Use of CD39 and CD103 to Identify Tumor-Reactive Human T Cells for the Treatment of Cancer

Abstract

Methods for treating a subject with a tumor are disclosed. These methods include administering a therapeutically effective amount of CD8+CD39+CD103+ T cells to the subject. Methods for isolating a nucleic acid encoding a T cell receptor (TCR) that specifically binds to a tumor cell antigen are also disclosed. These methods include isolating CD8+CD39+CD103+ T cells from a sample from a subject with a tumor that expresses the tumor cell antigen and cloning a nucleic acid molecule encoding the TCR from the CD8+CD39+CD103+ T cells. Additionally, methods for expanding CD8+CD39+CD103+ T cells are disclosed. In additional embodiments, methods for determining whether a subject with a tumor will respond to a checkpoint inhibitor are disclosed. The method includes detecting the presence of CD8+CD39+CD103+ T cells in a biological sample from the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 517,612, filed June 9, 2017, which is incorporated herein by reference.

[0002] The present invention relates to the field of cancer, specifically to the use of CD8 + CD39 + CD103 + The use of adoptive transfer of cells such as T cells, and CD8 + CD39 + CD103 + Regarding the use of T cell receptors (TCRs), such as those derived from T cells, and CD8 + CD39 + CD103 + The present invention relates to methods for evaluating treatment, such as by measuring T cells. [Background technology]

[0003] The immune system plays a major role in recognizing and killing tumor cells, and new therapeutic approaches have been focused on boosting and / or restoring its function in cancer patients. An effective immune response involves the concerted action of several different cell types, with CD8 T cells being key players that can specifically recognize and kill cancer cells by releasing cytotoxic molecules and cytokines (Klebanoff CA et al., 211:214-24, Immunol. Rev., 2006). Tumor-infiltrating CD8 T cells recognize tumor-associated antigens (TAA), which may include self-antigens overexpressed in neoplastic lesions, as well as tumor-specific antigens (TSAs) or neoantigens resulting from tumor-specific mutations (Finn OJ, Cancer Immunol Res, 2017). According to the current paradigm, tumor-specific CD8 T cells are primed in tumor-draining lymph nodes and then migrate via the blood to the tumor to exert their effector functions. Previous studies have shown that tumor-infiltrating CD8 T cells are a heterogeneous population of cells composed of tumor-specific T cells as well as bystander T cells without tumor specificity. The latter are recruited to tumor sites by inflammation associated with tumor progression (proliferation, angiogenesis, and metastasis). However, there is still a need for immunotherapy with increased efficacy / efficiency of adoptive transfer. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Klebanoff CA et al., 211:214-24, Immunol. Rev., 2006 [Non-Patent Document 2] Finn OJ, Cancer Immunol Res, 2017 Summary of the Invention [Means for solving the problem]

[0005] The phenotype and function of tumor-reactive CD8 T cells were evaluated, and it was confirmed that co-expression of CD103 and CD39 identifies a population of tumor-infiltrating CD8 T cells that are specifically induced in the tumor microenvironment. These cells, which are chronically stimulated within the tumor, were highly enriched for tumor reactivity and had the ability to kill autologous tumor cells. + CD39 + CD103 + Higher frequencies of CD8 T cells correlated with improved overall survival. + CD39 + CD103 + It has been determined that the presence of CD8 T cells may indicate that a therapy is effective in treating a tumor. + CD39 + CD103 + T cells can be adoptively transferred into a subject.

[0006] A method for treating a subject having a tumor, comprising administering to the subject a therapeutically effective amount of CD8 + CD39 + CD103 + A method is disclosed that includes administering T cells to a subject having a tumor, thereby treating the subject. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a programmed death (PD)-1 antagonist, a programmed death-ligand (PD-L) 1 antagonist, a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antagonist, a B- and T-lymphocyte attenuator (BTLA) antagonist, a T-cell immunoglobulin and mucin domain-containing-3 (TIM-3) antagonist, a lymphocyte-activation gene 3 (LAG3) antagonist, or a 4-1BB agonist.

[0007] In some embodiments, methods are disclosed for isolating nucleic acid sequences encoding T cell receptors (TCRs) that specifically bind to tumor cell antigens. These methods involve isolating CD8 T cells from a subject with a tumor that expresses the tumor cell antigen. + CD39 + CD103 +Isolating T cells and + CD39 + CD103 + and cloning the nucleic acid sequence encoding the TCR molecule from the T cell, thereby isolating a nucleic acid sequence encoding a tumor-specific TCR.

[0008] In a further embodiment, CD8 + CD39 + CD103 + Methods for expanding T cells are disclosed. These methods include + CD39 + CD103 + The method includes culturing T cells in tissue culture medium containing glutamine, serum, and antibiotics, stimulating the isolated T cells with an effective amount of irradiated allogeneic feeder cells and cytokines, and culturing the stimulated T cells in tissue culture medium and an effective amount of cytokines.

[0009] In yet another embodiment, a method is disclosed for determining whether a subject having a tumor will respond to a cancer therapeutic, such as a checkpoint inhibitor, a biological response modifier (e.g., cytokines and chemokines), a cancer vaccine, chemotherapy and / or radiation. Also disclosed is a method for determining whether a subject will respond to a surgical procedure. The method comprises detecting CD8 in a biological sample from a subject. + CD39 + CD103 + detecting the presence of CD8 T cells in the biological sample; + CD39 + CD103 + The presence of the T cells indicates that the cancer therapeutic agent or surgical procedure is effective in treating the tumor in the subject. In some non-limiting examples, the method can also include administering a cancer therapeutic agent to the subject or performing a surgical procedure.

