLF1, and Mart-1). [Figure 8B]

[0052] Figure 8B depicts exemplary results of a recall assay of antigen-specific memory CD8+ T cell responses to peptide-loaded antigen-presenting cells, then incubated with viral antigen-loaded and unloaded APCs. The percentage of CD8+ T cells releasing the indicated cytokines at two time points is charted. [Figure 9]

[0053] Figure 9 depicts exemplary results of a cytotoxicity assay used to evaluate whether induced T cell cultures can kill antigen-expressing tumor lines. The ratio of live and dead caspase-3-positive tumor cells to total tumor cells is shown. Live caspase-3-positive tumor cells represent cells undergoing early cell death. [Figure 10]

[0054] Figure 10 depicts an exemplary flow cytometry analysis of antigen-specific CD4+ T cell responses to peptide-loaded antigen-presenting cells, then incubated with PIN-loaded and unloaded APCs. The percentage of CD4+ T cells releasing IFNγ is shown. [Figure 11]

[0055] FIG. 11 depicts exemplary results of the percentage of antigen-specific CD4+ T cells releasing IFNγ after restimulation with mutant or wild-type peptides. [Figure 12]

[0056] 12 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV5 peptides, demonstrating both short-term and long-term induction. [Figure 13]

[0057] FIG. 13 depicts an exemplary flow cytometry analysis showing the percentage of antigen-specific CD8+ naive T cell responses to short ME1 peptides using whole PBMC samples from human donors. [Figure 14]

[0058] FIG. 14 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV3 peptides using a whole PBMC sample from a human donor. [Figure 15]

[0059] FIG. 15 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to long CSNK1A1 peptides using a whole PBMC sample from a human donor. [Figure 16]

[0060] FIG. 16 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to long CSNK1A1 peptides using a PBMC sample from a human donor depleted of CD25+ cells. [Figure 17]

[0061] FIG. 17 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short GAS7 peptides using a PBMC sample from a human donor depleted of CD25+ cells. [Figure 18]

[0062] FIG. 18 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short ACTN4 peptides using a PBMC sample from a human donor depleted of CD25+ cells. [Figure 19A]

[0063] Figure 19A depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short ACTN4 peptides using a PBMC sample from a human donor depleted of CD25+ cells. Short-term induction is shown. [Figure 19B]

[0064] Figure 19B depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV3 peptides using a PBMC sample from a human donor depleted of CD25+ cells. Long-term induction is shown. [Figure 20]

[0065] 20 depicts an exemplary flow cytometry analysis of antigen-specific CD8+ naive T cell responses to short HIV5 peptides using a whole PBMC sample from a human donor, showing both short-term and long-term induction. [Figure 21]

[0066] 21 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV3 peptides using a whole PBMC sample from a human donor. Short-term induction is shown. [Figure 22]

[0067] Figure 22 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short PRDX5 peptides using a PBMC sample from a human donor depleted of CD25+ cells, demonstrating both very short-term and long-term induction. [Figure 23]

[0068] Figure 23 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ naive T cell responses to short HIV5 peptides using a PBMC sample from a human donor that was depleted of CD25+ cell streams, demonstrating both short-term and long-term induction. [Figure 24]

[0069] FIG. 24 depicts a schematic of an example method for generating a therapeutic T cell composition, including expansion of memory T cells and induction of naive T cells. [Figure 25]

[0070] FIG. 25 depicts an exemplary method for testing the functionality, phenotype and / or function of T cells and / or T cell responses. [Figure 26]

[0071] FIG. 26 depicts an example of a recall assay for testing the functionality, phenotype and / or function of T cells and / or T cell responses. [Figure 27A]

[0072] Figure 27A depicts an exemplary flow cytometry analysis demonstrating the ability to deconvolute multiplexed samples with labeled samples acquired separately or as a mixture in a recall assay. Uniquely labeled samples were separated with minimal to no cross-contamination to other barcodes. [Figure 27B]

[0073] FIG. 27B depicts an exemplary flow cytometry analysis showing the detection of antigen-specific CD8+ T cells by multimer staining of a mixture of nine uniquely labeled samples in a recall assay. [Figure 28A]

[0074] FIG. 28A depicts an exemplary flow cytometry analysis of a recall assay using six uniquely barcoded samples recalled with unloaded DCs and neoantigen-loaded DCs. [Figure 28B]

[0075] Figure 28B depicts an exemplary bar graph of the percent of CD4+ T cells with functional counts incubated with DCs loaded with the indicated concentrations of peptide in a recall response assay. Samples from two induction cultures containing de novo CD4+ T cell responses were analyzed alone without barcoding or mixed with an unrelated sample. Barcoding did not alter detectable functionality. The functional counts and magnitude of responses elicited from the cells were not significantly altered by barcoding the samples. [Figure 29A]

[0076] Figure 29A depicts an exemplary bar graph showing the results of antigen-specific memory CD8+ T cell responses to viral antigens. CD8+ memory responses to CMV pp65, MART-1, EBV BRLF1 and BMLF1 epitopes could be increased from 0.23% of CD8+ T cells in the starting healthy donor material to over 60%. [Figure 29B]

[0077] Figure 29B depicts exemplary results of a recall assay of antigen-specific memory CD8+ T cell responses to viral antigens, followed by recall with viral antigen-loaded and unloaded DCs. The percentages of CD8+ T cells releasing the indicated cytokines at two time points are charted. [Figure 30A]

[0078] Figure 30A depicts exemplary results of hit identification by detection and functional characterization of de novo induced CD4+ responses with multiple specificities in the same culture. In the example shown, induction was performed in four replicate cultures targeting 10 HIV-derived epitopes that were naive targets in HIV-negative healthy donors. Antigen-specific responses were detected in 4 / 4 biological replicates, and the magnitude of the responses varied. [Figure 30B]

[0079] Figure 30B depicts exemplary results of pooled deconvolution by detection and functional characterization of de novo induced CD4+ responses with multiple specificities in the same culture. Multiple responses were detected in each replicate tested, with the same two epitopes (HIV#5 and HIV#7) producing the highest magnitude responses in each case. [Figure 30C]

[0080] Figure 30C depicts exemplary results of sensitivity determination by detection and functional characterization of de novo induced CD4+ responses with multiple specificities in the same culture. In pooled deconvolution assays, similar magnitudes were observed for each response. Responses to HIV #5, HIV #6, and HIV #4 showed EC50s of 0.45 μM, 0.43 μM, and 9.1 μM, respectively. [Figure 31]

[0081] FIG. 31 depicts an exemplary schematic of an antigen-specific T cell manufacturing protocol. [Figure 32]

[0082] FIG. 32 depicts an exemplary schematic of a T cell induction protocol. [Figure 33]

[0083] FIG. 33 depicts an exemplary schematic of a dendritic cell generation protocol. [Figure 34]

[0084] Figure 34 depicts exemplary pMHC multimer plots showing CD8+ T cell responses induced in leukapheresis material from melanoma patients targeting patient-specific epitopes: SRSF1E>K, ARAP1Y>H, and PKDREJG>R, and patient-specific epitopes (AASDH neoORF and seven model neoantigens: ACTN4K>N, CSNK1A1S>L, DHX40neoORF, GLI3P>L, QARSR>W, FAM178BP>L, and RPS26P>L). The plots in the first panel of the first and second rows show memory responses, while the remaining plots show de novo responses. [Figure 35]

[0085] Figure 35 depicts exemplary data (left panel) of pMHC multimer plots of SRSF1E>K and ARAP1Y>H before and after peptide stimulation, and the pie chart depicts neoantigen-specific T cell functionality upon rechallenge with neoantigen-loaded DCs, gating on pMHC multimer+CD8+ or CD4+ T cells. The polyfunctional profile of CD8+ memory, CD8+ de novo, and CD4+ de novo responses induced in patients with melanoma is demonstrated by a combination of one, two, or three functions (e.g., one or more functions is the production of one or more factors selected from IFNγ, TNFα, CD107a, and 4-1BB). [Figure 36]

[0086] Figure 36 depicts the specificity of memory and de novo responses induced in melanoma patients to mutant and wild-type peptides. SRSF1E>K- and ARAP1Y>H-specific T cell responses were challenged with DCs loaded with mutant or wild-type neoantigen peptides at different concentrations (X-axis: 0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM), and IFN-γ and / or TNFα and / or CD107a (Y-axis) were measured among total CD8 T cells in the samples. All responses showed significant differences relative to the 0 μM concentration, whereas responses to the wild-type neoantigen peptide were not. Statistical analysis: FDR adjusted p-values, P-values: *≦0.05, ***≦0.001, ****≦0.0001. [Figure 37A]

[0087] Figure 37A depicts the cytotoxicity profile of memory responses induced in melanoma patients, as quantified by the frequency of CD8+CD107a+ T cells. It also depicts the killing of target cells by these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The cytotoxic potential of the induced CD8+ T cell responses was assessed by rechallenge with mutant or wild-type neoantigen-transduced tumor cells. Untransduced tumor cells (parental A375 line) or tumor cells transduced with the 200aa construct were used. The constructs contained mutant or wild-type sequences with central mutations. Upregulation of CD107a on CD8+ T cells and activated caspase 3 on tumor cells were measured by coculture. Target ratio: 3.3:1 (SRSF1E>K). [Figure 37B]

[0088] Figure 37B depicts another example of the cytotoxic profile of a memory response induced in a patient with melanoma, as quantified by the frequency of CD8+CD107a+ T cells. It also depicts the killing of target cells by these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The cytotoxic potential of the induced CD8+ T cell response was assessed by rechallenge with tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The constructs contained either mutant or wild-type sequences with central mutations. Upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells were measured by coculture. Red circles highlight the pMHC+ fraction. The effector:target ratio was 5:1 (SRSF1E>K). Statistical analysis: unpaired t-test, P values ​​**≤0.01, ****≤0.0001. [Figure 37C]

[0089] Figure 37C depicts the cytotoxicity profile of de novo responses induced in melanoma patients, as quantified by the frequency of CD8+CD107a+ T cells. It also depicts target cell killing by these T cell responses, as quantified by the frequency of aCAS3+ tumor cells. The cytotoxic potential of the induced CD8+ T cell responses was assessed by rechallenge with tumor cells transduced with mutant or wild-type neoantigens. Untransduced tumor cells (parental A375 line) or tumor cells transduced with a 200aa construct were used. The constructs contained mutant or wild-type sequences with central mutations. Upregulation of CD107a on CD8+ T cells and active caspase 3 on tumor cells were measured by coculture. Circles highlight the pMHC+ fraction. Effector:target ratio: 0.66:1 (ARAP1Y>H). Statistical analysis: unpaired t-test, P values: **≤0.01, ****≤0.0001. [Figure 38A]

[0090] Figure 38A depicts the identification of neoantigen-specific CD4+ T cell responses in melanoma patients. Responses are identified based on the production of IFN-γ and TNFα (Y axis) upon rechallenge with DCs (0.8 μM) loaded with mutant neoantigen peptides. MKRN1S>L, CREBBPS>L, and TPCN1K>E were identified as positive responses. [Figure 38B]

[0091] Figure 38B depicts the specificity of the CD4+ T cell responses depicted in Figure 38A to the indicated mutant and wild-type peptides. In a validation study, the CD4 T cell responses shown in Figure 38A were challenged with different concentrations of mutant and wild-type neoantigen peptides (X-axis—0 μM, 0.05 μM, 0.2 μM, 0.8 μM, and 3.2 μM), and IFNγ and / or TNFα levels of total CD4+ (Y-axis) in the samples were measured. Two of the CD4+ T cell responses (MKRN1S>L and CREEBPS>L) showed significant differences relative to the 0 μM concentration and were unresponsive to the wild-type neoantigen peptide, whereas the TPCN1K>E response was responsive to both the mutant and wild-type neoantigen peptides. Statistical analysis: FDR adjusted p-values, p-value <0.05. [Figure 38C]

[0092] Figure 38C depicts the polyfunctional profiles of these CD4+ T cell responses, as indicated by a combination of one, two, three, or four functions (e.g., one or more functions are the production of one or more factors selected from IFNγ, TNFα, CD107a, and 4-1BB). The polyfunctionality of the identified CD4+ T cell responses was assessed by rechallenge with DCs (0.8 μm) loaded with mutant neoantigen peptides. The percentages in the pie chart represent the percentage of functional CD4+ T cells (one, two, and / or three functions). Representative data are shown from CD4+ T cell responses after stimulation induced in patients. [Figure 39]

[0093] Figure 39 depicts the functionality of memory responses induced in two healthy donors with or without the addition of epacadostat, as indicated by a combination of one, two, or three functions (e.g., the one or more functions are the production of one or more factors selected from IFNγ, TNFα, and CD107a). [Figure 40]

[0094] FIG. 40 depicts the percent of induced de novo CD8+ T cell responses ("hit rate," average of four healthy donors) in six replicate inductions with or without the addition of epacadostat. [Figure 41A]

[0095] Figure 41A depicts the absolute numbers of antigen-specific cells from healthy donors after induction with the T cell manufacturing protocol provided herein, with or without the addition of a PD-1 blocking antibody. [Figure 41B]

[0096] Figure 41B depicts the absolute number of antigen-specific cells from healthy donors after induction with the T cell manufacturing protocol provided herein, with or without the addition of a PD-1 blocking antibody. [Figure 42A]

[0097] FIG. 42A depicts multimer-positive frequency as a percentage of CD8+ T cells from the de novo CD8+ T cell compartment with or without the addition of IL-12. [Figure 42B]

[0098] FIG. 42B depicts an exemplary graphical representation of the percentage of CD8+ T cells from the de novo CD8+ T cell compartment with or without the addition of IL-12. [Figure 43]

[0099] Figure 43 shows an exemplary graphical representation of the hit rate percentage of highly immunogenic and less immunogenic antigens to which naive CD8 cells are responsive after performing different antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors. Also shows an exemplary graphical representation of the absolute number of antigen-specific cells after performing different antigen-presenting cell enrichment and antigen loading protocols using PBMCs from healthy donors using Mart-1 peptide or highly immunogenic and less immunogenic antigens. [Figure 44A]

[0100] FIG. 44A depicts exemplary flow cytometry results of CD123-positive cells after performing the indicated antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from three different healthy donors. [Figure 44B]

[0101] Figure 44B depicts an exemplary graphical representation of the absolute numbers of the indicated CD11c+ cell subsets after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from a healthy donor: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells. [Figure 45]

[0102] Figure 45 depicts an exemplary graphical representation of the total number of CD8 T cells and the indicated cell percentages after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from healthy donors. The treatments are as follows: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells. [Figure 46]

[0103] FIG. 46 depicts exemplary flow cytometry results of CD11b-positive cells after the indicated antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from three different healthy donors. [Figure 47]

[0104] FIG. 47 depicts exemplary flow cytometry results of CD19-positive cells after the indicated antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from three different healthy donors. [Figure 48]

[0105] Figure 48 depicts an exemplary graphical representation of the fold expansion of cells after three antigen-presenting cell enrichment and antigen-loading protocols. The treatments are as follows: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells. [Figure 49A]

[0106] Figure 49A depicts exemplary data showing the number of specific antigens to which naive CD8 T cells are responsive after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from healthy donors. Results are the average of three healthy donors. Treatments are as follows: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells. An exemplary graphical representation of the data is shown in the bottom graph. [Figure 49B]

[0107] Figure 49B depicts an exemplary graphical representation of the percent hit rates of highly immunogenic (left) and less immunogenic (right) antigens to which naive CD8 cells are responsive, after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from healthy donors. Results are the average of three healthy donors. Treatments are as follows: basal Flt3L, FLT3L treatment alone; CD11b, FLT3L treatment and depletion of CD11b-expressing cells; CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells. [Figure 50]

[0108] Figure 50 depicts an exemplary graphical representation of antigen-specific cell numbers in cell populations activated by highly and poorly immunogenic antigens to which T cells are responsive, using PBMCs from healthy donors after 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 CD11b- and CD19-expressing cells. [Figure 51A]

[0109] Figure 51A depicts an exemplary graphical representation of the percentage of viable cells after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from a healthy donor. The treatments are as follows: Base, FLT3L treatment alone; Base + CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells; + APC, an additional PBMC fraction added to Base + CD11b- / CD19-, where the additional fraction was depleted of CD3, CD19, CD11b, CD25, and CD14-expressing cells. [Figure 51B]

[0110] Figure 51B depicts an exemplary graphical representation of the percentage of viable cells after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from a healthy donor. The treatments are as follows: Base, FLT3L treatment alone; Base + CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells; + APC, an additional PBMC fraction added to Base + CD11b- / CD19-, where the additional fraction was depleted of CD3, CD19, CD11b, CD25, and CD14-expressing cells. [Figure 51C]

[0111] Figure 51C depicts an exemplary graphical representation of the percentage of viable cells after three antigen-presenting cell enrichment and antigen-loading protocols using PBMCs from a healthy donor. The treatments are as follows: Base, FLT3L treatment alone; Base + CD11b- / CD19-, FLT3L treatment and depletion of CD11b- and CD19-expressing cells; + APC, an additional PBMC fraction added to Base + CD11b- / CD19-, where the additional fraction was depleted of CD3, CD19, CD11b, CD25, and CD14-expressing cells. [Figure 51D]

[0112] FIG. 51D depicts exemplary data showing the number of specific antigens to which CD8 cells are responsive per donor using an exemplary antigen-presenting cell enrichment protocol. [Figure 51E]

[0113] FIG. 51E depicts an exemplary graphical representation of the percent hit rate of the indicated peptides to which CD8 cells were responsive, averaged across three healthy donors. [Figure 52A]

[0114] Figure 52A depicts exemplary flow cytometry analysis results from an experiment in which cell populations added to the culture process at different times were labeled with a membrane-permeable amine-reactive dye (e.g., carboxyfluorescein succinimidyl ester or TagIT Violet™) before stimulation with antigen-loaded APCs. In the second stimulation, a cell population already cultured for 14 days was labeled with one dye, while another cell population containing antigen-loaded APCs and a new preparation of T cells was labeled with a different dye. The two populations were mixed together for restimulation or expansion. The relative contribution of each of these populations to the overall antigen-specific T cell pool was confirmed by the presence and dilution of each dye. In all cases, cell populations were cultured for 14 days (first stimulation), labeled with one dye, and then added to another cell population labeled with another dye that had been antigen-stimulated 1 day (standard protocol), 4 days (5-day headstart), or 6 days (7-day headstart) earlier. [Figure 52B]

[0115] Figure 52B shows an exemplary schematic of three different T cell expansion protocols, each involving two stimulations that include a head start of antigen-loaded APCs 2, 5, or 7 days before contact with T cells. [Figure 52C]

[0116] Figure 52C shows an exemplary graph of antigen-specific T cell numbers over time using the three different T cell expansion protocols depicted in Figure 52B: 1, standard protocol; 2, 5-day headstart; 3, 7-day headstart. [Figure 53]

[0117] Figure 53 shows an exemplary graph of fold expansion of cultures treated with the indicated neo-antigen peptides (peps) or neo-antigen RNA. After CD3 lymphocyte isolation or depletion, CD14 / CD25-depleted PBMC cells were stimulated with antigen (peptide or antigen-encoding mRNA). CD3 lymphocytes were reintroduced and stimulated for 14 days. [Figure 54]

[0118] Figure 54 shows an exemplary graph of the number of multimer-positive antigen-specific cells in cultures nucleofected with the indicated neoantigen peptide (pep) or neoantigen RNA. Cultures were nucleofected in the presence or absence of T cells (-CD3). Irr, irradiation. [Figure 55]

[0119] FIG. 55 depicts an exemplary flow cytometry analysis showing antigen-specific CD8+ memory responses using viral peptides or RNA encoding peptides, and naive responses using peptides or RNA encoding neoantigens, in a short-term induction protocol. [Figure 56A]

[0120] FIG. 56A depicts a schematic of an exemplary process for generating RNAs containing sequences encoding neoantigens and using them to load PBMCs and activate T cells. [Figure 56B]

[0121] Figure 56B depicts a schematic of an exemplary process for generating RNAs containing sequences encoding neoantigens and using them to load PBMCs and activate T cells. [Figure 57A]

[0122] Figure 57A shows a schematic of an exemplary RNA concatemer construct encoding a string of neo-antigens. [Figure 57B]

[0123] Figure 57B depicts a schematic diagram of an exemplary arrangement of neo-antigen strings in a 5'-3' orientation within the construct shown in Figure 57A. [Figure 58A]

[0124] Figure 58A depicts a schematic diagram of an exemplary mRNA sequence for incorporating a 5'-CAP structure into an mRNA encoding a concatemerized neo-antigen string for expression in PBMCs. The addition of an "A" nucleotide in the mRNA string was used for compatibility with CleanCap® Technology. [Figure 58B]

[0125] FIG. 58B depicts an exemplary graphical representation of the percentage of viable cells 24 hours after expressing mRNA encoding concatemerized neo-antigen strings with different 5′-CAP structures in PBMCs. [Figure 58C]

[0126] FIG. 58C depicts an exemplary graphical representation of the total number of GFP-positive cells 24 hours after expressing mRNAs encoding concatemerized neo-antigen strings with different 5′-CAP structures in PBMCs. [Figure 59A]

[0127] Figure 59A depicts exemplary results showing the generation of mRNA using modified nucleotides. The mRNA was modified by substituting all (Full) or some (Part) of the uridine (U) and cytidine (C) residues within the mRNA. For example, the Part C set contains 30% of the C residues substituted with methylcytidine. Results show the effect on the expression of the mRNA-encoded peptide in transfected PBMCs over time. [Figure 59B]

[0128] Figure 59B depicts exemplary data comparing the effect of commercially available and in-house preparations of mRNA containing substituted uridines and / or cytidines on the generation of multimer-specific T cells stimulated with PBMCs loaded with the mRNA. [Figure 59C]

[0129] FIG. 59C depicts exemplary data comparing the expansion of stimulated T cells generated as described in FIG. 59B. [Figure 60A]

[0130] Figure 60A depicts an exemplary schematic diagram of an mRNA construct using shortmers (9-10 amino acids, top row) and longmers (25 amino acids, bottom row) used for expression in cells. [Figure 60B]

[0131] Figure 60B depicts an exemplary graph of multimer-specific CD8+ cells as a percentage of total CD8+ cells. The antigens used in the multimer assay are indicated. [Figure 60C]

[0132] Figure 60C depicts an exemplary flow cytometry analysis for the detection of multimer-positive CD8+ T cells comparing shortmer (9-10 amino acids) and longmer (25 amino acids) peptide-stimulated APCs with APCs containing those encoding the same shortmer (9-10 amino acids) and longmer (25 amino acids) peptides. [Figure 61A]

[0133] FIG. 61A depicts a schematic diagram of exemplary RNA constructs with which cells in the experiments shown in FIGS. 61B-61D were transfected. [Figure 61B]

[0134] Figure 61B depicts an exemplary graphical representation of the results obtained from the multimer assay. In all three PBMC handling conditions, RNA-transfected PBMCs were better than peptide-loaded PBMCs in generating antigen-specific T cells. In the case of the Gli3 antigen, a more than 10-fold increase in multimer-positive cells was observed compared to peptide-loaded PBMCs. [Figure 61C]

[0135] Figure 61C depicts exemplary flow cytometry data showing detection of Gli3 multimer-positive T cells in each indicated set, with or without depletion of CD3 cells. Direct transfection of CD25+ PBMCs results in an increase in multimer-positive cells compared to PBMCs depleted of CD14 and CD25 cells or PBMCs thawed from frozen stocks. [Figure 61D]

[0136] Figure 61D depicts an exemplary graphical representation of results from a multimer assay. PBMCs treated overnight with FTL3L cells or CD25-depleted PBMCs were electroporated with RNA encoding either 25 amino acid long neoantigen sequences (longmers) or epitope-length neoantigen sequences (shortmers). The percentage of neoantigen-positive cells in the cultures was assayed using multimer technology. [Figure 61E]

[0137] Figure 61E depicts an exemplary graphical representation of the fold expansion results obtained from the experiment described in Figure 61D. PBMCs or CD25-depleted PBMCs treated overnight with FTL3L cells were electroporated with RNA encoding either 25 amino acid long neoantigen sequences (longmers) or epitope-length neoantigen sequences (shortmers). The fold expansion of the cells after 26 days of culture and two stimulations is depicted. [Figure 62A]

[0138] Figure 62A (top) depicts a schematic diagram of exemplary RNA constructs with which cells in the experiments shown in Figures 62A-62C were transfected.

