Vaccinated T-cell therapy as combination immunotherapy for cancer

Abstract

The present application discloses a method of treating or preventing cancer in a mammal, the method comprising: (a) isolating T cells from a tumor sample from the mammal, where the isolated T cells are depleted and / or differentiated and have antigen specificity for tumor-specific antigens expressed by the tumor sample from the mammal, and wherein the isolated T cells have antigen specificity for tumor-specific antigens expressed by the tumor sample from the mammal; wherein the tumor-specific antigen is a tumor-specific neoantigen or an antigen having a tumor-specific driver mutation; and optionally increasing the number of the 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, the isolated T cells having antigen specificity for the tumor-specific antigen.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 295,762, filed on December 31, 2021, which is incorporated herein by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This application was made with government support from the National Institutes of Health, National Cancer Institute under Grant No. ZIABC010985. The government has certain rights in this application.

[0005] Electronically Submitted Materials Incorporated by Reference

[0006] Incorporated herein by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing filed concurrently with this document, which is identified as follows: a 2,650-byte XML file named "766239.xml" and dated December 28, 2022. Background of the Invention

[0008] Adoptive cell therapy (ACT) using T cells targeting tumor-specific antigens can produce positive clinical responses in some patients. However, there are still several obstacles to the successful treatment of cancer and other conditions using ACT. For example, the phenotype of exhausted anti-tumor T cells may not be able to generate a sustained immune response to the tumor. Therefore, there is a need to improve immunotherapy for cancer. Summary of the invention

[0009] One aspect of the present application provides a method for treating or preventing cancer in a mammal, the method comprising: (a) isolating T cells from a tumor sample from a mammal, wherein the isolated T cells are exhausted and / or differentiated, 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 neoantigen or an antigen with a tumor-specific driver mutation; and optionally expanding the number of the 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 a tumor-specific antigen, wherein the isolated T cells have antigen specificity for the tumor-specific antigen.

[0010] One aspect of the present application provides a method for treating or preventing cancer in a mammal suffering from a tumor, the method comprising: (a) isolating T cells from a biological sample of the mammal suffering from the tumor; (b) introducing a nucleic acid comprising a nucleotide sequence encoding an exogenous receptor into the isolated T cells, wherein the exogenous receptor has antigenic specificity for a tumor-specific antigen expressed by the tumor of the mammal, to produce T cells expressing the exogenous receptor, wherein the tumor-specific antigen is a tumor-specific neoantigen or an antigen with a tumor-specific driver mutation; and optionally expanding the number of T cells expressing the exogenous receptor; and (c) administering to the mammal (i) T cells expressing the exogenous receptor of (b) and (ii) a vaccine that specifically stimulates an immune response against the tumor-specific antigen, wherein the exogenous receptor has antigenic specificity for the tumor-specific antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a flow chart showing the steps of combining neoantigen-specific T cell ACT with neoantigen vaccination according to one aspect of the present application.

[0012] Figure 2A According to one aspect of the present application, an example is given in B16 + Schematic representation of the timeline of neoantigen-specific T cell ACT in combination with neoantigen vaccination in mice.

[0013] Figure 2B Flow cytometry images are shown, which show that the expression level of cluster of differentiation 39 (CD39) after 2 stimulations is divided into low (CD39 lo ), middle (CD39 med ) and high(CD39 hi ) + The number of T cells.

[0014] Figure 2C CD39 lo and CD39 hi Heat map of PD-1 and TIM3 expression in T cells and CD39 lo and CD39 hi Diagram of 4-1BB activation in T cells.

[0015] Figure 3A-3B It is shown that PBS, control (bulk) Pmel + T cells (control), with high levels (CD39 hi ), medium level (CD39 med ) and low levels (CD39 lo ) Pmel expressing CD39 + T cells ( Figure 3A) treated mice, as well as in PBS, control Pmel + T cells (control), CD39 lo 、CD39 hi +Isotype control antibody (IgG), CD39 hi + Anti-CD40 antibody or CD39 hi + Anti-PD1 Antibody( Figure 3B )-treated mice, B16 + Mouse tumor size (mm 2 )’s picture.

[0016] Figure 4A It shows that PBS and Pmel + T cells (control Pmel), low expression of CD39 Pmel + T cells (CD39 lo )+isotype control antibody (rat IgG), high expression of CD39 Pmel + T cells (CD39 hi )+anti-CD40 antibody, CD39 hi + Anti-PD1 antibody or CD39 hi + related vaccinia virus (r.VACV) targeting human glycoprotein 100 epitope sequence KVPRNQDWL (SEQ ID NO: 2) hgp100 )-treated group (n=5), B16 + Plot of mouse survival probability.

[0017] Figure 4B PBS, control, CD39 lo 、CD39 hi +IgG, CD39 hi + Anti-CD40 antibody, CD39 hi + Anti-PD1 antibody or CD39 hi +r.VACV hgp100 In treated mice, B16 + Tumor size of mice (mm 2 )’s picture.

[0018] Figures 5A-5C To show that mice treated with the following alone or in various combinations had increased B16 expression within days after ACT + Tumor size of mice (mm 2 ) as shown in the figure: PBS, low expression of CD39 Pmel + T cells (CD39 lo ), high expression of CD39Pmel + T cells (CD39hi ), isotype control antibody (IgG), human glycoprotein 100 peptide (Pep), vaccinia virus against human glycoprotein 100 (VACV hgp100 )( Figure 5A ), adenovirus against human glycoprotein 100 (ADV hgp100 )( Figure 5B ) or anti-CD40 antibody ( Figure 5C ).

[0019] Figure 5D It is shown that low expression of CD39 Pmel + T cells (CD39 lo ) or high expression of CD39 Pmel + T cells (CD39 hi ) and (VACV hgp100 ) or ADV hgp100 B16 in combination therapy + Plot of the probability of survival of mice (n=5) over several days following ACT.

[0020] Fig. 6A It is shown that PBS alone or low expression of CD39 Pmel + In mice treated with T cells or in combination with a related vaccinia virus targeting human glycoprotein 100 (r.VACVhgp100), B16 + Mouse tumor size (mm 2 )'s graph( Fig. 6A ).

[0021] Figure 6B It is shown that PBS alone or expressing Pmel + PD1 and TIM3 (PD1 + TIM3 + ) is a graph of tumor size in mice treated with or in combination with r.VACVhgp100.

[0022] Figure 6C It is shown that PBS alone or expressing Pmel + CD39 and CD69 on T cells (CD39 + CD69 + ) is a graph of tumor size in mice treated with or in combination with r.VACVhgp100.

[0023] Figure 7A-7B To show that PBS alone or CD39 Pmel + T cells (CD39 lo ) treatment or with vaccinia virus (VACV) directed against an unrelated HLA-A2 restricted human glycoprotein 100 (hgp100) epitopehgp100(209).irr ) or against the related hgp100 epitope 25 (hgp100 25 VACV hgp100(25) ) combination therapy ( Fig. 7A ), or use it alone to express Pmel + CD39 and CD69 (CD39 + CD69 + ) Treatment or with VACV hgp100(209).irr or VACV hgp100(25) Combination therapy ( Figure 7B ) mice, B16 + Tumor size of mice (mm 2 )’s picture.

[0024] Figures 8A-8F It shows that low expression of CD39 Pmel + T cells (CD39 lo ), CD39 lo and a related vaccinia virus directed against human glycoprotein 100 (rVACVhgp100) or rVACV alone hgp100 Therapeutic B16 + Mouse spleen ( Fig. 8A )、Draining lymph nodes (LN) Figure 8B ) and tumors ( Figure 8C ) in Thy1.1 at 3, 7, and 10 days after ACT + Vβ13 + CD8 + T cells account for the total CD8 + Bar graph showing the percentage of T cells and the expression of Pmel + CD39 and CD69 (CD39 + CD69 + ), CD39 + CD69 + and rVACV hgp100 or rVACV only hgp100 Therapeutic B16 + Mouse spleen ( Fig.8D )、Drainage LN( Fig. 8E ) and tumors ( Figure 8F ) in Thy1.1 at 3, 7, and 10 days after ACT + Vβ13 + CD8 + T cells account for the total CD8 + Bar graph of percentage of T cells.

