T cell therapy with vaccination as a combination immunotherapy for cancer

Abstract

A method of treating or preventing cancer in a mammal is disclosed, the method comprising: (a) isolating T cells from a tumor sample from the mammal, wherein the isolated T cells are in one or both of an exhausted state and a differentiated state, and the isolated T cells have antigen specificity for a tumor-specific antigen expressed by the tumor sample from the mammal, the tumor-specific antigen being a tumor-specific neo-antigen or an antigen having a tumor-specific driver mutation; and, optionally, expanding the number of isolated tumor antigen-specific T cells; and (b) administering to the mammal (i) the isolated T cells of (a), and (ii) a vaccine that specifically stimulates an immune response against the tumor-specific antigen for which the isolated T cells have antigen specificity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 295,762, filed December 31, 2021, which is incorporated by reference in its entirety herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Project No. ZIABC010985 awarded by the National Cancer Institute of the National Institutes of Health. The Government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Materials Incorporated herein by reference in its entirety is a computer readable nucleotide / amino acid sequence listing, submitted contemporaneously herewith, and identified as follows: one 2,650 byte XML file entitled "766239.xml", dated December 28, 2022. [Background technology]

[0004] Adoptive cell therapy (ACT) using T cells targeting tumor-specific antigens can produce good clinical responses in some patients. Nevertheless, several obstacles remain to the successful use of ACT for the treatment of cancer and other pathologies. For example, antitumor T cells with an exhausted phenotype may be unable to mount a sustained immune response against tumors. Thus, improved immunotherapies for cancer are needed. Summary of the Invention

[0005] One aspect of the invention provides a method of treating or preventing cancer in a mammal, the method comprising: (a) isolating T cells from a tumor sample from the mammal, wherein the isolated T cells are in one or both of an exhausted state and a differentiated state, and the isolated T cells have antigen specificity for a tumor-specific antigen expressed by the tumor sample from the mammal, wherein the tumor-specific antigen is a tumor-specific neo-antigen or an antigen having a tumor-specific driver mutation; and optionally expanding the number of isolated tumor antigen-specific T cells; and (b) administering to the mammal: (i) the isolated T cells of (a), and (ii) a vaccine that specifically stimulates an immune response against the tumor-specific antigen for which the isolated T cells have antigen specificity.