[0139] Figure 62A (bottom panel) depicts an exemplary graphical representation of the number of live ACTN4- and Gli3-responsive T cells from two donors at day 26 after maturation, with combinations shown on the X-axis. [Figure 62B]

[0140] Figure 62B depicts exemplary data of the percentage of Gli3-responsive T cells from live cells expanded in the presence of the indicated maturation mix. [Figure 62C]

[0141] Figure 62C depicts exemplary flow cytometry data showing detecting Gli3 multimer-positive T cells grown in the presence of the indicated maturation mixes. [Figure 63A]

[0142] Figure 63A depicts representative mass spectrometry data showing detection of presentation of the indicated Gli3 epitopes by PBMCs using radioisotope incorporation. PBMCs transfected with mRNA encoding multiple epitopes (including the Gli3 epitope), and peptide expression is detected using a reference peptide labeled with a heavier isotope. [Figure 63B]

[0143] Figure 63B depicts an exemplary graphical representation of the percentage of maximum presentation by HLA-A02:01 of the indicated epitopes over time after transfection of PBMCs with mRNA encoding each epitope. Each isotope-labeled epitope was detected by mass spectrometry. Maximum surface presentation was observed 6 hours after transfection. [Figure 64A]

[0144] Figure 64A depicts an exemplary graphical representation from a recall assay of the percentage change in TNFα and / or IFNγ production (left) or the percentage of CD107a positive cells (right) from neoantigen-specific CD8 T cells challenged with increasing concentrations of the indicated peptides used to load the APCs. [Figure 64B]

[0145] Figure 64B depicts an exemplary graphical representation from a multimer assay of the percentage change in TNFα and / or IFNγ production (left) or the percentage of CD107a-positive cells (right) from neoantigen-specific CD8 T cells challenged with increasing concentrations of the indicated peptides used to load the APCs. [Figure 65]

[0146] Figure 65 depicts an exemplary Venn diagram of criteria to consider to create optimal personalized T cell therapy using mRNA as the immunogen. [Figure 66]

[0147] FIG. 66 depicts an exemplary flow diagram showing the steps for selecting peptide sequences for preparing patient-specific T cell products. [Figure 67A]

[0148] Figures 67A and 67B illustrate several advantageous aspects of a clinical approach using T cells produced by the process shown in Figure 1A and Figure 67A. [Figure 67B] Figures 67A and 67B illustrate several advantageous aspects of a clinical approach using T cells produced by the process shown in Figure 1A and Figure 67A. [Figure 68]

[0149] Figure 68 depicts exemplary representative flow cytometry data showing the characterization of patient-specific T cell products prepared by multiple engineering runs. CD3+ as a percentage of live cells (top panel), and CD8+ and CD4+ as a percentage of live CD3+ T cells (bottom panel) are depicted. [Figure 69A]

[0150] Figure 69A depicts an exemplary graphical representation of data showing the characterization of patient-specific T cell products prepared by multiple engineering runs. The percentage of multimer-positive CD8-positive cells is shown. [Figure 69B]

[0151] Figure 69B depicts exemplary representative flow cytometry data showing the characterization of patient-specific T cell products prepared by multiple engineering runs. The percentages of multimer A- and multimer B-positive CD8 cells for the indicated epitopes are shown. [Figure 69C]

[0152] Figure 69C depicts an exemplary pie chart showing the polyfunctionality of identified pMHC+CD8+ T cells following re-challenge with mutant neoantigen-loaded DCs compared to unloaded DCs. [Figure 70]

[0153] Figure 70 depicts representative data showing the variation in IFNγ and / or TNFα production by CD4+ cells of patient-specific T cell products prepared by multiple engineering runs. Also depicted are exemplary representative data showing the characterization of IFNγ+ and / or TNFα+ and / or CD107a+ CD4+ cells of patient-specific T cell products prepared by multiple engineering runs. [Figure 71]

[0154] Figure 71 depicts an exemplary graphical representation showing the percentage of central memory T cells (Tcm), effector memory T cells (Tem), effector T cells (Teff), and naive T cells (Tnaive) in patient-specific T cell products prepared by multiple engineering runs. Central memory T cells (Tcm): CD62L+CD45RA-, effector memory T cells (Tem): CD62L-CD45RA-, effector T cells (Teff): CD62L-CD45RA+, naive T cells (Tnaive): CD62L+CD45RA+. [Figure 72]

[0155] Figure 72 depicts an exemplary graphical representation of data from a multimer assay showing the percentage of IFN-γ and / or TNFα and / or CD107a cells among total CD8 cells (top panel) or total CD4 T cells (bottom panel) measured upon challenge with DCs loaded with various concentrations of peptide in the sample. The peptide used is indicated on each graph. [Figure 73]

[0156] Figure 73 depicts an exemplary graphical representation of data showing upregulation of CD107a on CD8+ T cells (top) and activated caspase 3 on tumor cells (bottom). Measurements were obtained after co-culture with A375 tumor cell lines untransformed or transfected with the 200 amino acid construct, or with A375 tumor cell lines loaded or unloaded with peptide. [Figure 74]

[0157] Figure 74 depicts an exemplary graphical representation of data showing that induced T cells can kill antigen-expressing cells. Neoantigen-specific T cells were tested to recognize autologous tumors or peptide-loaded autologous tumors through a recall response assay. Readout: IFN-γ+ and / or TNFα+ and / or CD107a+ pMHC+ (CD8+%) and pMHC- (CD8+%) T cells (Y-axis). Significance was assigned using one-way ANOVA (P<0.05). [Figure 75]

[0158] Figure 75 depicts an exemplary schematic of cohorts and doses for use in a clinical trial (NEO-PTC-01). [Figure 76A]

[0159] FIG. 76A shows a schematic overview of the NEO-PTC-01 manufacturing process. [Figure 76B]

[0160] Figure 76B shows the characterization of the T cell products. [Figure 77ABCD]

[0161] Figure 77A shows the frequency of multimer-specific CD8+ T cells in each pool of either the NEO-PTC-01.pep or NEO-PTC-01.RNA process using patient PBMCs from patient 1.

[0162] Figure 77B shows the frequency of multimer-specific CD8+ T cells in each pool of either the NEO-PTC-01.pep or NEO-PTC-01.RNA process using patient PBMCs from patient 2.

[0163] Figure 77C shows a summary of responses in each patient separated by NEO-PTC-01.pep and NEO-PTC-01.RNA processes. The denominator indicates the total number of neoantigen sequences used to induce a T cell response.

[0164] Figure 77D shows an exemplary flow cytometry plot of pMHC+ T cells in the CD8+ population. [Figure 77E]

[0165] Figure 77E shows the frequency of multimer-specific CD8+ T cells generated from NEO-PTC-01. The black circles represent pMHC+-specific responses from NEO-PTC-01.pep performed at large scale in melanoma patient samples, the red circles represent pMHC+-specific responses from NEO-PTC-01.pep performed at small scale with ovarian patient samples, and the green circles represent pMHC+-specific responses from NEO-PTC-01.RNA performed at small scale with ovarian patient samples. [Fig. 78AB]

[0166] Figure 78A shows results demonstrating that neoantigen-specific CD8+ T cell responses from ovarian patient samples are polyfunctionally representative of the NEO-PTC-01.pep set. Data depict the expression of functional markers (IFN-γ, TNF-α, and CD107a) of identified pMHC+CD8+ T cells upon re-challenge with mutant neoantigen-loaded DCs compared to pMHC+CD8+ T cells challenged with DMSO-loaded DCs. Percentages above the bars represent the percentage of pMHC+CD8+ T cells present in the sample.

[0167] Figure 78B shows results demonstrating that neoantigen-specific CD8+ T cell responses from ovarian patient samples are polyfunctionally representative of data from the NEO-PTC-01.RNA set. Data depict the expression of functional markers (IFN-γ, TNF-α, and CD107a) of identified pMHC+CD8+ T cells upon re-challenge with mutant neoantigen-loaded DCs compared to pMHC+CD8+ T cells challenged with DMSO-loaded DCs. Percentages above the bars represent the percentage of pMHC+CD8+ T cells present in the sample. [Figure 79ABCD]

[0168] Figure 79A shows the frequency of neoantigen-specific CD4+ T cells producing IFN-γ+ and / or TNF-α+ when cocultured with dendritic cells loaded with cognate peptide above a threshold (the amount of IFN-γ+ and / or TNF-α+ produced when cocultured with dendritic cells alone). Ovarian patient sample 1 treated with NEO-PTC-01.pep.

[0169] Figure 79B shows the frequency of neoantigen-specific CD4+ T cells producing IFN-γ+ and / or TNF-α+ when co-cultured with dendritic cells loaded with cognate peptide above a threshold (the amount of IFN-γ+ and / or TNF-α+ produced when co-cultured with dendritic cells alone). Ovarian patient sample 1 induced with NEO-PTC-01.RNA.

[0170] Figure 79C shows the frequency of neoantigen-specific CD4+ T cells producing IFN-γ+ and / or TNF-α+ when cocultured with cognate peptide-loaded dendritic cells above a threshold (the amount of IFN-γ+ and / or TNF-α+ and / or CD107a produced when cocultured with dendritic cells alone). Ovarian patient sample 2 treated with NEO-PTC-01.pep.

[0171] Figure 79D shows the frequency of neoantigen-specific CD4+ T cells producing IFN-γ+ and / or TNF-α+ when cocultured with cognate peptide-loaded dendritic cells above a threshold (the amount of IFN-γ+ and / or TNF-α+ and / or CD107a produced when cocultured with dendritic cells alone) in NEO-PTC-01.RNA-induced ovarian patient sample 2. [Figure 79E]

[0172] Figure 79E tabulates the CD4+ T cell responses for each patient isolated by the NEO-PTC-01.pep and NEO-PTC-01.RNA processes. The data demonstrate the diversity of responses obtained with cells produced using peptide stimulation (left column) and RNA-mediated antigen expression (right column) processes from sample cells from ovarian cancer patients 1 and 2. The denominators represent the total number of neoantigens used to induce a response in each process. [Figure 79F]

[0173] Figure 79F shows flow cytometry plots of IFN-γ+ cells in the CD4+ population after restimulation with dendritic cells loaded with DMSO or their cognate neoantigens. [Figure 79G]

[0174] Figure 79G shows the frequency of antigen-specific activated (IFN-γ+ and / or TNF-α+) CD4+ T cells generated from NEO-PTC-01. The black circles represent pMHC+-specific responses from NEO-PTC-01.pep performed at large scale in melanoma patient samples, the red circles represent pMHC+-specific responses from NEO-PTC-01.pep performed at small scale in ovarian patient samples, and the green circles represent pMHC+-specific responses from NEO-PTC-01.RNA performed at small scale in ovarian patient samples. [Figure 80A]

[0175] Figure 80A shows results demonstrating that neoantigen-specific CD4+ T cell responses from ovarian patient samples are polyfunctional. Polyfunctionality of antigen-specific CD4+ T cells in the NEO-PTC-01.pep process re-challenged with neoantigen-loaded DCs was compared to DMSO-loaded dendritic cells. [Figure 80B]

[0176] Figure 80B shows results demonstrating that neoantigen-specific CD4+ T cell responses from ovarian patient samples are polyfunctional. Polyfunctionality of antigen-specific CD4+ T cells in the NEO-PTC-01.RNA process re-challenged with neoantigen-loaded DCs was compared to DMSO-loaded dendritic cells. [Figure 81A]

[0177] Figure 81A shows flow cytometry gating of the multimer-responding cell population in the Neo-PTC-01.pep process. The pMHC+ plot shows the frequency of multimer-positive cells. [Figure 81B]

[0178] Figure 81B shows the frequency of naive, central memory (CM), effector memory (EM) or effector T cells present in bulk CD4, bulk CD8 or multimer positive cells in the Neo-PTC-01.pep process. [Figure 81C]

[0179] Figure 81C shows representative data from the Neo-PTC-01.pep process cell population showing upregulation of CD107a neoantigen-specific CD8+ T cells measured 6 hours after co-culture of T cells with tumor cell lines loaded with mutant, wild-type, irrelevant peptide, or DMSO (no peptide). [Figure 81D]

[0180] Figure 81D shows flow cytometry gating of the multimer-responding cell population in the Neo-PTC-01.RNA process. The pMHC+ plot shows the frequency of multimer-positive cells. [Figure 81E]

[0181] Figure 81E shows the frequency of naive, central memory (CM), effector memory (EM) or effector T cells present in bulk CD4, bulk CD8 or multimer positive cells in the NEO-PTC-01.RNA process. [Figure 81F]

[0182] Figure 81F shows representative data from NEO-PTC-01.RNA process cell populations demonstrating upregulation of CD107a neoantigen-specific CD8+ T cells measured 6 hours after co-culture of T cells with tumor cell lines loaded with mutant, wild-type, irrelevant peptide, or DMSO (no peptide). [Figure 82]

[0183] Figure 82 shows a schematic diagram of the study design for a Phase 1, open-label, dose-finding and expansion study of the safety and activity of a personalized T-cell therapy. Part 1 of the study is designed for monotherapy with dose escalation, in which patients who have progressed on anti-PD-1 therapy and received anti-CTLA4 therapy receive ≥ 1 x 10 cells and ≤ 1 x 10 cells ± 25% (Dose 1) or ≥ 2 x 10 cells and ≤ 1 x 10 cells ± 25% (Dose 2). Dose expansion will then occur in patients in the highest dose cohort deemed safe and well-tolerated. Part 2 of the study is designed for combination therapy, in which patients deemed stable or asymptomatically progressing after 3 months of anti-PD-1 therapy (with or without anti-CTLA4 therapy) will receive the personalized T-cell therapy at the dose deemed safe in Part 1 and anti-PD1 therapy (with or without anti-CTLA4 therapy). Patients will be enrolled in this trial and receive anti-PD1 therapy (+ / - anti-CTLA4 therapy) when the personalized T-cell therapy is manufactured, and will continue anti-PD1 therapy during the follow-up period (until EOS). This part of the study will measure the efficacy of the combination of anti-PD1 and personalized T-cell therapy. [Figure 83]

[0184] Figure 83 shows a schematic timeline of the study described in Figure 82. As described, the prescreening and screening phases occur between days -20 and -16, respectively, relative to administration of the personalized T cell therapy on day 0. Selected subjects undergo leukapheresis on day -12, and the cells are processed for subsequent periods to generate the personalized T cell therapy. On day -1, subjects receive their last chemotherapy treatment prior to the personalized T cell therapy. The personalized T cell therapy is administered on day 0, followed by a 36-52 week follow-up period. Subjects receive anti-PD-1 therapy (nivolumab) every six weeks (Q6W). [Figure 84]

[0185] Figure 84 (top panel) shows an overview of small-scale induction performed on three ovarian cancer (OVC) patient samples. Patient samples (PBMCs) obtained from subjects with ovarian cancer were used to generate T cell products using the small-scale manufacturing NEO-STIM process, and the quality of the products was then analyzed. Antigen-specific T cell generation from ovarian cancer cells was performed according to the NEO-STIM manufacturing process, including using peptides (peptide-loaded APCs) to stimulate the cells and APCs expressing RNA encoding the peptides, and the results were compared. Patient characteristics and the mutated antigens (neoantigens) identified from each patient are shown within each box. Bottom panel: A graphical representation is provided showing the two RNA designs used in the CD8+ and CD4+ T cell stimulation processes. [Figure 85]

[0186] Figure 85 provides an overview of the assays and result highlights for the comparison and validation of peptide vs. RNA-mediated stimulation processes, demonstrating that both processes are suitable to meet the quality requirements of the manufactured products. [Figure 86]

[0187] Figure 86 shows in more detail the method for comparison and validation of peptide versus RNA-mediated stimulation processes in the NEOSTIM manufacturing process. [Figure 87A]

[0188] Figure 87A shows the results of the NEO-PTC-01.pep and NEO-PTC-01.RNA processes, demonstrating that both generate similar frequencies of neoantigen-specific CD8+ T cells (patient N16NEON-17, OVC#1). [Figure 87B]

[0189] Figure 87B shows the results of the NEO-PTC-01.pep and NEO-PTC-01.RNA processes, demonstrating that both generate similar frequencies of neoantigen-specific CD8+ T cells (patient N16NEON-19, OVC#2). [Figure 87C]

[0190] Figure 87C shows the results of the NEO-PTC-01.pep and NEO-PTC-01.RNA processes, demonstrating that both generate similar frequencies of neoantigen-specific CD8+ T cells (patient N16NEON-18, OVC#3). [Figure 88]

[0191] Figure 88 shows data demonstrating that similar frequencies of neoantigen-specific CD8+ T cells for peptide and RNA protocols are consistent with observations in engineering runs. [Figure 89]

[0192] Figure 89 shows data demonstrating successful generation of CD4+ responses in two OVC patients. Frequencies represent the (D) delta change of CD4 positive samples from no peptide controls. [Figure 90]

[0193] Figure 90 shows data demonstrating that both the NEO-PTC-01.pep and NEO-PTC-01.RNA protocols generate similar frequencies of neoantigen-specific CD4+ T cells in OVC patient samples. [Figure 91]

[0194] Figure 91 shows data demonstrating that the predominant phenotype of the NEO-PTC-01.RNA and NEO-PTC-01.Peptide protocols is T effector memory cells. [Figure 92]

[0195] Figure 92 shows data demonstrating that the use of IL-21 in NEO-STIM increases antigen-specific CD8+ T cell central memory phenotype. [Figure 93]

[0196] Figure 93 shows data demonstrating that IL-21 addition improves expansion of NEO-STIM.RNA cultures. [Figure 94]

[0197] Figure 94 shows data demonstrating that IL-21 addition improves the priming of specific T cells in NEO-STIM.RNA cultures. [Figure 95]

[0198] Figure 95 shows data demonstrating that IL-21 addition increases the Tcm population in NEO-STIM.RNA cultures. [Figure 96]

[0199] Figure 96 shows a diagram of the proposed interactions of different cell types. The black x's indicate protein mutations that result in neoantigens that are presented to CD4+ and CD8+ T cells by antigen-presenting cells such as dendritic cells. [Figure 97A]

[0200] Figure 97A shows a table containing a summary of induced neoantigen-specific T cell responses (study scale). Summary of induced neoantigen-specific CD8+ and CD4+ T cell responses. Targeted genes are indicated with the gene name and mutation type. For example, TENM3S>L represents a single nucleotide mutation in the TENM3 gene. neoORF: novel open reading frame, fs: frameshift, del: deletion. Percentage of neoantigen-specific T cells: for CD8+ T cell responses, percent (%) of CD8+ pMHC+ (among live cells); for CD4+ T cell responses, percent (%) of live CD4+ T cells (among live cells) delta IFNγ and / or TNFα+ (between negative control and test conditions). *SRSF1E>K T cell responses are memory responses; delta indicates induced responses in multiple cultures; typical frequencies of single cultures are shown. [Figure 97B]

[0201] Figure 97B shows a table containing a summary of induced neoantigen-specific T cell responses (therapeutic scale). Summary of induced neoantigen-specific CD8+ and CD4+ T cell responses. Targeted genes are shown with the gene name and mutation type. For example, TENM3S>L represents a single nucleotide mutation in the TENM3 gene. Percentage of neoantigen-specific T cells: for CD8+ T cell responses, percent (%) of CD8+ pMHC+ (among live cells); for CD4+ T cell responses, percent (%) of live CD4+ T cells (among live cells) + ΔIFNγ and / or TNFα+ (between negative control and test conditions). [Figure 98A]

[0202] Figures 98A and 98B show induced neoantigen-specific CD8+ T cell responses. Detection of induced neoantigen-specific CD8+ T cells using pMHC multimers. Dot plots of antigen-specific CD8+ T cell responses from (A) donors NV10, NV06, and NV15, and (B) lot #190710HD108, lot #190827ENG01, and lot #190924ENG02. CD8+ pMHC+ T cells (among live cells) are depicted in red circles. Numbers within the plots indicate the percentage of neoantigen-specific CD8+ T cells. Identified neoantigen-specific T cell responses were confirmed in all cases by independent staining with different fluorochrome combinations. (A) Pie chart depicting the diversity of TCRs identified in depleted leukapheresis and neoantigen-induced cultures of SRSF1E>K and ARAP1Y>HT cell responses from patient NV10. [Figure 98B] Figures 98A and 98B show induced neoantigen-specific CD8+ T cell responses. Detection of induced neoantigen-specific CD8+ T cells using pMHC multimers. Dot plots of antigen-specific CD8+ T cell responses from (A) donors NV10, NV06, and NV15, and (B) lot #190710HD108, lot #190827ENG01, and lot #190924ENG02. CD8+ pMHC+ T cells (among live cells) are depicted in red circles. Numbers within the plots indicate the percentage of neoantigen-specific CD8+ T cells. Identified neoantigen-specific T cell responses were confirmed in all cases by independent staining with different fluorochrome combinations. (A) Pie chart depicting the diversity of TCRs identified in depleted leukapheresis and neoantigen-induced cultures of SRSF1E>K and ARAP1Y>HT cell responses from patient NV10. [Figure 99]

[0203] Figures 99A and 99B show induced neoantigen-specific CD4+ T cell responses. Detection of induced neoantigen-specific CD4+ T cells using an antigen recall assay. Bar graphs of antigen-specific CD4+ T cell responses for (A) donors NV10, NV06, and NV15, and (B) lots #190710HD108, #190827ENG01, and #190924ENG02. Consider an increase of ≥ 5% between the negative control (measured in triplicate) and the test condition, and ≥ 2.5 times the standard deviation of the negative control. [Figure 100A]

[0204] Figures 100A and 100B show that neoantigen-induced T cells have a polyfunctional profile. Polyfunctionality of induced neoantigen-specific T cell responses was assessed for (A) donors NV10, NV06, and NV15, and (B) lots #190710HD108, #190827ENG01, and #190924ENG02. Profiles are shown for the negative control (unloaded DCs) and test conditions (neoantigen-loaded DCs). Polyfunctional profiles of pMHC+ CD8+ T cells (left panel) and CD4+ T cells (right panel) are shown. The numbers in the middle of the pie charts indicate the percentage (%) of cells with one, two, or three functions. [Figure 100B] Figures 100A and 100B show that neoantigen-induced T cells have a polyfunctional profile. Polyfunctionality of induced neoantigen-specific T cell responses was assessed for (A) donors NV10, NV06, and NV15, and (B) lots #190710HD108, #190827ENG01, and #190924ENG02. Profiles are shown for the negative control (unloaded DCs) and test conditions (neoantigen-loaded DCs). Polyfunctional profiles of pMHC+ CD8+ T cells (left panel) and CD4+ T cells (right panel) are shown. The numbers in the middle of the pie charts indicate the percentage (%) of cells with one, two, or three functions. [Figure 101]

[0205] Figure 101 provides insights into the functional state of neoantigen-specific T cells through single-cell RNA and TCR sequencing analysis. Single-cell whole-transcriptome analysis of SRSF1Y>H and ARAPE>K CD8+ T cell responses (patient NV10). tSNE maps overlaid with activation scores (red: high score, blue: low score) are shown (top panel, left and right). Activation score distribution is depicted, with Recall- indicating samples challenged with unloaded DCs and Recall+ indicating samples challenged with neoantigen-loaded DCs. STIM: Pre = starting material, 1 = Stim 1 (day 14), 2 = Stim 2 (day 21), 3 = Stim 3 (day 28). Specificity: Bulk = CD8+ T cells, SRSF1 and ARAP1 = pMHC+ CD8+ T cells, Bystander = pMHC- CD8+ T cells, from NEO-STIM endpoint (bottom panel, left and right). [Figure 102]

[0206] FIG. 102 shows a graph showing the correlation between effector function and proliferation ability. [Figure 103]

[0207] Figure 103 shows data demonstrating the differentiation status of neoantigen-induced cultures. The differentiation status of neoantigen-induced cultures of NV10, lot #190710HD108, and lot #190924ENG02 was evaluated. Bulk indicates the evaluation of the differentiation status of CD8+ or CD4+ T cells in a representative pool, and pMHC+ indicates the evaluation of the differentiation status of neoantigen-specific CD8+ pMHC+ T cells. Left panel: CD8+ fraction; right panel: CD4+ fraction. Blue: naive T cells (Tnaive); green: effector memory T cells (Tem); red: effector T cells (Teff); purple: central memory T cells (Tcm). [Figure 104]

[0208] Figure 104 shows data demonstrating that neoantigen-induced T cells specifically respond to mutant peptides. Antigen recall assays were used to assess the specificity profile of SRSF1Y>H and ARAPE>K CD8+ T cell responses. Graphs show the percent of IFNγ and / or TNFα and / or CD107a produced in response to mutant neoantigen peptides (dark blue; 0, 0.05, 0.2, 0.8, and 3.2 μM peptide) or wild-type peptides (light blue; 0, 0.05, 0.2, and 0.8 μM peptide). False discovery statistical analysis corrected for multiple comparisons was performed to assess peptide specificity (p<0.05). *: p≦0.05, **: p≦0.01, ***: p≦0.001, ****: p≦0.0001. [Figure 105]

[0209] Figure 105 shows a table displaying the assessment of the percentage of neoantigen-specific T cells specific for the mutant epitopes. [Figure 106A]