[0025] Fig. 9Three bar graphs are shown, which show the expression of low CD39 Pmel + T cells (CD39 lo )、(CD39 lo ) and related vaccinia viruses targeting human glycoprotein 100 (rVACVhgp100), expressing Pmel + CD39 and CD69 (CD39 + CD69 + ), or CD39 + CD69 + and rVACV hgp100 Therapeutic B16 + In mice, terminally exhausted PD1 isolated from tumors at days 3, 7, and 10 after ACT + TIM3 + Adoptively transferred Thy1.1 + Vβ13 + The percentage of neoantigen-specific T cells.

[0026] Fig.10 It is shown that PBS alone or expressing Pmel + CD39 and CD69 (CD39 + CD69 + ) treatment or with an unrelated peptide influenza nucleoprotein (Flu.NP), targeting the related hgp100 epitope 25 (hgp100 25 VACV hgp100(25) In mice treated with various combinations of one or more of ), anti-CD40 antibody, and isotype control antibody (rat IgG), B16 + Tumor size of mice (mm 2 )’s picture.

[0027] Fig.11 It is shown that PBS or Pmel alone + T cells were either treated with unrelated influenza peptides (Irr.Pep) or related neoepitopes (hgp100 KVP In mice treated with a combination of bone marrow-derived dendritic cells (DCs) and ACT, B16 + Tumor size of mice (mm 2 )’s picture.

[0028] Figures 12A-12C To show the efficacy of PBS alone or in combination with vaccinia virus (VACV hgp100 )Combination therapy( Fig. 12A ) or low expression of CD39 Pmel alone + T cells (CD39lo ) or with VACV hgp100 Combination therapy ( Fig. 12B ) or expressed as Pmel alone + CD39 and CD69 (CD39 + CD69 + ) or with VACV hgp100 Combination therapy ( Fig. 12C ) mice, the MC38 tumor size (mm) of C57BL / 6 mice within a few days after ACT 2 )’s picture.

[0029] Figures 13A-13B To show that PBS alone or in combination with hgp100 25 Vaccinia virus (VACV hgp100(25) ) combined treatment, and low expression of CD39 Pmel + T cells (CD39 lo ) or with VACV hgp100(25) combination( Fig.13A ) or treated with Pmel + CD39 and CD69 (CD39 + CD69 + ) or with VACV hgp100(25) Tumor size (mm) in β2M knockout (KO) mice in combination-treated mice within days after ACT 2 )’s picture.

[0030] Figures 14A-14B To show the B16KVP tumor size (mm) in C57BL / 6 mice treated with the following alone or in various combinations within days after ACT 2 ), as shown: PBS, anti-B7.1 and anti-B7.2 antibodies (anti-B7.1 / 2), isotype control antibody (IgG), 25 Vaccinia virus (VACV hgp100(25) ), and low expression of CD39Pmel + T cells (CD39 lo )( Fig.14A ), or express Pmel + CD39 and CD69 (CD39 + CD69 + )( Fig. 14B ).

[0031] Fig.15A is a flow chart illustrating the treatment process for a patient.

[0032] Fig. 15BFlow cytometry graphs showing the frequency of HLA-A0201-restricted GP100 tetramers as a percentage of CD8+ TILs in the infusion products (Rx1 and Rx3) are shown, indicating that there was no significant difference in the frequency of GP100 TILs administered to patients during the first intravenous treatment without vaccine (Rx1) and the second intravenous treatment combined with GP100 vaccine (Rx3).

[0033] Fig. 15C Scatter plots of the clonal frequencies of GP100 TCRs within Rx1 and Rx3 are shown, demonstrating that there were no apparent differences in the clonal distribution of therapeutic products labeled with the immunodominant GP100 TCR.

[0034] Figures 15D-15G Heatmaps of GP100 TILs in the infusion products (Rx1 and Rx3) are shown, indicating that there is no clear difference in the phenotypic status of the frequencies of GP100 TILs administered to patients during Rx1 and Rx3, with plots showing the expression of CD39 and CD69 ( Fig.15D ), CD62L and CD8( Fig.15E ), TIM3 and CD8( Fig.15F ) and CD39 and CD8 ( Figure 15G Thus, both Rx1 and Rx3 comprise similarly differentiated, dysfunctional antitumor TILs that were able to induce tumor regression only when the vaccine was administered in the presence of Rx3, but not in the absence of the vaccine (Rx1). DETAILED DESCRIPTION

[0035] Anti-cancer ACT involves in vitro expansion and in vivo administration of autologous anti-tumor T cells targeting the patient's own tumor. Anti-tumor T cells, such as those targeting tumor-specific mutations ("neoantigens"), may be in a terminally differentiated, exhausted state. These exhausted anti-tumor T cells may have limited efficacy against established tumors during ACT, and may have limited persistence in the patient's body after treatment. Conventional cell therapy using anti-tumor T cells may not produce a significant response. It has been found that combined immunotherapy includes (i) exhaustion and / or differentiation and T cells with antigen specificity for tumor-specific antigens, and (ii) vaccines that specifically stimulate immune responses to tumor-specific antigens, which can produce excellent anti-tumor treatments in vivo. Administration of vaccines that specifically stimulate immune responses to tumor-specific antigens can enhance the use of exhausted and / or differentiated T cells with antigen specificity for the same tumor-specific antigens. The anti-tumor effect of this combined immunotherapy may be superior to the anti-tumor effect mediated by ACT using vaccines alone or using exhausted and / or differentiated T cells alone. Therefore, the use of vaccines that specifically stimulate immune responses against the same tumor-specific antigens can synergistically enhance ACT using exhausted and / or differentiated T cells or genetically engineered (TCR-transduced T cells). The methods of the present application can advantageously overcome the challenges of developing effective immunotherapies using anti-tumor T cells with an exhausted phenotype, which otherwise may not be able to generate a sustained immune response to the tumor.

[0036] The methods of the present application can advantageously target metastatic cancers, such as epithelial cancers, which cause more than about 90% of all cancer deaths. These cancers may not respond to conventional immunotherapy and may not respond to anti-tumor vaccines as a single agent.

[0037] By combining a vaccine with the administration of exhausted and / or differentiated T cells, the method of the present application can save the anti-tumor activity of exhausted and / or differentiated T cells. In this regard, one aspect of the present application provides a method for treating or preventing mammalian cancer. The method may include separating T cells from a tumor sample from a mammal. The tumor sample may be, for example, a tissue from a primary tumor or a tissue from a metastatic tumor site. Therefore, a tumor sample may be obtained by any suitable means, including but not limited to aspiration, biopsy or excision.

[0038] The isolated T cells can be exhausted and / or differentiated. T cell exhaustion is a state of T cell dysfunction that occurs in response to chronic antigen stimulation. It is defined as poor effector function, persistent expression of inhibitory receptors, and a phenotype different from the phenotype of functional effector or memory T cells. As described herein, by combining exhausted T cells with a vaccine targeting the same antigen targeted by the exhausted T cells, the adverse effector functions commonly exhibited by the exhausted T cells can be improved or overcome in the present application method. In one aspect of the present application, 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 + As used herein, the symbol " + , ", including "high" in T cell exhaustion markers (" hi ”) and “medium” (“ med ”) expression, and means that the cell upregulates expression of the indicator marker compared to a less exhausted T cell. Upregulated expression can encompass, for example, a quantitative increase in expression of a T cell exhaustion indicator marker by 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 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 “ - ,” encompasses both lack of expression and “low” expression of an indicator marker (“ lo”), and means that the cell downregulates expression of an indicator marker compared to a less exhausted T cell. Downregulated expression can encompass, for example, a quantitative decrease in an indicator marker of T cell exhaustion by 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 -300, about -310, about -320, about -330, about -340, about -350, about -360, about -370 0, 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. Isotype controls can be used to distinguish marker expression. Within a gating for a given marker, the bottom tertile of expression can be designated as " lo ”, the middle tertile group can be designated as “ med ”, while the upper tertile can be designated as “ hi ”.

[0039] T cell differentiation refers to the process in which precursor cells acquire more mature T cell characteristics. T cell differentiation is linearly progressed along the continuum of the main cluster (for example, progressed in the following order: immature T cells (TN), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM) and terminal effector (TTE) cells), wherein the cells with lower differentiation respond to antigen stimulation and produce more offspring that differentiate. With the increase of differentiation, memory T cells gradually acquire or lose specific functions. For example, T cell differentiation increases and is considered to have a negative impact on the ability of T cells to function in vivo. As described herein, by combining differentiated T cells with vaccines targeting the same antigens targeted by differentiated T cells, the adverse effector functions that differentiated T cells usually show can be improved or overcome in the present application method. In one aspect of the present application, the T cells separated are terminally differentiated.