[0010] In a further embodiment, a method for determining whether a subject having a tumor will respond to a cancer therapeutic agent is disclosed. The method includes administering to the subject a first dose of a cancer therapeutic agent and detecting CD8 in a biological sample from the subject. + CD39 + CD103 + and determining the number of CD8 T cells in the biological sample. + CD39 + CD103 + An increase in the number of T cells compared to the control indicates that the first dose of the cancer therapeutic is effective in treating the tumor in the subject.

[0011] In a further embodiment, a method for treating a subject having a tumor is disclosed. The method includes administering to the subject a first dose of a cancer therapeutic agent and detecting CD8 in a biological sample from the subject. + CD39 + CD103 + and determining the number of CD8 T cells in the biological sample. + CD39 + CD103 + An increase in the amount of T cells compared to the control indicates that the first dose of the cancer therapeutic agent is effective in treating the tumor in the subject. A second dose of the cancer therapeutic agent is administered to the subject, where the first dose is the same as the second dose or the second dose is lower than the first dose.

[0012] In another embodiment, a method for treating a subject having a tumor is disclosed. The method includes administering to the subject a first dose of a cancer therapeutic agent and detecting CD8 in a biological sample from the subject. + CD39 + CD103 + and determining the number of CD8 T cells in the biological sample. + CD39 + CD103 +A decrease or no change in the amount of T cells compared to the control indicates that the first dose of the cancer therapeutic agent is not effective in treating the tumor in the subject. A second dose of the cancer therapeutic agent is administered to the subject, where the second dose is higher than the first dose or the second dose is the same as the first dose.

[0013] The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0014] [Figure 1A]Figures 1A-1F. Tumor-infiltrating CD103+CD8 T cells co-express the ectonucleotidase CD39. (A) Representative gating strategy for flow cytometry analysis of T cells infiltrating head and neck squamous cell carcinoma (HNSCC). Numbers in the plots indicate the percentage of cells in the respective gate. (B) viSNE analysis of tumor-infiltrating lymphocytes (TILs) gated for CD103+CD8 T cells shown in (A). Gates identify CD103+CD8 T cells with the highest expression of CD39. Gates are applied to all plots showing expression levels of PD-1, CD69 and CD127. (C) Flow cytometry analysis of CD8 TILs isolated from HNSCC, melanoma, ovarian and CRLM cancer patients. Numbers in each quadrant indicate the percentage of cells positive for CD39 and / or CD103 in CD3+CD8+ T cells. (D) Summary of the frequency of CD39+CD103+(DP)CD8 TILs among patients with different solid malignancies. 40 HNSCC patients, 2 lung cancer patients, 6 melanoma patients, 2 ovarian cancer patients, 3 rectal cancer patients, 3 colon cancer patients and 6 patients with CRLM are shown. The red circle highlights one colon patient with Lynch syndrome resulting in microsatellite instability (MSI). (E) Flow cytometry analysis of the percentage of DP CD8 T cells in peripheral blood, normal LNs, primary tumor and metastatic LNs from one HNSCC patient. Numbers in each quadrant indicate the percent of cells positive for CD39 and / or CD103 in CD3+CD8+ T cells. Data represent 40 HNSCC patients analyzed (only 9 patients for normal LNs and metastatic LNs). Percentages are summarized in (F). [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above.

[0015] [Figure 2AB] Figures 2A-2D. Gene expression profiling of CD8+CD39+CD103+ TILs reveals a profile suggestive of TRM cells. (A) Principal component analysis (PCA) of gene expression of sorted DN, SP, and DP CD8 TILs isolated from three HNSCC and two ovarian cancer patients. The analysis was performed on genes (n=372) differentially expressed between double positive (DP) and double negative (DN) CD8 TILs. (B) Unsupervised clustering of samples using 372 genes differentially expressed between DP and DN CD8 TILs. Dark grey indicates downregulated genes and light grey indicates upregulated genes. (C) Waterfall plots depict the fold change (log2) of the most upregulated and downregulated genes in DP and DN subsets. Light grey identifies genes with a chronic activation signature; dark grey indicates genes associated with the TRM phenotype. (D) Expression of the top differentially expressed genes in each of the five patients analyzed. The top row indicates genes associated with the TRM phenotype, and the bottom row indicates chronic activation signature genes. Each symbol represents a patient and is connected by a line. [Figure 2C] Same as above. [Figure 2D] Same as above.

[0016] [Figure 3A]Figures 3A-3E. Phenotypic and functional properties of DP CD8 TILs, SP CD8 TILs and DN CD8 TILs. (A) Ex vivo phenotypic analysis of the expression of surface proteins CD69, CCR7, CD127 and CD28 depicting the TRM phenotype in CD8 TILs (top). Summary of the frequency of the above markers among several cancer patients (n=6-13, bottom). *p<0.05; ***p<0.001; ****p<0.0001 (ANOVA). (B) Ex vivo phenotypic analysis of the expression of surface and intracellular proteins PD-1, CTLA-4, TIM-3, 4-1BB and Ki-67 illustrating the activated / chronically stimulated phenotype in CD8 TILs (top). Summary of the frequency of the above markers among several cancer patients (n=5-17, bottom). ***p<0.001; ****p<0.0001 (ANOVA). (C) Representative flow cytometry analysis of IFN-γ and TNF-α production by CD8 TIL subsets stimulated with PMA / ionomycin for 4 hours. Numbers in each quadrant indicate the percentage of cells positive for IFN-γ and / or TNF-α for CD3+CD8+ T cells. (D) Frequency of IFN-γ, TNF-α, and IFN-γ / TNF-α double positive cells by CD8 TIL subsets. Data are from six HNSCC patients. (E) Representative flow cytometry analysis of granzyme B production by CD8 TILs (left) and summary for six different HNSCC patients (right). *p<0.05; **p<0.01 (ANOVA). [Figure 3B] Same as above. [Figure 3C-D] Same as above. [Figure 3E] Same as above.