[0210] Figures 106A-106C show that neoantigen-specific CD8+ T cells can kill antigen-expressing tumor cells and recognize autologous tumors. The killing capacity of subsets of CD8+ T cell responses induced by neoantigens from donors NV10 and NV06, lot #190710HD108, lot #190827ENG01, and lot #190924ENG02 is assessed. (A, Study Scale) Black bars: parental A375 tumor cells transduced with relevant HLA; red bars: relevant HLA-expressing A375 tumor cells transduced with mutant epitopes; gray bars: relevant HLA-expressing A375 tumor cells transduced with wild-type epitopes. (A, Treatment Scale) Black bars: parental A375 tumor cells transduced with relevant HLA or transduced with relevant HLA and loaded with irrelevant peptide; red bars: relevant HLA-expressing A375 tumor cells transduced with mutant epitopes or loaded with neoantigenic peptides; gray bars: relevant HLA-expressing A375 tumor cells transduced with irrelevant epitopes. Top panel: CD107a+ CD8+ T cells; bottom panel: percent of live, active caspase-3+ A375 tumor cells. Statistical analysis: unpaired t-test; ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. (B) The ability of CD8+ T cell subsets induced with neoantigens from lot #190827ENG01 and lot #190924ENG02 to recognize autologous tumors was assessed. Gray bars: NEO-PTC-01 alone; red bars: NEO-PTC-01 + autologous tumor digest; or NEO-PTC-01 + autologous tumor digest loaded with neoantigen peptides. Statistical test: One-way ANOVA, adjusted for multiple comparisons; ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. [Figure 106B]Figures 106A-106C show that neoantigen-specific CD8+ T cells can kill antigen-expressing tumor cells and recognize autologous tumors. The killing capacity of subsets of CD8+ T cell responses induced by neoantigens from donors NV10 and NV06, lot #190710HD108, lot #190827ENG01, and lot #190924ENG02 is assessed. (A, Study Scale) Black bars: parental A375 tumor cells transduced with relevant HLA; red bars: relevant HLA-expressing A375 tumor cells transduced with mutant epitopes; gray bars: relevant HLA-expressing A375 tumor cells transduced with wild-type epitopes. (A, Treatment Scale) Black bars: parental A375 tumor cells transduced with relevant HLA or transduced with relevant HLA and loaded with irrelevant peptide; red bars: relevant HLA-expressing A375 tumor cells transduced with mutant epitopes or loaded with neoantigenic peptides; gray bars: relevant HLA-expressing A375 tumor cells transduced with irrelevant epitopes. Top panel: CD107a+ CD8+ T cells; bottom panel: percent of live, active caspase-3+ A375 tumor cells. Statistical analysis: unpaired t-test; ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. (B) The ability of CD8+ T cell subsets induced with neoantigens from lot #190827ENG01 and lot #190924ENG02 to recognize autologous tumors was assessed. Gray bars: NEO-PTC-01 alone; red bars: NEO-PTC-01 + autologous tumor digest; or NEO-PTC-01 + autologous tumor digest loaded with neoantigen peptides. Statistical test: One-way ANOVA, adjusted for multiple comparisons; ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. [Figure 106C]Figures 106A-106C show that neoantigen-specific CD8+ T cells can kill antigen-expressing tumor cells and recognize autologous tumors. The killing capacity of subsets of CD8+ T cell responses induced by neoantigens from donors NV10 and NV06, lot #190710HD108, lot #190827ENG01, and lot #190924ENG02 is assessed. (A, Study Scale) Black bars: parental A375 tumor cells transduced with relevant HLA; red bars: relevant HLA-expressing A375 tumor cells transduced with mutant epitopes; gray bars: relevant HLA-expressing A375 tumor cells transduced with wild-type epitopes. (A, Treatment Scale) Black bars: parental A375 tumor cells transduced with relevant HLA or transduced with relevant HLA and loaded with irrelevant peptide; red bars: relevant HLA-expressing A375 tumor cells transduced with mutant epitopes or loaded with neoantigenic peptides; gray bars: relevant HLA-expressing A375 tumor cells transduced with irrelevant epitopes. Top panel: CD107a+ CD8+ T cells; bottom panel: percent of live, active caspase-3+ A375 tumor cells. Statistical analysis: unpaired t-test; ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. (B) The ability of CD8+ T cell subsets induced with neoantigens from lot #190827ENG01 and lot #190924ENG02 to recognize autologous tumors was assessed. Gray bars: NEO-PTC-01 alone; red bars: NEO-PTC-01 + autologous tumor digest; or NEO-PTC-01 + autologous tumor digest loaded with neoantigen peptides. Statistical test: One-way ANOVA, adjusted for multiple comparisons; ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. [Figure 107]

[0211] Figure 107 shows a table displaying the major cell populations found in the starting material and induction cultures. Summary of the major cell populations found in the starting material and neoantigen induction cultures for lot #190710HD108, lot #190827ENG01, and lot #190924ENG02. Cell populations quantified as a percentage of total live cells: T cells (CD3), B cells (CD19), NK cells and other cells of the myeloid lineage, including monocytes (CD56 and CD56 / CD16 double positive cells), DCs (CD11c, CD11b, and CD11c / CD11b double positive cells). Cell populations quantified as a percentage of CD3+ cells: CD4+ T cells (CD4), CD8+ T cells (CD8), and gamma delta T cells (Vγ9 TCR). [Figure 108]

[0212] Figure 108 shows data demonstrating that CD11c+ DCs play an important role in generating neoantigen-specific T cells. Various cell subsets were depleted, and then their ability to induce neoantigen-specific T cells (as measured by percent hit rate) was assessed: Control: CD14- and CD25-depleted; CD11b-: CD14-, CD25-, and CD11b-depleted; CD11c-: CD14-, CD25-, and CD11c-depleted; CD11c- / CD11b-: CD14-, CD25-, CD11c-, and CD11b-depleted. HD46: healthy subject #46; HD66: healthy subject #66; HD67: healthy subject #67. [Figure 109]

[0213] Figure 109 shows a summary of induced neoantigen-specific CD4 and CD8 responses in patient NAC01 dp. Summary of induced neoantigen-specific CD8+ and CD4+ T cell responses. Percentage of neoantigen-specific T cells: For CD8+ T cell responses: Percent (%) of CD8+ pMHC+ (of live cells); For CD4+ T cell responses: Percent (%) of live CD4+ T cells (of live cells) ΔIFNγ+ and / or TNFα+ (between negative control and test conditions). [Figure 110]

[0214] Figure 110 shows the polyfunctionality profile of neoantigen-specific T cells in patient drug products. The polyfunctionality of induced neoantigen-specific T cell responses is evaluated. Profiles are shown for the negative control (unloaded DCs) and test conditions (neoantigen-loaded DCs). The polyfunctionality profiles of pMHC+ CD8+ T cells (left panel) and CD4+ T cells (right panel) are shown. The numbers in the middle of the pie charts indicate the percentage (%) of cells with functions 1, 2, or 3. [Figure 111]

[0215] Figure 111 shows the differentiation status of cultures before and after NEO-STIM. Assessment of the differentiation status of neoantigen-induced cultures from patient NAC01. Left panel: CD8+ fraction, right panel: CD4+ fraction. Purple: naive T cells (Tnaive), cyan: effector memory T cells (Tem), green: effector T cells (Teff), blue: central memory T cells (Tcm). [Figure 112]

[0216] Figure 112 shows the mutant-specific responses observed in neoantigen-specific T cell responses induced in patient NAC01 DP. The specificity profile of representative CD8+ and CD4+ T cell responses was assessed using an antigen recall assay. Graphs show the percentage of IFNγ and / or TNFα and / or CD107a relative to CD8+ responses, and the percentage of IFNγ and / or TNFα relative to CD4+ responses, generated in response to mutant neoantigen peptides (dark blue; 0, 0.05, 0.2, 0.8, and 3.2 μM peptide) or wild-type peptides (light blue; 0, 0.05, 0.2, and 0.8 μM peptide). Sidak's statistical analysis was performed to assess peptide specificity, corrected for multiple comparisons (p<0.05); ns: p>0.05, *: p≦0.05, **: p≦0.01, ***: p≦0.001, ****: p≦0.0001. [Figure 113]

[0217] Figure 113 shows that neoantigen-specific CD8+ T cells in patient NAC01 DP can kill tumor cells expressing the antigen. Evaluation of the killing capacity of neoantigen-induced CD8+ T cell responses in patient NAC01 DP using tumor A375 cell lines expressing surrogate antigens. Black bars: parental A375 tumor cells transduced with relevant HLA mutant or wild-type epitopes in the absence of T cells. Red bars: A375 tumor cells expressing relevant HLA transduced with mutant epitopes in the presence of T cells. The Y-axis represents the percentage of viable, active caspase 3+ A375 tumor cells. Statistical analysis: unpaired t-test; ns: p>0.05, *: p≦0.05, **: p≦0.01, ***: p≦0.001, ****: p≦0.0001. [Figure 114]

[0218] Figure 114 shows peripheral blood analysis of patient NAC01 4 weeks after DP infusion. Detection of induced neoantigen-specific CD8+ (top) and CD4+ (bottom) T cells using pMHC multimers. CD8+ or CD4+ pMHC+ T cells are depicted in red in the dot plots. Numbers within the plots indicate the percentage of antigen-specific CD8+ and CD4+ T cells. Identified neoantigen-specific T cell responses were confirmed in all cases by independent staining with different fluorescent dye combinations. [Figure 115]

[0219] Figure 115 is a schematic of interim clinical and translational data from NTC-001, showing the workflow of manufacturing and post-infusion analysis. [Figure 116]

[0220] Figure 116 shows a schematic diagram of the trial design. [Figure 117]

[0221] Figure 117 shows data showing the percent change in target lesion sum before and after injection of monotherapy NEO-PTC-01. [Figure 118A]

[0222] Figures 118A and 118B show data regarding responses in the drug product and responses detected in peripheral blood after infusion. Figure 118A shows example flow cytometry plots of pMHC-responsive (pMHC+) CD8+ T cells to the mutant S100A6 62:R>W neoantigen in patient NAC01 on drug product (DP) (top row) and 3-6 weeks after infusion (bottom row). Figure 118B shows data showing that a subset of responses detected in the DP were detected in the periphery 3-6 weeks later. Responses were detected in the DP using MHC class I tetramers for CD8+ responses, and CD4+ responses showed upregulation of IFN-gamma and / or TNF-alpha upon rechallenge with the mutant neoantigen peptide. After infusion, cells were detected using MHC class I tetramers, MHC class II tetramers, bulk TCR sequencing, and / or re-challenged with neoantigen peptides to assess degranulation and / or TNFα / IFNγ secretion. NAC: neoantigen cell administration received; NVD: not administered. [Figure 118B] Figures 118A and 118B show data regarding responses in the drug product and responses detected in peripheral blood after infusion. Figure 118A shows example flow cytometry plots of pMHC-responsive (pMHC+) CD8+ T cells to the mutant S100A6 62:R>W neoantigen in patient NAC01 on drug product (DP) (top row) and 3-6 weeks after infusion (bottom row). Figure 118B shows data showing that a subset of responses detected in the DP were detected in the periphery 3-6 weeks later. Responses were detected in the DP using MHC class I tetramers for CD8+ responses, and CD4+ responses showed upregulation of IFN-gamma and / or TNF-alpha upon rechallenge with the mutant neoantigen peptide. After infusion, cells were detected using MHC class I tetramers, MHC class II tetramers, bulk TCR sequencing, and / or re-challenged with neoantigen peptides to assess degranulation and / or TNFα / IFNγ secretion. NAC: neoantigen cell administration received; NVD: not administered. [Figure 119]

[0223] Figure 119 shows data demonstrating that the induced response is mutant-type reactive. DP cells were co-cultured overnight with APCs presenting a range of concentrations of either mutant or wild-type peptides. Upregulation of IFNγ, TNFα, and / or CD107α was measured using flow cytometry. Representative curves are shown. The graph on the left shows a representative CD4+ response. The graph on the right shows a representative CD8+ response. [Figure 120]

[0224] Figure 120 shows data on TCRs from patients. TCRs were sequenced from patients, cloned, and transduced into NFAT Jurkat cells, and functional avidity was measured to confirm specificity. The graph on the left shows EC50 values ​​calculated by the TCR avidity assay (black indicates EC50 for the saturation curve, gray indicates EC50 for the non-saturation curve). The graph on the right shows the minimum peptide concentration required for NFAT activation. [Figure 121]

[0225] Figure 121 shows data demonstrating that antigen-specific T cells kill antigen-expressing targets in vitro. Cytotoxicity assays were performed using A375 target cell lines (lentiviral transduction) expressing either wild-type or mutant neoantigens on relevant HLA alleles in co-culture with DP-derived CD8+ T cells (bead-isolated). Upregulation of caspase 3 in tumor cells was used to measure / assess killing capacity. The graph on the right shows the number of responses in five different patients. [Figure 122]

[0226] Figure 122 shows data demonstrating that a subset of post-infusion responses are functional. Patient NAC02 PBMCs were thawed and recalled with no peptide (DMSO) or a peptide pool consisting of all positive shortmer and longmer epitope peptides for which responses were detected in the DP. These cells were recalled for 24 hours and then subjected to flow staining to assess functionality. Responses were gated on HLA-DR+CD4+ T cells. [Figure 123]

[0227] Figure 123 shows a summary of sustained T cell responses examined in the periphery 3-6 weeks after infusion. When material was available, all responses in DPs where responses were obtained were evaluated in the periphery after infusion. [Figure 124]

[0228] Figure 124 shows data demonstrating that PD1 is upregulated after infusion. The graph on the left shows the MFI of PD-1 surface expression between XIRP1+ (red, CD8+ tetramer+) and XIRP1- (gray, CD8+ tetramer-). T cells from patient NAC03 are shown in a histogram. The data on the right is a heatmap showing the fold change in median fluorescence intensity (MFI) of tetramer+ relative to tetramer- CD8+ T cells. Patient PBMCs were thawed and stained with tetramer and surface antibodies. These cells were not stimulated or recalled. [Figure 125]

[0229] Figure 125 shows data from a case study of one patient (NAC09) demonstrating tumor shrinkage and histological data. The data on the left shows a computed tomography (CT) scan from the patient. On the right are multispectral IHC images of the tumor before and after injection, showing CD3+, CD8+, and SOX10+ in the bottom panel. [Figure 126]

[0230] Figure 126 shows neoantigen-specific clones in tumor and periphery. The top panel shows a schematic of the workflow. MAGEB2 78:S>F-specific clonotypes identified from the DP of patient NAC09 using multimer selection. The bottom left graph shows the frequency of each clonotype in the DP. The bottom right graph shows the frequency of MAGEB2 78:S>F-specific clonotypes in patient NAC09 PBMCs using bulk TCR sequencing. Clonotypes with frequencies below 0.0001% are plotted on the x-axis. [Figure 127]

[0231] Figure 127 shows UMAP analysis of phenotypes in DP cells (top) and recall cells (bottom). The top image shows multimodal single-cell data from patient NAC09 drug product (GEX, CITE, TCR). The bottom image shows UMAP of cells with MAGEB2 78:S>F-specific and HIST1H1B203P>L-specific clonotypes. This data demonstrates the phenotypic diversity of neoantigen-specific responses. [Figure 128]

[0232] Figure 128 shows the frequency of single cell phenotypes in NAC09 drug product by sample. [Figure 129]

[0233] FIG. 129 shows heat maps depicting the phenotypes of HIST1H1B-specific cells (left) and MAGEB2 78:S>F-specific cells (right) after antigen recall by clonotype. [Figure 130]

[0234] Figure 130 shows an overview of patient recruitment for the T cell therapy trial (NTC-001). As demonstrated in the NTC-001 pilot study and clinical trial, the NEOSTIM process for expanding patient T cells will be followed. Using the same framework, the NTC-001 trial will be directed toward T cell therapy using autologous T cells derived from patients. In this study, patients are heavily pre-treated prior to enrollment. All patients in this population have been treated with nivolumab or pembrolizumab and anti-CTLA4. In this study, NEO-PTC-01 is planned as a monotherapy regimen. [Figure 131]

[0235] Figure 131 shows the mutation profile of the patient identified using RECON. [Figure 132]

[0236] Figure 132 is a schematic diagram showing the timeline of different steps in the NEOSTIM process of this study. The top panel shows the timeline of the individual parts of the manufacturing process for each patient who was treated. The bottom panel shows the timeline for patients who were not treated. [Figure 133A]

[0237] Figure 133A shows a schematic diagram describing the T cell manufacturing protocol. [Figure 133B]

[0238] Figure 133B shows the fold expansion of T cells from each patient. For each patient, the first bar from the left shows the fold expansion following the first stimulation, the second bar shows the expansion following the second stimulation, and the third bar shows the overall fold expansion at the end of the process. [Figure 133C]

[0239] Figure 133C shows the percent viable cells in the drug product. For each patient designated by the number below, the first bar from the left is total PBMC and the second bar is drug product (DP); the % viable cell values ​​are sorted by cell type as indexed in the top right box. [Figure 134]

[0240] Figure 134 shows exemplary RECIST % changes in target lesions by patient. BOR, best overall response; SD, partial response; PD, progressive disease. [Figure 135]

[0241] Figure 135 shows the dosing schedule for each patient in NEO-PTC-01 therapy. DETAILED DESCRIPTION OF THE INVENTION

[0033]

[0242] T cell therapeutics are expected to be relatively safe and well-tolerated adoptive T cell products. However, based on an evaluation of the risks associated with the products, there are three general classes of potential toxicities associated with T cell therapeutics: (a) treatment-related toxicity due to lymphodepletion, cell infusion, or cytokine release syndrome; (b) off-tumor, off-target toxicity due to the expansion of autoreactive clones or cross-reactivity of neoantigen-specific T cells; and (c) off-tumor, on-target toxicity due to the presentation of neoantigens on non-tumor tissues. Novel immunotherapeutic agents and their uses based on the discovery of neoantigens arising from mutational events unique to individual tumors are described herein. Accordingly, the disclosure described herein provides methods and protocols for generating antigen-specific immune cells, e.g., T cells, for use in disease treatment.

[0034]

[0243] A composition of neoantigen-reactive T cells for cancer immunotherapy is presented herein. Adoptive T cell therapy is a promising new approach for cancer therapy, but requires numerous improvements. In general, T cells must be appropriately cytotoxic to cancer cells, avoid targeting non-cancerous cells in the body, not lose immunogenicity in the tumor environment, and provide long-term protection. In addition, the use of virally transduced cells presents its own problems. Therefore, finding the right balance to achieve a therapeutically effective composition that specifically targets cancer cells, avoids targeting healthy cells, slows disease progression, results in palliative or at least substantial tumor regression, and prevents cancer recurrence requires numerous improvements at almost every step of this complex process.

[0035]

[0244] To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0245] Antigens are substances foreign to the body that elicit an immune response. "Neoantigens" refer to a class of tumor antigens that arise from tumor-specific changes in proteins. Neoantigens include, but are not limited to, substitutions in protein sequences, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, and tumor antigens resulting from the expression of endogenous retroviral polypeptides.

[0036]

[0246] "Neoepitope" refers to an epitope that is not present in a reference, non-diseased cell, e.g., a non-cancerous cell or a germline cell, but is found in a diseased cell, e.g., a cancer cell. This includes situations where the corresponding epitope is found in a normal, non-diseased cell or a germline cell, but due to one or more mutations in the diseased cell, e.g., a cancer cell, the sequence of the epitope has been altered to create a neoepitope.

[0037]

[0247] "Mutation" refers to a change in or difference in a nucleic acid sequence (e.g., a nucleotide substitution, addition, or deletion) compared to a reference nucleic acid. "Somatic mutations" can occur in any cell of the body except germ cells (sperm and eggs) and are not passed on to offspring. These changes can (but do not necessarily) cause cancer or other diseases. In some embodiments, the mutation is a nonsynonymous mutation. "Nonsynonymous mutation" can also refer to a mutation (e.g., a nucleotide substitution) that results in an amino acid change, e.g., an amino acid substitution, in the translation product. "Frameshift" typically occurs when a mutation disrupts the normal phase of a gene's codon periodicity (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. The same altered reading frame can be achieved for different mutations in a gene.

[0038]

[0248] "Antigen processing" or "processing" can also refer to the degradation of a polypeptide or antigen into processing products that are fragments of the polypeptide or antigen (e.g., degradation of a polypeptide into peptides), and the association of one or more of these fragments with an MHC molecule (e.g., via binding) for cellular presentation to specific T cells, e.g., by an antigen-presenting cell.

[0039]

[0249] "Antigen-presenting cells" (APCs) refer to cells that present peptide fragments of protein antigens in association with MHC molecules on their cell surface. This term includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells), as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).

[0040]

[0250] The term "affinity" refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide and a class I or 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 refers to 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 off-rate constant of the two members of a binding pair (e.g., the off-rate constant of an HLA-binding peptide with class I or II HLA, or of a peptide-HLA complex with a TCR). on refers to the on-rate constant of the two members of a binding pair (eg, the on-rate constant of an HLA-binding peptide with class I or II HLA, or of a peptide-HLA complex with a TCR).

[0041]

[0251] Throughout this disclosure, the results of the "Combined Data" are referred to as "IC 50 Affinity can also be expressed in units of inhibitory concentration (IC 50 ), or the concentration at which 50% of the first member of the binding pair (e.g., a peptide) is displaced. Similarly, ln(IC 50 ) is IC 50 For example, IC 50 is the concentration of peptide tested in a binding assay at which 50% inhibition of binding of the labeled reference peptide is observed. Taking into account the conditions under which the assay is performed (e.g., limiting HLA protein concentration and / or labeled reference peptide concentration), these values ​​are used to calculate the K DThe value can be approximated. Assays for determining binding are well known in the art and are described in detail in, for example, PCT publications WO94 / 20127 and WO94 / 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 by referring to the binding of a reference standard peptide. Binding can also be measured using live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189 (1991); del Guercio et al., J. Immunol. 154:685 (1995)), cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high-flow 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)).

[0042]

[0252] The term "derived" may be used synonymously with "prepared" when discussing epitopes. Derived epitopes may be isolated from natural sources or synthesized according to standard protocols in the art. Synthetic epitopes may also include artificial amino acid residues, "amino acid mimetics," such as naturally occurring L-amino acid residues or D-isomers of unnatural amino acid residues, such as cyclohexylalanine. Derived or prepared epitopes may be analogs of native epitopes. The term "derived from" refers to origin or source, and examples include naturally occurring, recombinant, unpurified, purified, or differentiated molecules or cells. For example, expanded or induced antigen-specific T cells may be derived from T cells. For example, expanded or induced antigen-specific T cells may be derived from antigen-specific T cells in a biological sample. For example, mature APCs (e.g., professional APCs) may be derived from non-mature APCs (e.g., immature APCs). For example, APCs may be derived from monocytes (e.g., CD14 + For example, dendritic cells can be derived from monocytes (e.g., CD14 + For example, the APCs may be derived from bone marrow cells.

[0043]

[0253] An "epitope" can be the collective features of a molecule (e.g., the charge and primary, secondary, and tertiary structure of a peptide) 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 the set of amino acid residues involved in recognition by a particular immunoglobulin; a major histocompatibility complex (MHC) receptor; or, in the context of T cells, the residues recognized by a T cell receptor protein and / or a chimeric antigen receptor. Epitopes can be prepared by isolation from natural sources, or epitopes can be synthesized according to standard protocols in the art. Synthetic epitopes can include artificial amino acid residues, amino acid mimetics (e.g., naturally occurring L-amino acid residues or D-isomers of non-naturally occurring amino acid residues). Throughout this disclosure, epitopes may in some cases be referred to as peptides or peptide epitopes. In certain embodiments, the peptides of the present disclosure are limited in length. An embodiment with a limited length occurs when a protein or peptide containing an epitope described herein contains a region (i.e., a continuous series of amino acid residues) that has 100% identity with a native sequence. To avoid, for example, reading the definition of an epitope across the entire natural molecule, there is a limit to the length of any region that has 100% identity with a native peptide sequence. Thus, for a peptide containing an epitope described herein and a region that has 100% identity with a native peptide sequence, the region that has 100% identity with the native sequence generally has a length of less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues.In certain embodiments, an "epitope" as described herein is comprised of a peptide having an amino acid residue region that is 100% identical to a native peptide sequence of less than 51, e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues, with any variation of up to 5 amino acid residues.

[0044]

[0254] A "T cell epitope" refers to a peptide sequence bound to an MHC molecule in the form of a peptide-MHC (pMHC) complex that is capable of recognizing and binding to the TCR of a T cell (e.g., a cytotoxic T lymphocyte or a helper T cell).

[0045]

[0255] "T cells" are CD4 + T cells and CD8 +The term T cell may include T cells. The term T cell may also include both T helper type 1 T cells and T helper type 2 T cells. T cells can be generated by the methods described herein for clinical use. For example, the T cells or adoptive T cells referred to herein for clinical use may be cells isolated from a biological source, manipulated and cultured ex vivo, and prepared into a drug candidate for a specific therapy of cancer, such as melanoma. Once a drug candidate cell passes certain qualitative and quantitative criteria for suitability for clinical use, the drug candidate can be designated as a drug product. In some cases, a drug product is selected from a number of drug candidates. In the context of the present application, a drug product is a T cell, more specifically a population of T cells, or more specifically a population of T cells with heterogeneous characteristics and subtypes. For example, a drug product disclosed herein may have a population of T cells, including CD8+ T cells and CD4+ T cells, where the cells have at least a specific antigen specificity and a certain percentage of each exhibit, among other things, a memory phenotype.

[0046]

[0256] "Immune cells" can also refer to cells that play a role in immune responses. Immune cells are of hematopoietic origin, and examples include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0047]

[0257] An "immunogenic" peptide or "immunogenic" epitope or "immunogenic" peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated or humoral response, 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 subsequently inducing a cell-mediated or humoral response (e.g., a CTL (cytotoxic) response, or an HTL response) against the peptide.

[0048]

[0258] A "protective immune response" or "therapeutic immune response" can also refer to a CTL and / or HTL response against an antigen derived from a pathogenic antigen (e.g., a tumor antigen) that in some way prevents or at least partially suppresses disease symptoms, side effects, or progression. The immune response can include an antibody response promoted by stimulation of helper T cells.

[0049]

[0259] The term "T cell receptor" ("TCR") often refers to a molecule found on the surface of T lymphocytes (T cells) that recognizes antigens bound to major histocompatibility complex (MHC) molecules, whether natural or partially or wholly synthetically produced. The ability of a T cell to recognize antigens associated with various diseases (e.g., cancer) or infectious organisms is conferred by its TCR, which is composed of both alpha (α) and beta (β) chains or gamma (γ) and delta (δ) chains. The proteins that make up these chains are encoded by DNA and employ unique mechanisms to generate the vast diversity of TCRs. This multisubunit immune recognition receptor associates with the CD3 complex and binds peptides presented by MHC class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of the TCR to peptides on APCs is a central event in T cell activation.

[0050]

[0260] "Chimeric antigen receptor" or "CAR," as used herein, refers to an antigen-binding protein comprising an immunoglobulin antigen-binding domain (e.g., an immunoglobulin variable domain) and a T cell receptor (TCR) constant domain. In some cases, the "constant domain" of a TCR polypeptide can be used, often comprising the membrane-proximal TCR constant domain, the TCR transmembrane domain, and / or the TCR cytoplasmic domain, or fragments thereof. For example, in some embodiments, a CAR may be a monomer comprising a polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCR β constant domain. In some embodiments, a CAR may be a dimer comprising a first polypeptide comprising an immunoglobulin heavy or light chain variable domain linked to a TCR α or TCR β constant domain, and a second polypeptide comprising an immunoglobulin heavy or light chain variable domain (e.g., a kappa or lambda variable domain) linked to a TCR β or TCR α constant domain.