[0040] In one aspect of the present application, 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 + . As used herein, the symbol "+," for expression of a T cell differentiation indicator marker encompasses "high" ("hi") and "med" ("med") expression of a T cell differentiation indicator marker, and means that the cell upregulates expression of the indicator marker compared to a less differentiated T cell. Upregulated expression can encompass, for example, a quantitative increase in expression of an indicator marker for T cell differentiation by the following average log fold change (based on 2): 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 a marker indicative of T cell differentiation encompasses both the absence of expression and "low" expression of the marker. lo”), and means that the cell downregulates expression of an indicator marker compared to a less differentiated T cell. Downregulated expression can encompass, for example, a quantitative decrease in expression of an indicator marker of T cell differentiation by the following mean log fold change (base 2): 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 -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. Isotype controls can be used to distinguish marker expression, as described herein for other aspects of the application.

[0041] The isolated T cells may have antigen specificity for a tumor-specific antigen expressed by a tumor sample from a mammal. As used herein, the phrase "antigen-specific" means that a T cell can specifically bind to and immunologically recognize an antigen or an epitope thereof, such that the binding of the T cell to the antigen or an epitope thereof triggers an immune response.

[0042] As used herein, the term "tumor-specific antigen" or "tumor antigen" refers to any molecule (e.g., protein, polypeptide, peptide, lipid, carbohydrate, etc.) that is expressed or overexpressed solely or primarily by tumor cells, so that the antigen is associated with a tumor. Tumor-specific antigens can also be expressed by normal, non-tumor or non-cancerous cells. However, in this case, the expression of tumor-specific antigens of normal, non-tumor or non-cancerous cells is not as strong as that of tumors. In this regard, tumor cells can overexpress antigens, or express antigens at significantly higher levels, compared to the antigen expression of normal, non-tumor or non-cancerous cells. In addition, tumor-specific antigens can also be expressed by cells of different developmental or mature states. For example, tumor-specific antigens can also be expressed by cells in the embryonic or fetal stage, which are not usually present in adult hosts. Alternatively, tumor-specific antigens can also be expressed by stem cells or precursor cells, which are not usually present in adult hosts.

[0043] In one aspect of the present application, a tumor-specific antigen may be a tumor-specific neoantigen. Neoantigens are a class of tumor-specific antigens produced by cancer-specific mutations in expressed proteins. The term "neoantigen" relates to a peptide or protein expressed by a tumor cell including one or more amino acid modifications compared to a corresponding wild-type (non-mutated) peptide or protein expressed by a normal (non-cancerous) cell. Neoantigens may be patient-specific. In one aspect of the present application, a tumor-specific neoantigen is an individualized neoantigen encoded by one or more somatic mutations unique to a mammalian tumor, optionally wherein the tumor-specific neoantigen is not a tumor-specific driver mutation.

[0044] In one aspect of the present application, a tumor-specific antigen can be an antigen with a tumor-specific driver mutation. A tumor-specific driver mutation is a mutation that is present in tumor cells but not present in normal (non-cancerous) cells, and it induces cell proliferation and tumor growth. Driver mutations confer growth advantages to tumor cells carrying them. 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.

[0045] In one aspect of the present application, the method includes screening for tumors expressing tumor-specific antigens. Methods for screening tumors expressing antigens are known in the art. For example, screening for tumors expressing tumor-specific antigens may include sequencing the whole exome, whole genome or whole transcriptome of tumor cells. Sequencing may be performed in any suitable manner known in the art. Examples of sequencing techniques that can be used in the present application method include next generation sequencing (NGS) (also referred to as "massively parallel sequencing technology") or third generation sequencing.

[0046] In one aspect of the present application, the method optionally includes amplifying the number of tumor antigen-specific T cells isolated. The amplification of the number of T cells can be achieved by any of a variety of methods known in the art, such as described in, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent 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 number of T cells is amplified by culturing T cells with OKT3 antibodies, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs). In one aspect of the present application, the method also includes amplifying the number of tumor antigen-specific T cells isolated.

[0047] The method may include administering to a mammal (i) separated T cells and (ii) a vaccine that specifically stimulates an immune response to a tumor-specific antigen, wherein the separated T cells have antigen specificity for the tumor-specific antigen. In one aspect of the present application, the method includes administering the T cells to a mammal intravenously or intraperitoneally. In one aspect of the present application, the separated T cells are tumor infiltrating lymphocytes (TIL). In one aspect of the present application, the separated T cells are CD4 + In another aspect of the present application, the isolated T cells are CD8 + .

[0048] The method may include administering a pharmaceutical composition comprising isolated T cells and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier for the cells to be injected may include any isotonic carrier, such as normal saline (water containing about 0.90% w / v NaCl, water containing about 300 mOsm / L NaCl, or water containing about 9.0 g NaCl per liter), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), water containing about 5% glucose, or Ringer's lactate. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0049] The vaccine can be any type of vaccine that specifically stimulates an immune response to a tumor-specific antigen. Examples of vaccines include, but are not limited to, cancer cell vaccines, conjugated polysaccharide vaccines, dendritic cell vaccines, DNA vaccines, inactivated vaccines (of any type), live attenuated vaccines, nanoparticle vaccines, peptide vaccines, protein vaccines, recombinant vaccines, RNA vaccines, subunit vaccines, and viral vaccines. Examples of viral vaccines include, but are not limited to, adenovirus (ADV) vaccines, vaccinia virus (VACV) vaccines, and fowl pox virus vaccines.

[0050] In one aspect of the present application, the method includes administering a vaccine of no more than a single dose to a mammal. In other aspects of the present application, the method includes administering two, three or more doses of vaccine to a mammal. In one aspect of the present application, the method includes administering a vaccine to a mammal every other day starting from the first day of administering T cells to a mammal. In some aspects, the method may include administering a vaccine to a mammal by intramuscular, subcutaneous, intravenous or intraperitoneal means.

[0051] In one aspect of the present application, the method further comprises administering an adjuvant to the mammal. The adjuvant can enhance the strength and persistence of the immune response to the tumor-specific antigen. In one aspect of the present application, the adjuvant comprises an anti-CD40 antibody or an anti-PD-1 antibody.

[0052] In one aspect of the application, the method is included in the vaccine of the immunne response of the T cell and specific stimulation for tumor-specific antigens used in 30 days of each other. In one aspect of the application, the method can include using the T cell and vaccine of separation in 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 include using the T cell and vaccine of separation in 48 hours, in 36 hours, in 24 hours or in 12 hours of each other. In one aspect of the application, the method may include administering a vaccine that specifically stimulates an immune response to a tumor-specific antigen within 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 before administering the separated T cells. In one aspect of the application, the method may include administering a vaccine that specifically stimulates an immune response to a tumor-specific antigen within 48 hours, 36 hours, 24 hours, or 12 hours before administering the separated T cells.

[0053] In one aspect of the present application, the separated T cells and the vaccine are applied to mammals simultaneously. In one aspect of the present application, the separated T cells and the vaccine are applied to mammals together in the same composition. In one aspect of the present application, the separated T cells and the vaccine are applied to mammals simultaneously but separately.

[0054] In one aspect of the present application, the separated T cells and the vaccine are sequentially applied to mammals. For example, the separated T cells can be applied to mammals before the vaccine is applied to mammals. In one aspect of the present application, the separated T cells are applied to mammals within 24 hours before the vaccine is applied to mammals. In one aspect of the present application, before the separated T cells are applied to mammals, the vaccine is applied to mammals. For example, the separated T cells can be applied to mammals within 24 hours after the vaccine is applied to mammals.

[0055] The method of the present application can also advantageously enhance the anti-tumor activity of T cells that are not necessarily exhausted and / or differentiated. For example, the method of the present application can also enhance the anti-tumor activity of T cells that have been modified to express exogenous receptors, but are not necessarily exhausted and / or differentiated.

[0056] One aspect of the present application provides a method for treating or preventing cancer in a mammal with a tumor. The method may include separating T cells from a biological sample of a mammal with a tumor. In one aspect of the present application, the biological sample is a tumor sample. The tumor sample may be as described herein with respect to other aspects of the present application. The separated T cells may be TILs. In one aspect of the present application, the biological sample is a peripheral blood sample.