[0006] One aspect of the invention provides a method of treating or preventing cancer in a mammal having a tumor, the method comprising: (a) isolating T cells from a biological sample from the mammal having the tumor; (b) introducing into the isolated T cells a nucleic acid comprising a nucleotide sequence encoding a foreign receptor having antigen specificity for a tumor-specific antigen expressed by a tumor in the mammal to produce T cells expressing the foreign receptor, wherein the tumor-specific antigen is a tumor-specific neo-antigen or an antigen having a tumor-specific driver mutation; and optionally expanding the number of T cells expressing the foreign receptor; and (c) administering to the mammal the T cells expressing the foreign receptor of (i)(b) and (ii) a vaccine that specifically stimulates an immune response against the tumor-specific antigen for which the foreign receptor has antigen specificity. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 is a flow chart showing steps for neo-antigen specific T cell ACT in combination with neo-antigen vaccination according to one aspect of the present invention. [Figure 2A] FIG. 2A is a schematic diagram showing the timeline of neoantigen-specific T cell ACT in combination with neoantigen vaccination in B16+ mice according to one aspect of the present invention. [Figure 2B] FIG. 2B is a flow cytometry graph showing the number of Pmel+ T cells with expression levels of cluster of differentiation 39 (CD39) categorized into low (CD39lo), intermediate (CD39med), and high (CD39hi) groups after two rounds of stimulation. [Figure 2C] Figure 2C shows heat map graphs of PD-1 and TIM3 expression in CD39lo and CD39hi T cells, and graphs of 4-1BB activation in CD39lo and CD39hi T cells. [Figure 3A] FIG. 3A is a graph showing tumor size (mm2) in B16+ mice over days after ACT in mice treated with PBS, bulk Pmel+ T cells (bulk), Pmel+ T cells expressing high (CD39hi), intermediate (CD39med), and low (CD39lo) levels of CD39. [Figure 3B] Figure 3B is a graph showing tumor size (mm2) in B16+ mice over days after ACT in mice treated with PBS, bulk Pmel+ T cells (bulk), CD39lo, CD39hi+ isotype control antibody (IgG), CD39hi+ anti-CD40 antibody, or CD39hi+ anti-PD1 antibody. [Figure 4A] FIG. 4A is a graph showing survival of B16+ mice over days after ACT in groups (n=5) treated with PBS, bulk Pmel+ T cells (Bulk Pmel), low expressing CD39 Pmel+ T cells (CD39lo) + isotype control antibody (Rat IgG), high expressing CD39 Pmel+ T cells (CD39hi) + anti-CD40 antibody, CD39hi + anti-PD1 antibody, or CD39hi + related vaccinia virus against the human glycoprotein 100 epitope sequence KVPRNQDWL (SEQ ID NO:2) (r. VACVhgp100). [Figure 4B]Figure 4B is a graph showing tumor size (mm2) in B16+ mice over days after ACT in mice treated with PBS, bulk, CD39lo, CD39hi+IgG, CD39hi+anti-CD40 antibody, CD39hi+anti-PD1 antibody, or CD39hi+r.VACVhgp100. [Figure 5A] Figures 5A-5C are graphs showing tumor size (mm) in B16+ mice over days after ACT in mice treated alone or in various combinations with: PBS, low expressing CD39 Pmel+ T cells (CD39lo), high expressing CD39 Pmel+ T cells (CD39hi), isotype control antibody (IgG), human glycoprotein 100 peptide (Pep), vaccinia virus against human glycoprotein 100 (VACVhgp100) (Figure 5A), adenovirus against human glycoprotein 100 (ADVhgp100) (Figure 5B), or anti-CD40 antibody (Figure 5C). [Figure 5B] Figures 5A-5C are graphs showing tumor size (mm) in B16+ mice over days after ACT in mice treated alone or in various combinations with: PBS, low expressing CD39 Pmel+ T cells (CD39lo), high expressing CD39 Pmel+ T cells (CD39hi), isotype control antibody (IgG), human glycoprotein 100 peptide (Pep), vaccinia virus against human glycoprotein 100 (VACVhgp100) (Figure 5A), adenovirus against human glycoprotein 100 (ADVhgp100) (Figure 5B), or anti-CD40 antibody (Figure 5C). [Figure 5C]Figures 5A-5C are graphs showing tumor size (mm) in B16+ mice over days after ACT in mice treated alone or in various combinations with: PBS, low expressing CD39 Pmel+ T cells (CD39lo), high expressing CD39 Pmel+ T cells (CD39hi), isotype control antibody (IgG), human glycoprotein 100 peptide (Pep), vaccinia virus against human glycoprotein 100 (VACVhgp100) (Figure 5A), adenovirus against human glycoprotein 100 (ADVhgp100) (Figure 5B), or anti-CD40 antibody (Figure 5C). [Figure 5D] FIG. 5D is a graph showing survival of B16+ mice over days after ACT treated with low CD39 Pmel+ T cells (CD39lo) or high CD39 Pmel+ T cells (CD39hi) in combination with (VACVhgp100) or ADVhgp100 (n=5). [Figure 6A] FIG. 6A is a graph showing tumor size (mm2) in B16+ mice over days after ACT in mice treated with PBS or low expressing CD39 Pmel+ T cells (CD39lo) alone or in combination with the related vaccinia virus against human glycoprotein 100 (r.VACVhgp100) (FIG. 6A). [Figure 6B] FIG. 6B is a graph showing tumor size in mice treated with PBS or PD1- and TIM3-expressing Pmel+ T cells (PD1+TIM3+) alone or in combination with r.VACVhgp100. [Figure 6C] FIG. 6C is a graph showing tumor size in mice treated with PBS or Pmel+ T cells expressing CD39 and CD69 (CD39+CD69+) alone or in combination with r.VACVhgp100. [Figure 7A]FIG. 7A is a graph showing tumor size (mm2) in B16+ mice over days after ACT in mice treated with PBS or CD39 Pmel+ T cells (CD39lo) alone or in combination with a vaccinia virus against an irrelevant HLA-A2-restricted human glycoprotein 100 (hgp100) epitope (VACVhgp100(209).irr) or a vaccinia virus against the related hgp100 epitope 25 (hgp10025) (VACVhgp100(25)). [Figure 7B] Figure 7B is a graph showing tumor size (mm2) in B16+ mice over days after ACT in mice treated with CD39- and CD69-expressing Pmel+ T cells (CD39+CD69+) alone or in combination with VACVhgp100(209).irr or VACVhgp100(25). [Figure 8A] Figures 8A-8F are bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells at days 3, 7, and 10 after ACT in the spleen (Figure 8A), draining lymph nodes (LNs) (Figure 8B), and tumors (Figure 8C) of B16+ mice treated with CD39 low expressing Pmel+ T cells (CD39lo), CD39lo and related vaccinia virus against human glycoprotein 100 (rVACVhgp100), or rVACVhgp100 alone. Bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells on days 3, 7, and 10 after ACT in the spleen (Figure 8D), draining lymph node (Figure 8E), and tumor (Figure 8F) of B16+ mice treated with T cells (CD39+CD69+), CD39+CD69+ and rVACVhgp100, or rVACVhgp100 alone. [Figure 8B]Figures 8A-8F are bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells at days 3, 7, and 10 after ACT in the spleen (Figure 8A), draining lymph nodes (LNs) (Figure 8B), and tumors (Figure 8C) of B16+ mice treated with CD39 low expressing Pmel+ T cells (CD39lo), CD39lo and related vaccinia virus against human glycoprotein 100 (rVACVhgp100), or rVACVhgp100 alone. Bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells on days 3, 7, and 10 after ACT in the spleen (Figure 8D), draining lymph node (Figure 8E), and tumor (Figure 8F) of B16+ mice treated with T cells (CD39+CD69+), CD39+CD69+ and rVACVhgp100, or rVACVhgp100 alone. [Figure 8C] Figures 8A-8F are bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells at days 3, 7, and 10 after ACT in the spleen (Figure 8A), draining lymph nodes (LNs) (Figure 8B), and tumors (Figure 8C) of B16+ mice treated with CD39 low expressing Pmel+ T cells (CD39lo), CD39lo and related vaccinia virus against human glycoprotein 100 (rVACVhgp100), or rVACVhgp100 alone. Bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells on days 3, 7, and 10 after ACT in the spleen (Figure 8D), draining lymph node (Figure 8E), and tumor (Figure 8F) of B16+ mice treated with T cells (CD39+CD69+), CD39+CD69+ and rVACVhgp100, or rVACVhgp100 alone. [Figure 8D]Figures 8A-8F are bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells at days 3, 7, and 10 after ACT in the spleen (Figure 8A), draining lymph nodes (LNs) (Figure 8B), and tumors (Figure 8C) of B16+ mice treated with CD39 low expressing Pmel+ T cells (CD39lo), CD39lo and related vaccinia virus against human glycoprotein 100 (rVACVhgp100), or rVACVhgp100 alone. Bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells on days 3, 7, and 10 after ACT in the spleen (Figure 8D), draining lymph node (Figure 8E), and tumor (Figure 8F) of B16+ mice treated with T cells (CD39+CD69+), CD39+CD69+ and rVACVhgp100, or rVACVhgp100 alone. [Figure 8E] Figures 8A-8F are bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells at days 3, 7, and 10 after ACT in the spleen (Figure 8A), draining lymph nodes (LNs) (Figure 8B), and tumors (Figure 8C) of B16+ mice treated with CD39 low expressing Pmel+ T cells (CD39lo), CD39lo and related vaccinia virus against human glycoprotein 100 (rVACVhgp100), or rVACVhgp100 alone. Bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells on days 3, 7, and 10 after ACT in the spleen (Figure 8D), draining lymph node (Figure 8E), and tumor (Figure 8F) of B16+ mice treated with T cells (CD39+CD69+), CD39+CD69+ and rVACVhgp100, or rVACVhgp100 alone. [Figure 8F]Figures 8A-8F are bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells at days 3, 7, and 10 after ACT in the spleen (Figure 8A), draining lymph nodes (LNs) (Figure 8B), and tumors (Figure 8C) of B16+ mice treated with CD39 low expressing Pmel+ T cells (CD39lo), CD39lo and related vaccinia virus against human glycoprotein 100 (rVACVhgp100), or rVACVhgp100 alone. Bar graphs showing the percentage of Thy1.1+ Vβ13+ CD8+ T cells relative to total CD8+ T cells on days 3, 7, and 10 after ACT in the spleen (Figure 8D), draining lymph node (Figure 8E), and tumor (Figure 8F) of B16+ mice treated with T cells (CD39+CD69+), CD39+CD69+ and rVACVhgp100, or rVACVhgp100 alone. [Figure 9] FIG. 9 is a three bar graph showing the percentage of terminally exhausted PD1+TIM3+ adoptively transferred Thy1.1+Vβ13+ neo-antigen-specific T cells isolated from tumors on days 3, 7, and 10 after ACT in B16+ mice treated with either CD39 low expressing Pmel+ T cells (CD39lo), CD39lo and related vaccinia virus against human glycoprotein 100 (rVACVhgp100), Pmel+ T cells expressing CD39 and CD69 (CD39+CD69+), or CD39+CD69+ and rVACVhgp100. [Figure 10] FIG. 10 is a graph showing tumor size (mm2) in B16+ mice over days after ACT in mice treated with PBS or Pmel+ T cells expressing CD39 and CD69 (CD39+CD69+) alone or various combinations of one or more of an irrelevant peptide influenza nucleoprotein (Flu.NP), vaccinia virus against the relevant hgp100 epitope 25 (hgp10025) (VACVhgp100(25)), anti-CD40 antibody, and an isotype control antibody (Rat IgG). [Figure 11]FIG. 11 is a graph showing tumor size (mm2) in B16+ mice over days following ACT in mice treated with PBS or Pmel+ T cells alone or in combination with bone marrow-derived dendritic cells (DCs) loaded with an irrelevant influenza peptide (Irr.Pep) or a relevant neoepitope (hgp100KVP). [Figure 12A] Figures 12A-12C are graphs showing MC38 tumor size (mm2) in C57BL / 6 mice over days after ACT in mice treated with PBS alone or in combination with vaccinia virus against hgp100 (VACVhgp100) (Figure 12A), CD39 low expressing Pmel+ T cells (CD39lo) alone or in combination with VACVhgp100 (Figure 12B), or Pmel+ T cells expressing CD39 and CD69 (CD39+CD69+) alone or in combination with VACVhgp100 (Figure 12C). [Figure 12B] Figures 12A-12C are graphs showing MC38 tumor size (mm2) in C57BL / 6 mice over days after ACT in mice treated with PBS alone or in combination with vaccinia virus against hgp100 (VACVhgp100) (Figure 12A), CD39 low expressing Pmel+ T cells (CD39lo) alone or in combination with VACVhgp100 (Figure 12B), or Pmel+ T cells expressing CD39 and CD69 (CD39+CD69+) alone or in combination with VACVhgp100 (Figure 12C). [Figure 12C] Figures 12A-12C are graphs showing MC38 tumor size (mm2) in C57BL / 6 mice over days after ACT in mice treated with PBS alone or in combination with vaccinia virus against hgp100 (VACVhgp100) (Figure 12A), CD39 low expressing Pmel+ T cells (CD39lo) alone or in combination with VACVhgp100 (Figure 12B), or Pmel+ T cells expressing CD39 and CD69 (CD39+CD69+) alone or in combination with VACVhgp100 (Figure 12C). [Figure 13A]Figures 13A-13B are graphs showing tumor size (mm2) in β2M knockout (KO) mice over days after ACT in mice treated with PBS alone or in combination with the related vaccinia virus for hgp10025 (VACVhgp100(25)) and CD39 low expressing Pmel+ T cells (CD39lo) alone or in combination with VACVhgp100(25) (Figure 13A), or Pmel+ T cells expressing CD39 and CD69 (CD39+CD69+) alone or in combination with VACVhgp100(25) (Figure 13B). [Figure 13B] Figures 13A-13B are graphs showing tumor size (mm2) in β2M knockout (KO) mice over days after ACT in mice treated with PBS alone or in combination with the related vaccinia virus for hgp10025 (VACVhgp100(25)) and CD39 low expressing Pmel+ T cells (CD39lo) alone or in combination with VACVhgp100(25) (Figure 13A), or Pmel+ T cells expressing CD39 and CD69 (CD39+CD69+) alone or in combination with VACVhgp100(25) (Figure 13B). [Figure 14A] 14A-14B are graphs showing B16KVP tumor size (mm2) in C57BL / 6 mice over days after ACT in mice treated alone or in various combinations with either: PBS, anti-B7.1 and anti-B7.2 antibodies (anti-B7.1 / 2), isotype control antibody (IgG), the related vaccinia virus against hgp10025 (VACVhgp100(25)), and low expressing CD39 Pmel+ T cells (CD39lo) (FIG. 14A), or CD39 and CD69 expressing Pmel+ T cells (CD39+CD69+) (FIG. 14B). [Figure 14B]14A-14B are graphs showing B16KVP tumor size (mm2) in C57BL / 6 mice over days after ACT in mice treated alone or in various combinations with either: PBS, anti-B7.1 and anti-B7.2 antibodies (anti-B7.1 / 2), isotype control antibody (IgG), the related vaccinia virus against hgp10025 (VACVhgp100(25)), and low expressing CD39 Pmel+ T cells (CD39lo) (FIG. 14A), or CD39 and CD69 expressing Pmel+ T cells (CD39+CD69+) (FIG. 14B). [Figure 15A] FIG. 15A is a flow chart showing the treatment progress of a patient. [Figure 15B] FIG. 15B is a flow cytometry graph showing the frequency of HLA-A0201-restricted GP100 tetramers as a percentage of CD8+ TILs in the infusion formulations (Rx1 and Rx3), demonstrating no apparent difference in GP100 TIL frequency during the course of the first intravenous treatment without vaccine (Rx1) and the second intravenous treatment in combination with GP100 vaccine (Rx3) administered to patients. [Figure 15C] FIG. 15C is a scatter plot showing the clonal frequency of GP100 TCR within Rx1 and Rx3, showing no obvious differences in the clonal distribution of immunodominant GP100 TCR-tagged therapeutic products. [Figure 15D] Figures 15D-15G show heat map graphs of GP100 TILs in the infusion formulations (Rx1 and Rx3), showing that there is no obvious difference in the phenotypic status of GP100 TIL frequency administered to patients during the Rx1 and Rx3 treatment periods. The figures include plots showing expression of CD39 and CD69 (Figure 15D), CD62L and CD8 (Figure 15E), TIM3 and CD8 (Figure 15F), and CD39 and CD8 (Figure 15G). Thus, both Rx1 and Rx3 contained similarly differentiated dysfunctional antitumor TILs that were only able to induce tumor regression when the vaccine was administered at Rx3, but not when the vaccine was not administered (Rx1). [Figure 15E]Figures 15D-15G show heat map graphs of GP100 TILs in the infusion formulations (Rx1 and Rx3), showing that there is no obvious difference in the phenotypic status of GP100 TIL frequency administered to patients during the Rx1 and Rx3 treatment periods. The figures include plots showing expression of CD39 and CD69 (Figure 15D), CD62L and CD8 (Figure 15E), TIM3 and CD8 (Figure 15F), and CD39 and CD8 (Figure 15G). Thus, both Rx1 and Rx3 contained similarly differentiated dysfunctional antitumor TILs that were only able to induce tumor regression when the vaccine was administered at Rx3, but not when the vaccine was not administered (Rx1). [Figure 15F] Figures 15D-15G show heat map graphs of GP100 TILs in the infusion formulations (Rx1 and Rx3), showing that there is no obvious difference in the phenotypic status of GP100 TIL frequency administered to patients during the Rx1 and Rx3 treatment periods. The figures include plots showing expression of CD39 and CD69 (Figure 15D), CD62L and CD8 (Figure 15E), TIM3 and CD8 (Figure 15F), and CD39 and CD8 (Figure 15G). Thus, both Rx1 and Rx3 contained similarly differentiated dysfunctional antitumor TILs that were only able to induce tumor regression when the vaccine was administered at Rx3, but not when the vaccine was not administered (Rx1). [Figure 15G] Figures 15D-15G show heat map graphs of GP100 TILs in the infusion formulations (Rx1 and Rx3), showing that there is no obvious difference in the phenotypic status of GP100 TIL frequency administered to patients during the Rx1 and Rx3 treatment periods. The figures include plots showing expression of CD39 and CD69 (Figure 15D), CD62L and CD8 (Figure 15E), TIM3 and CD8 (Figure 15F), and CD39 and CD8 (Figure 15G). Thus, both Rx1 and Rx3 contained similarly differentiated dysfunctional antitumor TILs that were only able to induce tumor regression when the vaccine was administered at Rx3, but not when the vaccine was not administered (Rx1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] ACT against cancer involves the in vitro expansion and in vivo administration of autologous antitumor T cells that target the patient's own tumor. Antitumor T cells, for example those targeting tumor-specific mutations ("neo-antigens"), may exist in a terminally differentiated, exhausted state. Such exhausted antitumor T cells may have limited efficacy during ACT against established tumors and may also have limited persistence in vivo in patients after treatment. Conventional cell therapy using antitumor T cells may not produce significant responses. It has been found that combination immunotherapy including (i) exhausted and / or differentiated T cells with antigen specificity against tumor-specific antigens and (ii) a vaccine that specifically stimulates an immune response against the tumor-specific antigen results in superior antitumor effects in vivo. Administration of a vaccine that specifically stimulates an immune response against a tumor-specific antigen can enhance ACT with exhausted and / or differentiated T cells with antigen specificity against the same tumor-specific antigen. The antitumor effect of this combination immunotherapy may be superior to vaccine alone or ACT using exhausted and / or differentiated T cells alone. Thus, ACT using exhausted and / or differentiated T cells or genetically modified T cells (TCR-transduced T cells) can be synergistically enhanced with a vaccine that specifically stimulates an immune response against the same tumor-specific antigen. The method of the present invention may advantageously overcome the challenges of developing effective immunotherapy using antitumor T cells with an exhausted phenotype that would not be able to generate a sustained immune response against tumors.