[0051]

[0261] The "major histocompatibility complex" or "MHC" is often understood as a cluster of genes that play a role in controlling the interaction of cells involved in physiological immune responses. The term "major histocompatibility complex" and the abbreviation "MHC" may include any class of MHC molecule, such as MHC class I and MHC class II molecules, and refer to a complex of genes present in all vertebrates. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. Thus, "human leukocyte antigen" or "HLA" refers to human major histocompatibility complex (MHC) proteins (see, e.g., Stites et al., Immunology, 8th ed., Lange Publishing, Los Altos, Calif. (1994)). For a detailed description of the MHC and HLA complexes, see Paul, Fundamental Immunology, 3rd ed., Raven Press, New York (1993).

[0052]

[0262] The major histocompatibility complex in the genome may contain genetic regions whose gene products, expressed on the cell surface, are important for binding and presenting endogenous and / or foreign antigens, thus regulating immunological processes. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or diseased cells in the immune response. MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the surface of cells and can present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. MHC-binding peptides arise from proteolytic cleavage of protein antigens and can represent potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes). MHC transports peptides to the cell surface, where they can be presented to specific cells, such as cytotoxic T lymphocytes, helper T cells, or B cells. MHC regions can be divided into three subgroups: class I, class II, and class III. MHC class I proteins may contain an α chain and β2-microglobulin (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T cells. MHC class II proteins may contain α and β chains, which can present antigen fragments to helper T cells. The MHC class III region can encode other immune components, such as complement components and cytokines. MHCs can be both polygenic (there are numerous MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).

[0053]

[0263] A "receptor" may refer to a biological molecule or group of molecules capable of binding to a ligand. Receptors may be useful for transmitting information in cells, cell structures, or organisms. A receptor comprises at least one receptor unit, for example, where each receptor unit may be a protein molecule. A receptor has a structure complementary to that of a ligand, allowing it to complex with the ligand as a binding partner. Information is transmitted by a conformational change in the receptor, particularly after ligand complex formation on the cell surface. In some embodiments, a receptor is understood to mean, in particular, MHC class I and II proteins capable of forming a receptor / ligand complex with a ligand, particularly a peptide or peptide fragment having a suitable length. A "ligand" refers to a molecule having a structure complementary to that of a receptor and capable of forming a complex with the receptor. In some embodiments, a ligand is understood to mean a peptide or peptide fragment having a suitable length and a suitable binding motif in its amino acid sequence, such that the peptide or peptide fragment can form a complex with an MHC protein, for example, an MHC class I or MHC class II protein. In some embodiments, "receptor / ligand complex" shall also be understood to mean a "receptor / peptide complex" or "receptor / peptide fragment complex" comprising an MHC molecule, e.g., an MHC class I or MHC class II molecule, that presents a peptide or peptide fragment.

[0054]

[0264] A "native" or "wild-type" sequence may refer to a sequence found in nature. The term "naturally occurring," as used herein, refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that naturally occurs in an organism (including viruses), can be isolated from a source, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0055]

[0265] The terms "peptide" and "peptide epitope" are used herein synonymously with "oligopeptide" to refer to a series of residues typically connected to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues. A "synthetic peptide" refers to a peptide obtained from a non-natural source, e.g., an artificial peptide. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. A "synthetic peptide" includes a "fusion protein."

[0056]

[0266] The term "motif" can also refer to a pattern of residues in an amino acid sequence having a defined length, e.g., in the case of a class I HLA motif, a peptide having a length of less than about 15 amino acid residues, or less than about 13 amino acid residues, e.g., about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13), and in the case of a class II HLA motif, about 6 to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), which are recognized by a specific HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs differ in their pattern of primary and secondary anchor residues. In some embodiments, MHC class I motifs identify peptides having a length of 7, 8, 9, 10, 11, 12, or 13 amino acid residues. In some embodiments, the MHC class II motif identifies peptides that are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acid residues in length. A "cross-reactive binding" peptide refers to a peptide that binds to more than one member of a class of binding pair members (e.g., a peptide that binds to both class I and class II HLA molecules).

[0057]

[0267] The term "residue" often refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or encoded by a nucleic acid (DNA or RNA). The nomenclature used to describe a peptide or protein follows conventional practice. The amino group is provided on the left (amino or N-terminus), and the carboxyl group is provided on the right (carboxy or C-terminus) of each amino acid residue. When the positions of amino acid residues in a peptide epitope are described, they are numbered from amino to carboxyl, with the first position being the residue located at the amino terminus of the epitope or peptide or protein of which the epitope may be a part. In formulas representing selected specific embodiments of the present invention, the amino and carboxyl terminal groups are not specifically indicated, but are in the form they assume at physiological pH values ​​unless otherwise specified. In amino acid structural formulas, each residue is generally represented by a standard three-letter or one-letter symbol. The L-form of an amino acid residue is represented by a capital letter or the first letter of a capital letter in a three-letter symbol, and the D-form of an amino acid residue is represented by a lowercase letter or a lowercase three-letter symbol. However, when three-letter symbols or full names are used without capital letters, they may refer to L-amino acid residues. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or "G." The amino acid sequences of the peptides described herein are generally written using standard one-letter symbols (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine).

[0058]

[0268] A "conservative amino acid substitution" may be a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of phenylalanine with tyrosine is a conservative substitution. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate peptide function are well known in the art.

[0059]

[0269] "Pharmaceutically acceptable" may also refer to compositions or components of compositions that are generally non-toxic, inert, and / or physiologically compatible. "Pharmaceutical excipients" or "excipients" include materials such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. A "pharmaceutical excipient" is an excipient that is pharmaceutically acceptable.

[0060]

[0270] According to the present disclosure, the term "vaccine" can refer to a pharmaceutical preparation (composition) or product that, upon administration, induces an immune response, e.g., a cellular or humoral immune response, that recognizes and attacks pathogens or diseased cells, such as cancer cells. Vaccines can be used to prevent or treat disease. The terms "individualized 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.

[0061]

[0271] The terms "polynucleotide" and "nucleic acid" can be used interchangeably herein and refer to polymers of nucleotides of any length, including DNA and RNA, such as 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 polynucleotide administered using the methods of the present invention is mRNA.

[0062]

[0272] The terms "isolated" or "biologically pure" can also refer to material that is substantially or essentially free from components that normally accompany the material when found in its natural state. Thus, the isolated peptides described herein do not contain some or all of the materials that normally accompany the peptide in its in situ environment. For example, an "isolated" epitope may not contain the entire sequence of the protein from which it is derived. For example, a naturally occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide separated from some or all of the coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector, and / or such polynucleotides or peptides may be part of a composition, still "isolated" in that such vectors or compositions are not part of their natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, as well as synthetically produced such molecules. In some embodiments, an isolated polypeptide, antibody, polynucleotide, vector, cell, or composition is substantially pure. The term "substantially pure," as used herein, refers to material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0063]

[0273] The term "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides can refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps as necessary), without taking into account any conservative amino acid substitutions as part of the 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 align amino acid or nucleotide sequences are well known in the art. Examples of 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 means that they share 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%, or 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 residues, e.g., over a region of at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., over the entire length of the amino acid sequence of a peptide or the coding region of a nucleotide sequence.

[0064]

[0274] The term "subject" can refer to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, dogs, cats, rodents, etc., that is expected to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0065]

[0275] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" may refer to an amount of a therapeutic agent effective to "treat" a disease or disorder in a subject or mammal. A therapeutically effective amount of a drug has a therapeutic effect and may thus prevent the onset of a disease or disorder; slow down the onset of a disease or disorder; slow down the progression of a disease or disorder; alleviate to some extent one or more symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

[0066]

[0276] The terms "treating" or "treatment" or "treat" or "alleviating" or "alleviating" can refer to both (1) therapeutic measures that cure, slow down, lessen the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or slow the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to having the disorder; and those in whom the disorder is to be prevented.

[0067]

[0277] The term "depleted," when used to describe a cell sample (e.g., a peripheral blood mononuclear cell (PBMC) sample), can also refer 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 +Cells can be depleted or removed from a PBMC sample, for example, by using an antibody that binds to CD14.

[0068]

[0278] "Stimulation" refers to a response induced by the binding of a stimulatory molecule to its cognate ligand, thereby mediating a signal transduction event. For example, stimulation of a T cell can refer to the binding of a T cell's TCR to a peptide-MHC complex. For example, stimulation of a T cell can refer to the step in Protocol 1 or Protocol 2 in which PBMCs are cultured with peptide-loaded APCs.

[0069]

[0279] The term "enriched" refers to a composition or fraction in which a species of interest has been partially purified such that the concentration of the species of interest is substantially greater than the naturally occurring level of that species in the finished product without enrichment. The term "induced cells" refers to cells that have been treated with an inducing compound, cell, or population of cells that affects the protein expression, gene expression, differentiation state, shape, morphology, viability, etc. of the cell.

[0070]

[0280] A "reference" can be used to correlate and / or compare the results obtained in the disclosed methods with diseased samples. Typically, a "reference" can be based on one or more normal samples, particularly samples not affected by disease, either obtained from the individual or from one or more different individuals, such as individuals of the same species (e.g., healthy individuals). A "reference" can be empirically determined by testing a sufficiently large number of normal samples.

[0071]

[0281] As used herein, a tumor is a cancerous tumor unless otherwise specified, and the terms cancer and tumor are used interchangeably throughout the document. Although tumors are cancers of solid tissue, some of the compositions and methods described herein are, in principle, applicable to blood cancers, including leukemia.

[0072]

[0282] Overview of T-cell therapy

[0283] Generating 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 therapies). The present disclosure provides methods for producing T cells and therapeutic T cell compositions that can be used to treat subjects with cancer and other conditions, diseases, and disorders. The goal is to expand and induce antigen-specific T cells with favorable phenotypes and functions. The present disclosure provides compositions and methods for producing T cells that can be used for antigen-specific T cell therapy (e.g., personalized T cell therapy). The T cell compositions provided herein may be personalized antigen-specific T cell therapies. Figure 1 diagrammatically illustrates an overview of the process for T cell therapy, which includes, on the one hand, identifying cancer and cancer-specific antigens in a subject with cancer, which triggers the production of neoantigenic peptides; and, on the other hand, preparing activated antigen-specific cells for immunotherapy and administering the cell product.

[0073]

[0284] Neoantigens for T cell-based therapy

[0285] In one aspect, provided herein are therapeutic agents in which the active agent is a cell population comprising tumor antigen-specific T cells. In some embodiments, the active agent is an autologous personalized T cell product for adoptive cell therapy that is produced ex vivo and targets neoantigens presented in tumor cells and the tumor microenvironment. In some embodiments, the autologous personalized T cell product has a T cell count ranging from 75,000,000 to 125,000,000.

[0074]

[0286] In one embodiment, provided herein is a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject an expanded population of cells comprising tumor antigen-specific T cells, and a cytokine.

[0075]

[0287] A method of treating cancer in a human subject in need thereof, comprising the steps of: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a CD14- and / or CD25-depleted population of immune cells comprising a first population of APCs and T cells; (b) injecting the first population of APCs and T cells from step (a) into a first population of immune cells comprising (i) an FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of the human subject with cancer, or (B) a polynucleotide encoding said polypeptide, for a first period of time. (c) forming an expanded population of cells comprising tumor antigen-specific T cells by expanding the population of cells comprising stimulated T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) 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); (d) administering the expanded population of cells from (c) to the human subject; and (e) administering a cytokine to the human subject, wherein the cytokine is interleukin-2 (IL-2).

[0076]

[0288] In some embodiments, the tumor antigen is a neoantigen. Traditional antigen-targeted immunotherapy has focused on antigens such as tumor-associated antigens (TAAs), cancer-testis antigens (typically germline-restricted gene products aberrantly expressed in tumors), or antigens derived from genes that exhibit tissue-specific expression. However, tumors also present protein products of mutated genes called neoantigens. The number and type of mutations can be easily defined using next-generation sequencing approaches and include single amino acid missense mutations, fusion proteins, and novel open reading frames (neoORFs) of various lengths, ranging from 1 to 100 or more amino acids. Neoantigens are antigens that contain non-silent mutations in epitopes, meaning that the same antigen is not expressed in non-cancerous cells within the same human body. Mutation-based antigens are particularly useful because they bypass central tolerance (a process that occurs during normal thymic development and eliminates autoreactive T cells) and demonstrate excellent tumor specificity. Each nonsynonymous (i.e., protein-coding) mutation has the potential to generate neoantigens that can be recognized by a patient's T cells. T cells that recognize these neoantigens can function both to directly kill tumor cells and to 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 utilize these cells in adoptive cell therapy.

[0077]

[0289] In some embodiments, the neo-antigens used herein comprise point mutations.

[0290] In some embodiments, a neoantigen as used herein comprises a frameshift mutation.

[0078]

[0291] In some embodiments, a neoantigen as used herein comprises a cross mutation.

[0292] In some embodiments, a neoantigen as used herein comprises an insertional mutation caused by the insertion of one or more nucleotides.

[0079]

[0293] In some embodiments, neoantigens as used herein include deletion mutations caused by the deletion of one or more nucleotides.

[0294] In some embodiments, neoantigens may arise from insertion-deletion (in-del) mutations.

[0080]

[0295] 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 antigen or neoantigenic peptide has an IC of at least 5000 nM or less, at least 500 nM or less, at least 100 nM or less, or at least 50 nM or less. 50 Or K D , or lower IC 50 Or K D It has.

[0081]

[0296] In some embodiments, the antigen or neo-antigenic peptide may be about 8 to about 50 amino acid residues in length, or about 8 to about 30, about 8 to about 20, about 8 to about 18, about 8 to about 15, or about 8 to about 12 amino acid residues in length. In some embodiments, the antigen or neo-antigenic peptide may be about 8 to about 500 amino acid residues in length, or about 8 to about 450, about 8 to about 400, about 8 to about 350, about 8 to about 300, about 8 to about 250, about 8 to about 200, about 8 to about 150, about 8 to about 100, about 8 to about 50, or about 8 to about 30 amino acid residues in length.

[0082]

[0297] In some embodiments, the antigen or neo-antigen peptide may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid residues in length. In some embodiments, the neo-antigenic peptides may be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acid residues in length. In some embodiments, the antigen or neo-antigen 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 antigen or neo-antigen 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.

[0083]

[0298] In some embodiments, the antigen or neo-antigen peptide has an overall length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

[0084]

[0299] In some embodiments, the antigen or neo-antigen peptide has an overall length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids. In some embodiments, the peptide length is 8-15 amino acids in length. In some embodiments, the peptide length is 8-12 amino acids for targeting CD8+ T cells. In some embodiments, the peptide length is 8-11 amino acids for targeting CD8+ T cells. In some embodiments, the peptide length is 25 amino acids for targeting CD4+ T cells.

[0085]

[0300] In some embodiments, the neo-antigenic peptides may have a pI value of about 0.5 to about 12, about 2 to about 10, or about 4 to about 8. In some embodiments, the neo-antigenic peptides may have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or greater. In some embodiments, the neo-antigenic peptides may have a pI value of up to 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or lower.

[0086]

[0301] In some embodiments, the antigen or neo-antigenic 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 neo-antigenic peptide may have an HLA binding affinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 μM, or higher. In some embodiments, the antigen or neo-antigen peptide may have an HLA binding affinity of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 μM.

[0087]

[0302] In some embodiments, the antigen or neoantigen peptides described herein may comprise a carrier, such as those known in the art, e.g., thyroglobulin, albumin, e.g., human serum albumin, tetanus toxoid, polyamino acid residues, e.g., poly-L-lysine, poly-L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, etc.

[0088]

[0303] In some embodiments, the antigen or neoantigen peptides described herein may be acylated at the terminal NH2, e.g., alkanoyl (C1-C 20 ) or thioglycolyl acetylation, by amidation of the terminal carboxyl, e.g., with ammonia, methylamine, etc. In some embodiments, these modifications can provide sites for linkage to supports or other molecules.

[0089]

[0304] In some embodiments, the antigen or neo-antigen peptides described herein may contain modifications such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surface active materials, e.g., lipids, or may be chemically modified, e.g., acetylation. Furthermore, the bonds in the peptides may be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0090]

[0305] In some embodiments, the antigen or neoantigen peptides described herein may contain substitutions to alter the physical properties (e.g., stability or solubility) of the resulting peptide. For example, the antigen or neoantigen peptide may be modified by substituting cysteine ​​(C) with α-aminobutyric acid ("B"). Due to its chemical nature, cysteine ​​has the propensity to form disulfide bridges, sufficiently altering the peptide structure so that its binding capacity is reduced. Substituting C with α-aminobutyric acid not only alleviates this problem, but may actually improve binding and cross-linking capacity in certain cases. Substitution of cysteine ​​with α-aminobutyric acid may occur at any residue in the antigen or neoantigen peptide, for example, at either anchor or non-anchor positions of the epitope or analog within the peptide, or at other positions in the peptide.

[0091]

[0306] In some embodiments, the antigenic peptides or neo-antigenic peptides described herein contain amino acid mimetics or unnatural 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-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-alanine; D-(methyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-ρ-fluorophenylalanine; D- or L-ρ-biphenyl-phenylalanine; D- or L-ρ-methoxybiphenylphenylalanine; D- or L-2-indole(allyl)alanine; and D- or L-alkylalanine, where the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isobutyl, isopentyl, or a non-acidic amino acid residue. Examples of aromatic rings of unnatural amino acids include thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Modified peptides containing various amino acid mimetics or unnatural amino acid residues are particularly useful because they tend to exhibit increased stability in vivo. Such peptides may also have improved shelf life or manufacturing properties.

[0092]

[0307] In some embodiments, the peptides contact immune cells and activate the cells, making them antigen-responsive.

[0308] In some embodiments, the peptide is contacted with the immune cells ex vivo.

[0093]

[0309] In some embodiments, the peptide is contacted with an immune cell in a biological system, for example, in the human body.

[0310] In some embodiments, the immune cell is an antigen-presenting cell.

[0094]

[0311] In some embodiments, the immune cell is a T cell.

[0312] The present disclosure relates to methods for producing T cells specific for an immunogenic antigen.

[0313] The present disclosure also relates to a composition comprising antigen-specific T cells stimulated with APCs. In some embodiments, one or more antigen peptides are loaded onto APCs, and then the peptide-loaded APCs are used to stimulate T cells to produce antigen-specific T cells. In some embodiments, the antigen is a neoantigen. In some embodiments, the APCs used for peptide loading are dendritic cells.

[0095]

[0314] In some embodiments, the peptide sequence comprises a mutation that does not exist in the non-cancer cells of the subject.In some embodiments, the peptide is encoded by a gene or expressed gene in the cancer cells of the subject.In some embodiments, the peptide sequence has a length of at least 8;9;10;11;12;13;14;15;16;17;18;19;20;21;22;23;24;25;26;27;28;29;30;40;50;60;70;80;90;100;150;200;250;300;350;400;450;500;600;700;800;900;1,000;1,500;2,000;2,500;3,000;4,000;5,000;7,500; or 10,000 or more naturally occurring amino acids.

[0096]

[0315] In some embodiments, the peptide sequence binds to proteins encoded by class I HLA alleles and has a length of 8 to 12 naturally occurring amino acids. In some embodiments, the peptide sequence binds to proteins encoded by class II HLA alleles and has a length of 16 to 25 naturally occurring amino acids. In some embodiments, the peptide sequence comprises a plurality of antigenic peptide sequences. In some embodiments, the plurality of antigenic peptide sequences comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 antigenic peptide sequences. In some embodiments, the antigenic peptide sequences comprise unmodified peptide bonds between amino acids in the sequence. In some embodiments, multiple antigenic peptide sequences are linked together by a linker. In some embodiments, the linker sequence contains G and S amino acids, such as GSS, GSSS, or GGGS. In some embodiments, the linker may be a modified linker having a cleavable sequence. Exemplary cleavable sequences include self-cleaving sequences, such as T2A and P2A. In some embodiments, the antigenic peptide sequence may contain modifications, such as an MITD sequence or an SP1 signaling domain.

[0097]

[0316] In some embodiments, APCs are transfected or transduced with a nucleic acid encoding a peptide sequence comprising one or more antigenic peptide sequences. The APCs express the antigenic peptide sequences and present the antigen associated with MHC to T cells, thereby activating T cells. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA.

[0098]

[0317] In some embodiments, the antigens described herein are neo-antigens. Candidate immunogenic neo-antigen sequences can be identified by any suitable method known in the art. The methods of the present disclosure may be useful, for example, to produce therapeutics specific to a disease of a subject or to produce a vaccine against a disease. Candidate immunogenic neo-antigens may be previously identified neo-antigens. In some embodiments, candidate immunogenic neo-antigens may not have been previously identified. Candidate immunogenic neo-antigens for use in the methods and compositions described herein may be specific to a subject. In some embodiments, candidate neo-antigens for use in the methods and compositions described herein may be specific to multiple subjects.

[0099]

[0318] In both animals and humans, mutated epitopes may be effective in inducing immune responses or activating T cells, etc. In one embodiment, potentially immunogenic epitopes of an infectious agent, such as a virus, in a subject may be determined. In one embodiment, potentially immunogenic mutated epitopes of a subject with a disease, such as cancer, may be determined. In some embodiments, potentially immunogenic antigens or neoantigens for use in the methods described herein may be differentiation antigens expressed in cells of the tumor and the tissue type from which they were generated. In some embodiments, potentially immunogenic antigens or neoantigens for use in the methods described herein may be cancer / germline antigens that are not expressed in other differentiated tissues. In some embodiments, potentially immunogenic antigens or neoantigens for use in the methods described herein may be mutated antigens. For example, candidate immunogenic antigen or neoantigen peptides for use in the methods described herein may include antigens or neoantigens of fusion proteins generated via missense point mutations or tumor-specific translocation of gene segments. In some embodiments, potentially immunogenic antigens or neoantigens for use in the methods described herein may be overexpressed antigens. In some embodiments, potentially immunogenic antigens or neoantigens can be found in tumors. For example, potentially immunogenic antigens or neoantigens for use in the methods described herein can include proteins whose expression is tightly regulated in cells of differentiated normal tissues.

[0100]

[0319] Potentially immunogenic mutant epitopes can be determined by genomic or exomic sequencing of tumor tissues and healthy tissues from cancer patients using next-generation sequencing technology. For example, genes selected based on their mutation frequency and ability to act as antigens or neoantigens can be sequenced using next-generation sequencing technology. In one embodiment, the sequencing data can be analyzed to identify potentially immunogenic mutant peptides that can bind to the target HLA molecule. In one embodiment, the data can be analyzed using a computer. In another embodiment, the sequence data can be analyzed for the presence of antigen or neoantigen peptides. In one embodiment, potentially immunogenic antigen or neoantigen peptides can be determined by their affinity for MHC molecules.

[0101]

[0320] Potentially immunogenic antigens or neo-antigenic peptides can be determined by direct protein sequencing. For example, protein sequencing of enzymatic protein digests using multidimensional mass spectrometry techniques (e.g., tandem mass spectrometry (MS / MS)) may be used to identify potentially immunogenic antigens or neo-antigenic peptides for use in the methods described herein.

[0102]

[0321] 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.

[0103]

[0322] Potentially immunogenic antigens or neoantigen peptides can also be identified using MHC multimers to identify antigen-specific T cell responses. For example, high-throughput analysis of antigen-specific T cell responses in patient samples can be performed using MHC tetramer-based screening technology. Tetramer-based screening technology can be used to initially identify potentially immunogenic tumor-specific antigens, or alternatively, can be used as a secondary screening protocol to evaluate which potentially immunogenic antigens a patient has already been exposed to, thereby facilitating the selection of potentially immunogenic antigens for use in the methods described herein.