[0057] Although in aspects of the present application, T cells isolated from a biological sample may be exhausted and / or differentiated, preferably, T cells isolated from a biological sample are not exhausted or differentiated, but may have a less differentiated phenotype.

[0058] The method may include introducing a nucleic acid comprising a nucleotide sequence encoding an exogenous receptor into an isolated T cell to produce a T cell expressing an exogenous receptor, wherein the exogenous receptor has antigen specificity for a tumor-specific antigen expressed by a mammalian tumor. "Exogenous" means that the receptor is not native to the T cell (not naturally occurring). The tumor-specific antigen may be a tumor-specific neoantigen or an antigen with a tumor-specific driver mutation. Antigen specificity and tumor-specific antigens may be as described herein with respect to other aspects of the present application.

[0059] In one aspect of the present application, an exogenous receptor with antigen-specificity for a tumor-specific antigen is a T cell receptor (TCR). Exogenous TCR may be a recombinant TCR. Recombinant TCR is a TCR produced by recombinantly expressing one or more exogenous TCR α-, β-, γ- and / or δ-chain encoding genes. Recombinant TCR may include a polypeptide chain completely derived from a single mammalian species, or recombinant TCR may be a chimeric or hybrid TCR composed of an amino acid sequence of a TCR derived from two different mammalian species. For example, an antigen-specific TCR may include a variable region derived from a human TCR and a constant region of a mouse TCR, so that the TCR is "murinized". Exogenous TCRs with antigen-specificity for tumor-specific antigens may be used in the methods of the present application. TCRs generally include two polypeptides (i.e., polypeptide chains), such as an α chain of a TCR, a β chain of a TCR, a γ chain of a TCR, a δ chain of a TCR, or a combination thereof. Such TCR polypeptide chains are known in the art. TCRs may include any amino acid sequence, provided that the TCR is able to specifically bind to and immunorecognize a tumor-specific antigen or an epitope thereof. Examples of exogenous TCRs that can be used in the methods of the present application include, but are not limited to, those disclosed in, for example, U.S. Patents 7,820,174; 7,915,036; 8,088,379; 8,216,565; 8,431,690; 8,613,932; 8,785,601; 9,128,080; 9,345,748; 9,487,573; 9,879,065; 11,306,131 and U.S. Patent Application Publication No. 2013 / 0116167, each of which is incorporated herein by reference.

[0060] In one embodiment of the present application, the exogenous 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), which is fused to the transmembrane domain and intracellular domain of TCR. Therefore, the antigen specificity of CAR can be encoded by a scFv that specifically binds to a cancer antigen or its epitope. Any CAR with antigen specificity for a tumor-specific antigen can be used in the method of the present application. Examples of CARs that can be used in the method of the present application include, but are not limited to, those disclosed in, for example, U.S. Patents 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.

[0061] Nucleic acids comprising nucleotide sequences encoding exogenous receptors can be introduced into isolated T cells by any suitable technique (such as, for example, gene editing, transfection, transformation or transduction, as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press (2012)). Various transfection techniques are known in the art and include, for example, calcium phosphate DNA coprecipitation; DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment; and strontium phosphate DNA coprecipitation. Phage or viral vectors can be introduced into T cells after the infectious particles are grown in suitable packaging cells, many of which are commercially available.

[0062] The method may optionally include expanding the number of T cells expressing the exogenous receptor. The number of T cells expanded can be performed as described herein with respect to other aspects of the application. In one embodiment of the application, the method includes expanding the number of T cells expressing the exogenous receptor.

[0063] The method also includes administering to the mammal (i) T cells expressing exogenous receptors and (ii) a vaccine that specifically stimulates an immune response against a tumor-specific antigen, wherein the exogenous receptor has antigenic specificity for the tumor-specific antigen. The vaccine may be as described herein with respect to other aspects of the present application.

[0064] The method may include administering (i) T cells and (ii) vaccines expressing exogenous receptors to mammals within 30 days of each other. In one aspect of the application, the method may include administering T cells and vaccines expressing exogenous receptors 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 T cells and vaccines expressing exogenous receptors within 48 hours, within 36 hours, within 24 hours or within 12 hours of each other. The administration of T cells and the administration of vaccines may be as described herein with respect to other aspects of the application. In one aspect of the present application, the method may include administering a vaccine that specifically stimulates an immune response to a tumor-specific antigen within 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 before administering T cells expressing exogenous receptors. In one aspect of the present application, the method may include administering a vaccine that specifically stimulates an immune response to a tumor-specific antigen within 48 hours, 36 hours, 24 hours, or 12 hours before administering T cells expressing exogenous receptors.

[0065] As used herein, the terms "treatment" and "prevention" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. On the contrary, there are different degrees of treatment or prevention, which are considered by those of ordinary skill in the art to have potential benefits or therapeutic effects. In this regard, the method of the present application can provide any amount or any level of cancer treatment or prevention in mammals. In addition, the treatment or prevention provided by the method of the present application may include treating or preventing one or more cancers or symptoms of the cancer being treated or prevented. For example, treatment or prevention may include promoting tumor regression. In addition, for the purposes of this article, "prevention" may include delaying the occurrence of cancer, or its symptoms, conditions or recurrence.

[0066] Advantageously, the cancer can be any cancer, including acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nose cancer, nasal cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, bile duct cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, stomach cancer, gastrointestinal carcinoid tumor, nerve cancer, Through glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer (such as non-small cell lung cancer), malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental and 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, cancer is epithelial cancer. In one embodiment, cancer is bile duct cancer, melanoma, colon cancer, rectal cancer, breast cancer, lung cancer, anal cancer, esophageal cancer or gastric cancer. Preferably, cancer expresses tumor-specific antigens. In one aspect of the present application, cancer is a virus-related cancer. Virus-related cancers include but are not limited to HBV+, HCV+, HIV+, HPV+, HTLV+, HHV8+, MCPyV+ and EBV+ related cancers.

[0067] The mammal mentioned in the method of the present application 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 lagomorpha 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 (cows) and suids (pigs) or the order Perssodactyla, including equines (horses). Preferably, the mammal is a primate, ape (Ceboids) order or monkey (Simoids) order (monkeys) or anthropoid (Anthropoids) order (humans and apes). A more preferred mammal is a human. In a particularly preferred embodiment, the mammal is a patient expressing a tumor-specific antigen.

[0068] The following examples further illustrate the present application but, of course, should not be construed as limiting its scope in any way.

[0069] Example

[0070] The experiments described in Examples 1-13 utilized the following materials and methods.

[0071] Mice and tumor cell lines

[0072] The B16 melanoma mouse tumor line was modified by inserting a human GP100 epitope sequence into its mouse counterpart, making it a neo-epitope (EGSRNQDWL (SEQ ID NO: 1) -> KVPRNQDWL (SEQ ID NO: 2)) that can be targeted by Pmel TCR transgenic mouse T cells (Hanada et al., JCI Insight, 4(10): e124405 (2019)).

[0073] Use mice from Pmel mouse line to obtain exhausted T cells, the mice express T cell receptors that recognize peptide epitopes 25-33 derived from mouse and human gp100. Mouse tumor line B16KVP (or "B16") (human melanoma model) and MC38KVP (or "MC38") mouse tumor line (human colon cancer model) are used to test the efficacy of treatment on tumor progression. B16 and MC38 are both derived from C57BL / 6 background mice and express antigenic epitopes that can be recognized by Pmel-1 T cells. Wild-type C57BL / 6 and b2M KO mice are used in tumor treatment experiments.

[0074] B16 and MC38 cell lines were cultured in complete medium (CM): RPMI 1640 containing 10% FBS, 1 mM sodium pyruvate (Thermo Fisher Scientific), 1× non-essential amino acids (Thermo Fisher Scientific), 55 μM 2-mercaptoethanol (Gibco), 1× antibiotic-antimycotic (Thermo Fisher Scientific), and 50 μg / ml gentamicin (Gibco). The modified B16 cell line was maintained in CM containing blasticidin S (10 μg / ml; Invivogen) or puromycin (5 μg / ml; Invivogen).

[0075] Cell culture

[0076] Pmel TCR transgenic mice were used as T cells for ACT. Splenocytes 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 10ug / mL and cultured in CM containing 30IU / ml recombinant human IL-2 (rhIL-2; Prometheus Laboratories). On the fifth day, cells were isolated in 24-well plates and restimulated with anti-CD3 and anti-CD28 antibodies. On the 10th day (treatment day), exhausted T cells were obtained from Pmel mice and sorted by FACS based on CD39 expression, high CD39 and CD69 co-expression, or PD1 and TIM3 co-expression. By sorting CD39 lo and negative T cells, or by sorting CD39 / CD69 low-expressing T cells to obtain less exhausted T cells.