[0017] [Figure 4]Figure 4. Expression of CD39 and CD103 in CD8 T cells requires sustained stimulation in a TGF-β-rich environment. Kinetics of CD39, CD103 and PD-1 expression in sorted naive CD8 T cells from peripheral blood. Cells were stimulated with CD3 / CD28-coated beads at a bead:T cell ratio of 1:2 in the presence or absence of TGF-β (2 ng / ml) and analyzed by flow cytometry for CD39, CD103 and PD-1 expression at the indicated time points. Data are representative of four independent experiments.

[0018] [Figure 5A] Figures 5A-5D. DP CD8 TILs are highly clonal and have little overlap with any other CD8 T cell subset. (A) Diversity of TCRβ repertoires within blood memory CD8 T cells, DN CD8 TILs, SP CD8 TILs and DP CD8 TILs. Frequencies of the most frequent clonotype, the second to fifth most frequent clonotypes, the sixth to thirtieth most frequent clonotypes and the remaining clonotypes are shown for HPV+ and HPV- HNSCC patients and one ovarian cancer patient. (B) The 500 most frequent CD8 TIL clonotypes are plotted based on their frequency in DN CD8 TIL, SP CD8 TIL and DP CD8 TIL subsets. Each dot represents a distinct TCR clonotype. Dots on the axis indicate clonotypes detected within a single repertoire. (C) Same analysis as in (B) comparing the frequency of the top 500 clonotypes in blood and normal LN memory CD8 T cells with the frequency of the top 500 clonotypes in DN and DP CD8 TIL subsets. (D) TCRβ sequence overlap between DP CD8 TILs and blood memory CD8 T cells, DN CD8 TILs, and SP CD8 TILs analyzed using the Morisita-Horn index. A TCRβ sequence overlap of 1 indicates 100% similarity between the two populations. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above.

[0019] [Figure 6A] Figures 6A-6F. DP CD8 TILs recognize and kill autologous tumor cells, and their frequency correlates with improved overall survival. (A) Expanded CD8 TILs were tested for tumor reactivity by culturing with increasing numbers of autologous tumor cells for 20 h and assessing recognition by measuring the frequency of 4-1BB expression (closed symbols) and IFN-γ secretion (open symbols). Results are shown for three cancer patients. (B) Reactivity of DP CD8 TILs was confirmed by culturing autologous tumor cells with or without MHC-I blocking antibodies, allogeneic tumor cells, and plate-bound anti-CD3. Upregulation of 4-1BB after 20 h for three cancer patients is shown. (C) Tumor cell killing was measured by assessing the amount of caspase 3 / 7+ events per well monitored hourly over a 20 h period. (D) Representative images for DN CD8 TILs and DP CD8 TILs taken at the beginning (T=0) and end (T=20 h) of coculture. (E) Overall survival for a cohort of 62 HNSCC patients based on the frequency of DP CD8 TILs among total CD8 TILs at the time of surgery. (F) A similar analysis was performed on a cohort of 30 HPV-negative HNSCC patients. [Figure 6BC] Same as above. [Figure 6D] Same as above. [Figure 6EF] Same as above.

[0020] [Figure 7A]Figures 7A-7C. DP CD8 TILs are enriched for tumor-reactive cells, and primary tumor TCR repertoires overlap with metastatic LN TCR repertoires within the same individual. (A) and (B) DP CD8 TILs were sorted and expanded in vitro. After expansion, DP CD8 TILs were co-cultured with autologous tumor cells for 24 h. At the end of the culture, cells were sorted based on 4-1BB and CD25 expression. The TCR repertoires of DP CD8 TILs before co-culture were compared with those of 4-1BB+CD25+DP CD8 TILs (tumor-reactive). The top 15 clonotypes in DP CD8 TILs before co-culture are shown along with their respective frequencies before and after co-culture with tumor cells. Results from two cancer patients are shown. (C) The 500 most frequent DP CD8 TIL clonotypes are plotted based on their frequency in primary tumors and metastatic LNs. Results from two HNSCC patients are shown. In Figure 7A, SEQ ID NOs: 1 to 15 are shown in order from top (SEQ ID NO: 1) to bottom (SEQ ID NO: 15). In Figure 7B, SEQ ID NOs: 16 to 30 are shown in order from top (SEQ ID NO: 16) to bottom (SEQ ID NO: 30). [Figure 7B] Same as above. [Figure 7C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Sequence Listing The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 C.FR1.822. Only one strand of each nucleic acid sequence is shown, but it should be understood that any reference to the strand shown includes the complementary strand. The sequence listing has been submitted as an ASCII text file [Sequence_Listing, May 4, 2018, 7,113 bytes] and is incorporated herein by reference. In the attached sequence listing: SEQ ID NOs: 1-30 are exemplary nucleotide sequences of TCRB CDR3 regions. Detailed Description