[0009] The method of the present invention can advantageously target metastatic cancers, such as epithelial cancers, which cause more than about 90% of cancer deaths, and which may not respond to conventional immunotherapy, or to antitumor vaccines as single agents.

[0010] By combining the administration of vaccines with exhausted and / or differentiated T cells, the method of the present invention can rescue the anti-tumor activity of exhausted and / or differentiated T cells. In this regard, one aspect of the present invention provides a method of treating or preventing cancer in a mammal. The method can include isolating T cells from a tumor sample from a mammal. The tumor sample can be, for example, tissue from a primary tumor or tissue from a site of a metastatic tumor. Thus, the tumor sample can be obtained by any suitable means, including, but not limited to, aspiration, biopsy, and resection.

[0011] The isolated T cells may be exhausted and / or differentiated. T cell exhaustion is a state of T cell dysfunction in response to chronic antigenic stimulation. T cell exhaustion is defined by reduced effector function, persistent expression of inhibitory receptors, and a phenotype distinct from functional effector and memory T cells. In the methods of the invention, the reduced effector function typically exhibited by exhausted T cells can be improved or overcome by combining exhausted T cells with a vaccine that targets the same antigen as that targeted by the exhausted T cells, as described herein. In one aspect of the invention, the isolated T cells express any one or more of the following T cell exhaustion markers: (a) RNA encoding any one or more of: 4-1BB + , CCL3 + , CD28 - , CD39 + , CD62L - (SELL - ), CD69 + , CTLA4 + , CX3CR1 + , CXCL13 + , CXCR6 + , GZMA + , G.Z.M.B. + , GZMK + , IL7R - , LAG-3 + , LAYN +, LEF1 - , PD-1 + , PRF1 + , TCF7 - , TIGIT + , TIM-3 + , and TOX + and (b) any one or more of the following proteins: 4-1BB + , CCL3 + , CD28 - , CD39 + , CD62L - (SELL - ), CD69 + , CTLA4 + , CX3CR1 + , CXCL13 + , CXCR6 + , GZMA + , G.Z.M.B. + , GZMK + , IL7R - , LAG-3 + , LAYN + , LEF1 - , PD-1 + , PRF1 + , TCF7 - , TIGIT + , TIM-3 + , and TOX + As used herein, the symbol "" refers to expression of the indicated markers of T cell exhaustion. + " indicates high (" hi " ) and "Medium" (" med" expression, meaning that the cells upregulate expression of the indicated markers compared to less exhausted T cells. Upregulated expression can include, for example, a mean log fold change (base 2) of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or a quantitative increase of any two or more of the foregoing values ​​in expression of the indicated markers of T cell exhaustion. As used herein, the symbol " " refers to expression of the indicated markers of T cell exhaustion. - ” indicates the absence and “low” ( lo ") expression, meaning that the cells downregulate expression of the indicated markers compared to less exhausted T cells. Downregulated expression can refer to, for example, the average log fold change (base 2) of about -1, about -2, about -3, about -4, about -5, about -6, about -7, about -8, about -9, about -10, about -20, about -30, about -40, about -50, about -60, about -70, about -80, about -90, about -100, about -110, about -120, about -130, about -140, about -150, about -160, about -170, about -180, about -190, about -200, about -210, about 220, about -230, about -240, about -250, about -260, about The present invention may include a quantitative reduction in the range of any two or more of the foregoing values, such as -270, about -280, about -290, about -300, about -310, about -320, about -330, about -340, about -350, about -360, about -370, about -380, about -390, about -400, about -410, about -420, about -430, about -440, about -450, about -460, about -470, about -480, about -490, about -500, about -510, about -520, about -530, about -540, about -550, about -560, about -570, about -580, about -590, about -600, or more. Isotype controls can be used to distinguish expression of the markers. Within the gate for a given marker, the lower tertile expression was designated as lo" and the middle tertile " med " and the upper tertile " hi " can be specified.

[0012] T cell differentiation refers to the process by which precursor cells acquire the characteristics of more mature T cells. T cell differentiation proceeds in the following order: naive T cells (T N ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), and terminal effector (T TE (memory T cells) follow a linear progression along a continuum, where less differentiated cells give rise to more differentiated progeny cells in response to antigenic stimulation. As differentiation progresses, memory T cells gradually gain or lose certain functions. For example, it is believed that the increased differentiation of T cells adversely affects the ability of T cells to function in vivo. The reduced effector function that differentiated T cells typically exhibit can be improved or overcome in the methods of the invention by combining the differentiated T cells with a vaccine that targets the same antigen as the differentiated T cells target, as described herein. In one aspect of the invention, the isolated T cells are terminally differentiated.

[0013] In one aspect of the invention, the isolated T cells express any one or more of the following differentiation markers: (a) RNA encoding any one or more of the following: CCR7 - , CD27 - , CD45RA + , CD45RO - , CD95 + , EOMES - , FOXO1 - , KLRG1 + , T-BET + , TCF7 - , T.O.X. + , and ZEB2 + and (b) any one or more of the following proteins: CCR7 - , CD27 -, CD45RA + , CD45RO - , CD95 + , EOMES - , FOXO1 - , KLRG1 + , T-BET + , TCF7 - , T.O.X. + , and ZEB2 + As used herein, the symbol " + " is a T cell differentiation marker that is highly expressed (" hi " ) and "Medium Expression" (" med "), meaning that the cells upregulate expression of the indicated markers compared to less differentiated T cells. Upregulated expression can include, for example, a quantitative increase in expression of the indicated markers of T cell differentiation of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, or a range of any two of the foregoing values, or more. As used herein, the symbol " " with respect to expression of the indicated markers of T cell differentiation. - ” indicates the absence and “low” ( lo") expression, meaning that the cells down-regulate expression of the indicated markers compared to less differentiated T cells. Down-regulated expression can, for example, be an average log fold change (base 2) of about -1, about -2, about -3, about -4, about -5, about -6, about -7, about -8, about -9, about -10, about -20, about -30, about -40, about -50, about -60, about -70, about -80, about -90, about -100, about -110, about -120, about -130, about -140, about -150, about -160, about -170, about -180, about -190, about -200, about -210, about 220, about -230, about -240, about -250, about -260, about -270, about -280, about -290, about -310, about -320, about -330, about -340, about -350, about -360, about -370, about -380, about -390, about -410, about -420, about -430, about -440, about -450, about -460, about -470, about -480, about -490, about -510, about -520, about -530, about -540, about -550, about -560, about -570, about -580, about -610, about -620, about -630, about -640, about -650, about -660, about -700, about -800, about -900, about -1000, about -1100, about -1200, about -1300, about -1400, about -1500, about -650, about -7 The range of the quantitative reduction may include about -270, about -280, about -290, about -300, about -310, about -320, about -330, about -340, about -350, about -360, about -370, about -380, about -390, about -400, about -410, about -420, about -430, about -440, about -450, about -460, about -470, about -480, about -490, about -500, about -510, about -520, about -530, about -540, about -550, about -560, about -570, about -580, about -590, about -600, or a range of any two of the foregoing values, or more. As described herein with respect to other aspects of the invention, isotype controls can be used to distinguish marker expression.

[0014] The isolated T cells can have antigen specificity for tumor-specific antigens expressed by tumor samples from mammals. As used herein, the phrases "antigen-specific" and "antigen specificity" mean that the T cells can specifically bind to and immunologically recognize an antigen or its epitope, and the binding of the T cells to the antigen or its epitope elicits an immune response.