[0077] Adoptive cell therapy

[0078] Adoptive cell therapy (ACT) is based on Figure 2A Pmel mice were used to obtain exhausted T cells, and B16 tumor-bearing mice were used + , MC38 or β2M KO mice were used as recipients. Mice were injected subcutaneously with 5×10 5 Ten days later, the tumor-bearing mice received 5 Gy of whole-body irradiation. On day 11 after tumor inoculation, the mice were treated with cultured Pmel T cells (1E6 T cells per mouse) that were either depleted or less depleted (with or without 2 × 10 7 Mice were treated with 1E8 PFU of recombinant human gp100-vaccinia (rVVhgp100), or with 1E8 PFU of recombinant adenovirus expressing the hgp100 neoepitope, or with peptide vaccines (intraperitoneally or intravenously) and anti-CD40 agonist antibodies (intraperitoneally or intravenously). In addition, mice were intraperitoneally injected with 180,000 IU of rhIL-2 every day for three consecutive days after cell transfer.

[0079] ACT supplements vaccination or immunomodulator therapy

[0080] In combination with ACT, mice were injected with a combination of PBS, vaccine, antibody, and peptide, e.g. Figure 2A The neoantigen vaccine used was vaccinia virus (r.VACV) against human glycoprotein 100. hgp100 ), vaccinia virus (VACV hgp100(25) ), vaccinia virus (VACV hgp100(209)irr.), adenovirus against human glycoprotein 100 (ADV hgp100 ), by four hours of peptide pulse loading of the relevant human glycoprotein 100 amino acid 25-33 epitope (DC+hgp100 KVP Bone marrow-derived dendritic cells (BMDC) with either Irr.Pep) or an irrelevant influenza virus peptide (DC+Irr.Pep) were injected intravenously (iv) once at a concentration of 2E7 PFU on the day of infusion. Anti-CD40, anti-PD1, anti-B7.1 and anti-B7.2 (anti-B7.1 / 2) and isotype control (IgG) antibodies were administered intraperitoneally (ip) at a dose of 100 μg, 3 times every other day starting on the day of infusion, or once intravenously (iv) on the day of infusion. The peptides used were human glycoprotein 100 (hgp100) peptide or influenza nucleoprotein (Flu.NP) peptide, which were injected subcutaneously (sc) or intravenously (iv) as a single dose on the day of infusion at a concentration of 100 μg.

[0081] Tumor size assessment

[0082] Tumor treatment and measurements were performed by independent investigators in a double-blind manner. Two perpendicular diameters were measured using calipers, and tumor size was calculated based on the product of the two diameters.

[0083] Survival probability assessment

[0084] The probability of survival was assessed by measuring the percentage of surviving mice (n=5) per treatment group each day after ACT.

[0085] Statistics

[0086] The Wilcox rank-sum test was used for comparisons between groups. In the figures, NS, *, **, and *** indicate not significant, p < 0.05, < 0.01, and < 0.001, respectively.

[0087] Example 1

[0088] This example demonstrates the use of CD39 hi Pmel T cells as a model of terminally exhausted T cells.

[0089] To establish a model of terminally exhausted T cells, mice of the melanocyte protein (Pmel) mutant line were injected with 100 μg / ml human glycoprotein 100 (hgp100) peptide. Five days later, these mice were injected with anti-CD3 / anti-CD28 antibodies. On day 11, the low-expressing (CD39 lo ), middle (CD39 med ) or high (CD39 hi ) Pmel T cells with CD39 level, B16 +ACT treatment was performed. Isotype control was used to distinguish CD39 expression. Within the CD39 gate, the bottom tertile of expression was designated as CD39 lo , the middle tertile was designated as CD39 med , and the upper tertile was designated as CD39 hi .

[0090] Then, the transferred CD39 cells extracted from Pmel mice were used to med or high CD39 hi T cell response to B16 as a melanoma model + Tumor-bearing mice were infused, followed by three injections of interleukin 2 (IL-2), and tumor growth was monitored ( Figure 2A ). Two stimulations were performed in vitro and B16 + After tumor-bearing mice were administered a moderate cell dose of stimulator cells (7.5e5-1e6), CD39 low 、CD39 med and CD39 hi Phenotype of exhausted T cells T cell exhaustion. Isolation of Pmel + T cells were divided into low (CD39 lo ), middle (CD39 med ) or high (CD39 hi ),like Figure 2B CD39 lo Cells express TIM3 and PD1 at lower levels than CD39 hi Cells. CD39 lo and CD39 hi T cells and B16 hgp100 Mel cells were co-cultured and 4-1BB activation was measured as an indicator of cell exhaustion. lo 4-1BB activation of T cells is higher than CD39 hi T cells, and therefore less exhausted ( Figure 2C ).

[0091] Monitor tumor size within a few days after ACT treatment. med 、CD39 hi , PBS and bulk control treatment failed to control 16 hgp100 Tumor progression. However, CD39 lo Treatment significantly slowed tumor progression ( Figure 3A ). CD39 hi + Anti-PD1 Antibodies and CD39 hi + anti-CD40 antibody treatment is better than CD39 alone hiT cells better controlled tumor progression, but not as well as less depleted CD39 alone lo Good cells ( Figure 3B ). These results indicate that T cells from Pmel mice can serve as an effective model for T cell exhaustion.

[0092] Example 2

[0093] This example demonstrates that hgp100 neoantigen vaccine can rescue CD39 hi Exhaustion of anti-tumor T cells.

[0094] Repeat the ACT treatment described in Example 1 and increase CD39 hi + hgp100 neoantigen vaccination (r.VACV hgp100 ) treatment group. Figure 4A and 4B The treatment groups shown in the application were administered with the following doses: VACV: 2E7 PFU 1 dose; anti-CD40 antibody 100 μg (ip) 3 doses; anti-PD1 antibody 100 μg (ip) 3 doses. hi +r.VACV hgp100 The treatment group had a 100% survival probability after 40 days, while CD39 lo 80% chance of survival, while the remaining treatment groups had a survival probability of 20% or less ( Figure 4A ). CD39 hi +r.VACV hgp100 The treatment was also more effective than all other treatment groups in controlling tumor progression and was the only treatment that shrank tumors ( Figure 4B ). The results showed that the control tumor treatment effect could be largely eliminated by depletion of less CD39 lo T cell capture. The results also showed that VACV.hgp100 iv also rescued CD39 hi Pmel anti-tumor T cells. The mice in these experiments had small tumors.

[0095] Example 3

[0096] This example demonstrates the use of CD39 hi ACT of depleted antitumor T cells to test different neoantigen vaccine paradigms.

[0097] To further test the neoantigen vaccine format of ACT, additional experiments were performed in mice bearing large tumors (approximately 200 mm). + Mouse infusion with CD39 hi (more depleted) or CD39 lo(less depleted) T cells, and in combination with (i) a single intravenous (iv) dose of VACV on the day of cell infusion 100hgp , (ii) a single intravenous (iv) dose of ADV on the day of cell infusion hgp100 , (iii) three intraperitoneal (ip) doses of 100 μg of anti-CD40 antibody every other day starting on the day of cell infusion, or (iv) a single subcutaneous (sc) dose of 100 μg hgp100 peptide on the day of infusion and three intraperitoneal (ip) doses of 100 μg anti-CD40 antibody every other day starting on the day of cell infusion.

[0098] Compared with the control CD39 alone hi Treatment, CD39 hi +VACV 100hgp Treatment can significantly slow tumor progression ( Figure 5A ). CD39 hi +ADV hgp100 treat( Figure 5B ) and various anti-CD40 antibody treatments ( Figure 5C ) can delay tumor progression to a certain extent, but the effect is not as good as CD39 hi +VACV 100hgp Treatment. The CD39 lo +VACV 100hgp (80%) and CD39 hi +VACV 100hgp (60%) treated, but CD39lo+ADV hgp100 (40%) and CD39 hi +ADV hgp100 (20%) treatment also improved survival relative to all other treatments (0%) Figure 5D ). T cells with less CD391o depletion alone were used as a control. These results suggest that neoantigen-specific vaccines can rescue the capacity of exhausted T cells, thereby slowing tumor progression and improving survival probability.