[0022] There is a need to improve the efficacy of immunotherapy and adoptive T cell transfer for cancer patients, as well as a need to characterize CD8 T cells involved in anti-tumor responses in order to use specific subpopulations in diagnostic methods. + CD8 + T cells include CD39+CD103+CD8+ T cells and CD39+CD103-CD8+ T cells. It is disclosed herein that the phenotype and function of tumor-reactive CD8 T cells are determined, and it is demonstrated that the co-expression of CD103 and CD39 identifies a unique population of tumor-infiltrating CD8 T cells that are specifically induced in tumor microenvironment. These cells, which are chronically stimulated in tumors, are highly enriched in tumor reactivity and have the ability to kill autologous tumor cells. The high frequency of CD103+CD39+CD8 T cells in human tumors correlates with prolonged overall survival, indicating that the evaluation of these cells in biological samples provides a method for determining the effectiveness of treatment and for evaluating whether tumor-bearing subjects respond to treatment. term

[0023] Unless otherwise stated, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0024] In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[0025] 4-1BB: Also called CD137 and TNFRSF9, it is a transmembrane protein and a member of the tumor necrosis factor receptor superfamily (TNFRS). 4-1BB expression is generally activation-dependent and is present in a wide range of immune cell subsets, including activated NK and NKT cells, regulatory T cells, activated CD4 and CD8 T cells, dendritic cells (DCs), stimulated mast cells, differentiating myeloid cells, monocytes, neutrophils, and eosinophils (Wang, 2009, Immunological Reviews 229: 192-215). 4-1BB expression has also been demonstrated in tumor vasculature (Broll, 2001, Amer. J. Clin. Pathol. 115(4):543-549; Seaman, 2007, Cancer Cell 11: 539-554) and in sites of inflammation or atherosclerotic endothelium (Drenkard, 2007 FASEB J. 21: 456-463; Olofsson, 2008, Circulation 117: 1292-1301). The ligand that stimulates 4-1BB, i.e., 4-1BB ligand (4-1BBL), is expressed in activated antigen-presenting cells (APCs), myeloid progenitor cells, and hematopoietic stem cells. Human 4-1BB is a 255 amino acid protein (see GENBANK Accession Nos. NM_001561 and NP_001552, available on June 1, 2017, which are incorporated herein by reference). Agonistic antibodies of 4-1BB are disclosed, for example, in U.S. Patent No. 8,337,850, which is incorporated herein by reference.

[0026] Altered level: A change, either by increase or decrease, in the number of cells of a particular cell type, or the level of production or expression of a nucleic acid or amino acid sequence (e.g., polypeptide, siRNA, miRNA, mRNA, gene) compared to a control level.

[0027] Antisense, Sense, and Antigene: DNA has two antiparallel strands, a 5' to 3' strand called the plus strand and a 3' to 5' strand called the minus strand. The minus strand of DNA serves as a template for RNA during transcription as nucleic acids are added in the 5' to 3' direction by RNA polymerase. Thus, the RNA transcript has a sequence complementary to the minus strand and a sequence identical to the plus strand (except for the substitution of U for T).

[0028] Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the positive strand of DNA. Sense molecules are molecules that are specifically hybridizable or specifically complementary to the negative strand of DNA. Antigene molecules are either antisense or sense molecules directed against a DNA target. Antisense RNA (asRNA) is a molecule of RNA that is complementary to a sense (coding) nucleic acid molecule.

[0029] Antibody: A polypeptide ligand comprising at least a light or heavy chain immunoglobulin variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an epitope of an antigen, such as a PD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG3 or 4-1BB polypeptide, or a fragment thereof. Immunoglobulin molecules are composed of heavy and light chains, each of which is made up of a variable heavy chain (V H ) region and the variable light chain (V L ) region. H Area and V L Together the regions are involved in binding to the antigen that is recognized by the antibody.

[0030] Antibodies include intact immunoglobulins, as well as variants and portions of antibodies known in the art, such as single domain antibodies (e.g., VH domain antibodies), Fab fragments, Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which the variable region of an immunoglobulin light chain and the variable region of an immunoglobulin heavy chain are linked by a linker, while dsFvs are mutated chains such that disulfide bonds are introduced to stabilize the association of the chains. The term also encompasses genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and the like. Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0031] Generally, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0032] Each heavy and light chain contains a constant region and a variable region (regions are also known as "domains"). The heavy and light chain variable regions combine to specifically bind to an antigen. The light and heavy chain variable regions contain a "framework" region interspersed with three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs are defined according to Kabat et al. (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991) and the ImMunoGeneTics database (IMGT) (see Lefranc, Nucleic Acids Res 29:207-9, 2001; and imgt.cines.fr / IMGT_vquest / vquest?_livret=0&Option=humanIg). The Kabat database is maintained online at ncbi.nlm.nih.gov / igblast / . The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, which is the combination of the constituent light and heavy chain framework regions, serves to position and align the CDRs in three-dimensional space.

[0033] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are generally numbered sequentially starting from the N-terminus and are referred to as CDR1, CDR2, and CDR3, and are generally also identified by the chain in which the particular CDR is located. Thus, V H CDR3 (or H-CDR3) is located in the variable domain of the heavy chain of the antibody in which it is found and L CDR1 (or L-CDR1) is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds to PD-1, PD-L1, or PD-L2 has, for example, a specific V H Region array and V LEach CDR has a specific CDR sequence and therefore a specific CDR sequence. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions in the CDRs are directly involved in binding to the antigen. These positions in the CDRs are called specificity determining residues (SDRs).