[0015] The term "tumor-specific antigen" or "tumor antigen" as used herein refers to any molecule (e.g., protein, polypeptide, peptide, lipid, carbohydrate, etc.) that is expressed or overexpressed, either singly or predominantly, by tumor cells such that the antigen is associated with the tumor. A tumor-specific antigen may also be expressed by normal, non-tumor, or non-cancer cells. However, in such cases, the expression of the tumor-specific antigen by normal, non-tumor, or non-cancer cells is not as robust as the expression by the tumor. In this regard, the tumor cells may overexpress the antigen or express the antigen at a significantly higher level compared to the expression of the antigen by normal, non-tumor, or non-cancer cells. A tumor-specific antigen may also be expressed by cells of a different developmental or maturational state. For example, a tumor-specific antigen may also be additionally expressed by cells at the embryonic or fetal stage that are not normally found in the adult host. Alternatively, a tumor-specific antigen may also be additionally expressed by stem or progenitor cells that are not normally present in the adult host.

[0016] In one aspect of the invention, the tumor-specific antigen may be a tumor-specific neoantigen. A neoantigen is a type of tumor-specific antigen resulting from cancer-specific mutations of expressed proteins. The term "neoantigen" refers to a peptide or protein expressed by a tumor cell that contains one or more amino acid modifications compared to the corresponding wild-type (non-mutated) peptide or protein expressed by a normal (non-cancerous) cell. The neoantigen may be patient-specific. In one aspect of the invention, the tumor-specific neoantigen is a personal neoantigen encoded by one or more somatic mutations specific to a mammalian tumor, and optionally, the tumor-specific neoantigen is not a tumor-specific driver mutation.

[0017] In one aspect of the present invention, the tumor-specific antigen can be an antigen that has a tumor-specific driver mutation. A tumor-specific driver mutation is a mutation that is found in tumor cells but not in normal (non-cancerous) cells and induces cell proliferation and tumor growth. A driver mutation confers a growth advantage to the tumor cells that carry it. Examples of tumor-specific driver mutations include, but are not limited to, mutated ALK, mutated APC, mutated ATRX, mutated BRAF, mutated CDKN2A, mutated DDX3X, mutated DNMT3A, mutated EGFR, mutated ESR1, mutated EWSR1, mutated FGFR1, mutated FLI1, mutated HRAS, mutated IDH1, mutated IDH2, mutated KMT2C, mutated KRAS, mutated MYC, mutated NOTCH1, mutated NRAS, mutated PIK3CA, mutated PTCH1, mutated PTEN, mutated RB1, mutated RUNX1, mutated SETD2, mutated SMARCA4, mutated STK11, and mutated TP53.

[0018] In one aspect of the present invention, the method comprises screening tumor for the expression of tumor-specific antigen. Methods of screening tumor for the expression of antigen are known in the art. For example, screening tumor for the expression of tumor-specific antigen can comprise sequencing the whole exome, whole genome, or whole transcriptome of the cells of tumor. Sequencing can be carried out by any suitable method known in the art. Examples of sequencing technology that can be useful in the method of the present invention include next-generation sequencing (NGS) (also called "massively parallel sequencing technology") or third-generation sequencing.

[0019] In one aspect of the present invention, the method optionally includes expanding the number of isolated tumor antigen-specific T cells. The expansion of the number of T cells can be achieved by any of many methods known in the art, for example, as described in U.S. Patent No. 8,034,334; U.S. Patent No. 8,383,099; U.S. Patent No. 11,401,503; Dudley et al., J. Immunother. 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). For example, the expansion of the number of T cells is carried out by culturing T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs). In one aspect of the present invention, the method further includes expanding the number of isolated tumor antigen-specific T cells.

[0020] The method can include administering to the mammal (i) an isolated T cell and (ii) a vaccine that specifically stimulates an immune response to a tumor-specific antigen for which the isolated T cell has antigen specificity. In one aspect of the invention, the method includes administering to the mammal the T cell intravenously or intraperitoneally. In one aspect of the invention, the isolated T cell is a tumor infiltrating lymphocyte (TIL). In one aspect of the invention, the isolated T cell is a CD4 + In another aspect of the invention, the isolated T cells are CD8 + It is.

[0021] The method can include administering a pharmaceutical composition comprising isolated T cells and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers for injectable cells can include any isotonic carrier, such as, for example, normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's. In one embodiment, the pharma-ceutically acceptable carrier is supplemented with human serum albumin.

[0022] Vaccine can be any type of vaccine that specifically stimulates immune response against tumor-specific antigens.Examples of vaccines include, but are not limited to, cancer cell vaccines, conjugate polysaccharide vaccines, dendritic cell vaccines, DNA vaccines, inactivated vaccines (any type), live-attenuated vaccines, nanoparticle vaccines, peptide vaccines, protein vaccines, recombinant vaccines, RNA vaccines, subunit vaccines, and virus vaccines.Examples of virus vaccines include, but are not limited to, adenovirus (ADV) vaccines, vaccinia virus (VACV) vaccines, and avian pox virus vaccines.

[0023] In one aspect of the invention, the method comprises administering to the mammal less than a single dose of the vaccine. In another aspect of the invention, the method comprises administering to the mammal two, three, or more doses of the vaccine. In one aspect of the invention, the method comprises administering to the mammal the vaccine every other day from the first day that the T cells are administered to the mammal. In an aspect, the method can comprise administering to the mammal the vaccine intramuscularly, subcutaneously, intravenously, or intraperitoneally.

[0024] In one aspect of the invention, the method further comprises administering to the mammal an adjuvant. The adjuvant can enhance the magnitude and durability of the immune response to the tumor-specific antigen. In one aspect of the invention, the adjuvant comprises an anti-CD40 antibody or an anti-PD-1 antibody.

[0025] In one aspect of the present invention, the method comprises administering isolated T cells and a vaccine that specifically stimulates an immune response against a tumor-specific antigen within 30 days of each other.In one aspect of the present invention, the method can comprise administering isolated T cells and a vaccine within 30 days, 29 days, 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day of each other.For example, the method can comprise administering isolated T cells and a vaccine within 48 hours of each other, within 36 hours of each other, within 24 hours of each other, or within 12 hours of each other. In one aspect of the invention, the method can include administering a vaccine that specifically stimulates an immune response against a tumor-specific antigen within 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days prior to administering the isolated T cells. In one aspect of the invention, the method can include administering a vaccine that specifically stimulates an immune response against a tumor-specific antigen within 48, 36, 24, or 12 hours prior to administering the isolated T cells.

[0026] In one aspect of the invention, the isolated T cells and the vaccine are administered to the mammal simultaneously. In one aspect of the invention, the isolated T cells and the vaccine are administered to the mammal together in the same composition. In one aspect of the invention, the isolated T cells and the vaccine are administered to the mammal simultaneously but separately.

[0027] In one aspect of the present invention, the isolated T cells and the vaccine are administered to the mammal sequentially. For example, the isolated T cells can be administered to the mammal before the vaccine is administered to the mammal. In one aspect of the present invention, the isolated T cells are administered to the mammal within 24 hours before the vaccine is administered to the mammal. In one aspect of the present invention, the vaccine is administered to the mammal before the isolated T cells are administered to the mammal. For example, the isolated T cells can be administered to the mammal within 24 hours after the vaccine is administered to the mammal.

[0028] The methods of the present invention can also advantageously enhance the anti-tumor activity of T cells that are not necessarily exhausted and / or differentiated. For example, the methods of the present invention can enhance the anti-tumor activity of T cells that are modified to express an exogenous receptor, but are not necessarily exhausted and / or differentiated.

[0029] An aspect of the present invention provides a method for treating or preventing cancer in a mammal having a tumor. The method may include isolating T cells from a biological sample from a mammal having a tumor. In an aspect of the present invention, the biological sample is a tumor sample. The tumor sample may be as described herein with respect to another aspect of the present invention. The isolated T cells may be TILs. In an aspect of the present invention, the biological sample is a peripheral blood sample.

[0030] In one aspect of the invention, the T cells isolated from the biological sample may be in one or both of an exhausted and differentiated state, but preferably the T cells isolated from the biological sample are not exhausted and differentiated, but instead have a less differentiated phenotype.

[0031] The method may include introducing a nucleic acid comprising a nucleotide sequence encoding a foreign receptor having antigen specificity for a tumor-specific antigen expressed by a mammalian tumor into the isolated T cells to produce T cells expressing the foreign receptor. By "foreign" it is meant that the receptor is not native (naturally occurring) to the T cell. The tumor-specific antigen may be a tumor-specific neo-antigen or an antigen with a tumor-specific driver mutation. The antigen specificity and tumor-specific antigen may be as described herein with respect to another aspect of the invention.

[0032] In one aspect of the invention, the foreign receptor with antigen specificity for a tumor-specific antigen is a T cell receptor (TCR). The foreign TCR may be a recombinant TCR. A recombinant TCR is a TCR generated by recombinant expression of one or more foreign TCR alpha, beta, gamma, and / or delta chain encoding genes. A recombinant TCR can comprise a polypeptide chain entirely derived from a single mammalian species, or the recombinant TCR can be a chimeric or hybrid TCR composed of amino acid sequences derived from TCRs from two different mammalian species. For example, an antigen-specific TCR can comprise a variable region derived from a human TCR and a constant region from a mouse TCR, making the TCR "mouse-ized." Any foreign TCR with antigen specificity for a tumor-specific antigen is useful in the methods of the invention. A TCR generally comprises two polypeptides (i.e., polypeptide chains), such as a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or a combination thereof. Such TCR polypeptide chains are known in the art. TCR can comprise any amino acid sequence, as long as it can specifically bind to tumor-specific antigen or its epitope and immunologically recognize it.Examples of foreign TCR useful in the present method include, but are not limited to, those disclosed in the following documents: U.S. Patent No. 7,820,174; U.S. Patent No. 7,915,036; U.S. Patent No. 8,088,379; U.S. Patent No. 8,216,565; U.S. Patent No. 8,431,690; U.S. Patent No. 8,613,932; U.S. Patent No. 8,785,601; U.S. Patent No. 9,128,080; U.S. Patent No. 9,345,748; U.S. Patent No. 9,487,573; U.S. Patent No. 9,879,065; U.S. Patent No. 11,306,131 and U.S. Patent Application Publication No. 2013 / 0116167, each of which is incorporated herein by reference.