[0099] Example 4

[0100] This example demonstrates that neoantigen vaccines rescue distinct subsets of exhausted anti-tumor T cells.

[0101] Vaccine-mediated rescue is not limited to CD39 hi Exhaustion of anti-tumor T cells.

[0102] With PD1 + TIM3 + Cells (alone or with rVACV 100hgpcombination) or PBS (alone or with rVACV 100hgp Combination) to treat the tumor-bearing mice described in Example 1. PD1 + TIM3 + Terminally exhausted neoantigen-specific T cells were also rescued with neoantigen vaccines ( Figure 6B ).

[0103] Using CD39 + CD69 + Cells (alone or with rVACV 100hgp combination) or PBS (alone or with rVACV 100hgp The neoantigen vaccine also rescued CD39 + CD69 + Tumor progression in terminally exhausted neoantigen-specific T cells Figure 6C ).

[0104] CD39lo cells (alone or with rVACV 100hgp combination) or PBS (alone or with rVACV 100hgp The neoantigen vaccine also rescued CD39 lo Depletion of fewer antitumor cells, despite CD39 alone lo Treatment also inhibited tumor progression ( Fig. 6A ).

[0105] Example 5

[0106] This example demonstrates that vaccine rescue of exhausted T cells during ACT requires relevant neo-epitopes.

[0107] Testing for irrelevant HLA-A2 restricted hgp100 epitopes (hgp100 209 ) and related hgp100 epitopes (hgp100 25 ) to identify the neoepitopes required for rescue of exhausted T cells. The tumor-bearing mice described in Example 1 were treated with PBS alone or with hgp100 209 or 100 25 Combination therapy or CD39 alone lo T cells or hgp100 209 or hgp100 25 Combination treatment.

[0108] CD39 lo Less exhausted T cells are only able to associate with the relevant VACV hgp100(25) Combination inhibits tumor progression ( Fig. 7A ). In this experiment, CD39 alone loT cell therapy could not significantly inhibit tumor progression ( Fig. 7A ). CD39 + CD69 + Exhausted neoantigen-specific T cells can only associate with the relevant VACV hgp100(25) Combination inhibits tumor progression ( Figure 7B ). These results suggest that relevant neoepitopes are required in neoantigen vaccines to rescue exhausted T cell antitumor activity.

[0109] Example 6

[0110] This example demonstrates that neoantigen vaccines increase CD8 + Frequency of exhausted T cells.

[0111] The tumor-bearing mice described in Example 1 were treated with CD39 alone. lo T cells, rVACV alone hgp100 Or use CD39 lo T cells and rVACV hgp100 Combination therapy ( Figures 8A-8C ), or CD39 alone + CD69 + T cells, rVACV alone hgp100 Or use CD39 + CD69 + Cells and rVACV hgp100 Combination therapy ( Figures 8D-8F ).

[0112] To further investigate the effects of neoantigen vaccines on exhausted antitumor T cells of different subsets, we isolated Thy1.1 in spleen, draining lymph nodes, and tumors of mice after ACT. + Vβ13 + CD8 + The percentage of transferred T cells was compared with total CD8+ T cells.

[0113] ACT infusion of CD39 lo After depletion of fewer T cells, rVACV hgp100 Treatment with increased B16 + The spleen of mice on days 3, 7, and 10 after ACT ( Fig. 8A ), draining lymph nodes ( Figure 8B ) and tumors ( Figure 8C ) in the adoptively transferred T cells. lo When using a functionally impaired differentiated CD39 hi or CD39 + CD69 +The difference was even more pronounced when ACT was performed with T cells and vaccines. + 69 + After highly depleted T cells, rVACV hgp100 Treatment also increases B16 + The spleen of mice at 3, 7 and 10 days after ACT ( Fig.8D ), draining lymph nodes ( Fig. 8E ) and tumors ( Figure 8F ) The frequency of adoptively transferred T cells in . + The fold changes of cells were significantly higher in terminally exhausted T cells than in less exhausted T cells.

[0114] Example 7

[0115] This example demonstrates that neoantigen vaccines reduce PD1 after ACT + TIM3 + Frequency of terminally exhausted T cells.

[0116] The tumor-bearing mice described in Example 1 were treated with CD39 lo rVACV hgp100 Combination therapy or CD39 alone + CD69 + rVACV hgp100 Combination treatment.

[0117] To further investigate the effect of neoantigen vaccines on exhausted anti-tumor T cells, Thy1.1 in mouse tumors was detected after ACT. + Vβ13 + Percentage of neoantigen-specific transferred T cells. hgp100 Treatment significantly reduced PD1 + TIM3 + Adoptive transfer of Thy1.1 + Vβ13 + hgp100 neoantigen-specific CD39 lo and CD39 + CD69 + The frequencies of T cells accounted for the total viable CD8 T cells isolated from mouse tumors on days 3 and 10 after ACT. + The percentage of T cells ( Fig. 9 ). These results suggest that neoantigen vaccination combined with ACT reduces the frequency of exhausted T cells that migrate into tumors after ACT.

[0118] Example 8

[0119] This example demonstrates that anti-CD40 antibody and neoepitope intravenous vaccination delays tumor progression during ACT with exhausted T cells.

[0120] To further investigate how the route of vaccine administration affects the success of vaccine-mediated rescue of exhausted T cells in the ACT, additional experiments were performed. + CD69 + T cell ACT had a modest effect. + Antibodies and peptides were tested.

[0121] Results showed that vaccination delayed the onset of terminally exhausted CD39 + CD69 + Effects of ACT on T cells Fig.10 The tumor-bearing mice described in Example 1 were treated with CD39 + CD69 + ACT treatment of hgp100 neoantigen-specific Pmel T cells, or those T cells co-administered with vaccinia virus vaccine encoding hgp100(25-33), or the relevant neoepitope (hgp100(25)) with or without co-administered intravenous anti-CD40 or isotype control (rat IgG), or an unrelated peptide (Flu.NP) with or without co-administered intravenous anti-CD40 or isotype control (rat IgG). The inability of the unrelated peptide to rescue exhausted T cell ACT further suggests that the relevant neoepitope is a determinant of vaccine-mediated rescue of terminally differentiated dysfunctional T cells ACT (see Flu NP vs. hgp100). Furthermore, this suggests that the route of peptide or antibody administration (intravenous rather than intraperitoneal) may play a role in the success of vaccine-mediated rescue of exhausted T cell ACT. The effects of peptides and anti-CD40 antibodies on tumor size were similar to those of vaccinia virus vaccine on tumor size, suggesting that non-vaccinia virus vaccines can also mediate rescue of exhausted T cells due to terminal exhaustion of CD39 + CD69 + ACT rescue caused by T cells.

[0122] Example 9

[0123] This example demonstrates that neo-epitope-loaded dendritic cells administered as a vaccine together with ACT delay tumor progression.

[0124] To investigate non-vaccinia vaccines, bone marrow-derived dendritic cells (BMDC) were tested. Tumor progression was measured in tumor-bearing mice described in Example 1 that were treated with bulk, unsorted, transgenic hgp100 neoantigen-specific Pmel T cells (Pmel) alone or with Pmel T cells co-administered with BMDC. BMDC had been treated with either an unrelated influenza virus peptide (Pmel+DC+Irr.Pep) or a related neoepitope (Pmel+DC+hgp100 KVP Pep) was pulsed with peptide for 4 hours. Untreated mice (PBS) served as controls. Treatment with BMDCs pulsed with the relevant neoepitope significantly delayed tumor progression compared with hgp100 neoantigen-specific Pmel T cells alone or co-administered with BMDCs pulsed with an irrelevant peptide (P<0.01) ( Fig.11 ). These data suggest that DC-based vaccines can also mediate tumor regression via differentiated T cell ACT.

[0125] Example 10

[0126] This example demonstrates that neoantigen vaccines and depleted T cell ACT delay tumor progression in a colon tumor model.