[0034] "V H References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including the variable region of an Fv, scFv, dsFv or Fab. L " or "VL" refers to the variable region of an immunoglobulin light chain, including the variable region of an Fv, scFv, dsFv or Fab.

[0035] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with a single antibody light and / or heavy chain gene. Monoclonal antibodies are produced by methods known to those of skill in the art, for example, by creating hybrid antibody-forming cells from the fusion of a myeloma cell and an immune spleen cell. Monoclonal antibodies include humanized monoclonal antibodies.

[0036] A "chimeric antibody" has framework residues derived from one species, such as human, and CDRs (which generally confer antigen binding) derived from another species, such as a murine antibody that specifically binds to a PD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG3 or 4-1BB polypeptide.

[0037] A "human" antibody (also referred to as a "fully human" antibody) is an antibody that comprises a human framework region and all of the CDRs derived from a human immunoglobulin. In one example, the framework and CDRs are derived from the same originating human heavy and / or light chain amino acid sequence. However, a framework from one human antibody can be engineered to contain CDRs from a different human antibody. A "humanized" immunoglobulin is an immunoglobulin that comprises a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor" and the human immunoglobulin providing the framework is referred to as the "acceptor". In one embodiment, all of the CDRs in a humanized immunoglobulin are derived from the donor immunoglobulin. Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized immunoglobulin are substantially identical to the corresponding parts of a natural human immunoglobulin sequence, except perhaps for the CDRs. A "humanized antibody" is an antibody that contains a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions with amino acids taken from the donor framework. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by genetic engineering (see, for example, U.S. Patent No. 5,585,089).

[0038] Antigen: A compound, composition, or substance capable of stimulating the production of antibodies or a T-cell response in an animal, including compositions injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes. "Epitope" or "antigenic determinant" refers to a site on an antigen to which B cells and / or T cells respond. In one embodiment, T cells respond to an epitope when the epitope is presented in conjunction with an MHC molecule. Epitopes may be formed of contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed of contiguous amino acids are generally retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost on treatment with denaturing solvents. An epitope typically includes at least 3 amino acids, more usually, at least 5 amino acids, about 9 amino acids, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

[0039] Antigen-presenting cell (APC): A cell that can present antigens bound to MHC class I or class II molecules to T cells. APCs include, but are not limited to, monocytes, macrophages, dendritic cells, B cells, T cells, and Langerhans cells. T cells that can present antigens to other T cells (including CD4+ and / or CD8+ T cells) are antigen-presenting T cells (T-APCs).

[0040] B and T lymphocyte attenuator (BTLA): a protein also known as CD272. BTLA expression is induced during T cell activation, and BTLA remains expressed on Th1 cells. BTLA interacts with B7H4, a B7 homolog, and plays a role in T cell inhibition through interaction with tumor necrosis family receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). The BTLA-HVEM complex negatively regulates T cell immune responses. Specific, non-limiting BTLA amino acid sequences, and mRNA sequences encoding BTLA are provided in GENBANK® Accession No. NM_001085357, September 1, 2016, which are incorporated herein by reference. BTLA antagonists include agents that reduce the expression or activity of BTLA, or inhibit the T cell inhibitory function of BTLA, for example, by specifically binding to BTLA and inhibiting the binding of BTLA to the tumor necrosis factor receptor. Exemplary compounds include antibodies (e.g., anti-BTLA antibodies), RNAi molecules, antisense molecules, and dominant negative proteins.

[0041] Binding or stable binding (oligonucleotide): An oligonucleotide binds or stably binds to a target nucleic acid when a sufficient amount of the oligonucleotide base pairs or hybridizes with its target nucleic acid to allow for detection of the binding. Binding can be detected by either the physical or functional properties of the target:oligonucleotide complex. The binding of the target and the oligonucleotide can be detected by any procedure known to those skilled in the art, including both functional and physical binding assays. For example, binding can be functionally detected by determining whether the binding has an observable effect on biosynthetic processes, such as gene expression, DNA replication, transcription, translation, etc.

[0042] Physical methods for detecting binding of complementary strands of DNA or RNA are well known in the art and include methods such as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures. For example, one method that is widely used because it is simple and reliable involves slowly increasing the temperature and observing the change in light absorption at 220-300 nm of a solution containing an oligonucleotide (or analog) and a target nucleic acid. If the oligonucleotide or analog is bound to its target, there will be a sudden increase in absorption as the oligonucleotide (or analog) and target dissociate, or melt, from each other at a characteristic temperature.

[0043] The binding of an oligomer to its target nucleic acid occurs at the temperature at which 50% of the oligomer is melted from its target (T m ) is often characterized by (T m ) is higher, the complex is (T m ) is stronger or more stable than a lower complex.

[0044] Binding affinity: The affinity of an antibody for an antigen. In one embodiment, affinity is calculated by a modified version of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by competitive radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. An antibody that "specifically binds" to an antigen (such as a PD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG3 or 4-1BB polypeptide) is an antibody that binds to the antigen with high affinity and does not bind significantly to other unrelated antigens.

[0045] Cancer therapeutic agent: Any agent useful for treating cancer in a subject. Cancer therapeutic agents include checkpoint inhibitors, biological response modifiers (e.g., cytokines and chemokines), cancer vaccines, chemotherapy and / or radiation.

[0046] Chemotherapeutic agent: Any chemical agent having therapeutic utility in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancers, as well as diseases characterized by hyperplastic growth, such as psoriasis. In one embodiment, the chemotherapeutic agent is a radioactive compound. Those of skill in the art can readily identify useful chemotherapeutic agents (see, e.g., Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2002). nd ed., (C) 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an antibody that binds to PD-1, PD-L1, or CTLA-4 polypeptides, used in combination with cells, radioactive compounds, or chemical compounds.