[0033] In one embodiment of the present invention, the foreign receptor is a chimeric antigen receptor (CAR). Typically, CAR comprises an antigen-binding domain of an antibody, such as a single-chain variable fragment (scFv) fused with the transmembrane and intracellular domains of a TCR. Thus, the antigen specificity of CAR can be encoded by an scFv that specifically binds to a cancer antigen or its epitope. Any CAR that has antigen specificity for a tumor-specific antigen can be useful in the method of the present invention. Examples of CARs that are useful in the method of the present invention include, but are not limited to, those disclosed in, for example, U.S. Patent Nos. 8,465,743; 9,266,960; 9,765,342; 9,359,447; 9,868,774; and 10,287,350, each of which is incorporated herein by reference.

[0034] Nucleic acid comprising a nucleotide sequence encoding a foreign receptor can be introduced into isolated T cells by any suitable technique, such as gene editing, transfection, transformation, or transduction, as described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Ed.), Cold Spring Harbor Laboratory Press (2012).Many transfection techniques are known in the art, including calcium phosphate DNA co-precipitation; DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment; and strontium phosphate DNA co-precipitation.Phage or viral vectors can be introduced into T cells after propagating infectious particles in suitable packaging cells, many of which are commercially available.

[0035] The method may optionally include expanding the number of T cells expressing the foreign receptor. Expanding the number of T cells can be performed as described herein for other aspects of the invention. In one embodiment of the invention, the method includes expanding the number of T cells expressing the foreign receptor.

[0036] The method further comprises administering to the mammal a vaccine that specifically stimulates an immune response to (i) T cells expressing the foreign receptor and (ii) a tumor-specific antigen for which the foreign receptor has antigenic specificity. The vaccine may be as described herein with respect to another aspect of the invention.

[0037] The method may include administering (i) the T cells expressing a foreign receptor and (ii) the vaccine to the mammal within 30 days of each other. In one aspect of the invention, the method may include administering the T cells expressing a foreign receptor and the vaccine within 30 days, 29 days, 28 days, 27 days, 26 days, 25 days, 24 days, 23 days, 22 days, 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day of each other. For example, the method may include administering the T cells expressing a foreign receptor and the vaccine within 48 hours of each other, within 36 hours of each other, within 24 hours of each other, or within 12 hours of each other. The administration of the T cells and the administration of the vaccine may be as described herein for other aspects of the invention. In one aspect of the invention, the method can include administering a vaccine that specifically stimulates an immune response against a tumor-specific antigen within 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day prior to administering the T cells that express a foreign receptor. In one aspect of the invention, the method can include administering a vaccine that specifically stimulates an immune response against a tumor-specific antigen within 48, 36, 24, or 12 hours prior to administering the T cells that express a foreign receptor.

[0038] The terms "treat" and "prevent" as used herein, and words derived therefrom, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that those skilled in the art recognize as having potential benefits or therapeutic effects. In this regard, the method of the present invention may provide any amount or level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the method of the present invention may include treatment or prevention of one or more cancers or symptoms of cancer that are treated or prevented. For example, the treatment or prevention may include promoting tumor regression. Also, for purposes of this specification, "prevention" may include delaying the onset of cancer, or its symptoms, conditions, or recurrence.

[0039] The cancer may advantageously be any cancer, including any of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain tumor, breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, bile duct cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, cancer of the esophagus, cancer of the cervix, gastric cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma. , hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, bladder cancer, solid tumors, and liquid tumors. Preferably, the cancer is an epithelial cancer. In one embodiment, the cancer is bile duct cancer, melanoma, colon cancer, rectal cancer, breast cancer, lung cancer, anal cancer, esophageal cancer, or gastric cancer. Preferably, the cancer expresses a tumor-specific antigen. In one aspect of the invention, the cancer is a virus-associated cancer. Virus-associated cancers include HBV, + , HCV + , H.I.V. + , HPV + , HTLV+ , HHV8 + , MCPyV + and EBV + These include, but are not limited to, related cancers.

[0040] The mammal referred to in the method can be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to rodent mammals such as mice and hamsters, and logomorpha mammals such as rabbits. Preferably, the mammal is from the order Carnivora, including felines (cats) and canines (dogs). Preferably, the mammal is from the order Artiodactyla, including bovines (cattle) and porcines (pigs), or from the order Perssodactyla, including equines (horses). Preferably, the mammal is from the order Primates, Felidae, Simianes (monkeys), or Anthropoids (humans and apes). More preferably, the mammal is a human. In a particularly preferred embodiment, the mammal is a patient expressing a tumor-specific antigen.

[0041] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0042] The following materials and methods were employed in the experiments described in Examples 1-13.

[0043] Mice and tumor lines The B16 melanoma mouse tumor line was modified by inserting the human GP100 epitope sequence into its mouse counterpart, resulting in a neoepitope that could be targeted by Pmel TCR transgenic mouse T cells (EGSRNQDWL(SEQ ID NO:1)→KVPRNQDWL(SEQ ID NO:2) (Hanada et al, JCI Insight, 4(10): e124405 (2019)).

[0044] Mice of the Pmel mouse strain, which express a T cell receptor that recognizes peptide epitopes 25-33 derived from both mouse and human gp100, were used to obtain shed T cells. The mouse tumor lines B16KVP (or "B16") (a human melanoma model) and MC38KVP (or "MC38") (a human colon cancer model) were used to test the efficacy of treatments on tumor progression. Both B16 and MC38 are derived from C57BL / 6 background mice and express antigenic epitopes recognized by Pmel-1 T cells. Wild-type C57BL / 6 mice and b2M KO mice were used for tumor treatment experiments.

[0045] B16 and MC38 cell lines were cultured in complete medium (CM): RPMI 1640 supplemented with 10% FBS, 1 mM sodium pyruvate (Thermo Fisher Scientific), 1X non-essential amino acids (Thermo Fisher Scientific), 55 μM 2-mercaptoethanol (Gibco), 1X antibiotic-antimycotic (Thermo Fisher Scientific), and 50 μg / ml gentamicin (Gibco). Modified B16 cell lines were maintained in CM supplemented with blasteidin S (10 μg / ml; Invivogen) or puromycin (5 μg / ml; Invivogen).

[0046] cell culture Pmel TCR transgenic mice were used as T cells for ACT. Spleen cells from mice were isolated by single cell suspension. Hgp100 neoepitope (KVPRNQDWL) (SEQ ID NO: 2) was added to the cell suspension at a concentration of 10 μg / mL and cultured in CM with 30 IU / ml recombinant human IL-2 (rhIL-2; Prometheus Laboratories). On day 5, cells were split and restimulated with anti-CD3 and anti-CD28 antibodies in 24-well plates. On day 10 (treatment day), exhausted T cells were obtained from Pmel mice and sorted by FACS based on CD39 expression, high co-expression of CD39 and CD69, or co-expression of PD1 and TIM3. CD39lo We obtained less exhausted T cells by sorting CD39+ / CD69-negative or CD39 / CD69-low expressing T cells.

[0047] Adoptive Cell Therapy Adoptive cell therapy (ACT) was performed as shown in Figure 2A. Pmel mice were used to obtain exhausted T cells, and tumor-bearing B16 + Either β2M KO or MC38 mice were used as recipients. Mice were inoculated with 5 × 10 5 Tumor cells were subcutaneously injected. Ten days later, tumor-bearing mice received 5 Gy total body irradiation. On the 11th day after tumor inoculation, 2 × 10 7 Mice were treated iv with exhausted or non-exhausted cultured Pmel T cells (1E6 T cells per mouse) with 1PFU of recombinant human gp100 vaccinia (rVVhgp100), or with 1E8 PFU of recombinant adenovirus expressing hgp100 neoepitopes, or with peptide vaccines (ip or iv) with anti-CD40 agonist antibodies (ip or iv). Additionally, mice were injected ip with 180,000 IU of rhIL-2 daily for 3 days after cell transfer.

[0048] ACT complementary vaccination or immunomodulatory therapy In combination with ACT, mice were injected with a combination of PBS, vaccine, antibody, and peptide, as shown in Figure 2A. The neoantigen vaccine used was a vaccinia virus against human glycoprotein 100 (r.VACV hgp100 ), vaccinia virus (VACV) against human glycoprotein 100 amino acid 25-33 epitope hgp100(25) ), and vaccinia virus (VACV) directed against an unrelated HLA-A2-restricted hgp100 epitope hgp100(209)irr. ), adenovirus against human glycoprotein 100 (ADV hgp100 ), related human glycoprotein 100 amino acid 25-33 epitope (DC+hgp100 KVPBone marrow-derived dendritic cells (BMDCs) loaded via 4-hour peptide pulsing with either DC+Irr.Pep or an irrelevant influenza virus peptide (DC+Irr.Pep) were injected intravenously (iv) once on the day of infusion at a concentration of 2E7 PFU. Anti-CD40, anti-PD1, anti-B7.1, anti-B7.2 (anti-B7.1 / 2), and isotype control (IgG) antibodies were administered intraperitoneally (ip) at 100 μg, three times every other day from the day of infusion, or intravenously (iv) once on the day of infusion. The peptides used were human glycoprotein 100 (hgp100) peptide or influenza nucleoprotein (Flu.NP) peptide, injected subcutaneously (sc) or intravenously (iv) at a concentration of 100 μg once on the day of infusion.

[0049] Tumor size assessment Tumor treatments and measurements were performed by independent investigators in a double-blind manner. Two perpendicular diameters were measured with a caliper, and tumor size was calculated as the product of the two diameters.

[0050] Survival assessment Survival rates were assessed by determining the proportion of surviving mice (n=5) on each day after ACT in each treatment group.