[0104]

[0323] In some embodiments, specific neo-antigens are targeted for immunotherapy. In some embodiments, neo-antigen peptides are synthetic. The neo-antigen peptides used herein are designed so that each peptide is specific to an HLA antigen and can bind to the HLA antigen with high binding affinity and specificity. In some embodiments, the peptides used herein are designed based on a high-performance HLA binding prediction model created by the inventors, as described, for example, in the following patent applications / publications: WO2011143656, WO2017184590, and US Provisional Application Nos. 62 / 783,914 and 62 / 826,827, all of which are incorporated herein by reference. NetMHCIIpan may be the current prediction standard, but may not be considered accurate. Of the three class II loci (DR, DP, and DQ), data may exist only for certain common alleles of HLA-DR. Briefly, the newly created predictive model can be used to aid in the identification of immunogenic antigen peptides, develop drugs such as personalized drugs, and isolate and characterize antigen-specific T cells. In this case, the machine learning HLA-peptide presentation predictive model includes multiple predictor variables identified based on at least training data, where the training data includes sequence information of peptides expressed in cells and presented by HLA proteins identified by mass spectrometry; training peptide sequence information including amino acid position information, where the training peptide sequence information is associated with HLA proteins expressed in cells; and a function representing the relationship between the amino acid position information received as input and the presentation probability generated as output based on the amino acid position information and the predictor variables. CD4+ T cell responses may have antitumor activity. Existing prediction methods may demonstrate a high percentage of CD4+ T cell responses even without using class II prediction (e.g., 60% of SLP epitopes in the NeoVax study (49% in NTC-001) and 48% of mRNA epitopes in the BioNTech study). It may not be clear whether these epitopes are typically presented naturally (by tumors or by phagocytic DCs).Therefore, it is desirable to translate high CD4+ T cell response rates into therapeutic efficacy by improving the identification of naturally occurring class II epitopes. The roles of gene expression, enzymatic cleavage, and pathway / localization bias may not have been robustly quantified. While 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, it can be assumed that most existing MS data originates from autophagy. Various data generation approaches exist for learning the rules of class II presentation, including standard and proposed approaches in the field. Standard approaches in the field may include affinity measurements, which may be the basis for NetMHCIIpan predictors, but these offer low throughput, require radioactive reagents, and are subject to processing limitations. New approaches include mass spectrometry, where data from cell lines / tissues / tumors can help determine processing rules for autophagy (much of this data is already publicly available), and monoallelic MS allows for the determination of allele-specific binding rules (multiallelic MS data is speculated to be overly complex for efficient learning). The newly generated prediction method involves training a machine learning HLA-peptide presentation prediction model, where the training involves using a computer processor to generate amino acid positional sequences of HLA-peptides isolated from one or more HLA-peptide complexes from cells expressing HLA class II alleles. The machine learning HLA-peptide presentation prediction model includes inputting the amino acid position information received as input to a machine learning HLA-peptide presentation prediction model, the machine learning HLA-peptide presentation prediction model including a plurality of predictor variables identified based on training data including at least sequence information of peptide sequences presented by HLA proteins expressed in cells and identified by mass spectrometry; training peptide sequence information including amino acid position information of the peptides to be trained, the training peptide sequence information being associated with the HLA proteins expressed in the cells; and, based on the amino acid position information and the predictor variables, generating a function representing the relationship between the amino acid position information received as input and the presentation probability.In some embodiments, the proposed model has a positive predictive value of at least 0.25 at a recall rate of 0.1% to 10%. In some embodiments, the proposed model has a positive predictive value of at least 0.4 at a recall rate of 0.1% to 10%. In some embodiments, the proposed model has a positive predictive value of at least 0.6 at a recall rate of 0.1% to 10%. In some embodiments, the mass spectrometry is monoallelic mass spectrometry. 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 enhanced by using multiple quality metrics. In some embodiments, the multiple quality metrics include removal of common contaminant peptides, high scored peak intensities, high scores, and high mass accuracy. In some embodiments, the scored peak intensities are at least 50%. In some embodiments, the scored peak intensities are at least 70%. In some embodiments, the peptides presented by HLA proteins expressed in cells are peptides presented by HLA proteins expressed in a single immunoprecipitated cell. In some embodiments, the plurality of predictor variables includes a predictor variable for peptide-HLA affinity. In some embodiments, the plurality of predictor variables includes a predictor variable for source protein expression level. In some embodiments, the plurality of predictor variables includes a predictor variable for peptide cleavability. In some embodiments, the peptides presented by HLA proteins include peptides identified by searching a peptide database using a reverse database search strategy. In some embodiments, the HLA protein is an HLA-DR protein, and an HLA-DP or HLA-DQ protein. In some embodiments, the HLA protein is an HLA-DR protein selected from the group consisting of an HLA-DR protein, and an HLA-DP or HLA-DQ protein.In some embodiments, the HLA proteins are HLA-DPB1*01:01 / HLA-DPA1*01:03, HLA-DPB1*02:01 / HLA-DPA1*01:03, HLA-DPB1*03:01 / HLA-DPA1*01:03, HLA-DPB1*04:01 / HLA-DPA1*01:03, HLA-DPB1*04:02 / HLA-DPA1*01:03, HLA-DPB1*06:01 / HLA-DPA1*01:03, HLA-DQB1*02:01 / HL A-DQA1*05:01, HLA-DQB1*02:02 / HLA-DQA1*02:01, HLA-DQB1*06:02 / HLA-DQA1*01:02, HLA-DQB1*06:04 / HLA-DQA1*01:02, HLA-DR B1*01:01, HLA-DRB1*01:02, HLA-DRB1*03:01, HLA-DRB1*03:02, HLA-DRB1*04:01, HLA-DRB1*04:02, HLA-DRB1*04:03, HLA-DRB1*04 :04, HLA-DRB1*04:05, HLA-DRB1*04:07, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*08:02, HLA-DRB1*08:03, HLA-DRB1*08:04, HLA-DRB1*09:01, HLA-DRB1*10:01, HLA-DRB1*11:01, HLA-DRB1*11:02, HLA-DRB1*11:04, HLA-DRB1*12:01, HLA-DRB1*12:02, HLA-D In some embodiments, the peptides presented by the HLA proteins include peptides identified by comparing the MS / MS spectrum of the HLA-peptide with the MS / MS spectrum of one or more HLA-peptides in a peptide database.

[0105]

[0324] 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.

[0106]

[0325] In some embodiments, the peptides presented by the HLA proteins have a length of 15 to 40 amino acids. In some embodiments, the peptides presented by the HLA proteins 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, from a peptide database, peptide sequences corresponding to the MS / MS spectra of the one or more isolated HLA-peptides, wherein the one or more sequences obtained from step (d) identify the sequences of the one or more isolated HLA-peptides.

[0107]

[0326] A variety of antigenic peptides can be used to induce or expand T cells. A variety of antigenic peptides can be used to activate antigen-presenting cells (APCs), which in turn activate T cells by contacting them with antigen-loaded APCs.

[0108]

[0327] In some embodiments, the peptide comprises a mutation selected from (A) a point mutation, (B) a splice site mutation, (C) a frameshift mutation, (D) a readthrough mutation, (E) a gene fusion mutation, and combinations thereof. In some embodiments, the peptide comprises a point mutation and binds to an HLA protein of interest with greater affinity than the corresponding wild-type peptide.

[0109]

[0328] In some embodiments, the peptide has an IC of less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. 50 In some embodiments, the peptide binds to an HLA protein of interest with an IC of less than 500 nM, less than 250 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, or less than 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, less than 250 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, or less than 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, less than 250 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. 50 or K D and bind to a peptide-HLA complex. In some embodiments, each of the at least one antigenic peptide sequence comprises a mutation that is not present in non-cancer cells of the subject. In some embodiments, each of the at least one antigenic peptide sequence is encoded by a gene or expressed gene of a cancer cell of the subject.

[0110]

[0329] In some embodiments, the peptide has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 or more naturally occurring amino acids. In some embodiments, the peptide binds to a protein encoded by a class I HLA allele and has a length of 8 to 12 naturally occurring amino acids. In some embodiments, the peptide binds to a protein encoded by a class II HLA allele and has a length of 16 to 25 naturally occurring amino acids. In some embodiments, the peptide 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.

[0111]

[0330] In some embodiments, the present disclosure provides peptides (e.g., peptides with tumor-specific mutations, viral peptides, or peptides associated with non-cancerous diseases) or polynucleotides encoding peptides identified using the methods briefly described herein above.

[0112]

[0331] 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 the nucleic acid sequence of the target polynucleotide.

[0113]

[0332] Preparation of activated antigen-specific T cells

[0333] Generating T cells for therapeutic purposes, such as cancer therapy, has been pursued for decades without overwhelming success. Adoptive T cell therapy is conceptually simple but extremely difficult to achieve in practice. The present methods and compositions have become highly functional through numerous innovative improvements in the process. Stimulating patient T cells with neoantigens expressed in the patient is considered an exciting pathway to redirect the immune response to the tumor and bypass T cell anergy and tolerance to highly expressed, endogenous cancer antigens. However, at the same time, achieving the desired drug product procedurally requires a long time. The goal of this research is to shorten the time from laboratory to clinical setting. The goal of this research is to create a method that is flawless in design, produces consistent results, is feasible, and can be completed predictably with minimal errors. The resulting drug product will have a high safety profile and patients will tolerate DP better than earlier attempts.

[0114]

[0334] In one embodiment, the method provided herein is a monotherapy. The patient may be pretreated with other therapies. If enrolled in an immediate treatment regimen, the patient may receive the T cell therapy described herein at the start of therapy.

[0115]

[0335] In some embodiments, it is anticipated that the T cell therapy of the present disclosure will be administered in conjunction with one or more additional therapies, as determined by a medical professional or clinical trial administrator to be in the best interest of the patient.

[0116]

[0336] In some embodiments, it is expected that the T cell therapy will be administered in addition to an ongoing therapy.

[0337] In some embodiments, the T cell therapy is administered alone for the duration of the therapy, for example, from the start of therapy until the end of the clinical trial.

[0117]

[0338] In some embodiments, the patient has been pretreated with one or more anti-cancer therapeutic agents, such as nivolumab, pembrolizumab, or both, and anti-CTLA4 therapy, or anti-CTLA4 therapy and nivolumab therapy, or anti-CTLA4 therapy plus nivolumab and / or pembrolizumab therapy. In some embodiments, the patient has stable disease. In some embodiments, the patient may have demonstrated relatively low tolerance to one or more of the pretreatment drugs. In some embodiments, the patient may have been refractory to one or more drugs with which the patient was pretreated. In some embodiments, the patient is refractory to nivolumab or pembrolizumab, or anti-CTLA4 therapy, or all of them.

[0118]

[0339] Provided herein are methods for stimulating T cells. For example, the methods provided herein can be used to stimulate antigen-specific T cells. The methods provided herein can be used to 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 stimulate 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. + It can be used to expand T cells. For example, the therapeutic composition can include antigen-specific CD8+ T cells. For example, the therapeutic composition can include antigen-specific memory T cells.

[0119]

[0340] In some embodiments, T cell therapy is administered only to patients with highly advanced but relatively stable tumors, hi some embodiments, therapy is directed to stable patients who can tolerate new therapies.

[0120]

[0341] T cells can be activated ex vivo with a composition comprising a neoantigenic peptide or a polynucleotide encoding the neoantigenic peptide.

[0342] T cells can be activated ex vivo with a composition comprising antigen-loaded antigen-presenting cells.

[0121]

[0343] In some embodiments, the APCs and / or T cells are derived from a biological sample obtained from a subject.

[0344] In some embodiments, the APCs and / or T cells are derived from a biological sample that is a peripheral blood mononuclear cell (PBMC).

[0122]

[0345] In some embodiments, the subject is administered FLT3L prior to obtaining a biological sample for preparing APCs and / or T cells.

[0346] In some embodiments, the APCs and / or T cells are derived from a biological sample that is a leukapheresis sample.

[0123]

[0347] In some embodiments, antigen-presenting cells are first loaded with neoantigenic peptides ex vivo and used to prepare neoantigen-activated T cells. In some embodiments, the compositions provided herein comprise T cells stimulated by APCs, e.g., by APCs preloaded with antigenic peptides. The compositions can include a population of immune cells, including T cells, from a sample (e.g., a biological sample), where the T cells include T cells stimulated by APCs. In some embodiments, mRNA encoding one or more neoantigenic peptides is introduced into the APCs for expression of the neoantigenic peptides. Such APCs are used to stimulate or activate T cells.

[0124]

[0348] In some embodiments, the biological sample comprises a percentage of at least one antigen-specific T cell in the composition that is at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%. In some embodiments, the biological sample comprises less than 0.0001%, less than 0.0005%, less than 0.001%, less than 0.005%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, or less than 10% of antigen-activated T cells of the total number of cells in a biological sample derived from peripheral blood or leukapheresis. In some embodiments, the biological sample comprises antigen-activated T cells that are less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% of the total number of cells in the biological sample derived from peripheral blood or leukapheresis. In some embodiments, the biological sample comprises antigen-naive T cells.In some embodiments, the biological sample comprises more than about 0.00001%, more than 0.00002%, more than 0.00005%, more than 0.0001%, more than 0.0005%, more than 0.001%, more than 0.005%, more than 0.01%, more than 0.05%, more than 0.1%, more than 0.5%, more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8% of the total cell count in a biological sample derived from peripheral blood or leukapheresis. In some embodiments, the composition comprises more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, more than 15%, more than 16%, more than 17%, more than 18%, more than 19%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% antigen-naive cells. In some embodiments, the composition comprises at least one antigen-specific CD8. + The percentage of T cells in a biological sample derived from peripheral blood or leukapheresis is less than about 0.00001%, less than 0.00002%, less than 0.00005%, less than 0.0001%, less than 0.0005%, less than 0.001%, less than 0.005%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 3%, less than 4%, or less than 5%. In some embodiments, the composition contains at least one antigen-specific CD4 +The percentage of T cells in a biological sample derived from peripheral blood or leukapheresis 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%. In some embodiments, the percentage of 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, at least one antigen-specific CD8 + The percentage of T cells is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total immune cells. In some embodiments, at least one antigen-specific CD4 + The percentage of T cells is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of 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%. + In some embodiments, the percentage of T cells is at most about 0.5%. + The percentage of T cells is at most about 0.5% in a biological sample.

[0125]

[0349] In some embodiments, the biological sample is depleted of CD25+ cells prior to cell culture and T cell expansion.

[0350] In some embodiments, the biological sample is depleted of CD56+ cells prior to cell culture and T cell expansion.

[0126]

[0351] In some embodiments, the biological sample is depleted of CD25+ cells and CD56+ cells prior to cell culture and T cell expansion.

[0352] In some embodiments, the biological sample is depleted of CD19+ cells and CD56+ cells prior to cell culture and T cell expansion.

[0127]

[0353] In some embodiments, the biological sample is depleted of CD25+, CD19+ and CD56+ cells prior to cell culture and T cell expansion.

[0354] In some embodiments, the biological sample is depleted of CD14+ cells, CD25+ cells, CD19+ cells and CD56+ cells prior to cell culture and T cell expansion.

[0128]

[0355] In some embodiments, the biological sample is depleted of CD14+ cells, CD25+ cells, and CD56+ cells prior to cell culture and T cell expansion.

[0356] In some embodiments, the biological sample is depleted of CD14+ cells and CD25+ cells prior to cell culture and T cell expansion.

[0129]

[0357] Preparation of neoantigen-loaded APCs

[0358] In some embodiments, the composition comprises a population of immune cells incubated with one or more cytokines, growth factors, or ligands, such as ligands that bind to cell surface receptors on APCs or T cells. Non-limiting examples of such cytokines, growth factors, and ligands include, but are not limited to, GM-CSF, IL-4, IL-7, FLT3L, TNF-α, IL-1β, IL-15, PGE1, IL-6, IFN-α, IFN-γ, R848, LPS, ss-RNA40, and poly I:C. In some embodiments, the composition comprises a population of immune cells incubated with one or more APCs or APC preparations. For example, the composition may comprise APCs stimulated with one or more cytokines, growth factors, and / or ligands, or a population of immune cells incubated with an APC preparation stimulated with cytokines, growth factors, and / or ligands. For example, the composition may comprise a population of immune cells incubated with APCs stimulated with one or more cytokines, growth factors, and / or ligands, or a population of immune cells incubated with an APC preparation stimulated with cytokines, growth factors, and / or ligands. For example, the composition may comprise a population of immune cells incubated with APCs stimulated by one or more growth factors or a growth factor-stimulated APC preparation. For example, the composition may comprise a population of immune cells incubated with APCs stimulated by one or more ligands or a ligand-stimulated APC preparation.

[0130]

[0359] In some embodiments, the APCs are autologous, allogeneic, or artificial APCs.

[0360] Immune cells are characterized by cell surface molecules.In some embodiments, immune cells are selected based on cell surface markers, for example, cell surface markers from biological samples, preferably by 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 that cause the cells to be negatively selected.

[0131]

[0361] In some embodiments, antigen-presenting cells (APCs) are prepared from a biological sample by selecting from APCs or precursor cells that can be cultured in the presence of neoantigen peptides to generate neoantigen-loaded APCs used to activate T cells. Some relevant cell surface markers for selecting and / or enriching sets of cells are described below.

[0132]

[0362] CD1 (cluster of differentiation 1) is a family of glycoproteins expressed on the surface of various human antigen-presenting cells. They associate with class I MHC molecules and are involved in the presentation of lipid antigens to T cells.

[0133]

[0363] CD11b or integrin alpha M (ITGAM) is a heterodimeric integrin alpha-M beta-2 (α M ITGAM is one protein subunit that forms the macrophage-1 antigen (Mac-1) or complement receptor 3 (CR3). ITGAM is also known as CR3A and cluster of differentiation molecule 11b (CD11b). M The second chain of β2 is the common integrin β2 subunit known as CD18, and therefore integrin α M β2 belongs to the β2 subfamily (or leukocyte) integrins. M Integrin β2 is expressed on the surface of many leukocytes involved in the innate immune system, such as monocytes, granulocytes, macrophages, and natural killer cells. It mediates inflammation by regulating leukocyte adhesion and migration, and has been shown to be involved in numerous immune processes, including phagocytosis, cell-mediated cytotoxicity, chemotaxis, and cell activation. It is involved in the complement system due to its ability to bind inactivated complement component 3b (iC3b). Integrin α M The ITGAM (alpha) subunit of β2 is directly involved in initiating cell adhesion and spreading, but cannot mediate cell migration without the presence of the β2 (CD18) subunit.

[0134]

[0364] CD11c, also known as integrin, alpha X (complement component 3 receptor subunit 4) (ITGAX), is the gene encoding CD11c. CD11c is an integrin alpha X chain protein. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. This protein combines with a beta 2 chain (ITGB2) to form a leukocyte-specific integrin called inactivated C3b (iC3b) receptor 4 (CR4). The alpha X beta 2 complex appears to overlap with the properties of alpha M beta 2 integrin in neutrophil and monocyte adhesion to stimulated endothelial cells and in the 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 initiate the respiratory burst of neutrophils; it is expressed in hairy cell leukemia, acute nonlymphocytic leukemia, and some B-cell chronic lymphocytic leukemias.

[0135]

[0365] CD14 is a surface antigen preferentially expressed on monocytes / macrophages. In cooperation with other proteins, CD14 mediates the innate immune response to bacterial lipopolysaccharide. Alternative splicing results in multiple transcript variants encoding the same protein. CD14 exists in two forms: one membrane-anchored by a glycosylphosphatidylinositol tail (mCD14) and the other soluble form (sCD14). Soluble CD14 either appears after shedding of mCD14 (48 kDa) or is directly secreted from intracellular vesicles (56 kDa). CD14 acts as a coreceptor for the detection of bacterial lipopolysaccharide (LPS) (together with the Toll-like receptors TLR4 and MD-2). CD14 can bind LPS only in the presence of lipopolysaccharide-binding protein (LBP). Although LPS is considered its primary ligand, CD14 also recognizes other pathogen-associated molecular patterns such as lipoteichoic acid.

[0136]

[0366] CD25 is expressed by conventional T cells after stimulation and in human peripheral blood, CD4 + CD25 hi Only T cells have been shown to be "suppressors."

[0367] In some embodiments, the APC comprises a dendritic cell (DC). In some embodiments, the APC comprises 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 from a biological sample from the subject that contains PBMCs. For example, CD14 + Monocytes may be isolated, enriched, or purified from a biological sample from a subject that contains PBMCs. + The monocytes are stimulated with one or more cytokines or growth factors. In some embodiments, the one or more cytokines or growth factors include GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, 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 from a second biological sample containing PBMCs.

[0137]

[0368] In some embodiments, the isolated population of APCs may be enriched or substantially enriched. In some embodiments, the isolated population of APCs is at least 30%, at least 50%, at least 75%, or at least 90% homogeneous. In some embodiments, the isolated population of APCs is at least 60%, at least 75%, or at least 90% homogeneous. APCs, e.g., APCs, can include, for example, APCs derived from cultures of monocytic dendritic cell precursors, as well as internally derived APCs present in tissues such as peripheral blood, umbilical cord blood, skin, spleen, bone marrow, thymus, and lymph nodes.

[0138]

[0369] APCs and cell populations substantially enriched in APCs can be isolated by methods also provided by the present invention, which generally involve obtaining a population of cells containing APC precursors, differentiating the APC precursors into immature or mature APCs, and optionally further isolating the APCs from the population of differentiated immature or mature APCs.

[0139]

[0370] APC precursor cells can be obtained by methods known in the art. APC precursors can be isolated, for example, by density gradient separation, fluorescence-activated cell sorting (FACS), immunological cell separation techniques, such as panning, complement lysis, rosetting, magnetic cell separation techniques, nylon wool separation, and combinations of these methods. Methods for immunoselecting APCs include, for example, using antibodies against cell surface markers associated with APC precursors, such as anti-CD34 and / or anti-CD14 antibodies coupled to a substrate.

[0140]

[0371] It is also possible to obtain a concentrated population of APC precursors.The method for obtaining such a concentrated precursor population is known in the art.For example, the concentrated population of APC precursors can be isolated from tissue sources by selectively removing the cells that adhere to the substrate.For example, using tissue sources such as bone marrow or peripheral blood, adhesive monocytes can be removed from cell preparations using commercially treated plastic substrates (for example, beads or magnetic beads), to obtain a concentrated population of non-adherent APC precursors.

[0141]

[0372] Monocyte APC precursors can also be obtained from tissue sources by using a substrate to which the APC precursors adhere. For example, peripheral blood leukocytes isolated, e.g., by leukapheresis, are contacted with a substrate having a high surface area-to-volume ratio to which the monocytic APC precursors adhere, and the adherent monocytic APC precursors are isolated. In additional embodiments, the coupled substrate can be a particulate or fibrous substrate having a high surface-to-volume ratio, such as microbeads, microcarrier beads, pellets, granules, powders, capillaries, microvillous membranes, etc. Furthermore, the particulate or fibrous substrate can be glass, polystyrene, plastic, glass-coated polystyrene microbeads, etc.

[0142]

[0373] APC precursors may also be cultured in vitro for differentiation and / or expansion. Methods for differentiation / expansion of APC precursors are known in the art. Generally, expansion can be achieved by culturing the precursors in the presence of at least one cytokine that induces differentiation / proliferation of APCs (e.g., dendritic cells). Typically, these cytokines are granulocyte colony-stimulating factor (G-CSF) or granulocyte / macrophage colony-stimulating factor (GM-CSF). In addition, other agents can be used to inhibit the proliferation and / or maturation of non-APC cell types in culture, thereby further enriching the population of APC precursors. Typically, such agents include cytokines such as IL-13, IL-4, or IL-15.

[0143]

[0374] The isolated population of APC precursors is cultured and differentiated to obtain immature or mature APCs. Suitable tissue culture media include, but are not limited to, AIM-V®, RPMI1640, DMEM, X-VIVO, etc. The tissue culture medium is typically supplemented with amino acids, vitamins, divalent cations, and cytokines to promote the differentiation of precursors into APC phenotypes. Typically, the cytokines that promote differentiation are GM-CSF and / or IL-4.

[0144]

[0375] Furthermore, cultures of APC precursors during expansion, differentiation, and maturation to the APC phenotype may contain plasma to promote APC development. A typical plasma concentration is about 5%. In addition, for example, when APC precursors are isolated by adhesion to a substrate, CD14 + Plasma may be included in the culture medium during the adhesion step to promote phenotype. Typical plasma concentrations during adhesion are about 1% or more.

[0145]

[0376] The monocytic APC precursors may be cultured for any suitable period of time. In certain embodiments, a suitable culture period for differentiation of the precursors into immature APCs may be about 1 to about 10 days, for example, about 4 to about 7 days. Differentiation of the precursors into immature APCs can be monitored by methods known to those skilled in the art, for example, by detecting cell surface markers (e.g., CD11c). + , CD83 low , CD86 - / low , HLA-DR + Immature APCs can be monitored by the presence or absence of IL-4 and GM-CSF. Immature APCs can also be cultured in appropriate tissue culture media to maintain them in a state suitable for further differentiation or antigen uptake, processing, and presentation. For example, immature APCs can be maintained in the presence of GM-CSF and IL-4.

[0146]

[0377] In some embodiments, APC precursors may be isolated prior to differentiation. In some embodiments, the isolated population may be enriched or substantially enriched for APC precursors. In some embodiments, APC precursors are isolated with a CD14-specific probe. In one exemplary embodiment, CD14-expressing cells are detected by FACS using a CD14-specific probe directly conjugated to a fluorescent molecule (e.g., FITC or PE), or by using an unlabeled antibody specific for CD14 and a labeled second antibody specific to the first antibody. CD14 +Cells were also identified by FACS sorting as CD14 low and CD14 - It can also be isolated from cells. high Gating for the positive rate can be determined, for example, by reference to CD14 staining of monocytes derived from PBMCs. Typically, the CD14-specific binding agent is, for example, an anti-CD14 antibody (e.g., a monoclonal antibody or an antigen-binding fragment thereof). Many anti-CD14 antibodies suitable for use in the present invention are known to those skilled in the art, and many can be purchased commercially. Differentiation into immature APCs (CD14 negative) can occur after isolation.

[0147]

[0378] In another embodiment, a probe specific for CD14 is coupled to the substrate and detects CD14 + The cells are isolated by affinity selection. + A population of cells containing CD14 cells is exposed to the coupled substrate. + The cells are then specifically adhered to the non-adherent CD14 - The cells are washed off the substrate, and then the adherent cells are eluted to obtain an isolated cell population that is substantially enriched in 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 isolation of cell populations using antibodies coupled to substrates specific for surface markers are generally known.

[0148]

[0379] During the culture, the immature APCs may optionally be exposed to a predetermined antigen. Suitable predetermined antigens include any antigen for which T cell modulation is desired. In one embodiment, the immature APCs are cultured in the presence of prostate-specific membrane antigen (PSMA) for cancer immunotherapy and / or tumor growth inhibition. Other antigens may include, for example, bacterial cells, viruses, partially purified or purified bacterial or viral antigens, tumor cells, tumor-specific or tumor-associated antigens (e.g., tumor cell lysates, tumor cell membrane preparations, antigens isolated from tumors, fusion proteins, liposomes, etc.), recombinant cells expressing antigens on their surface, autoantigens, and any other antigens. Any of the antigens may be presented as peptides or recombinantly produced proteins or portions thereof. After contact with the antigen, the cells may be cultured for any suitable time to allow for antigen uptake and processing, expand the population of antigen-specific APCs, etc.

[0149]

[0380] For example, in one embodiment, after antigen uptake, immature APCs may be cultured to promote maturation of the immature APCs into mature APCs that present the antigen in the context of MHC molecules. Methods for APC maturation are known. Such maturation can be carried out, for example, by culturing in the presence of known maturation factors, such as cytokines (e.g., TNF-α, IL-1β, or CD40 ligand), bacterial products (e.g., LPS or BCG), etc. 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 by testing for the expression of mRNA or proteins specific to mature APCs, for example, using oligonucleotide arrays.

[0150]

[0381] Optionally, immature APCs may be cultured in an appropriate tissue culture medium to expand the cell population and / or maintain the immature APCs in a context for further differentiation or antigen uptake. For example, immature APCs may be maintained and / or expanded in the presence of GM-CSF and IL-4. Immature APCs may also be cultured in the presence of anti-inflammatory molecules, such as, for example, anti-inflammatory cytokines (e.g., IL-10 and TGF-β), to inhibit maturation of the immature APCs.