[0047] CD39 (ENTPD1): An integral membrane protein with two transmembrane domains and a large extracellular region (Maliszewski et al, 1994). CD39 (ENTPD1) was first identified on human lymphocytes as an activation marker and as a vascular ecto-ADPase (Kaczmarek E et al. (1996) Biol Chem). The term "CD39" refers to the CD39 protein, also called ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1). In vivo, CD39 is expressed on regulatory T cells (Treg cells), B cells and some innate immune cells. CD39 plays an important role in immune suppression by hydrolyzing adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to adenosine monophosphate (AMP), which is then processed to adenosine by the ecto-5'-nucleotidase CD73. Adenosine is a potent immune regulator that enhances intracellular cAMP accumulation by binding to the A2A receptor on T cells, thereby preventing T cell activation (Deaglio S et al., JEM, 2007). Both CD39 and CD73 expression is increased in some human solid malignant lesions (Antonioli Luca et al., Trends Mol Med, 2013). Upregulation of both enzymes is favorable in a hypoxic environment, and their sequential coordinated action may play a role in tumor immune escape (Eltzschig HK et al., Blood 2009; Ghiringhelli F et al., J Biomed Biotech, 2012). Specific, non-limiting CD39 amino acid sequences and mRNA sequences encoding CD39 are provided in GENBANK® Accession No. NM_001776, May 1, 2017, which is incorporated herein by reference.

[0048] CD103: Known as integrin alpha E (ITGAE), CD103 binds to integrin beta 7 (β7-ITGB7) to form the heterodimeric integrin molecule, αEβ7. The primary ligand for αEβ7 is E-cadherin, an adhesion molecule found on epithelial cells. It mediates T cell homing to sites in the intestine and tissue-resident memory (T RM In vivo, CD103 is important for the maintenance of T cells. RM CD103 is expressed on a population of T cells present on peripheral tissues characterized as CD8+ T cells (Schenkel JM et al., Immunity, 2014;Mueller SN et al., Nat Rev Immunol, 2015). CD103 is also expressed on a subset of CD8+ T cells in high-grade serous ovarian cancer, lung cancer, urothelial cell carcinoma of the bladder, and endometrial cancer (Webb JR et al., Clin Cancer Res, 2014;Webb JR et al., Cancer Immunol Res, 2015;Komdeur FL et al., Oncotarget, 2016;Djenidi F et al., J Immunol, 2015;Wang B et al., J Urol, 2015;Workel HH et al, EJC, 2015). In these malignant lesions, CD103+CD8 T cells are preferentially localized within tumors, and thus favor direct interaction with tumor cells. CD103+CD8 T cells express high levels of the activation / exhaustion surface molecule PD-1, which, when interacting with its ligand PD-L1, leads to inhibition of T cell proliferation, survival and effector function (Webb JR et al., Cancer Immunol Res, 2015). Specific non-limiting CD103 amino acid sequences and mRNA sequences encoding CD103 are provided in GENBANK® Accession No. NM_002208, May 20, 2017, which is incorporated herein by reference.

[0049] Conservative variant: A "conservative" amino acid substitution is one that does not substantially affect or reduce the affinity of a protein, such as an antibody. For example, a human antibody that specifically binds to PD-1, PD-L1, BTLA, TIM-3, LAG3, CTLA-4 or 4-1BB can contain up to about 1, up to about 2, up to about 5, and up to about 10, or up to about 15 conservative substitutions and specifically bind to a PD-1, PD-L1, BTLA, TIM-3, LAG3, CTLA-4 or 4-1BB polypeptide. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the antibody specifically binds to a PD-1, PD-L1, BTLA, TIM-3, LAG3, CTLA-4 or 4-1BB polypeptide. Non-conservative substitutions are those that reduce activity or binding to a PD-1, PD-L1, BTLA, TIM-3, LAG3, CTLA-4, or 4-1BB polypeptide.

[0050] Conservative amino acid substitution tables, in which functionally similar amino acids are provided, are well known to those of skill in the art. The following six groups are examples of amino acids which are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0051] Contacting: Placing in direct physical association, including both solid and liquid forms.

[0052] Control level (immune parameter): baseline level of an immune parameter. In some embodiments, the control level is the level of a component of the immune system, e.g., a particular type of cell, in the absence of a therapeutic agent. The control level can be measured in a sample from a subject not treated with the agent of interest, or from a subject treated with a control agent. The control level can also be a standard value, such as a value determined from the average of multiple samples over time. The control level can also be measured in a sample from a subject treated with a particular dose of a therapeutic agent, where the dose has not been administered to the subject at the time the subject is currently being evaluated. The control can be from the subject being evaluated or from a different subject.

[0053] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4): a protein also known as CD152. CTLA-4 is a member of the immunoglobulin superfamily. CTLA-4 is a protein receptor that functions as an immune checkpoint and thus downregulates immune responses. CTLA-4 is constitutively expressed in regulatory T cells (Treg) and upregulated after activation in conventional T cells. CLTA4 binds to CD80 or CD86 on the surface of antigen-presenting cells and is an inhibitor of T cells. Specific non-limiting examples of CTLA-4 proteins and mRNAs encoding CTLA-4 are disclosed, for example, in GENBANK® Accession No. NM_001037631, October 7, 2016, which is incorporated herein by reference. CTLA-4 antagonists include agents that reduce the expression or activity of CTLA-4, or inhibit the T cell inhibitory function of CTLA-4, for example, by specifically binding to CTLA-4 and inhibiting its binding to CD80 or CD86 on the surface of antigen-presenting cells. Exemplary compounds include antibodies (e.g., anti-CTLA-4 antibodies), RNAi molecules, antisense molecules, and dominant negative proteins.