[0051] statistics Comparisons between groups were performed using the Wilcox rank-sum test. In the figures, NS, *, **, and *** indicate not significant, p<0.05, <0.01, and <0.001, respectively.

[0052] Example 1 This example describes the use of CD39 as a model for terminally exhausted T cells. hi The use of Pmel T cells is shown.

[0053] To establish a model of terminally exhausted T cells, mice of a line carrying a melanocyte protein (Pmel) mutation were injected with 100 μg / ml of human glycoprotein 100 (hgp100) peptide. Five days later, these mice were injected with anti-CD3 / anti-CD28 antibodies. On day 11, low levels (CD39 lo ), intermediate level (CD39 med ) or high levels (CD39 hi ) by transplanting CD39-expressing Pmel T cells, + Mice were treated with ACT. Isotype controls were used to differentiate CD39 expression. Within the CD39 gate, the bottom tertile of expression was determined as CD39 lo , the middle tertile was CD39 med , the top third was CD39 hi It was decided.

[0054] Next, we investigated the melanoma model B16 + Tumor-bearing mice were treated with transfected CD39 cells extracted from Pmel mice. med or high CD39 hi T cells were injected, followed by three injections of interleukin 2 (IL-2), and tumor growth was monitored (Figure 2A). low , CD39 med , and CD39 hi Exhausted T cells from B16 were stimulated twice in vitro and subsequently + Tumor-bearing mice were administered moderate doses of stimulated cells, 7.5e5-1e6, to test for phenotypic T cell deficiency. + T cells were isolated and classified into low (CD39) T cells by their expression levels of CD39 as shown in Figure 2B. lo ), medium (CD39 med ), high (CD39 hi ) were classified into CD39 lo The cells are CD39 hi The expression levels of TIM3 and PD1 were lower in CD39 cells than in CD39 cells. lo and CD39 hi T cells of B16 hgp100 They were co-cultured with Mel cells and 4-1BB activation was measured as an indicator of cell exhaustion.lo T cells are CD39 hi were more 4-1BB activated than T cells and therefore less exhausted (Fig. 2C).

[0055] Tumor size was monitored for several days after ACT treatment. med , CD39 hi , PBS, and bulk control treatments were B16 hgp100 However, CD39 lo Treatment significantly delayed tumor progression (Figure 3A). hi + Anti-PD1 and CD39 hi + Anti-CD40 antibody treatment inhibited CD39 hi suppressed tumor progression more than exhausted CD39 T cells alone, but not as exhausted lo These results indicate that T cells from Pmel mice serve as a valid model of exhausted T cells.

[0056] Example 2 This example shows that the hgp100 neoantigen vaccine inhibits CD39 hi We demonstrate that it can rescue exhausted anti-tumor T cells.

[0057] CD39 hi + hgp100 neoantigen vaccination (r.VACV hgp100 The ACT treatment described in this Example 1 was repeated with the addition of an additional treatment group of VACV, 2E7 PFU (1 dose); anti-CD40 antibody, 100 μg (ip), 3 doses; anti-PD1 antibody, 100 μg (ip), 3 doses. CD39 hi +r.VACV hgp100 The treatment group had a 100% chance of survival after 40 days and CD39 lo The CD39 group had an 80% chance of survival, whereas all remaining treatment groups had a 20% or lower chance of survival (Figure 4A). hi +r.VACV hgp100Treatment with CD39 was the only treatment that suppressed tumor progression and shrank tumors more effectively than all other treatment groups (Figure 4B). This result suggests that the efficacy of treatment in bulk tumors was due to the low CD39 lo This result also indicates that VACV.hgp100 iv expresses CD39 hi They also showed that it rescued Pmel anti-tumor T cells, and mice in these experiments had smaller tumors.

[0058] Example 3 This example demonstrates the testing of different neo-antigen vaccine modalities using ACT of CD39hi exhausted anti-tumor T cells.

[0059] To further test the neo-antigen vaccine modality using ACT, further experiments were performed using mice bearing large tumors of approximately 200 mm. + Mice, CD39 hi (more exhausted) or CD39 lo infusion of (less exhausted) T cells and in combination with either: (i) VACV on the day of cell infusion; 100hgp (ii) ADV on the day of cell infusion hgp100 (iii) 100 μg of anti-CD40 antibody given intraperitoneally three times every other day starting on the day of cell injection; or (iv) 100 μg of hgp100 peptide given subcutaneously (sc) once on the day of injection followed by 100 μg of anti-CD40 antibody given intraperitoneally (ip) three times every other day starting on the day of cell injection.

[0060] CD39 hi +VACV 100hgp Treatments were as follows: control, CD39 hi Compared to treatment alone, tumor progression was significantly delayed (Figure 5A). hi +ADV hgp100 Treatment with CD39 (Fig. 5B) and various anti-CD40 antibodies (Fig. 5C) both delayed tumor progression somewhat, but neither treatment with CD39 hi +VACV 100hgpThe survival rate at 50 days after ACT was significantly higher than that of CD39 lo +VACV 100hgp (80%) and CD39 hi +VACV 100hgp (60%) treatment was the most effective, but CD39 lo +ADV hgp100 (40%) and CD39 hi +ADV hgp100 (20%) treatment also increased survival compared to all other treatments (0%) (Figure 5D). lo "We used only non-exhausted T cells as a control. These results indicate that neoantigen-specific vaccines can rescue the ability of exhausted T cells to slow tumor progression and increase the chances of survival."

[0061] Example 4 This example shows that neo-antigen vaccines rescue distinct subsets of exhausted anti-tumor T cells.

[0062] Vaccine rescue is CD39 hi This is not limited to exhausted anti-tumor T cells.

[0063] Tumor-bearing mice as described in Example 1 were cultured with PD1 + TIM3 + Cells (alone or rVACV 100hgp ) or PBS (alone or in combination with rVACV 100hgp In combination with PD1 + TIM3 + Terminally exhausted neoantigen-specific T cells were also rescued by neoantigen vaccine (Figure 6B).

[0064] Tumor-bearing mice, as described in Example 1, were cultured using CD39 + CD69 + Cells (alone or rVACV 100hgp ) or PBS (alone or in combination with rVACV 100hgp The neoantigen vaccine also inhibited CD39 + CD69 +Rescued tumor progression of terminally exhausted neoantigen-specific T cells (Figure 6C).

[0065] Tumor-bearing mice, as described in Example 1, were cultured using CD39 lo Cells (alone or rVACV 100hgp ) or PBS (alone or in combination with rVACV 100hgp In combination with CD39 lo Although treatment alone inhibited tumor progression, the neoantigen vaccine also inhibited the CD39 lo Rescued less exhausted anti-tumor cells from the tumor (Figure 6A).

[0066] Example 5 This example shows that relevant neoepitopes are required for the rescue of exhausted T cells by vaccines during ACT.

[0067] To determine the neoepitopes required for the rescue of exhausted T cells, we used an unrelated HLA-A2-restricted hgp100 epitope (hgp100 209 ) and related hgp100 epitopes (hgp100 25 The tumor-bearing mice described in this Example 1 were treated with PBS alone or hgp100 209 or hgp100 25 or in combination with CD39 lo T cells alone or hgp100 209 or hgp100 25 was treated in combination with

[0068] CD39 lo Less exhausted T cells in hgp100(25) Only in combination with CD39 were tumor progression inhibited (Figure 7A). lo Treatment with CD39 T cells alone failed to significantly inhibit tumor progression in this experiment (Figure 7A). + CD69 + Exhausted neoantigen-specific T cells from the VACV-associated hgp100(25)Only in combination with IgG1 could tumor progression be suppressed (Figure 7B). These results demonstrated that the relevant neoepitopes are required in neoantigen vaccines to rescue the antitumor activity of exhausted T cells.

[0069] Example 6 This example shows that neoantigen vaccines increase CD8 + We demonstrate that IFN-γ increases the frequency of exhausted T cells in mice.

[0070] The tumor-bearing mice described in this Example 1 were cultured using CD39 lo T cells alone, rVACV hgp100 Alone or CD39 lo T cells and rVACV hgp100 (Figures 8A-8C), or CD39 + CD69 + T cells alone, rVACV hgp100 Alone or CD39 + CD69 + T cells and rVACV hgp100 and treated with a combination of the above (Figures 8D-8F).

[0071] To further explore the effect of neoantigen vaccination on different subgroups of exhausted antitumor T cells, Thy1.1 + Vβ13 + CD8 + The percentage of transferred T cells was compared to total CD8+ T cells in the spleen, draining lymph nodes, and tumors of mice following ACT.

[0072] CD39 lo rVACV following ACT infusion of less exhausted T cells hgp100 Treatment consisted of B16 on days 3, 7, and 10 after ACT. + Increased the frequency of adoptively transferred T cells in the spleen (Figure 8A), draining lymph nodes (Figure 8B) and tumors (Figure 8C) of mice. lo There is no difference in the transferred T cells 10 days after ACT when using less exhausted T cells. hi or CD39 + CD69+ The difference becomes even more apparent when T cells are used for ACT with a vaccine. + CD69 + rVACV following ACT infusion of highly exhausted T cells hgp100 Treatment also included B16 on days 3, 7, and 10 after ACT. + ACT increased the frequency of adoptively transferred T cells in the spleen (Figure 8D), draining lymph nodes (Figure 8E), and tumors (Figure 8F) of mice. The fold change in transferred CD8+ cells after ACT was significantly higher in terminally exhausted T cells than in less exhausted T cells.

[0073] Example 7 In this example, the neo-antigen vaccine is + TIM3 + Showing that the frequency of terminally exhausted T cells is reduced following ACT.