[0127] To test the efficacy of neoantigen vaccines and T cell depletion ACT in non-melanoma tumor models, 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 concurrent neoantigen vaccine. When administered alone, the neoantigen vaccine had no significant effect on tumor progression compared to no treatment (PBS) ( Fig. 12A ). When used alone to deplete less CD39 lo MC38 tumor progression was also measured after ACT with Pmel transgenic T cells or co-administered with neoantigen vaccines, both of which mediated long-term tumor control. Fig. 12B ). In neoantigen vaccines and terminal depletion of CD39 + CD69 + After ACT with Pmel transgenic T cells, MC38 tumor progression was tested, which was comparable to CD39 alone. + CD69 + This significantly reduced tumor progression compared with Pmel transgenic T cells (P<0.01). + CD69 + Pmel transgenic T cells alone are unable to mediate tumor control ( Fig. 12C ). These data demonstrate that ACT with neoantigen vaccines mediates durable tumor regression of large tumors in both melanoma and non-melanoma tumor models.

[0128] Embodiment 11

[0129] This example demonstrates that antigen presentation by host cells is required for vaccine rescue of exhausted T cells.

[0130] To investigate the role of host cell antigen presentation in the rescue of exhausted T cells by neoantigen-specific vaccines, experiments were performed in β2-microglobulin knockout (β2M KO) mice. lo Tumor progression was measured in Pmel transgenic T cells, T cells co-administered with vaccinia virus vaccine encoding hgp100(25-33), β2M KO mice treated with vaccinia virus alone or untreated (PBS). lo Less exhausted tumor neoantigen-specific T cells were not affected ( Fig.13A ). However, when using terminally depleted CD39 + CD69 + When similar experiments were performed with Pmel transgenic T cells, they could not be rescued by neoantigen vaccines ( Fig. 13B ). This suggests that antigen presentation by host cells may be required to rescue exhausted T cell ACT.

[0131] Example 12

[0132] This example demonstrates that exhausted T cells rescued by vaccine are affected by B7.1 / B7.2 blockade.

[0133] To further investigate the mechanism of rescue of exhausted T cells by neoantigen vaccines, the effects of B7.1 (CD80) and B7.2 (CD86) were tested. Anti-B7.1 and anti-B7.2 (anti-B7.1 / 2) antibodies (100 μg) or isotype control (100 μg) were used, or left untreated (PBS) or not treated with B7.1 / B7.2 blocking (PBS+anti-B7.1 / 2). lo Tumor progression was tested after ACT with Pmel transgenic T cells or co-administered with neoantigen vaccine (with or without B7.1 and B7.2 blockade) Fig.14A ). Using terminally depleted CD39 + CD69 + The same experiment was performed with Pmel transgenic T cells ( Fig. 14B ). Vaccinia virus-mediated targeting of CD39 encoding neoantigens + CD69 +The rescue of terminally exhausted neoantigen-specific T cells was significantly affected by B7.1 / B7.2 blockade (P < 0.01), indicating that costimulation of host antigen-presenting cells (APCs) may be involved in vaccine-mediated rescue of terminally exhausted T cell ACT. lo Less exhausted neoantigen-specific T cells had less impact on ACT.

[0134] Embodiment 13

[0135] This example demonstrates retrospective evidence for rescue of exhausted T cells by vaccine-based treatment of human patients with TIL ACT.

[0136] The clinical course of melanoma patient 2463 treated with tumor infiltrating lymphocyte (TIL) therapy with or without the use of vaccines provides further evidence of the efficacy of neoantigen vaccines in salvaging exhausted T cells. Patient 2463 initially received TIL treatment only (intravenous TIL (Rx1) and intra-arterial TIL (Rx2)). After disease progression, the patient was again treated with TIL + fowlpox vaccine encoding the tumor antigen GP100. Rx1 and Rx3 refer to the first and third treatments with TIL infusion products, respectively. The patient received intra-arterial TIL as infusion product 2 (Rx2), but had no response. Since Rx2 was administered by intra-arterial TIL administration, it was excluded from this analysis. The clinical course was published in Smith et al, J. Immunother., 32 (8): 870-874 (2009) ( Fig.15A ).

[0137] After failure of two previous TIL therapies, the TIL infusion products administered in the first intravenous administration (Rx1) and the second intravenous administration simultaneously with GP100 fowlpox vaccine (Rx3) had comparable GP100-specific TILs by tetramer staining ( Fig. 15B ). These numbers represent the frequency of HLA-A0201 restricted GP100 tetramers as a percentage of CD8+ TILs in Rx1 and Rx3. These data suggest that TIL infusions administered with the vaccine that mediated responses did not have more anti-tumor TILs compared to Rx1, which did not mediate clinical responses.

[0138] TCR clone frequencies were highly correlated between Rx1 and Rx3 ( Fig. 15C ). The marked immunodominant GP100 TCR-1 identified from patient 2463 TILs showed that the clonal repertoire was identical between Rx1 and Rx3, suggesting that the GP100 fowlpox vaccine was effective against similar frequencies of anti-tumor TILs in the infused products.

[0139] The phenotypic status of antitumor GP100 TILs was comparable between Rx1 and Rx3. The phenotypes in GP100 tetramer-positive antitumor TILs between Rx1 and Rx3 were expressed based on the numbers expressed as percentages of tetramer-positive TILs. Therefore, fowlpox GP100 vaccines co-administered with TILs may work against dysfunctional antitumor TILs, as evidenced by the presence of tetramer-positive TILs in the infusion product. + Very low frequency of CD39 in TILs - CD69 - and higher frequencies of CD39 + CD69 + ( Fig.15D ), and a single marker CD62L + ( Fig.15E )、TIM3 - ( Fig.15F ) or CD39 - ( Figure 15G ) cells. This clinical case demonstrates that neoantigen vaccine rescue of exhausted T cells is effective in mediating long-term control in humans.

[0140] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference in its entirety and were set forth in its entirety herein.

[0141] In the context of describing the present application (especially in the context of the following claims), the use of the terms "a", "an", "the", and "at least one", and similar indicators should be interpreted as covering the singular and the plural, unless otherwise specified herein or clearly contradicted by the 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") should be interpreted as selecting one item from any combination of two or more of the listed items (A or B) or the listed items (A and B), unless otherwise specified herein or clearly contradicted by the context. Unless otherwise specified, the terms "comprising", "having", "including", and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise specified herein, the numerical ranges listed herein are intended merely as a shorthand method of individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually listed herein. Unless otherwise specified herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present application and will not limit the scope of the present application unless otherwise stated. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0142] The preferred aspects of the present application are described herein, including the best mode of implementing the present application known to the inventor. After reading the above description, changes in these preferred aspects will become apparent to those of ordinary skill in the art. The inventor expects that skilled technicians will adopt such changes as appropriate, and the inventor intends to practice the present application in a manner different from that specifically described herein. Therefore, the present application includes all modifications and equivalents of the subject matter described in the appended claims as allowed by applicable law. In addition, unless otherwise stated herein or clearly contradictory to the context, the present application covers any combination of the above-mentioned elements in all possible variations thereof.

Claims

1. A combination for use in treating or preventing cancer in a mammal, comprising: (i) T cells isolated from a tumor sample from a mammal, wherein the isolated T cells are exhausted and / or differentiated, 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 neoantigen or an antigen with a tumor-specific driver mutation; and the number of the isolated tumor antigen-specific T cells is optionally expanded; and (ii) a vaccine that specifically stimulates an immune response against the tumor-specific antigen, wherein the isolated T cells have antigen specificity for the tumor-specific antigen.

2. The combination for use according to claim 1, wherein the number of the isolated tumor antigen-specific T cells is expanded.

3. A combination for treating or preventing cancer in a mammal with a tumor, comprising: (i) T cells isolated from a biological sample from a mammal bearing a tumor, wherein a nucleic acid has been introduced into the isolated T cell, wherein the nucleic acid comprises a nucleotide sequence encoding an exogenous receptor having antigenic specificity for a tumor-specific antigen expressed by the mammalian tumor, and wherein the T cell expresses the exogenous receptor, wherein the tumor-specific antigen is a tumor-specific neoantigen or an antigen having a tumor-specific driver mutation; and wherein the number of T cells expressing the exogenous receptor is optionally expanded; and (ii) a vaccine that specifically stimulates an immune response against the tumor-specific antigen, wherein the exogenous receptor has antigenic specificity for the tumor-specific antigen.

4. The combination for use according to claim 3, wherein the number of T cells expressing the exogenous receptor is expanded.

5. The combination for use according to claim 3 or 4, wherein the T cells isolated from the biological sample are exhausted and / or differentiated.

6. The combination for use according to any one of claims 3 to 5, wherein the biological sample is a sample of the tumor.