[0151]

[0382] In another embodiment, the isolated population of APCs is enriched for mature APCs. The isolated population of mature APCs can be obtained by culturing a differentiated population of immature APCs in the presence of maturation factors (e.g., bacterial products and / or pro-inflammatory cytokines) as described above, thereby inducing maturation. The immature APCs can be isolated by removing CD14+ cells.

[0152]

[0383] According to yet another aspect of the present invention, APCs may be preserved, for example, by cryopreservation, either before or after exposure to a suitable antigen. Cryopreservatives that can be used include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate, and inorganic salts. A controlled, slow cooling rate may be important. Different cryoprotectants and different cell types typically have different optimal cooling rates. The thermal melting phase, during which water turns to ice, should typically be minimized. The cooling procedure may be performed, for example, by using a programmable freezing device or a methanol bath procedure. A programmable freezing device allows for the determination of the optimal cooling rate and facilitates standard, reproducible cooling. Programmable rate-controlled freezers, such as Cryomed or Planar, allow adjustment of the freezing regimen to a desired cooling rate curve.

[0153]

[0384] After complete freezing, the APCs may be rapidly transferred to a container for long-term cryogenic storage. In a typical embodiment, the samples may be cryogenically stored in liquid nitrogen (-196°C) or its vapor (-165°C). Most of the considerations and procedures for the manipulation, cryopreservation, and long-term storage of hematopoietic stem cells, particularly hematopoietic stem cells from bone marrow or peripheral blood, are applicable to the APCs of the present invention.

[0154]

[0385] Frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37-41°C) and immediately cooled upon thawing. It may be desirable to treat the cells to prevent aggregation upon thawing. Various procedures can be used to prevent aggregation, including, but not limited to, the addition of DNase, low molecular weight dextran and citrate, hydroxyethyl starch, and the like, before and / or after freezing. Cryoprotectants, if toxic in humans, should be removed before therapeutic use of thawed APCs. One method for removing the cryoprotectant is by dilution to an insignificant concentration. Once frozen APCs are thawed and recovered, they can be used to activate T cells as described herein for unfrozen APCs.

[0155]

[0386] In one embodiment, a composition for T cell activation comprises a population of immune cells depleted of one or more types of immune cells. For example, the composition may comprise a population of immune cells depleted of one or more types of immune cells expressing one or more proteins, such as one or more cell surface receptors. In some embodiments, the composition comprises 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 CD14 and / or CD25 expressing immune cells in the population is proportionally different from the amount of CD14 and / or CD25 expressing immune cells in the biological sample. For example, the 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 CD14 expressing immune cells in the population is proportionally different from the amount of CD14 expressing immune cells 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 CD25 expressing immune cells 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 CD14 and CD25 expressing immune cells 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.

[0156]

[0387] 1. A method for preparing a cell composition for cancer immunotherapy, comprising: I. preparing antigen-loaded antigen-presenting cells (APCs), the method comprising: (a) obtaining peripheral blood mononuclear cells (PBMCs) from a subject pretreated with fms-like tyrosine kinase 3 ligand (FLT3L); (b) ex vivo loading the PBMCs with: (i) a plurality of cancer neoantigen peptides or one or more polynucleotides encoding a plurality of cancer neoantigen peptides, wherein each of the cancer neoantigen peptides or a portion thereof binds to a protein encoded by an HLA allele expressed in the subject; (ii) a stimulatory agent for activating the cells; (iii) an agent for promoting the growth and maintenance of the cells ex vivo to obtain a cell population; and (iv) CD11b low or II. contacting the isolated T cells ex vivo with an agent to reduce or deplete CD11b+ cells from the cell population to obtain CD11b-depleted antigen-loaded APCs; low or a step of contacting CD11b-depleted T cells with antigen-loaded APCs; III. preparing antigen-stimulated T cells for a cell composition for cancer immunotherapy.

[0157]

[0388] 1. An improved method for preparing tumor antigen-specific T cells ex vivo, comprising: (a) depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells to form a population of immune cells comprising a first population of CD14- and / or CD25-depleted APCs and T cells, wherein the population of immune cells is from a biological sample from a human subject; (b) inducing the first population of APCs and T cells from step (a) for a first period of time to express (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) a small molecule expressed by cancer cells of (A) the human subject having cancer. Provided herein are methods comprising the steps of (A) forming a population of cells comprising stimulated T cells by incubating the stimulated T cells in the presence of (B) a polypeptide comprising at least one tumor antigen epitope sequence, or a polynucleotide encoding said polypeptide; (c) forming an expanded population of cells comprising tumor antigen-specific T cells by expanding the stimulated T cells from step (b), wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence from step (b)(ii) and (ii) an MHC protein expressed by cancer cells or APCs of the human subject of (b)(ii). The method comprises administering the expanded population of cells from (c) to a human subject, wherein the expanded population of cells from step (c) is greater than or equal to 1 x 10 8 ~1×10 11

[0013] Provided herein are methods for preparing antigen-specific T cells comprising activated CD8+ T cells and CD4+ T cells from a naive T cell compartment; the process comprises depleting CD14+ cells from PBMCs prior to antigen stimulation and expansion; or depleting CD14+ cells and CD25+ cells, CD14+, CD25+, and CD11b+ cells.

[0014] Provided herein are methods for preparing antigen-specific T cells comprising activated CD8+ T cells and CD4+ T cells from a naive T cell compartment; the process comprises depleting CD25+ cells from PBMCs prior to antigen stimulation and expansion; or depleting CD14+ cells and CD25+ cells, CD14+, CD25+, and CD11b+ cells. In some embodiments, provided herein is a method for preparing antigen-specific T cells, including activated CD8+ T cells and CD4+ T cells, from the naive T cell compartment; the process includes depleting CD11b+ cells from PBMCs prior to antigen stimulation and expansion; or depleting CD14+ and CD25+ cells, or CD14+, CD25+ and CD11b+ cells.

[0158]

[0389] In some embodiments, the subject is pretreated with FLT3L for 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 for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks prior to isolation of PBMCs or leukapheresis.

[0159]

[0390] In some embodiments, the cell population is enriched for CD11c+ cells. In some embodiments, the antigen-loaded APCs comprise dendritic cells (DCs). In some embodiments, the antigen-loaded APCs comprise plasmacytoid dendritic cells (pDCs). In some embodiments, the antigen-loaded APCs comprise CD1c+ DCs. In some embodiments, the antigen-loaded APCs comprise CD141+ DCs. In some embodiments, the cell population comprises macrophages. In some embodiments, the method further comprises reducing or depleting CD19+ cells from the cell population to activate or enrich T cells activated by the neoantigen. In some embodiments, the method further comprises reducing or depleting both CD11b+ cells and CD19+ cells from the cell population to activate or enrich T cells activated by the neoantigen.

[0160]

[0391] In some embodiments, the method further comprises reducing or depleting CD14+ cells from the cell population to prepare and enrich for antigen-activated T cells. In some embodiments, the method further comprises reducing or depleting CD25+ cells from the cell population to prepare and enrich for 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 to activate or enrich for neoantigen-activated T cells.

[0161]

[0392] In some embodiments, the stimulatory agent for activating the cells comprises FL3TL.

[0393] In some embodiments, agents that promote cell growth and maintenance ex vivo include growth factors, cytokines, amino acids, supplements, or combinations thereof.

[0162]

[0394] In some embodiments, antigen-loaded APCs are capable of stimulating T cells for 2, 3, 4, 5, 6, or 7 days.

[0395] In some embodiments, each of the plurality of cancer neo-antigen peptides is 8-30 amino acids in length.

[0163]

[0396] 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; each of the plurality of neoantigenic peptides has the characteristics of the neoantigenic peptide as described in the previous section.

[0164]

[0397] In some embodiments, the neo-antigen 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 in between. In some embodiments, the neo-antigen peptide used to prepare antigen-loaded APCs comprises amino acids on either side of mutations that facilitate endogenous processing of the neo-antigen peptide to increase the rate of presentation to T cells.

[0165]

[0398] Longer immunogenic peptides can also be designed in a number of ways. In some embodiments, when HLA-binding peptides are predicted or known, longer immunogenic peptides can consist of (1) individual binding peptides with 2-5 amino acid extensions toward the N- and C-termini of their respective gene products; or (2) a concatenation of some or all of the binding peptides with their respective extended sequences. In other embodiments, when sequencing reveals long (more than 10 residues) epitope sequences, such as neoepitopes present in tumors (e.g., due to frameshifts, readthrough, or intron inclusion resulting in novel peptide sequences), longer neoantigenic peptides can consist of the entire novel tumor-specific stretch of amino acids, either as a single longer peptide or as several overlapping longer peptides. In some embodiments, the use of longer peptides is expected to allow for endogenous processing by the patient's cells, resulting in more effective antigen presentation and induction of T cell responses. In some embodiments, two or more peptides can be used, where the peptides are overlapping and tiled onto the long neoantigenic peptide.

[0166]

[0399] 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.

[0167]

[0400] In some embodiments, the one or more polynucleotides encoding the multiple cancer neo-antigenic peptides are DNA.

[0401] In some embodiments, one or more polynucleotides encoding multiple cancer neoantigenic peptides are inserted into one or more mammalian expression vectors.

[0168]

[0402] In some embodiments, the one or more polynucleotides encoding the multiple cancer neoantigenic peptides are messenger RNA.

[0403] In some embodiments, the present invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising modified nucleosides.

[0169]

[0404] In some embodiments, the present invention provides gene therapy vectors comprising RNA, oligoribonucleotides, and polyribonucleotides.

[0405] In some embodiments, the present invention provides methods of gene therapy and gene transcription silencing methods including same.

[0170]

[0406] In some embodiments, the polynucleotide encodes a single neo-antigenic peptide.

[0407] In some embodiments, one polynucleotide encodes more than one neoantigenic peptide.

[0171]

[0408] In some embodiments, the polynucleotide is a messenger RNA. In some embodiments, each messenger RNA comprises a coding sequence for two or more neo-antigenic peptides in tandem.

[0172]

[0409] In some embodiments, each messenger RNA contains coding sequences for two, three, four, five, six, seven, eight, nine, or ten or more neoantigen peptides in tandem. Typically, the mRNA contains a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In some embodiments, the mRNA is a self-amplifying mRNA. In the context of the present invention, mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription techniques are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0173]

[0410] The stability and translation efficiency of RNA may be modified. For example, RNA can be stabilized and its translation increased by one or more modifications that have a stabilizing effect on RNA and / or increase translation efficiency. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. To increase the expression of RNA used in the present invention, RNA may be modified within the coding region, i.e., within the sequence encoding the expressed peptide or protein, without changing the sequence of the expressed peptide or protein, to increase mRNA stability and perform codon optimization to increase GC content for enhanced translation in cells.

[0174]

[0411] In some embodiments, the mRNA may contain multiple neoantigen epitopes. In some embodiments, a long polyribonucleotide sequence capable of encoding a neo-ORF can be used, for example, a mutated GATA3 sequence encoding a neo-ORF. In some embodiments, the mRNA of most of the gene containing the sequence encoding the neoantigen peptide, or even the mRNA of the entire coding region of the gene, is delivered to immune cells for endogenous processing and antigen presentation.

[0175]

[0412] In some embodiments, the coding sequence for each neo-antigenic peptide is between 24 and 120 nucleotides in length.

[0413] In some embodiments, the mRNA is 50 to 10,000 nucleotides in length. In some embodiments, the mRNA is 100 to 10,000 nucleotides in length. In some embodiments, the mRNA is 200 to 10,000 nucleotides in length. In some embodiments, the mRNA is 50 to 5,000 nucleotides in length. In some embodiments, the mRNA is 100 to 5,000 nucleotides in length. In some embodiments, the mRNA is 100 to 1,000 nucleotides in length. In some embodiments, the mRNA is 300 to 800 nucleotides in length. In some embodiments, the mRNA is 400 to 700 nucleotides in length. In some embodiments, the mRNA is 450 to 600 nucleotides in length. In some embodiments, the mRNA is at least 200 nucleotides in length. 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.

[0176]

[0414] In some embodiments, the mRNA encoding one or more neoantigenic peptides is modified, where the modification relates to the 5'-UTR. In some embodiments, the modification relates to providing the RNA with a 5'-cap or 5'-cap analog in the 5'-UTR. The term "5'-cap" refers to the cap structure found at the 5' end of an mRNA molecule, generally consisting of a guanosine nucleotide attached to the mRNA via an unusual 5'-5' triphosphate linkage. In some embodiments, this guanosine is methylated at position 7. The term "conventional 5'-cap" refers to the naturally occurring 5'-cap of RNA, i.e., the 7-methylguanosine cap (mG). In the context of the present invention, the term "5'-cap" includes 5'-cap analogs that resemble the RNA cap structure and are modified to have the ability to stabilize RNA and / or enhance RNA translation when bound to it in vivo and / or in cells. In some embodiments, the mRNA is co-transcriptionally capped.

[0177]

[0415] In some embodiments, the mRNA encoding one or more neo-antigenic 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 polyA tail is longer than 170 nucleotides. In some embodiments, the polyA tail is longer than 180 nucleotides. In some embodiments, the polyA tail is longer than 190 nucleotides. In some embodiments, the polyA tail is longer than 200 nucleotides. In some embodiments, the polyA tail is longer than 210 nucleotides. In some embodiments, the polyA tail is longer than 220 nucleotides. In some embodiments, the polyA tail is longer than 230 nucleotides. In some embodiments, the polyA tail is longer than 100 nucleotides. In some embodiments, the polyA tail is longer than 240 nucleotides. In some embodiments, the polyA tail is longer than 100 nucleotides. In some embodiments, the polyA tail is about 250 nucleotides.

[0178]

[0416] 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 has 129 bases.

[0179]

[0417] In some embodiments, the coding sequences for two consecutive neoantigenic peptides are separated by a spacer or linker.

[0418] 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 is a sequence encoding lysine, such as AAA or AAG. Another exemplary spacer sequence is CAACTGGGATTG.

[0180]

[0419] In some embodiments, the mRNA comprises one or more additional structures to enhance antigen epitope processing and presentation by APCs.

[0420] In some embodiments, the linker or spacer region may contain a cleavage site. The cleavage site allows a protein product containing a continuous epitope sequence to be cleaved into another epitope sequence for presentation. A preferred cleavage site is located adjacent to a specific epitope to avoid inadvertent cleavage of the epitope within the sequence. In some embodiments, the design of the epitope and cleavage region on the mRNA encoding a series of epitopes is non-random.

[0181]

[0421] In certain embodiments, mRNA encoding the neo-antigenic peptides of the present invention is administered to a subject in need thereof. In some embodiments, the mRNA to be administered contains at least one modified nucleoside phosphate.

[0182]

[0422] In some embodiments, T cells are activated with neoantigenic peptides by artificial antigen-presenting cells. In some embodiments, an artificial scaffold is used to activate T cells with neoantigenic peptides, the artificial scaffold being loaded with neoantigenic peptides coupled to MHC antigens to which the neoantigenic peptides can bind with high affinity.

[0183]

[0423] In some embodiments, the additional structure comprises an encoded specific domain from a protein selected from the group of MITD, SP1, and the tenth fibronectin domain: 10FnIII.

[0184]

[0424] In some embodiments, peripheral blood-derived or leukapheresis-derived cells are contacted one or more times with multiple cancer neoantigen peptides or one or more polynucleotides encoding multiple cancer neoantigen peptides to prepare antigen-loaded APCs.

[0185]

[0425] In some embodiments, the method comprises incubating one or more of the APCs or APC preparations with a first culture medium comprising at least one cytokine or growth factor for a first period of time.

[0186]

[0426] In some embodiments, the method includes incubating one or more of the APC preparations with at least one peptide for a second period of time.

[0427] In some embodiments, the enriched cells further comprise CD1c+ cells.

[0187]

[0428] In some embodiments, the cell population is enriched for CD11c+ and CD141+ cells.

[0429] In some embodiments, the cell population comprising antigen-loaded APCs comprises more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% or more CD11c+ cells.

[0188]

[0430] In some embodiments, the cell population comprising antigen-loaded APCs comprises less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 20%, less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4% or less cells expressing CD11b+.

[0189]

[0431] In some embodiments, the cell population comprising antigen-loaded APCs comprises more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% of cells expressing the neo-antigen peptide are CD11c+.

[0190]

[0432] In some embodiments, the cell population comprising antigen-loaded APCs comprises more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% of cells expressing the neo-antigen peptide are CD11c+CD1c+ or CD141+ cells.

[0191]

[0433] In some embodiments, the neo-antigen-loaded APCs comprise mature APCs.

[0434] In some embodiments, the methods include obtaining a biological sample from the subject that includes at least one APC and at least one PBMC or at least one T cell.

[0192]

[0435] In some embodiments, the method comprises depleting cells expressing CD14 and / or CD25 and / or CD19 from the biological sample, thereby obtaining a sample depleted of CD14 and / or CD25 and / or CD19 cells.

[0193]

[0436] In some embodiments, the method comprises incubating a sample depleted of CD14 and / or CD25 and / or CD19 cells with FLT3L for a first period of time.

[0194]

[0437] In some embodiments, the method includes incubating at least one peptide with a sample depleted of CD14 and / or CD25 and / or CD19 cells for a second period of time, thereby obtaining a sample loaded with a first mature APC peptide.

[0195]

[0438] Using neoantigen-loaded APCs to prepare neoantigen-activated T cells

[0439] In some embodiments, neo-antigen-loaded APCs (APCs) prepared by the above-described 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 neo-antigen. In some embodiments, generating at least one antigen-specific T cell includes generating a plurality of antigen-specific T cells.

[0196]

[0440] In some embodiments, the T cells are obtained from a biological sample from the subject.

[0441] In some embodiments, the T cells are obtained from a biological sample from the same subject from which the APCs are derived. In some embodiments, the T cells are obtained from a biological sample from a different subject from which the APCs are derived.

[0197]

[0442] 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.

[0198]

[0443] In some embodiments, the APC comprises a dendritic cell (DC).

[0444] In some embodiments, the APCs are derived from CD14+ monocytes, or are CD14-enriched APCs, or are CD141-enriched APCs.

[0199]

[0445] In some embodiments, CD14+ monocytes are enriched from a biological sample from a subject that contains peripheral blood mononuclear cells (PBMCs).

[0446] 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.

[0200]

[0447] In some embodiments, PBMCs are loaded with antigens, which may be peptides, polypeptides, or polynucleotides, such as mRNAs encoding peptides and polypeptides. PBMCs (monocytes, DC phagocytes) can ingest antigens through phagocytosis and process them and present them on their surface for T cell activation. The peptides or polypeptides loaded onto PBMCs may be supplemented with adjuvants to increase immunogenicity. In some embodiments, PBMCs are loaded with nucleic acid antigens. The nucleic acid antigens may be in the form of mRNA containing sequences encoding one or more antigens. In some embodiments, loading with mRNA antigens does not require the supplementation of adjuvants, for example, because RNA can act as a self-adjuvant. In some embodiments, APCs are loaded with 20 to 40 antigens. In some embodiments, APCs express 20 to 40 antigens. In some embodiments, the antigens are neoantigens. In some embodiments, at least the majority of the antigens are neoantigens. In some embodiments, APCs (or PBMCs) are loaded with or express nucleic acid sequences encoding short peptides (each 8-12 amino acids in length) for CD8+ T cell stimulation. In some embodiments, APCs (or PBMCs) are loaded with or express nucleic acid sequences encoding short peptides (each 16-25 amino acids in length) for CD4+ T cell stimulation. In some embodiments, APCs (e.g., PBMCs) may be loaded with both short and long antigenic peptide sequences; or APCs express both short and long antigenic peptide sequences. In some embodiments, APCs are loaded with up to or about 40 short antigenic peptide sequences and up to or about 20 long antigenic peptide sequences. In some embodiments, APCs are transduced or transfected with nucleic acids comprising up to or about 40 short antigenic peptide sequences and up to or about 20 long antigenic peptide sequences.

[0201]

[0448] In some embodiments, PBMCs are directly isolated or thawed from a frozen sample and subjected to incubation with one or more antigens, e.g., neoantigens, or compositions comprising neoantigens, or one or more nucleic acids or polynucleotides encoding one or more antigens. In some embodiments, the PBMC sample is not further cultured for differentiation, or one or more cellular components within the PBMCs are subjected to further maturation (e.g., maturation of antigen-presenting cells or differentiation of monocytes into dendritic cells) before exposing the PBMCs to one or more antigens or nucleic acids encoding one or more antigens. In some embodiments, one or more cell types are depleted or removed from a freshly isolated or thawed PBMC cell population before exposing or incubating the cells with one or more antigens or nucleic acids encoding one or more antigens. In some embodiments, CD14+ cells are depleted from PBMCs. In some embodiments, CD25+ cells are depleted from PBMCs. In some embodiments, CD11b+ cells are depleted from PBMCs. In some embodiments, CD14+ and CD25+ cells are depleted from PBMCs before incubation with one or more antigens or one or more nucleic acids encoding one or more antigens. In some embodiments, CD11b+, and / or CD14+ and / or CD25+ cells are depleted from PBMCs. In some embodiments, the method provided herein comprises preparing tumor antigen-specific T cells by depleting CD14+ cells and / or CD25+ cells from a PBMC sample from a human subject containing immature dendritic cells (DCs) at a percentage approximately equal to the percentage of immature DCs in the peripheral blood of the human subject. In some embodiments, the method provided herein comprises preparing tumor antigen-specific T cells by depleting CD14+ cells and / or CD25+ cells from a PBMC sample from a human subject containing mature DCs at a percentage approximately equal to the percentage of mature DCs in the peripheral blood of the human subject.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 contains immature DCs and mature DCs in a ratio approximately the same as the ratio of immature DCs to mature DCs in the peripheral blood of the human subject. 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 been subjected to a step of maturing immature DCs into mature DCs.

[0202]

[0449] In some embodiments, the CD14+ monocytes are stimulated with one or more cytokines or growth factors.

[0450] 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.

[0203]

[0451] In some embodiments, the CD14+ monocytes are from a second biological sample that includes PBMCs.

[0452] In some embodiments, the second biological sample is from the same subject.

[0204]

[0453] In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0454] In some embodiments, at least one antigen-specific T cell is stimulated in 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.

[0205]

[0455] In some embodiments, the IDO inhibitor is epacadostat, navoximod, 1-methyltryptophan, or a combination thereof.

[0456] In some embodiments, the subject is administered FLT3L prior to obtaining the biological sample for preparing APCs and / or T cells.

[0206]

[0457] In some embodiments, the T cells are obtained from a biological sample from a subject, as described in the previous section of this disclosure.

[0458] In some embodiments, the biological sample is a freshly obtained from a subject or a frozen sample.

[0207]

[0459] In some embodiments, the incubating step is in the presence of at least one cytokine or growth factor including GM-CSF, IL-4, FLT3L, TNF-α, IL-1β, PGE1, IL-6, IL-7, IL-15, IFN-γ, IFN-α, IL-15, R848, LPS, ss-rna40, poly I:C, or any combination thereof.

[0208]

[0460] In some embodiments, the method comprises stimulating T cells with IL-7, IL-15, or a combination thereof. In some embodiments, the method comprises stimulating T cells with IL-7, IL-15, or a combination thereof in the presence of an IDO inhibitor, a PD-1 antibody, or IL-12. In some embodiments, the stimulated T cells are expanded in the presence of one or more tumor antigen epitope sequences, or APCs loaded with one or more tumor antigen epitope sequences, or APCs loaded with (e.g., expressing) a nucleic acid sequence (e.g., mRNA sequence) encoding one or more tumor antigen epitope sequences, and 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, FLT3L, under suitable ex vivo T cell expansion conditions. In some embodiments, the method further comprises administering antigen-specific T cells to the subject.

[0209]

[0461] 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.

[0210]

[0462] In some embodiments, the incubating step comprises incubating a first APC preparation of the APC preparation with the T cells for more than 7 days, hi some embodiments, the incubated T cells are stimulated T cells expanded in vitro in the presence of the APC preparation, cytokines and growth factors for more than 7 days.

[0211]

[0463] In some embodiments, the incubating step comprises incubating a first APC preparation of the APC preparation with the T cells for more than 7 days, more than 8 days, more than 9 days, more than 10 days, more than 11 days, more than 12 days, more than 13 days, more than 14 days, more than 15 days, more than 16 days, more than 17 days, more than 18 days, more than 19 days, or more than 20 days.

[0212]

[0464] In some embodiments, the first of the one or more periods is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 days.

[0465] In some embodiments, the total duration of the separate periods is less than 28 days. In some embodiments, the total duration of the separate periods is 20 to 27 days. In some embodiments, the total duration of the separate periods is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 days.

[0213]

[0466] In some embodiments, the method comprises incubating a first APC preparation of the APC preparation with T cells for more than 7 days. In some embodiments, the method comprises incubating a first APC preparation of the APC preparation with T cells for more than 7 days, more than 8 days, more than 9 days, more than 10 days, more than 11 days, more than 12 days, more than 13 days, more than 14 days, more than 15 days, more than 16 days, more than 17 days, more than 18 days, more than 19 days, or more than 20 days. In some embodiments, the method comprises incubating a first APC preparation of the APC preparation with T cells for 7 to 20, 8 to 20, 9 to 20, 10 to 20, 11 to 20, or 12 to 20 days. In some embodiments, the method comprises incubating a first APC preparation of the APC preparation with T cells for about 10 to 15 days.

[0214]

[0467] In some embodiments, the method includes incubating a second APC preparation of the APC preparation with T cells for 5 to 9 days. In some embodiments, the method includes incubating a second APC preparation of the APC preparation with T cells for 5, 6, 7, 8, or 9 days. In some embodiments, the method further includes removing one or more cytokines or growth factors of the second culture medium after the third period and before the start of the fourth period.

[0215]

[0468] In some embodiments, the method comprises incubating a third one of the APC preparations with the T cells for 5 to 9 days, hi some embodiments, the method comprises incubating a third one of the APC preparations with the T cells for 5, 6, 7, 8, or 9 days.