[0074] Tumor-bearing mice, as described in Example 1, were cultured using CD39 lo T cells alone or rVACV hgp100 or in combination with CD39 + CD69 + T cells alone or rVACV hgp100 was treated in combination with

[0075] To further investigate the effect of neoantigen vaccination on exhausted antitumor T cells, Thy1.1 + Vβ13 + The percentage of neoantigen-specific transferred T cells was examined in the tumors of mice after ACT. hgp100 Treatment was performed on days 3 and 10 after ACT using total viable CD8 + PD1 as a percentage of T cells + TIM3 + Adoptively transferred Thy1.1 + Vβ13 + hgp100 neoantigen-specific CD39 lo T cells and CD39 + CD69 +ACT significantly reduced the frequency of exhausted T cells in tumors after ACT (Figure 9). These results suggested that the combination of neoantigen vaccination and ACT reduced the frequency of exhausted T cells transferred in tumors after ACT.

[0076] Example 8 This example shows that intravenous vaccination with anti-CD40 antibodies and neoepitopes delays tumor progression during ACT with exhausted T cells.

[0077] Additional experiments were performed to further explore how the route of vaccine administration influences the success of vaccine-mediated rescue of exhausted T cells in ACT. Previous experiments using intraperitoneal administration of anti-CD40 antibodies in combination with peptide vaccination demonstrated that terminally exhausted CD39 T cells were rescued. + CD69 + The effect of T cells on ACT was minimal. + Intravenous administration of antibodies and peptides was tested.

[0078] As a result, vaccination reduces the risk of terminally exhausted CD39 + CD69 + The tumor-bearing mice described in this Example 1 were shown to have a delayed effect on T cell ACT (Figure 10). + CD69 + CD39 hgp100 neoantigen-specific Pmel T cells alone or with coadministration of a vaccinia virus vaccine encoding hgp100(25-33) + CD69 +hgp100 neoantigen-specific Pmel T cells were subjected to ACT with a relevant neoepitope (hgp100(25)) with or without intravenous co-administration of anti-CD40 antibody or isotype control (Rat IgG), or an irrelevant peptide (Flu.NP) with or without intravenous co-administration of anti-CD40 antibody or Rat IgG. The inability of the irrelevant peptide to rescue exhausted T cell ACT further suggests that the relevant neoepitope is a determinant of vaccine rescue of terminally exhausted and differentiated T cell-based ACT (see Flu NP vs. hgp100). Furthermore, it suggests that the route of administration of peptide or antibody, i.e., intravenous or intraperitoneal, may play a role in the success or failure of vaccine-mediated rescue of exhausted T cell ACT. The peptide and anti-CD40 antibody showed a similar effect on tumor size as the vaccinia virus vaccine, suggesting that non-vaccinia virus vaccines may also be effective in suppressing terminally exhausted CD39 + CD69 + It is suggested that this may mediate ACT rescue by T cells.

[0079] Example 9 This example shows that neoepitope-loaded dendritic cells administered as a vaccine with ACT delay tumor progression.

[0080] To investigate non-vaccinia virus vaccines, bone marrow derived dendritic cells (BMDCs) were tested. Tumor progression was measured in tumor-bearing mice as described in Example 1 treated with bulk, unsorted, transgenic hgp100 neoantigen-specific Pmel T cells alone (Pmel) or Pmel T cells co-administered with BMDCs. BMDCs were transfected with either an irrelevant influenza virus peptide (Pmel+DC+Irr.Pep) or a relevant neoepitope (Pmel+DC+hgp100). KVPPep) for 4 h. Untreated mice (PBS) served as control. Treatment with BMDCs pulsed with the relevant neoepitope significantly delayed tumor progression more than hgp100 neoantigen-specific Pmel T cells alone or co-administration with BMDCs pulsed with irrelevant peptide (P<0.01) (Figure 11). These data suggested that DC-based vaccines could also mediate tumor regression via differentiated T cell ACT.

[0081] Example 10 This example demonstrates that a neo-antigen vaccine coupled with exhausted T cell ACT delays tumor progression in a colon tumor model.

[0082] To test the efficacy of neoantigen vaccines with exhausted T cell ACT in a non-melanoma tumor model, experiments were performed to test tumor progression in MC38 colon cancer tumors expressing the hgp100KVP neoepitope. Vaccinia virus expressing the same hgp100KVP neoepitope was used as a co-neoantigen vaccine. When administered alone, the neoantigen vaccine did not significantly affect tumor progression compared to untreated (PBS) (Figure 12A). MC38 tumor progression was associated with increased expression of CD39 lo were also measured after ACT with less exhausted T cells alone or in combination with neoantigen vaccine, both of which mediated long-term tumor control (Figure 12B). + CD69 + After co-administration of Pmel transgenic T cells with neo-antigen vaccines and ACT, MC38 tumor progression was examined, and CD39 + CD69 + Tumor progression was significantly reduced compared with Pmel transgenic T cells alone (P<0.01). + CD69 + Pmel transgenic T cells alone failed to control tumors (Figure 12C). These data suggested that ACT with neoantigen vaccines mediated durable tumor regression of large tumors in both melanoma and non-melanoma tumor models.

[0083] Example 11 This example shows that antigen presentation by host cells is required for the rescue of exhausted T cells by a vaccine.

[0084] To investigate the role of antigen presentation by host cells in the rescue of exhausted T cells by neoantigen-specific vaccines, we performed experiments using β2 microglobulin knockout (β2M KO) mice. lo Tumor progression was measured after ACT with Pmel transgenic T cells alone, co-administration of T cells with a vaccinia virus vaccine encoding hgp100(25-33), vaccinia virus alone, or no treatment (PBS). lo Less exhausted neoantigen-specific T cells were not affected during ACT in β2M KO mice (Fig. S13A). However, terminally exhausted CD39 + CD69 + Similar experiments using Pmel transgenic T cells failed to rescue them with neoantigen vaccines (Fig. S13B), suggesting that antigen presentation from host cells may be required for rescue of exhausted T cell ACT.

[0085] Example 12 This example shows that vaccine rescue of exhausted T cells is affected by B7.1 / B7.2 blockade.

[0086] To further explore the mechanism of neo-antigen vaccine rescue of exhausted T cells, the role of B7.1 (CD80) and B7.2 (CD86) was examined. CD39 was upregulated by anti-B7.1 and anti-B7.2 (anti-B7.1 / 2) antibodies (100 μg) or isotype control (100 μg), or by naïve (PBS) or naïve with B7.1 / B7.2 blockade (PBS+anti-B7.1 / 2). loTumor progression was examined after ACT of Pmel transgenic T cells alone or with co-administration of neo-antigen vaccine with or without blockade of B7.1 and B7.2 (Figure 14A). + CD69 + The same experiment was performed using Pmel transgenic T cells (Figure 14B). + CD69 + Neoantigen-encoding vaccinia virus-mediated rescue of terminally exhausted neoantigen-specific T cells was significantly (P<0.01) affected by B7.1 / B7.2 blockade, suggesting that costimulation of host antigen-presenting cells (APCs) may be involved in vaccine-mediated rescue of terminally exhausted T cell ACT. B7.1 / B7.2 blockade upregulates CD39 lo There was less impact on the ACT of less exhausted neoantigen-specific T cells.

[0087] Example 13 This example provides retrospective evidence of rescue of exhausted T cells by vaccine-based therapy in human patients with TIL ACT.

[0088] Further evidence of the efficacy of exhausted T cell rescue by neoantigen vaccines was provided by the clinical course of melanoma patient 2463, who was treated with vaccine- and non-vaccine-assisted tumor infiltrating lymphocyte (TIL) therapy. Patient 2463 initially received TIL-only therapy (intravenous TIL (Rx1) and intra-arterial TIL (Rx2)). After disease progression, the patient was retreated with TIL + avian pox vaccine encoding the tumor antigen GP100. Rx1 and Rx3 refer to the first and third treatments with TIL infusion formulations, respectively. The patient received intra-arterial TIL as infusion formulation 2 (Rx2) but did not respond. Rx2 was excluded from this analysis because it was an intra-arterial TIL administration. This clinical course has been published in Smith et al, J. Immunother: 870-874 (2009) (Figure 15A).

[0089] After the first two TIL therapies failed, TIL infusions administered in the first intravenous dose (Rx1) and in the second intravenous dose (Rx3) concomitant with GP100 avian pox vaccine had comparable GP100-specific TILs by tetramer staining (Figure 15B). Numbers show HLA-A0201-restricted GP100 tetramer frequencies in Rx1 and Rx3 as a percentage of CD8+ TILs. These data suggested that TIL infusions administered with vaccines that mediated responses did not have more antitumor TILs compared to Rx1, which did not mediate clinical responses.

[0090] TCR clonal frequencies were highly correlated between Rx1 and Rx3 (Figure 15C). Labeled immunodominant GP100 TCR-1 identified from TILs of patient 2463 showed that the clonal repertoire was the same between Rx1 and Rx3, suggesting that the GP100 avian pox vaccine acted on similar frequencies of antitumor TILs in the infusion formulation.

[0091] The phenotype of antitumor GP100 TILs was comparable between Rx1 and Rx3. The phenotype of GP100 tetramer-positive antitumor TILs between Rx1 and Rx3 is based on the percentage count within the tetramer-positive TILs. Thus, avian GP100 vaccine coadministered with TILs suppressed the CD39 expression within the tetramer-positive TILs in the infusion formulation. - CD69 - The frequency of CD39 is very low. + CD69 + (Figure 15D), and the single marker CD62L + cells (Figure 15E), TIM3 - cells (Figure 15F), or CD39 - As shown in Fig. 15G, the tumor-specific activation of TILs likely affected dysfunctional antitumor TILs. This clinical case suggested that neo-antigen vaccine rescue of exhausted T cells is effective in mediating long-term control in humans.

[0092] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0093] The use of the terms "a" and "an," as well as "the" and "at least one," and similar reference words in the context of describing the invention (particularly in the context of the claims below) are to be construed as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") is to be construed as meaning one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. Reference to ranges of values ​​herein is intended to serve merely as a shorthand method for individually referring to each separate value falling within the range, and each separate value is incorporated herein as if it were individually set forth herein, unless otherwise indicated herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better illustrate the invention, and do not limit the scope of the invention unless otherwise asserted. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0094] A preferred aspect of the present invention is described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred aspects will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that such variations will be adopted by those skilled in the art as appropriate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, combinations of the above-described elements in all possible variations thereof are encompassed by the present invention, whether as described herein or unless otherwise clearly contradicted by context.