7. The combination for use according to any one of claims 1 to 6, wherein the isolated T cells are TILs.

8. The combination for use according to any one of claims 3 to 5, wherein the biological sample is a peripheral blood sample.

9. The combination for use according to any one of claims 3 to 8, wherein the exogenous receptor is a T cell receptor (TCR).

10. The combination for use according to any one of claims 3-8, wherein the exogenous receptor is a chimeric antigen receptor (CAR).

11. The combination for use according to any one of claims 1 to 10, wherein (i) and (ii) are administered to the mammal simultaneously.

12. The combination for use according to any one of claims 1 to 11, wherein (i) and (ii) are administered to the mammal together in the same composition.

13. The combination for use according to any one of claims 1 to 10, wherein (i) and (ii) are administered sequentially to the mammal.

14. The combination for use according to claim 13, wherein (i) is administered to the mammal before (ii).

15. The combination for use according to claim 13, wherein (ii) is administered to the mammal prior to (i).

16. The combination for use according to claim 13, wherein (i) is administered to the mammal within 24 hours prior to administration of (ii) to the mammal.

17. The combination for use according to claim 13, wherein (i) is administered to the mammal within 24 hours of administering (ii) to the mammal.

18. The combination for use according to any one of claims 1-17, 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. A combination for use according to any one of claims 1-18, wherein the vaccine is: a cancer cell vaccine, a conjugated 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. The combination for use according to any one of claims 1 to 19, 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 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 + ;as well as (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. The combination for use according to any one of claims 1 to 20, wherein the tumor has been screened for expression of the tumor-specific antigen.

22. The combination for use according to any one of claims 1-21, wherein no more than a single dose of the vaccine is administered to the mammal.

23. The combination for use according to any one of claims 1-21, wherein two, three or more doses of the vaccine are administered to the mammal.

24. The combination for use according to claim 23, wherein the vaccine is administered to the mammal every other day starting from the first day of administration of the T cells to the mammal.

25. The combination for use according to any one of claims 1 to 24, 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 proteins: CCR7 - 、CD27 - 、CD45RA + 、CD45RO - 、CD95 + 、EOMES - 、FOXO1 - 、KLRG1 + 、T-BET + 、TCF7 - 、TOX + and ZEB2 + ;as well as (b) any one or more of the following proteins: CCR7 - 、CD27 - 、CD45RA + 、CD45RO - 、CD95 + 、EOMES - 、FOXO1 - 、KLRG1 + 、T-BET + 、TCF7 - 、TOX + and ZEB2 + .

26. The combination for use according to any one of claims 1 to 25, wherein the vaccine is administered to the mammal intramuscularly, subcutaneously, intravenously or intraperitoneally.

27. The combination for use according to any one of claims 1-26, wherein the T cells are administered to the mammal intravenously or intraperitoneally.

28. The combination for use according to any one of claims 1 to 27, wherein the isolated T cells are CD4 + .

29. The combination for use according to any one of claims 1 to 27, wherein the isolated T cells are CD8 + .

30. The combination for use according to any one of claims 1 to 29, wherein the mammal is a human.

31. The combination for use according to any one of claims 1-30, wherein (i) and (ii) are administered to the mammal within 24 hours of each other.

32. The combination for use according to any one of claims 1 to 31, wherein an adjuvant is administered to the mammal.

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

34. The combination for use of any one of claims 1-33, wherein the tumor-specific neoantigen is a personalized neoantigen encoded by one or more somatic mutations unique to the tumor of the mammal, optionally wherein the tumor-specific neoantigen is not a tumor-specific driver mutation.

35. The combination for use according to any one of claims 1-34, wherein the isolated T cells are terminally differentiated.

36. A method for treating or preventing cancer in a mammal, the method comprising: (a) isolating T cells from a tumor sample from said mammal, wherein the isolated T cells are exhausted and / or differentiated, and 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 optionally expanding the number of the 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, the isolated T cells having antigen specificity for the tumor-specific antigen.

37. The method of claim 36, comprising expanding the number of the isolated tumor antigen-specific T cells.

38. 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 a mammal having a tumor; (b) introducing a nucleic acid comprising a nucleotide sequence encoding an exogenous receptor into the isolated T cell to produce a T cell expressing the exogenous receptor, wherein the exogenous receptor has antigenic specificity for a tumor-specific antigen expressed by a tumor of the mammal, wherein the tumor-specific antigen is a tumor-specific neoantigen or an antigen having a tumor-specific driver mutation; and optionally expanding the number of T cells expressing the exogenous receptor; and (c) administering to the mammal: (i) T cells expressing the exogenous receptor of (b), and (ii) a vaccine that specifically stimulates an immune response against the tumor-specific antigen, wherein the exogenous receptor has antigenic specificity for the tumor-specific antigen.

39. The method of claim 38, comprising expanding the number of T cells expressing the exogenous receptor.

40. The method of claim 38 or 39, wherein the T cells isolated from the biological sample are exhausted and / or differentiated.

41. The method of any one of claims 38-40, wherein the biological sample is a sample of the tumor.

42. The method of any one of claims 36-41, wherein the isolated T cells are TILs.

43. The method of any one of claims 38-40, wherein the biological sample is a peripheral blood sample.

44. The method of any one of claims 38-43, wherein the exogenous receptor is a T cell receptor (TCR).

45. The method of any one of claims 38-43, wherein the exogenous receptor is a chimeric antigen receptor (CAR).

46. ​​The method according to any one of claims 36 to 45, wherein (i) and (ii) are administered to the mammal simultaneously.

47. The method of any one of claims 36-46, wherein (i) and (ii) are administered to the mammal together in the same composition.

48. The method of any one of claims 36-45, wherein (i) and (ii) are administered sequentially to the mammal.

49. The method of claim 48, wherein (i) is administered to the mammal prior to (ii).

50. The method of claim 48, wherein (ii) is administered to the mammal prior to (i).

51. The method of claim 48, wherein (i) is administered to the mammal within 24 hours prior to administering (ii) to the mammal.

52. The method of claim 48, wherein (i) is administered to the mammal within 24 hours of administering (ii) to the mammal.

53. The method of any one of claims 36-52, 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.

54. A method according to any one of claims 36-53, wherein the vaccine is: a cancer cell vaccine, a conjugated 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.

55. The method of any one of claims 36-54, 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 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 + ;as well as (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 + .

56. The method of any one of claims 36-55, further comprising screening for tumors that express the tumor-specific antigen.

57. The method of any one of claims 36-56, comprising administering no more than a single dose of the vaccine to the mammal.

58. The method of any one of claims 36-56, comprising administering two, three or more doses of the vaccine to the mammal.

59. The method according to claim 58, comprising administering the vaccine to the mammal every other day starting from the first day of administering the T cells to the mammal.

60. The method according to any one of claims 36 to 59, 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 proteins: CCR7 - 、CD27 - 、CD45RA + 、CD45RO - 、CD95 + 、EOMES - 、FOXO1 - 、KLRG1 + 、T-BET + 、TCF7 - 、TOX + and ZEB2 + ;as well as (b) any one or more of the following proteins: CCR7 - 、CD27 - 、CD45RA + 、CD45RO - 、CD95 + 、EOMES - 、FOXO1 - 、KLRG1 + 、T-BET + 、TCF7 - 、TOX + and ZEB2 + .

61. The method of any one of claims 36-60, comprising administering the vaccine to the mammal intramuscularly, subcutaneously, intravenously or intraperitoneally.

62. The method of any one of claims 36-61, comprising administering the T cells to the mammal intravenously or intraperitoneally.

63. The method according to any one of claims 36 to 62, wherein the isolated T cells are CD4 + .

64. The method according to any one of claims 36-62, wherein the isolated T cells are CD8 + .

65. The method of any one of claims 36-64, wherein the mammal is a human.

66. The method according to any one of claims 36 to 65, wherein (i) and (ii) are administered to the mammal within 24 hours of each other.

67. The method of any one of claims 36-66, further comprising administering an adjuvant to the mammal.

68. The method of claim 67, wherein the adjuvant comprises an anti-CD40 antibody or an anti-PD-1 antibody.

69. The method according to any one of claims 36-68, wherein the tumor-specific neoantigen is a personalized neoantigen encoded by one or more somatic mutations unique to the tumor of the mammal, optionally wherein the tumor-specific neoantigen is not a tumor-specific driver mutation.

70. The method according to any one of claims 36 to 69, wherein the isolated T cells are terminally differentiated.

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