[0216]

[0469] In some embodiments, the method comprises incubating a first APC preparation of the APC preparations with T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days, incubating a second APC preparation of the APC preparations with T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days, and incubating a third APC preparation of the APC preparations 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.

[0217]

[0470] In some embodiments, the method is carried out ex vivo. In some embodiments, the T cells are cultured in a medium containing cytokines. In some embodiments, an example of a cytokine includes IL-7. In some embodiments, an example of a cytokine includes IL-15. In some embodiments, an example of a cytokine includes IL-7 and IL-15. In some embodiments, the T cells are cultured in a medium containing IL-7 and / or IL-15. In some embodiments, the cytokines in the T cell culture or medium have a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, IL-7 in the T cell culture or medium has a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, IL-15 in the T cell culture or medium has a final concentration of at least 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, or 20 ng / mL. In some embodiments, the T cells are cultured in medium further containing FLT3L.In some embodiments, FLT3L in the T cell culture or medium has a final concentration of at least 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, or 200 ng / mL. In some embodiments, T cells are incubated, induced, or stimulated in medium containing FLT3L for a first period of time. In some embodiments, T cells are incubated, induced, or stimulated in medium containing additionally added FLT3L for a second period of time. In some embodiments, the T cells are incubated, induced, or stimulated in medium containing additional added FLT3L for a third period of time, hi some embodiments, the T cells are incubated, induced, or stimulated in medium containing additional added FLT3L for a fourth, fifth, or sixth period of time, with freshly added FLT3L in each period.

[0218]

[0471] In some embodiments, T cells are cultured in the presence of a neoantigen, e.g., a neoantigen presented by an APC, in which case the medium contains high potassium [K] + In some embodiments, the T cells are cultured in a medium containing a high [K] + In some embodiments, the medium is cultured with APCs or T cells in the presence of [K] for at least the incubation period. + The content is varied over a period of time during incubation with at least APCs or T cells. In some embodiments, the content in the medium is maintained constant over the period of ex vivo culture of T cells. In some embodiments, the [K] in the T cell culture medium is + In some embodiments, the [K] content in the T cell culture medium is ≧5 mM. +In some embodiments, the [K] content in the T cell culture medium is ≧6 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧7 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧8 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧9 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧10 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧11 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧12 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧13 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧14 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧15 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧16 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧17 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧18 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧19 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧20 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧22 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧25 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧30 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧35 mM. + In some embodiments, the [K] content in the T cell culture medium is ≧40 mM. +The content is about 40 mM.

[0219]

[0472] In some embodiments, the [K] in the T cell culture medium + The content is about 40 mM for at least the period during incubation of the T cells with the neoantigen. In some embodiments, the neoantigen can be presented by APCs loaded with the neoantigen. In some embodiments, [K] + In some embodiments, the T cells in the presence of high [K] are tested for T effector function, CD8+ cytotoxicity, cytokine production, and for memory phenotype. + In some embodiments, T cells expanded in the presence of a high [K] express an effector T cell phenotype. + In some embodiments, T cells expanded in the presence of a high [K] express memory cell markers. + T cells expanded in the presence of do not express T cell exhaustion markers.

[0220]

[0473] In some embodiments, the stimulated T cells are a population of immune cells comprising activated T cells stimulated with APCs comprising the neoantigen peptide-MHC complex. In some embodiments, the method may include the steps of incubating a population of immune cells from a biological sample with APCs comprising the peptide-MHC complex, thereby obtaining a stimulated immune cell sample; determining the expression of one or more cell markers of at least one immune cell of the stimulated immune cell sample; and determining binding of at least one immune cell of the stimulated immune cell sample to the peptide-MHC complex, wherein the steps of determining expression of specific cell surface markers or other determinant markers, such as intracellular factors, or released agents, such as cytokines, and determining binding to the neoantigen-MHC complex are performed simultaneously. In some embodiments, the one or more cell markers comprise TNF-α, IFN-γ, LAMP-1, 4-1BB, IL-2, IL-17A, Granzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3, or any combination thereof. In some embodiments, the one or more cell markers comprise a cytokine. In some embodiments, the one or more cell markers comprise a degranulation marker. In some embodiments, the one or more cell markers comprise a cell surface marker. In some embodiments, the one or more cell markers comprise a protein. In some embodiments, determining binding of at least one immune cell of the stimulated immune cell sample to a peptide-MHC complex comprises determining binding of at least one immune cell of the stimulated immune cell sample to an MHC tetramer comprising the peptide and MHC of the peptide-MHC complex. In some embodiments, the MHC is class I MHC or class II MHC. In some embodiments, the peptide-MHC complex comprises one or more labels.

[0221]

[0474] In some embodiments, T cell activation is verified by detecting cytokine release by activated T cells. In some embodiments, the cytokine is one or more of TNF-α, IFN-γ, or IL-2. In some embodiments, T cell activation is verified by its specific antigen binding and cytokine release. In some embodiments, T cell activation is verified by its ability to kill tumor cells in vitro. A sample of activated T cells can be used to verify the activation status of T cells. In some embodiments, a sample from T cells is drawn from a T cell culture to determine the cell composition and activation status by flow cytometry.

[0222]

[0475] 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 cells in the composition is about 12%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 15%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 20%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 25%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 30%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 40%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 50%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 60%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 70%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 80%. In some embodiments, the percentage of at least one antigen-specific T cells in the composition is about 90%.

[0223]

[0476] In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 5%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 7%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 10%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 12%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 15%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 20%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 25%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 30%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 40%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 50%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 60%. In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the composition is about 70% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0224]

[0477] 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 the total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells.

[0225]

[0478] In some embodiments, the percentage of at least one antigen-specific T cells in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0226]

[0479] In some embodiments, the percentage of at least one antigen-specific CD8+ T cells in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, or 0.5% of the total CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0227]

[0480] In some embodiments, the percentage of at least one antigen-specific CD4+ 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 CD4+ T cells, total CD8+ T cells, total T cells, or total immune cells.

[0228]

[0481] 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.

[0482] In some embodiments, the number of at least one antigen-specific CD8+ T cell in the composition is at least about 1 x 10 6 , 2 × 10 6 , 5×10 6 , 1×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , or 5 × 10 8 In some embodiments, the number of at least one antigen-specific CD4+ T cell in the composition is at least about 1 x 10 6 , 2 × 10 6 , 5×10 6 , 1×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , or 5 × 10 8 antigen-specific CD4+ T cells.

[0229]

[0483] Pharmaceutical Composition

[0484] Provided herein is a composition (e.g., a pharmaceutical composition) comprising a population of immune cells. The composition may comprise at least one antigen-specific T cell comprising a T cell receptor (TCR). The composition may comprise at least one antigen-specific T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence.

[0230]

[0485] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, such as excipients or auxiliaries, which facilitate the processing of active agents into pharmaceutically usable preparations.Suitable formulations may depend on the route of administration selected.All well-known techniques, carriers and excipients can be used as appropriate and as understood in the art.

[0231]

[0486] In some cases, the pharmaceutical composition is formulated as a cell-based therapy, e.g., a T cell therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapy, a nucleic acid-based therapy, an antibody-based therapy, and / or a cell-based therapy. In some embodiments, the pharmaceutical composition comprises a peptide-based therapy or a nucleic acid-based therapy, wherein the nucleic acid encodes a polypeptide. In some embodiments, the pharmaceutical composition comprises a peptide-based therapeutic or a nucleic acid-based therapeutic, wherein the nucleic acid encodes a polypeptide; the peptide-based therapeutic or nucleic acid-based therapeutic is contained in a cell, in which case 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 neoantigen peptides.

[0232]

[0487] In one aspect, provided herein is a pharmaceutical composition comprising: (a) a population of immune cells comprising T cells from a biological sample, wherein the T cells comprise at least one antigen-specific T cell, which is a T cell stimulated by an APC and comprises a T cell receptor (TCR) specific for at least one antigenic peptide sequence, wherein the APC is an APC stimulated with FLT3L; and (b) a pharmaceutically acceptable excipient.

[0233]

[0488] In one aspect, provided herein is a pharmaceutical composition comprising: (a) a population of immune cells from a biological sample, the population comprising at least one antigen-specific T cell, the T cell comprising a T cell receptor (TCR) specific for at least one antigenic peptide sequence; and (b) a pharmaceutically acceptable excipient, wherein the amount of immune cells expressing CD14 and / or CD25 in the population is proportionally different from the 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 T cells stimulated by at least one APC. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the population is proportionally less than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the amount of immune cells expressing CD14 and / or CD25 in the population is proportionally greater than the amount of immune cells expressing CD14 and / or CD25 in the biological sample. In some embodiments, the at least one antigen-specific T cell comprises at least one CD4+ T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD8+ T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD4-enriched T cell. In some embodiments, the at least one antigen-specific T cell comprises at least one CD8-enriched T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory CD4+ T cell. In some embodiments, the at least one antigen-specific T cell comprises a memory CD8+ T cell. In some embodiments, the percentage of 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 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 the total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells.

[0234]

[0489] In addition to the active ingredient, the pharmaceutical composition may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other materials will depend on the route of administration.

[0235]

[0490] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and examples include buffers such as phosphate buffers, citrate buffers, and other organic acid buffers; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, 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 such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0236]

[0491] An acceptable carrier is one that is physiologically acceptable to the patient to whom it is administered and that retains the therapeutic properties of the compound with which it is administered. Acceptable carriers and their formulations are generally described, for example, in Remington's Pharmaceutical Sciences (18th ed., A. Gennaro, Mack Publishing Co., Easton, PA 1990). One example of a carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that carries or transports the compound of interest from the administration site in one organ or body part to another organ or body part, or that participates in an in vitro assay system. An acceptable carrier is compatible with the other ingredients of the formulation and is not harmful to the subject to which it is administered. An acceptable carrier should also not alter the specific activity of the neoantigen.

[0237]

[0492] In one aspect, provided herein are pharmaceutically acceptable or physiologically acceptable compositions that include solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonicity, and agents that promote or delay absorption, suitable for pharmaceutical administration. Thus, a pharmaceutical composition or pharmaceutical formulation refers to a composition suitable for pharmaceutical use in a subject. The composition can be formulated to be compatible with a specific administration route (i.e., systemic or local). Thus, the composition includes carriers, diluents, or excipients suitable for administration by various routes.

[0238]

[0493] In some embodiments, the composition may further contain an acceptable additive to improve the stability of immune cells in the composition. Acceptable additives may not alter the specific activity of immune cells. Examples of acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. Acceptable additives may be combined with acceptable carriers and / or excipients, such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 to increase peptide stability and reduce solution gelation. Surfactants may be added to the composition in amounts of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

[0239]

[0494] Pharmaceutical compositions may be administered, for example, by injection. Compositions for injection include aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, may also be included in the composition. The resulting solution may be packaged for immediate use or lyophilized; the lyophilized preparation may be combined with a sterile solution prior to administration. For intravenous injection or injection at the affected site, the active ingredient is expected to be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability.If those skilled in the art have relevant capabilities, suitable solutions can be prepared using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection.If necessary, preservatives, stabilizers, buffers, antioxidants and / or other additives can be included.Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount into a suitable solvent, optionally with one or a combination of the above-listed ingredients, and then sterilizing by filtration.Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from the above-listed ones.In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0240]

[0495] The composition may be conventionally administered intravenously, for example, by injection of a unit dose.For injection, the active ingredient may be in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has suitable pH, isotonicity and stability.For example, suitable solutions can be prepared using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection.Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed.In addition, the composition may be administered via aerosolization.

[0241]

[0496] When a composition is contemplated for use in any of the medicaments or methods provided herein, it is expected that the composition may be substantially pyrogen-free so that it does not provoke an inflammatory or dangerous allergic reaction when administered to a human patient. Testing compositions for pyrogens and preparing substantially pyrogen-free compositions is well understood by those of skill in the art and can be accomplished using commercially available kits.

[0242]

[0497] Acceptable carriers may contain compounds that act as stabilizers, compounds that increase or delay absorption, or compounds that increase or delay clearance. Such compounds include, for example, carbohydrates, such as glucose, sucrose, or dextran; low-molecular-weight proteins; compositions that reduce peptide clearance or hydrolysis; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can also be used to stabilize, increase, or decrease the absorption of pharmaceutical compositions, such as liposome carriers. To protect against digestion, the compound can be complexed with the composition to make it resistant to acid and enzymatic hydrolysis, or the compound can be complexed in a suitable resistant carrier, such as liposomes. Means for protecting compounds from digestion are known in the art (eg, Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135; and US Pat. No. 5,391,377).

[0243]

[0498] The composition may be administered in a therapeutically effective amount in a manner compatible with the dosage formulation.The amount to be administered depends on the subject to be treated, the ability of the subject's immune system to utilize the active ingredient, and the desired degree of binding ability.The exact amount of the active ingredient required to be administered depends on the judgment of the physician and is unique to each individual.Suitable regimens for initial administration and booster vaccination also vary, but typically involve an initial administration, followed by repeated doses at one or more time intervals by subsequent injections or other administrations.Alternatively, continuous intravenous infusion sufficient to maintain blood concentration is expected.

[0244]

[0499] In some embodiments, the present invention is directed to immunogenic compositions, e.g., pharmaceutical compositions capable of generating a neoantigen-specific response (e.g., a humoral or cell-mediated immune response). In some embodiments, the immunogenic compositions comprise a tumor-specific antigen or a neoantigen therapeutic agent described herein (e.g., a peptide, polynucleotide, TCR, CAR, cells containing a TCR or CAR, dendritic cells containing a polypeptide, dendritic cells containing a polynucleotide, an antibody, etc.) corresponding to the neoantigen.

[0245]

[0500] In some embodiments, the pharmaceutical compositions described herein are capable of generating a specific cytotoxic T cell response, a specific helper T cell response, or a B cell response.

[0246]

[0501] In some embodiments, antigenic polypeptides or polynucleotides can be provided as antigen-presenting cells (e.g., dendritic cells) containing such polypeptides or polynucleotides. In other embodiments, such antigen-presenting cells are used to stimulate T cells for use in a patient. In some embodiments, the antigen-presenting cells are dendritic cells. In related embodiments, the dendritic cells are autologous dendritic cells pulsed with neoantigenic peptides or nucleic acids. The neoantigenic peptides can be any suitable peptide that generates an appropriate T cell response. In some embodiments, the T cells are CTLs. In some embodiments, the T cells are HTLs. Thus, one embodiment of the present disclosure is an immunogenic composition containing at least one antigen-presenting cell (e.g., dendritic cell) pulsed or loaded with one or more neoantigenic 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 neoantigenic peptides or polynucleotides ex vivo. In related embodiments, such APCs or PBMCs are injected back into the patient. The polynucleotide may be any suitable polynucleotide capable of transducing dendritic cells, resulting in presentation of neo-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.

[0247]

[0502] In some embodiments, CTLs are injected into a patient. In some embodiments, HTLs are injected into a patient. In some embodiments, both CTLs and HTLs are injected into a patient. Administration of any of the therapeutic agents may be performed simultaneously or sequentially in any order.

[0248]

[0503] In some embodiments, pharmaceutical compositions (e.g., immunogenic compositions) described herein for therapeutic treatment can be formulated for parenteral, topical, nasal, oral, or local administration. In some embodiments, pharmaceutical compositions described herein are administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. In some embodiments, the compositions may be administered intratumorally. The compositions may be administered at a surgical resection site to induce a local immune response against the tumor. In some embodiments, compositions for parenteral administration are described herein, comprising a solution of a neo-antigenic peptide and an immunogenic composition, dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques or sterile filtered. The resulting aqueous solutions can be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain, as necessary, pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0249]

[0504] The ability of an adjuvant to increase the immune response to an antigen is typically demonstrated by a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity can be demonstrated by a significant increase in the titer of antibodies raised against the antigen, and an increase in T cell activity can be demonstrated by increased cell proliferation, cellular cytotoxicity, or cytokine secretion. Adjuvants can also modify the immune response, for example, by shifting a primarily humoral or T helper 2 response to a primarily cellular or T helper 1 response.

[0250]

[0505] Suitable adjuvants are known in the art (see WO2015 / 095811), and examples 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 ISA206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel® Vector System, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox.Quil or Superfos. A number of immunological adjuvants specific for dendritic cells (e.g., MF59) and their preparations 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.Numerous cytokines are directly involved in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), promoting dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1β, IL-4, IL-6, and CD40L) (U.S. Pat. No. 5,849,589, 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).

[0251]

[0506] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in therapeutic settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-initiated TLR9 activation enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live or immortalized viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cell immunogenic pharmaceutical compositions, and polysaccharide conjugates, in both prophylactic and therapeutic immunogenic pharmaceutical compositions. Importantly, TLR9 activation enhances dendritic cell maturation and differentiation, resulting in increased CD4 expression. +Even in the absence of T cell help, this leads to enhanced TH1 cell activation and the generation of strong cytotoxic T lymphocytes (CTLs). The TH1 bias induced by TLR9 stimulation is maintained even in the presence of adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or administered with other adjuvants, or in the form of compositions such as microparticles, nanoparticles, lipid emulsions, or similar formulations; these forms are particularly useful for inducing strong responses when the antigen is relatively weak. This can also enhance immune responses, and in some experiments, allows for a reduced antigen dose with an antibody response comparable to that of a full dose of an immunogenic pharmaceutical composition without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, June 5, 2006, pp. 471-484). US Patent No. 6,406,705 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens to induce antigen-specific immune responses.A commercially available CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) by Mologen (Berlin, DE), which is a component of the pharmaceutical composition described herein.Other TLR binding molecules, such as RNA binding TLR7, TLR8 and / or TLR9, can also be used.

[0252]

[0507] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly(I and / or polyC) (e.g., polyI:CI2U), non-CpG bacterial DNA or RNA, ssRNA40 for TLR8, as well as immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as an adjuvant. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of skill in the art without undue experimentation. Additional adjuvants include colony-stimulating factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0253]

[0508] In some embodiments, immunogenic compositions according to the present disclosure may include more than one different adjuvant. Additionally, the present invention encompasses pharmaceutical compositions comprising any adjuvant agent, including any of the above or a combination thereof. In some embodiments, the immunogenic composition includes a neoantigen therapeutic agent (e.g., a peptide, a polynucleotide, a TCR, a CAR, cells containing a TCR or a CAR, dendritic cells containing a polypeptide, dendritic cells containing a polynucleotide, an antibody, etc.), and the adjuvant can be administered separately in any suitable sequence.

[0254]

[0509] Lipidation can be classified into several different types, such as N-myristoylation, palmitoylation, GPI-anchor attachment, prenylation, and several additional types of modifications. N-myristoylation is the covalent attachment of myristate, a C14 saturated acid, to glycine residues. Palmitoylation is the thioester linkage of a long-chain fatty acid (C16) to a cysteine ​​residue. GPI-anchor attachment is the linkage of glycosyl-phosphatidylinositol (GPI) via an amide bond. Prenylation is the thioether linkage of an isoprenoid lipid (e.g., farnesyl (C-15), geranylgeranyl (C-20)) to a cysteine ​​residue. Additional types of modifications include the attachment of S-diacylglycerol via the sulfur atom of cysteine, O-octanoyl conjugation via serine or threonine residues, S-archeol conjugation to cysteine ​​residues, and cholesterol attachment.

[0255]

[0510] Fatty acids for generating lipidated peptides can include C2-C30 saturated, monounsaturated, or polyunsaturated fatty acid acyl groups. Exemplary fatty acids can include palmitoyl, myristoyl, stearoyl, and decanoyl groups. In some cases, lipid moieties with adjuvant properties can be attached to a polypeptide of interest to elicit or enhance immunogenicity in the absence of an exogenous adjuvant. Lipidated peptides or lipopeptides can be referred to as self-adjuvanting lipopeptides. Any of the fatty acids described above and elsewhere herein can elicit or enhance the immunogenicity of a polypeptide of interest. Fatty acids capable of eliciting or enhancing immunogenicity can include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, and decanoyl groups.

[0256]

[0511] A polypeptide, such as a naked peptide or a lipidated peptide, can be incorporated into a liposome. Sometimes, a lipidated peptide can be incorporated into a liposome. For example, the lipid portion of a lipidated peptide can spontaneously incorporate 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 made by the reverse-phase evaporation method (REVs), multilamellar vesicles made by the reverse-phase evaporation method (MLV-REVs), stable plurilamellar vesicles (SPLVs), and the like. These include vesicles (MV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods (VET), vesicles prepared by French press (FPV), vesicles prepared by fusion (FUV), dehydration-rehydration vesicles (DRV), and bubblesomes (BSV).

[0257]

[0512] Depending on the preparation method, liposomes may be unilamellar or multilamellar, and their diameters can vary from about 0.02 μm to more than about 10 μm. Liposomes can adsorb many types of cells and then release the incorporated drug (e.g., peptides described herein). In some cases, when liposomes fuse with target cells, the contents of the liposomes are released into the target cells. Liposomes may be taken up by endocytosis by phagocytic cells. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated drug.

[0258]

[0513] The liposomes provided herein may also include a carrier lipid. In some embodiments, the carrier lipid is a phospholipid. Carrier lipids capable of forming liposomes include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Other suitable phospholipids further include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic acid (DP PA); dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidylserine (DPPS), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidylserine (DSPS), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), etc., or combinations thereof.In some embodiments, liposome further comprises sterol (e.g., cholesterol) that modulates liposome formation.Carrier lipid can be any known non-phosphate polar lipid.

[0259]

[0514] The pharmaceutical compositions may also be encapsulated in liposomes using well known technology.Biodegradable microspheres may also be employed as carriers for the pharmaceutical compositions of this invention.

[0260]

[0515] The pharmaceutical composition may be administered in the form of liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to patients are well known to those skilled in the art. Basically, the material is dissolved in an aqueous solution, and appropriate phospholipids and lipids are added, if necessary, together with a surfactant, and the material is dialyzed or sonicated as necessary.

[0261]

[0516] Microspheres formed from polymers or proteins are well known to those skilled in the art and can be designed to pass through the gastrointestinal tract directly into the bloodstream. Alternatively, compounds may be incorporated and the microspheres, or composites of microspheres, may be implanted for delayed release over periods ranging from days to months.

[0262]

[0517] Cell-based immunogenic pharmaceutical compositions can also be administered to subjects.For example, antigen-presenting cell (APC)-based immunogenic pharmaceutical compositions can be formulated using any of well-known techniques, carriers, and excipients, as understood in the art, if appropriate.APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells.Sometimes, APC-based immunogenic pharmaceutical compositions can be dendritic cell-based immunogenic pharmaceutical compositions.

[0263]

[0518] 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 via ex vivo or in vivo methods. Ex vivo methods may include the use of autologous DCs pulsed ex vivo with a polypeptide described herein to activate or load the DCs before administration to a patient. In vivo methods may include targeting specific DC receptors using an antibody coupled to a polypeptide described herein. DC-based immunogenic pharmaceutical compositions may further comprise a DC activator, such as a TLR3, TLR-7-8, or CD40 agonist. DC-based immunogenic pharmaceutical compositions may further comprise an adjuvant and a pharmaceutically acceptable carrier.

[0264]

[0519] Adjuvants can be used to enhance the immune response (humoral and / or cellular) elicited in patients receiving the immunogenic pharmaceutical composition. Sometimes, adjuvants can elicit a Th1-type response. On the other hand, adjuvants can elicit a Th2-type response. Th1-type responses can be characterized by the production of cytokines such as IFN-γ, whereas Th2-type responses can be characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

[0265]

[0520] In some embodiments, 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 preparations described herein.

[0266]

[0521] Adjuvants can also include stimulatory molecules, such as cytokines, including, but not limited to, CCL20, α-interferon (IFNα), β-interferon (IFNβ), γ-interferon (IFNγ), platelet-derived growth factor (PDGF), TNFα, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), and mucosae-associated epithelial chemokine (MEC). chemokine), 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-la, MIP-1-, IL-8, L-selectin, P- Selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL- 18 mutant forms, 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.

[0267]

[0522] Additional adjuvants include MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic factors, and fibroblasts. Blast 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, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0268]

[0523] In some embodiments, the adjuvant may be a toll-like receptor modulator. Examples of toll-like receptor modulators include TLR9 agonists, but are not limited to small molecule toll-like receptor modulators such as imiquimod. Sometimes, the adjuvant is selected from bacterial toxoids, polyoxypropylene-polyoxyethylene 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 contain at least one oil and at least one surfactant, and the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion may have a diameter of less than 5 μm, and can even have a diameter at the submicron level, and these small sizes are achieved by using a microfluidizer to provide a stable emulsion. Droplets with a size of less than 220 nm may be subjected to filtration sterilization.

[0269]

[0524] In some cases, immunogenic pharmaceutical compositions may include carriers and excipients (examples of which include, but are not limited to, buffers, carbohydrates, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents, and / or preservatives), water, oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, saline, aqueous dextrose and glycerol solutions, flavors, colorants, detackifying agents, and other acceptable additives, adjuvants, or binders, as needed, and other pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, wetting agents, and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene, glycol, water, ethanol, etc. In another example, the pharmaceutical preparation is substantially free of preservatives. In another example, the pharmaceutical preparation may contain at least one preservative. Any suitable carrier known to those skilled in the art can be employed to administer the pharmaceutical compositions described herein, although it will be recognized that the type of carrier will vary depending on the mode of administration.

[0270]

[0525] The immunogenic pharmaceutical composition may contain a preservative, such as thiomersal or 2-phenoxyethanol. In some cases, the immunogenic pharmaceutical composition is substantially free of mercury-containing materials (e.g., <10 μg / mL), e.g., thiomersal-free. α-Tocopherol succinate can be used as an alternative to mercury-containing compounds.

[0271]

[0526] To control tonicity, physiological salts, such as sodium salts, may be included in the immunogenic pharmaceutical composition. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride.