Claims

1. A set of agents for use in treating or preventing cancer in a mammal, comprising: (i) T cells isolated from a mammalian tumor sample; wherein the isolated T cells have antigen specificity for a tumor-specific antigen expressed by a tumor sample from the mammal; wherein the tumor-specific antigen is a tumor-specific neoantigen or an antigen with a tumor-specific driver mutation; and (ii) A vaccine that specifically stimulates an immune response against a tumor-specific antigen to which isolated T cells have antigen specificity, The isolated T cells express one or both of (I) and (II): (I) Any one or more of the following T cell exhaustion markers: (a) RNA encoding any one or more of the following: 4-1BB + , CCL3 + , CD28 − , CD39 + , CD62L − (SELL − ), CD69 + , CTLA4 + , CX3CR1 + , CXCL13 + , CXCR6 + , GZMA + , GZMB + , GZMK + , IL7R − , LAG-3 + , LAYN + , LEF1 − , PD-1 + , PRF1 + , TCF7 − , TIGIT + , TIM-3 + , and TOX + ; and (b) any one or more of the following proteins: 4-1BB + , CCL3 + , CD28 − , CD39 + , CD62L − (SELL − ), CD69 + , CTLA4 + , CX3CR1 + , CXCL13 + , CXCR6 + , GZMA + , GZMB + , GZMK + , IL7R − , LAG-3 + , LAYN + , LEF1 − , PD-1 + , PRF1 + , TCF7 − , TIGIT + , TIM-3 + , and TOX + ; and (II) any one or more of the following T cell differentiation markers: (a) RNA encoding any one or more of the following: CCR7 − , CD27 − , CD45RA + , CD45RO − , CD95 + , EOMES − , FOXO1 − , KLRG1 + , T-BET + , TCF7 − , TOX + , and ZEB2 + ; and (b) any one or more of the following proteins: CCR7 − , CD27 − , CD45RA + , CD45RO − , CD95 + , EOMES − , FOXO1 − , KLRG1 + , T-BET + , TCF7 − , TOX + , and ZEB2 + .

2. 2. The set of agents of claim 1, wherein the number of isolated tumor antigen-specific T cells is expanded.

3. Use of a set of agents in the preparation of a medicament or product for the treatment or prevention of cancer in a mammal, comprising: (i) T cells isolated from a biological sample from a mammal bearing a tumor; wherein a nucleic acid has been introduced into an isolated T cell, wherein the nucleic acid comprises a nucleotide sequence encoding a foreign receptor having antigen specificity for a tumor-specific antigen expressed by a mammalian tumor, and wherein the T cell expresses the foreign receptor, wherein the tumor-specific antigen is a tumor-specific neo-antigen or an antigen with a tumor-specific driver mutation; and (ii) A vaccine that specifically stimulates an immune response against a tumor-specific antigen for which a foreign receptor has antigen specificity, The set is to be administered to a mammal having a tumor.

4. 4. The use of claim 3, wherein the number of T cells expressing the foreign receptor is expanded.

5. 5. The use according to claim 3 or 4, wherein the T cells isolated from the biological sample are in one or both of an exhausted state and a differentiated state.

6. 5. The use according to claim 3 or 4, wherein the biological sample is a tumor sample.

7. 5. The set of agents according to claim 1 or 2, or the use according to claim 3 or 4, wherein the isolated T cells are tumor infiltrating lymphocytes (TILs).

8. 5. The use according to claim 3 or 4, wherein the biological sample is a peripheral blood sample.

9. 5. The use according to claim 3 or 4, wherein the exogenous receptor is a T cell receptor (TCR).

10. 5. The use according to claim 3 or 4, wherein the exogenous receptor is a chimeric antigen receptor (CAR).

11. A set of agents described in claim 1 or 2, or a use described in claim 3 or 4, wherein the isolated T cells and the vaccine are administered simultaneously to a mammal.

12. A set of agents described in claim 1 or 2, or a use described in claim 3 or 4, wherein the isolated T cells and the vaccine are administered to a mammal together in the same composition.

13. A set of agents described in claim 1 or 2, or a use described in claim 3 or 4, wherein the isolated T cells and the vaccine are administered sequentially to a mammal.

14. The set or use of the agent of claim 13, wherein the isolated T cells are administered to a mammal prior to vaccination.

15. The set or use of agents according to claim 13, wherein the vaccine is administered to the mammal before the isolated T cells.

16. The set or use of the agent of claim 13, wherein the isolated T cells are administered to the mammal within 24 hours before the vaccine is administered to the mammal.

17. The set or use of the agent of claim 13, wherein the isolated T cells are administered to the mammal within 24 hours of the vaccine being administered to the mammal.

18. 5. The set of agents according to claim 1 or 2, or the use according to claim 3 or 4, wherein the tumor-specific driver mutation is mutated ALK, mutated APC, mutated ATRX, mutated BRAF, mutated CDKN2A, mutated DDX3X, mutated DNMT3A, mutated EGFR, mutated ESR1, mutated EWSR1, mutated FGFR1, mutated FLI1, mutated HRAS, mutated IDH1, mutated IDH2, mutated KMT2C, mutated KRAS, mutated MYC, mutated NOTCH1, mutated NRAS, mutated PIK3CA, mutated PTCH1, mutated PTEN, mutated RB1, mutated RUNX1, mutated SETD2, mutated SMARCA4, mutated STK11, or mutated TP53.

19. 5. The set of agents according to claim 1 or 2, or the use according to claim 3 or 4, wherein the vaccine is a cancer cell vaccine, a conjugate polysaccharide vaccine, a dendritic cell vaccine, a DNA vaccine, an inactivated vaccine, a live-attenuated vaccine, a nanoparticle vaccine, a peptide vaccine, a protein vaccine, a recombinant vaccine, an RNA vaccine, a subunit vaccine, or a viral vaccine.

20. 5. The use according to claim 3 or 4, wherein the isolated T cells express any one or more of the following T cell exhaustion markers: (a) RNA encoding any one or more of the following: 4-1BB ＋ , CCL3 ＋ , CD28 - , CD39 ＋ , CD62L - (SELL - ), CD69 ＋ , CTLA4 ＋ , CX3CR1 ＋ , CXCL13 ＋ , CXCR6 ＋ , GZMA ＋ , GZMB ＋ , GZMK ＋ , IL7R - , LAG-3 ＋ , LAYN ＋ , LEF1 - , PD-1 ＋ , PRF1 ＋ , TCF7 - , TIGIT ＋ , TIM-3 ＋ , and TOX ＋ and (b) Any one or more of the following proteins: 4-1BB ＋ , CCL3 ＋ , CD28 - , CD39 ＋ , CD62L - (SELL - ), CD69 ＋ , CTLA4 ＋ , CX3CR1 ＋ , CXCL13 ＋ , CXCR6 ＋ , GZMA ＋ , GZMB ＋ , GZMK ＋ , IL7R - , LAG-3 ＋ , LAYN ＋ , LEF1 - , PD-1 ＋ , PRF1 ＋ , TCF7 - , TIGIT ＋ , TIM-3 ＋ , and TOX ＋ .

21. 5. The set of agents according to claim 1 or 2 or the use according to claim 3 or 4, wherein the tumor is screened for the expression of a tumor-specific antigen.

22. 5. The set of agents according to claim 1 or 2 or the use according to claim 3 or 4, wherein not more than a single dose of the vaccine is administered to the mammal.

23. 5. The set of agents according to claim 1 or 2 or the use according to claim 3 or 4, wherein two, three or more doses of the vaccine are administered to the mammal.

24. The set or use of the agent of claim 23, wherein the vaccine is administered to the mammal every other day from the first day the isolated T cells are administered to the mammal.

25. 5. The use according to claim 3 or 4, wherein the isolated T cells express any one or more of the following differentiation markers: (a) RNA encoding any one or more of the following: CCR7 - , CD27 - , CD45RA ＋ , CD45RO - , CD95 ＋ , EOMES - , FOXO1 - , KLRG1 ＋ , T-BET ＋ , TCF7 - , TOX ＋ , and ZEB2 ＋ and (b) Any one or more of the following proteins: CCR7 - , CD27 - , CD45RA ＋ , CD45RO - , CD95 ＋ , EOMES - , FOXO1 - , KLRG1 ＋ , T-BET ＋ , TCF7 - , TOX ＋ , and ZEB2 ＋ .

26. 5. The set of agents according to claim 1 or 2 or the use according to claim 3 or 4, wherein the vaccine is administered to a mammal intramuscularly, subcutaneously, intravenously or intraperitoneally.

27. ​​A set of agents described in claim 1 or 2, or a use described in claim 3 or 4, wherein the isolated T cells are administered intravenously or intraperitoneally to a mammal.

28. Isolated T cells are CD4 ＋ 5. The set of agents according to claim 1 or 2, or the use according to claim 3 or 4, which are T cells.

29. Isolated T cells are CD8 ＋ 5. The set of agents according to claim 1 or 2, or the use according to claim 3 or 4, which are T cells.

30. 5. The set of agents according to claim 1 or 2, or the use according to claim 3 or 4, wherein the mammal is a human.

31. A set of agents described in claim 1 or 2, or a use described in claim 3 or 4, wherein the isolated T cells and the vaccine are administered to a mammal within 24 hours of each other.

32. 5. The set of agents according to claim 1 or 2, or the use according to claim 3 or 4, wherein the adjuvant is administered to a mammal.

33. 33. The set or use of agents according to claim 32, wherein the adjuvant comprises an anti-CD40 antibody or an anti-PD-1 antibody.

34. 5. The set of agents according to claim 1 or 2 or the use according to claim 3 or 4, wherein the tumor-specific neoantigen is an individual neoantigen encoded by one or more somatic mutations specific to a mammalian tumor, and the tumor-specific neoantigen is not a tumor-specific driver mutation.

35. 5. The set of agents according to claim 1 or 2 or the use according to claim 3 or 4, wherein the isolated T cells are terminally differentiated.

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