[0054] Detecting or detection (cell or biomolecule): The biomolecule or CD8 under investigation. + CD39 + CD103 + It refers to the quantitative or qualitative determination of the presence of a particular cell type, such as CD8 T cells, in a sample from a subject. + CD39 + CD103 + Quantitatively or qualitatively determining the presence of T cells. In general, detection of biological molecules such as proteins, nucleic acids, or detecting specific cell types or cell proliferation requires performing biological assays rather than simple observation. For example, assays utilizing antibodies or nucleic acid probes (either of which may be labeled) can be used to detect alternative proteins or cells, respectively. Diagnosing or diagnosing the effectiveness of a treatment, such as with a checkpoint inhibitor, includes the detection of CD8 + CD39 + CD103 + It involves detecting significant changes in cells, such as T cells, or biomolecules.

[0055] DNA (deoxyribonucleic acid): DNA is a long-chain polymer that contains the genetic material of most living organisms (some viruses have genes that contain ribonucleic acid (RNA)). The repeating units in a DNA polymer are four different nucleotides, each of which contains one of the four bases, adenine, guanine, cytosine, and thymine, linked to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (called codons) code for each amino acid in a polypeptide or a stop signal. The term codon is also used for the corresponding (and complementary) sequence of three nucleotides in the mRNA into which the DNA sequence is transcribed.

[0056] Unless otherwise specified, any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. Except where required herein to be single-stranded, a DNA molecule encompasses both strands of a double-stranded DNA molecule, even if written to show only a single strand.

[0057] Diagnosis: Identifying the presence or nature of a pathology, such as, but not limited to, a tumor. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of individuals who test positive and have the disease (percent true positives). The "specificity" of a diagnostic assay is one minus the false positive rate, where the false positive rate is defined as the proportion of individuals who test positive and do not have the disease. A particular diagnostic method does not necessarily provide a definitive diagnosis of a condition, but suffices if the method provides a positive indication that aids in diagnosis. "Prognosis" is the probability of development (e.g., severity) of a pathology, such as a tumor or metastasis.

[0058] Encode: A polynucleotide is said to encode a polypeptide if, in its natural state or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce the mRNA for the polypeptide or fragment thereof and / or the polypeptide or fragment thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0059] Feeder cells: A layer of cells at the bottom of, for example, a culture dish. Feeder cells can release nutrients, growth factors and / or cytokines into the culture medium and provide a substrate with which other cells, such as T cells, can interact. The cells can be irradiated. In one embodiment, the feeder cells are irradiated allogeneic peripheral blood mononuclear cells.

[0060] Immune checkpoint inhibitors: A class of agents that block biological pathways in certain types of immune system cells, including but not limited to T cells, and some cancer cells. These inhibitors prevent T cells from killing cancer cells. When checkpoint inhibitors are blocked, there is less "blockage" to the immune system and T cells are activated against cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1, PD-L1, CTLA-4, BTLA, and TIM-3.

[0061] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, such as a CD4+ or CD8+ response. In another embodiment, the response is a B cell response, resulting in the production of a specific antibody.

[0062] "Unresponsiveness", in reference to immune cells, includes the unresponsiveness of immune cells to stimuli, such as stimulation by an activating receptor or cytokine. Unresponsiveness can occur, for example, due to exposure to immunosuppressants or exposure to high doses of antigen. As used herein, the term "anergy" or "tolerance" includes unresponsiveness to activating receptor-mediated stimuli. Such unresponsiveness is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy (as opposed to unresponsiveness) in T cells is characterized by a lack of cytokine production (e.g., IL-2). T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, the cells cannot produce cytokines and therefore cannot proliferate even when re-exposed to the same antigen (even if the exposure occurs in the presence of a costimulatory molecule). However, anergic T cells can mount a response to unrelated antigens and can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by proliferation assays using indicator cell lines. Alternatively, reporter gene constructs can be used. For example, anergic T cells cannot initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5'IL-2 gene enhancer or by multimers of AP1 sequences that can be found within the enhancer (Kang et al. Science 257:1134, 1992). Anergic antigen-specific T cells can have at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% reduced cytotoxic activity compared to corresponding control antigen-specific T cells.

[0063] Inhibiting or treating a disease: Inhibiting a disease, such as tumor growth, refers to inhibiting the complete onset of the disease or alleviating the physiological effects of the disease process. In some examples, inhibiting or treating a disease refers to alleviating the symptoms of a tumor or infection by a pathogen. For example, treating a cancer can prevent the onset of paraneoplastic syndromes in a person known to have cancer, or alleviate the signs or symptoms of a tumor. In another embodiment, treating an infection can refer to inhibiting the onset of an infection or alleviating the symptoms of an infection. "Treatment" refers to a therapeutic intervention that reverses the signs or symptoms of a disease or disease-related pathology. Therapeutic vaccination refers to administering an agent to a subject already infected with a pathogen. The subject may be asymptomatic, and thus treatment prevents the onset of symptoms.