[0272]

[0527] The immunogenic pharmaceutical composition may have an osmolality of 200 mOsm / kg to 400 mOsm / kg, 240 to 360 mOsm / kg, or an osmolality within the range of 290 to 310 mOsm / kg.

[0273]

[0528] The immunogenic pharmaceutical composition may also contain one or more buffers, such as Tris buffer, borate buffer, succinate buffer, histidine buffer (particularly with an aluminum hydroxide adjuvant), or citrate buffer, in some cases in the range of 5-20 or 10-50 mM.

[0274]

[0529] The pH of the immunogenic pharmaceutical composition may 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.

[0530] 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.

[0275]

[0531] The immunogenic pharmaceutical composition may also contain detergents, such as polyoxyethylene sorbitan ester surfactants (known as "Tweens"), or octoxynol (e.g., octoxynol-9 (Triton X-100), or t-octylphenoxypolyethoxyethanol). The detergents may be present only in trace amounts. The immunogenic pharmaceutical composition may contain less than 1 mg / mL each of octoxynol-10 and polysorbate 80. Other remaining trace components may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0276]

[0532] The immunogenic pharmaceutical composition can be formulated as a sterile solution or suspension in a suitable vehicle as known in the art. The pharmaceutical composition can be sterilized by conventional, well-known sterilization techniques or sterile filtered. The resulting aqueous solution can be packaged for immediate use or lyophilized, and the lyophilized preparation can be combined with a sterile solution before administration.

[0277]

[0533] For example, pharmaceutical compositions comprising an active agent, such as immune cells described herein, in combination with one or more adjuvants can be formulated to contain a specific molar ratio. For example, a molar ratio of about 99:1 to about 1:99 can be used between an active agent, such as immune cells described herein, and one or more adjuvants. In some cases, the molar ratio range of the combination of an active agent, such as immune cells described herein, and one or more adjuvants can be selected from about 80:20 to about 20:80; about 75:25 to about 25:75; about 70:30 to about 30:70; about 66:33 to about 33:66; about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90. The molar ratio of the combination of an active agent, such as immune cells described herein, and one or more adjuvants can be about 1:9, and in some cases, about 1:1. Active agents, such as immune cells described herein, may be combined with one or more adjuvants and formulated together in the same dosage unit, e.g., in the form of one vial, suppository, tablet, capsule, aerosol spray; or each agent, form, and / or compound may be formulated in separate units, e.g., in the form of two vials, suppositories, tablets, two capsules, a tablet and a vial, aerosol spray, etc.

[0278]

[0534] In some cases, the immunogenic pharmaceutical composition may be administered together with an additional agent. The choice of additional agent may depend, at least in part, on the condition being treated. The additional agent may include, for example, a checkpoint inhibitor, such as an anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 agent (e.g., an anti-PD1, anti-CTLA4, anti-PD-L1, anti-CD40, or anti-TIM3 antibody); or any agent that has a therapeutic effect on pathogen infection (e.g., a viral infection), such as a drug used to treat an inflammatory condition, such as an NSAID, e.g., ibuprofen, naproxen, acetaminophen, ketoprofen, or aspirin. For example, the checkpoint inhibitor may be a PD-1 / PD-L1 antagonist selected from the group consisting of nivolumab (ONO-4538 / BMS-936558, MDX1 106, Opdivo), pembrolizumab (MK-3475, Keytruda), pidilizumab (CT-011), and MPDL328OA (ROCHE). As another example, the formulation may additionally contain one or more supplements, such as vitamins C, E, or other antioxidants.

[0279]

[0535] Pharmaceutical compositions comprising the combination of active agents such as immune cells described herein and one or more adjuvants can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients, diluents, and / or auxiliary agents, for example, those that facilitate the processing of active agents into administrable preparations.Suitable formulations can depend at least in part on the selected administration route.The agents described herein can be delivered to patients using multiple administration routes or modes, such as oral, buccal, topical, rectal, transdermal, transmucosal, subcutaneous, intravenous, and intramuscular application, and also by inhalation.

[0280]

[0536] Active agent can be formulated for parenteral administration (for example, by injection, for example, by bolus injection or continuous infusion), and can be provided in the form of unit dose of ampule, pre-filled syringe, small amount of infusion, or can be provided in a container for multiple doses with added preservative.Composition can take this form as suspension, solution, or emulsion in oily or aqueous medium, for example, solution in aqueous polyethylene glycol.

[0281]

[0537] In some embodiments, the pharmaceutical composition comprises a preservative or stabilizer. In some embodiments, the preservative or stabilizer is selected from cytokines, growth factors, or adjuvants or chemicals. In some embodiments, the composition comprises at least one agent that helps preserve cell viability through at least one freeze-thaw cycle. In some embodiments, the composition comprises at least one agent that helps preserve cell viability through at least one or more freeze-thaw cycles.

[0282]

[0538] For injectable formulations, the vehicle may be selected from those known in the art to be suitable, such as sesame oil, corn oil, cottonseed oil, or peanut oil, as well as aqueous solutions or oil suspensions or emulsions containing elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles. The formulation may also contain biocompatible, biodegradable polymer compositions, such as poly(lactic-co-glycolic acid). These materials can be fabricated into drug-loaded micro- or nanospheres that are coated or derivatized to provide excellent sustained-release performance. Suitable vehicles for periocular or intraocular injection include, for example, suspensions of therapeutic agents in injection-grade water, liposomes, and vehicles suitable for lipophilic substances. Other vehicles for periocular or intraocular injection are known in the art.

[0283]

[0539] In some cases, pharmaceutical compositions are formulated according to conventional procedures as pharmaceutical compositions adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may contain a solubilizing agent and a local anesthetic, such as lidocaine, to reduce pain at the injection site. Generally, these ingredients are supplied in unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate, either separately or mixed together, in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active ingredient. If the composition is to be administered by infusion, it can be supplied in an infusion bottle containing sterile pharmaceutical-grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

[0284]

[0540] Interleukin-2 Composition and Administration

[0541] In one aspect, the disclosure provides a therapy for treating cancer in a subject, the therapy comprising administering an expanded population of autologous cells comprising a tumor antigen-specific T cell product comprising T cells responsive to a neoantigen in the cancer in the subject in combination with a therapy comprising a cytokine, wherein the subject is administered the cytokine interleukin-2 (IL-2). In some embodiments, the subject is administered a composition comprising the cytokine interleukin-2 (IL-2). In some embodiments, the cytokine is administered on the same day as the expanded population of cells is administered to the human subject. In some embodiments, the cytokine is administered after the expanded population of cells is administered to the human subject. In some embodiments, the cytokine is administered on a day after the expanded population of cells is administered to the human subject.

[0285]

[0542] In some embodiments, the cytokine is administered 0 to 2 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered 1 to 2 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered 2 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered 2 to 4 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered 3 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered 4 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered 5 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered 6 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered 8 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered 10 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered about 12 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered about 14 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered about 16 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered about 18 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered about 20 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered about 24 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered every 8-12 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered every 6-24 hours after administration of the expanded population of cells to the human subject. In some embodiments, the cytokine is administered every 8-24 hours after administration of the expanded population of cells to the human subject.In some embodiments, the cytokine is administered every 12 to 24 hours after administration of the expanded population of cells to the human subject.

[0286]

[0543] In some embodiments, the cytokine is administered at a dose of 200,000 IU / kg to 1,000,000 IU / kg, hi some embodiments, the cytokine is administered at a dose of about 600,000 IU / kg.

[0287]

[0544] In some embodiments, the cytokine is administered at least once. In some embodiments, at least two doses of cytokine are administered. In some embodiments, at least three doses of cytokine are administered. In some embodiments, at least three doses of cytokine are administered. In some embodiments, at least four doses of cytokine are administered. In some embodiments, at least five doses of cytokine are administered. In some embodiments, up to six doses of cytokine are administered. In some embodiments, up to eight doses of cytokine are administered.

[0288]

[0545] In some embodiments, the cytokine is administered intravenously, ie, at a dose of 600,000 IU / kg, up to a maximum of six doses, every 8-12 hours after administration of the expanded population of cells to the human subject, if tolerated.

[0289]

[0546] In some embodiments, the cytokine is in the form of a pharmaceutical composition suitable for administration. In some embodiments, the cytokine is in the form of an aqueous solution. In some embodiments, the cytokine is in the form of an aqueous composition and is administered intravenously.

[0290]

[0547] In some embodiments, the subject is administered a polynucleic acid encoding a cytokine. In some embodiments, the polynucleic acid encoding a cytokine is DNA. In some embodiments, the polynucleic acid encoding a cytokine is mRNA. In some embodiments, the subject is administered a ribocytokine encoding IL-2. Ribocytokine compositions, particularly ribocytokine encoding IL-2, and methods of use thereof are disclosed at least in application PCT / EP2019 / 053134, filed February 8, 2019, published August 15, 2019 as WO2019154985; application PCT / EP2021 / 086761, filed December 20, 2021, published June 30, 2022 as WO2022136255; and application PCT / EP2021 / 086778, filed December 20, 2021, published June 30, 2022 as WO2022136266, all of which are incorporated by reference in their entireties. The ribocytokine platform technology addresses major limitations of recombinant cytokine therapy: short serum half-life, low bioavailability, and the resulting need for high and frequent dosing. Controlled release of cytokines via ribocytokine platform technology is likely to improve efficacy in addition to safety compared to recombinant cytokines.

[0291]

[0548] Ribocytokine mRNA can be produced by in vitro transcription using N1-methyl-pseudouridine substitution for the nucleoside uridine, based on the method described by Kreiter et al. (Kreiter, S. et al., Cancer Immunol. Immunother. 56, 1577-87 (2007)). The resulting mRNA is equipped with a Cap1 structure and depleted of double-stranded (dsRNA) molecules by cellulose purification (Baiersdorfer et al., Mol. Ther. (2019)). The purified mRNA is then eluted in HO and stored at -60 to -80 °C until further use. In vitro transcription of all described mRNA constructs was performed at BioNTech RNA Pharmaceuticals GmbH, BioNTech, and ribocytokines are expected to have a favorable safety profile and increased clinical efficacy compared to their recombinant counterparts.

[0292]

[0549] Immune Checkpoint Inhibitor Compositions and Administration

[0550] Checkpoint inhibitors are a class of drugs that block immune checkpoint proteins produced by some immune system cells, such as T cells, and some cancer cells. This prevents the immune response from becoming too strong and sometimes prevents T cells from killing cancer cells. Blocking these checkpoints allows T cells to better kill cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2. Cytotoxic T lymphocyte-associated antigen (CTLA-4), also known as CD152, is a co-inhibitory molecule that functions to regulate T cell activation. CTLA-4 was first identified as a negative regulator on the surface of T cells, and it is upregulated immediately after stimulation of a de novo immune response or a pre-existing response to reduce subsequent immune T cell responses and prevent autoimmunity or uncontrolled inflammation. Therefore, the magnitude of the developing immune response is closely linked to the action of CTL.A4. In certain embodiments, the anti-CTLA.4 antibody is ipilumumab or tremelimumab.

[0293]

[0551] Checkpoint inhibitors function by modulating the immune system's intrinsic mechanisms of T cell regulation. Ipilimumab (YERVOY, Bristol-Meyers Squibb, New York, NY) is a monoclonal antibody. It was the first such checkpoint inhibitor to be approved by the U.S. Food and Drug Administration (FDA) and is becoming the standard treatment for metastatic melanoma (Hodi et al., N. Engl. J. Med. 363:711–23, 2010; Robert et al., N. Engl. J. Med. 364:2517–26, 2011). Ipilimumab binds to the T cell surface co-inhibitory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) and blocks the inhibitory signaling mediated by it. Ipilimumab is under investigation for the treatment of patients with prostate, lung, kidney, and breast cancer, among other tumor types, as its mechanism of action is not specific to one tumor type and abundant preclinical data supports a role for tumor immune surveillance across multiple malignancies (Andre et al., Clin. Cancer Res. 19:28-33. 2013; May et al., Clin. Cancer Res. 17:5233-38. 2011). Ipilimumab works by activating the immune system by targeting C'FLA-4.

[0294]

[0552] Thus, in exemplary embodiments, the present disclosure features novel combinations of expanded T cell compositions and one or more anti-CTLA4 antibodies. In other exemplary embodiments, the present disclosure also features novel combinations of expanded autologous T cell therapeutic compositions, ipilimumab and / or nivolumab, and one or more anti-CTLA4 antibodies.

[0295]

[0553] In contrast, CTLA-4 helps regulate early T cell activation, while programmed death-1 (PD-1) signaling functions partially to regulate T cell activation in peripheral tissues. The PD-1 receptor refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed on numerous cell types, including T'rep, activated B cells, and natural killer (NK) cells, and is predominantly expressed on activated T cells in vivo. It binds to two ligands, PD-L1 and PD-L2. PD-1's endogenous ligands, PD-L1 and PD-L2, are expressed on activated immune cells as well as non-hematopoietic cells, including tumor cells. As used herein, PD-1 is meant to include human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one epitope with hPD-1. The complete hPD-1 sequence can be found under GENBANK Accession No. U64863. Programmed death-ligand-1 (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, PD-L1 includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-11, as well as analogs that share at least one epitop...

Claims

1. 1. A method of treating cancer in a human subject in need thereof, comprising: a. administering to a human subject an expanded population of cells comprising tumor antigen-specific T cells, the expanded population of cells being from a population of immune cells comprising APCs and a first population of T cells, the first population having been depleted of CD25+ and / or CD14+ cells and incubated 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 cancer cells of the human subject, or (B) a polynucleotide encoding said polypeptide; the tumor antigen-specific T cells comprising T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by cancer cells or APCs of the human subject; and b. Administering a cytokine to a human subject A method comprising:

2. 1. A method of treating cancer in a human subject in need thereof, comprising: (a) forming a CD14- and / or CD25-depleted population of immune cells comprising a first population of antigen-presenting cells (APCs) and T cells by depleting CD25+ cells and / or CD14+ cells from a population of immune cells comprising APCs and T cells; (b) treating the first population of APCs and T cells from step (a) for a first period of time; i. FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and ii. (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding said polypeptide. forming a population of cells comprising stimulated T cells by incubating in the presence of (c) expanding the population of cells comprising the stimulated T cells to form an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) 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); (d) administering the expanded population of cells from (c) to a human subject; and (e) administering the cytokine to a human subject A method comprising:

3. The method of claim 1 or 2, wherein the cytokine is interleukin-2 (IL-2).

4. 4. The method of any one of claims 1 to 3, wherein the cytokine is administered on the same day as or after administering the expanded population of cells to the human subject.

5. 5. The method of claim 4, wherein the cytokine is administered 6 to 24 hours after administration of the expanded population of cells to the human subject.

6. 5. The method of claim 4, wherein the cytokine is administered about 12 hours after administering the expanded population of cells to the human subject.

7. 7. The method of any one of claims 1 to 6, wherein the cytokine is administered at a dose of 200,000 IU / kg to 1,000,000 IU / kg.

8. 8. The method of claim 7, wherein the cytokine is administered at a dose of about 600,000 IU / kg.

9. 3. The method of claim 1 or 2, wherein the administering step comprises administering a polynucleic acid encoding a cytokine.

10. The method of claim 9, wherein the polynucleic acid is mRNA.

11. 11. The method of any one of claims 1 to 10, wherein the cytokine is administered every 8 to 12 hours after administration of the expanded population of cells to the human subject.

12. The method of any one of claims 1 to 11, wherein at least 2, 3, 4, 5 or 6 doses of cytokine are administered.

13. 13. The method of claim 12, wherein up to six doses of cytokines are administered.

14. 13. The method of claim 12, wherein six doses of cytokines are administered.

15. The method of any one of claims 1 to 14, wherein the cytokine is administered intravenously.

16. 16. The method of any one of claims 1-15, wherein the cytokine is administered intravenously at a dose of 600,000 IU / kg, up to a maximum of 6 doses if tolerated, every 8-12 hours after administration of the expanded population of cells to the human subject.

17. 17. The method of any one of claims 1 to 16, further comprising administering an immune checkpoint inhibitor to the human subject.

18. The expanded population of administered cells is 0.75 x 10 ８ ~1.25 x 10 １０ The method of any one of claims 1 to 17, comprising a total of 100 cells.

19. The expanded population of administered cells is 1.5 x 10 ９ ~1.25 x 10 １０ 19. The method of claim 18, comprising total cells.

20. The expanded population of administered cells is 5 x 10 ８ ~1 x 10 １０ 19. The method of claim 18, comprising total cells.

21. The expanded population of administered cells is 5 x 10 ８ ~1 x 10 ９ 19. The method of claim 18, comprising total cells.

22. The expanded population of administered cells is 5 x 10 ８ ~2 x 10 ９ 19. The method of claim 18, comprising total cells.

23. The expanded population of administered cells is 0.75 x 10 ８ ~1.25 x 10 ９ 19. The method of claim 18, comprising total cells.

24. The expanded population of administered cells is a. 0.75 x 10 ８ ~1 x 10 ９ total cells, b. 0.75 x 10 ８ ~0.75 x 10 ９ of total cells, c. 1 x 10 ８ ~1.25 x 10 ９ of total cells, d. 1 x 10 ８ ~1x10 ９ of total cells, e. 1 x 10 ８ ~0.75 x 10 ９ of total cells, f. 1.25 x 10 ８ ~1.25 x 10 ９ of total cells, g. 1.25 x 10 ８ ~1x10 ９ of total cells, h. 1.25 x 10 ８ ~0.75 x 10 ９ of total cells, i. 4x10 ８ ~1.25 x 10 ９ of total cells, j. 4x10 ８ ~1x10 ９ of total cells, k. 4x10 ８ ~0.75 x 10 ９ of total cells, l. 5x10 ８ ~1.25 x 10 ９ of total cells, m. 5x10 ８ ~0.75 x 10 ９ Total cells n. 6x10 ８ ~1.25 x 10 ９ of total cells, o. 6x10 ８ ~0.75 x 10 ９ Total cells p. 6x10 ８ ~1x10 ９ of total cells, q. 4 x 10 ８ ~2.5 x 10 ９ of total cells, r. 4x10 ８ ~2x10 ９ of total cells, s. 4x10 ８ ~1.5 x 10 ９ of total cells, t. 5x10 ８ ~2.5 x 10 ９ of total cells, u.5x10 ８ ~1.5 x 10 ９ Total cells v. 6x10 ８ ~2.5 x 10 ９ of total cells, w. 6 x 10 ８ ~1.5 x 10 ９ total cells, or x. 6 x 10 ８ ~2 x 10 ９ total cells 20. The method of claim 18, comprising:

25. The expanded population of administered cells is a. 1.5 x 10 ９ ~1 x 10 １０ total cells, b. 1.5 x 10 ９ ~0.75 x 10 １０ of total cells, c. 2 x 10 ９ ~1.25 x 10 １０ of total cells, d. 2 x 10 ９ ~1x10 １０ of total cells, e. 2 x 10 ９ ~0.75 x 10 １０ of total cells, f. 2.5 x 10 ９ ~1.25 x 10 １０ of total cells, g. 2.5 x 10 ９ ~1 x 10 １０ total cells, or h. 2.5 x 10 ９ ~0.75 x 10 １０ total cells 20. The method of claim 18, comprising:

26. The method of any one of claims 18 to 25, wherein the immune checkpoint inhibitor comprises an anti-PD1 agent.

27. 27. The method of claim 26, wherein the immune checkpoint inhibitor comprises an anti-PD1 antibody.

28. 27. The method of claim 26, wherein the immune checkpoint inhibitor comprises pembrolizumab or nivolumab.

29. 29. The method of any one of claims 26-28, wherein the immune checkpoint inhibitor is administered after the expanded population of cells is administered.

30. 30. The method of any one of claims 26-29, wherein the immune checkpoint inhibitor is administered before the expanded population of cells is administered.

31. 31. The method of claim 29 or 30, wherein the immune checkpoint inhibitor is administered at a dose of 200-400 mg, 2 mg / kg-4 mg / kg, 200 mg, 2 mg / kg, 400 mg or 4 mg / kg.

32. 32. The method of any one of claims 26 to 31, wherein the immune checkpoint inhibitor further comprises an anti-CTLA4 agent.

33. 33. The method of claim 32, wherein the anti-CTLA4 agent is an anti-CTLA4 antibody.

34. 34. The method of claim 33, wherein the anti-CTLA4 antibody comprises ipilimumab.

35. 35. The method of any one of claims 29-34, wherein the immune checkpoint inhibitor is administered Q3W or Q6W.

36. 36. The method of claim 35, wherein the immune checkpoint inhibitor is administered Q6W.

37. 37. The method of any one of claims 29-36, wherein an immune checkpoint inhibitor is not administered for up to one week following administration of the expanded population of cells.

38. 38. The method of any one of claims 27-37, wherein the immune checkpoint inhibitor is administered 1 to 2 weeks after the expanded population of cells is administered.

39. 39. The method of any one of claims 27-38, wherein the immune checkpoint inhibitor is administered Q6W for up to 36 or 52 weeks after the expanded population of cells is administered.

40. 40. The method of any one of claims 27-39, wherein the immune checkpoint inhibitor is not administered 36 weeks or 52 weeks after the time the expanded population of cells is administered.

41. 41. The method of any one of claims 1 to 40, further comprising administering filgrastim to the human subject.

42. 42. The method of claim 41, wherein filgrastim is administered after the expanded population of cells is administered.

43. Subject's neutrophil count >1.0 x 10 for 3 days ９ / L level or >5.0 × 10 ９ 43. The method of claim 41 or 42, wherein the filgrastim is daily until a level of 1 / L is reached.

44. Human subjects a. have unresectable melanoma; b. have previously received a regimen containing a PD-1 inhibitor or a PD-L1 inhibitor and a CTLA-4 inhibitor and have disease progression; c. have received or are currently receiving a PD-1 inhibitor or a PD-L1 inhibitor for at least 3 months and have stable disease or asymptomatic progressive disease; or d. Discontinuing a PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor due to toxicity, or e. The method of any one of claims 1 to 43, wherein the patient is not considered suitable to receive a CTLA-4 inhibitor.

45. The method of any one of claims 1 to 43, wherein the cancer is melanoma.

46. The method of any one of claims 1 to 43, wherein the cancer is ovarian cancer.

47. The method of any one of claims 1 to 43, wherein the cancer is non-small cell lung cancer (NSCLC).

48. The method of any one of claims 1 to 47, wherein the polynucleotide encoding the polypeptide is mRNA.

49. 49. The method of claim 48, wherein the polypeptide encoded by the mRNA comprises at least two tumor antigen epitope sequences.

50. 50. The method of any one of claims 2 to 49, comprising incubating the APCs and the first population of T cells in the presence of IL-21 for a first period of time.

51. 51. The method of any one of claims 2 to 50, comprising expanding the population of cells comprising the stimulated T cells in the presence of IL-21.

52. 52. The method of any one of claims 1 to 51, wherein the population of immune cells is from a biological sample from a human subject.

53. 1. A method of treating cancer in a human subject in need thereof, comprising: administering to a human subject an expanded population of cells comprising tumor antigen-specific T cells, the expanded population of cells being from a population of immune cells comprising APCs and a first population of T cells, the first population being depleted of CD14+ and / or CD25+ cells, over a first period of time, (i) an 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, or (B) incubated in the presence of a polynucleotide encoding said polypeptide; The tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) at least one tumor antigen epitope sequence and (ii) an MHC protein expressed by a cancer cell or APC of a human subject; the expanded population of cells comprises 5×10 ８ ~1 x 10 １０ Total cells, 5.0 x 10 ８ 1.0 x 10 from 10 pieces or more １０ cells or less, 5 x 10 ８ ~1 x 10 ９ total cells, or 5 x 10 ８ ~2 x 10 ９ Total cells included, step A method comprising:

54. 54. The method of claim 53, wherein the human subject has discontinued a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor due to toxicity; or is deemed unsuitable to receive a CTLA-4 inhibitor.

55. 1. A method of treating cancer in a human subject in need thereof, comprising: (a) forming a CD14- and / or CD25-depleted population of immune cells comprising a first population of antigen-presenting cells (APCs) and T cells by depleting CD14+ cells and / or CD25+ cells from a population of immune cells comprising APCs and T cells; (b) treating the first population of APCs and T cells from step (a) for a first period of time; FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding said polypeptide. forming a population of cells comprising stimulated T cells by incubating in the presence of (c) expanding the population of cells comprising the stimulated T cells to form an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells specific for a complex comprising (i) 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); and (d) administering the expanded population of cells from (c) to a human subject. Including, The method, wherein the human subject has discontinued a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor due to toxicity; or is deemed unsuitable to receive a CTLA-4 inhibitor.

56. The expanded population of cells was 5 x 10 ８ ~1 x 10 １０ Total cells, 5 x 10 ８ ~1 x 10 ９ total cells, or 5 x 10 ８ ~2 x 10 ９ 56. The method of claim 55, comprising a total of 56 cells.

57. 57. The method of any one of claims 53 to 56, further comprising administering a cytokine to the human subject.

58. 58. The method of claim 57, wherein the cytokine is interleukin-2 (IL-2) or a recombinant polynucleic acid encoding the cytokine IL-2.

59. 59. The method of any one of claims 55 to 58, wherein depleting comprises depleting only CD25+ cells.

60. 59. The method of any one of claims 55 to 58, wherein depleting comprises depleting CD25+ cells and CD56+ cells.

61. an anti-cancer monotherapy comprising autologous T cells from a subject who has previously been treated with anti-PD1, anti-PDL1, or anti-CTLA4 therapy, and ７ ~2 x 10 ９ Monotherapy comprising one or more doses of total cells.

62. 62. The monotherapy of claim 61, wherein the cancer is metastatic melanoma.

63. 62. The monotherapy of claim 61, wherein the cancer is ovarian cancer.

64. 62. The method of claim 61, wherein the cancer is non-small cell lung cancer (NSCLC).

65. 65. The method of any one of claims 61 to 64, wherein no other therapeutic agent is administered to the subject during the period of T cell therapy.

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