[0064] Isolated: An "isolated" biological component, e.g., a nucleic acid, protein (including an antibody), or cell, has been substantially separated or purified from other biological components, i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles, in the environment in which the component naturally occurs (e.g., other cells). "Isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0065] Lymphocyte: a type of white blood cell involved in the body's immune defenses. There are two major types of lymphocytes: B cells and T cells.

[0066] Lymphocyte activation gene 3 (LAG3): Also called CD223, this protein is encoded by the LAG3 gene in humans. LAG-3 is a cell surface molecule with diverse biological effects on T cell function and is an immune checkpoint receptor. LAG3 has been reported to negatively regulate T cell proliferation, activation, and homeostasis, and play a role in Treg suppressive function. Exemplary amino acids and mRNA encoding human LAG3 are provided in GENBANK® Accession No. NM_002286.5, April 9, 2017, which is incorporated herein by reference.

[0067] Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

[0068] Mean Fluorescence Intensity (Flow Cytometry): Flow cytometry is concerned with the measurement of light intensity on a cell or particle, whether it is scattered laser light or fluorescent light emitted by a fluorochrome. The light is detected by a photomultiplier tube (PMT), which converts the light through an amplifier into a voltage proportional to the original fluorescent intensity and the voltage on the PMT. These voltages, which are continuous distributions, are converted into discrete distributions by an analog-to-digital converter (ADC) which puts each signal into a specific channel depending on its level of fluorescence. The greater the resolution of the ADC, the more closely it reflects the continuous distribution.

[0069] Flow cytometry data can be displayed using either a linear or log scale. The use of a log scale is indicated in the majority of biological situations where the distribution is skewed to the right. In this case, the effect is to normalize the distribution - it is said to be log-normal and the data are log transformed. A linear signal is obtained by a linear amplifier, while a log transformation can be achieved by a log amplifier or by using a look-up table (LUT). The majority of ADCs in analytical cytometers are 10-bit, i.e. the data is split into 2e10 or 1024 channels, but there is an increasing trend towards the use of 12-bit or 14-bit ADCs to provide greater resolution of the data.

[0070] Data from a single data channel (scattering or fluorescence) is displayed as a histogram where the x-axis is divided into 1024 channels (for a 10-bit ADC). If the data is in a linear scale, the channel number and linear value for that channel are easily obtained. In a log scale, the x-axis is displayed as a 5log10 (log decade) scale even though it is divided into 1024 channels (commonly, 5log10 is used).

[0071] Measures of population distribution are used to quantify flow cytometry data. Commonly, measures of central tendency are the mean and the median. The mean is the "average" and can be the arithmetic mean or the geometric mean. The arithmetic mean is calculated as Sigma(x) / n, whereas the geometric mean is calculated as the n-th root (a1×a2×a3....an). Commonly, the geometric mean is used for logarithmic amplification data, since the geometric mean takes into account the weighting of the data distribution, whereas the arithmetic mean is used for linear data or data displayed on a linear scale. The median is the middle value, i.e., the 50th percentile, where half of the values ​​are above and half are below. Cells with "high" and "low" expression can be compared and determined according to the fluorescence of the entire population; these parameters are easily visualized on a plot of the flow cytometry data.

[0072] Culture medium (tissue culture or cell culture): A synthetic set of culture conditions with nutrients necessary to support the growth (cell proliferation / expansion) of a particular population of cells. Culture medium generally includes a carbon source, a nitrogen source, and a buffer to maintain pH. In one embodiment, the growth medium contains a minimum essential medium, such as RPMI, supplemented with various nutrients to enhance cell growth. Additionally, the minimum essential medium can be supplemented with additives such as human, calf, or fetal bovine serum.

[0073] Neoplasia, Malignancy, Cancer or Tumor: A neoplasm is an abnormal growth of tissue or cells resulting from excessive cell division. Neoplastic growth can result in a tumor. A "cancer" or "tumor" is a neoplasm that has undergone characteristic anaplasia with loss of differentiation, increased growth rate, invasion of surrounding tissues, and is capable of metastasis. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors. Tumors that do not metastasize are termed "benign." Tumors that can invade surrounding tissues and / or metastasize are termed "malignant." Metastatic cancer is cancer at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer originated.

[0074] Examples of hematological tumors include leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, as well as myeloblastic leukemia, proosteatotic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0075] Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, and papillary carcinoma. , papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, and CNS tumors (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0076] In some embodiments, the tumor is head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma, or rectal cancer.

[0077] Parenteral: Administration outside the intestine, e.g., administration not via the digestive tract. In general, parenteral formulations are formulations that are administered in any possible manner other than oral ingestion. This term specifically refers to injection, whether intravenous, intrathecal, intramuscular, intraperitoneal, intraarticular, or subcutaneous, as well as application to various surfaces, including, for example, intranasal, intradermal, and topical application.

[0078] Pharmaceutical Product: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or cell.

[0079] Pharmaceutically acceptable carriers: Useful pharma- ceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the antibodies disclosed herein.

[0080] Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually contain injectable fluids that contain pharma- ceutically and physiologically acceptable fluids as vehicles, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. For solid compositions (e.g., in powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as, for example, sodium acetate or sorbitan monolaurate.

[0081] Polynucleotide: The term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotides that is at least 10 bases in length. A recombinant polynucleotide includes a polynucleotide that is not immediately adjacent to either of the coding sequences (one on the 5' end and one on the 3' end) that are immediately adjacent to it in the naturally occurring genome of the organism from which it is derived. Thus, the term includes recombinant DNA that is, for example, incorporated into a vector; incorporated into an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryote or eukaryote, or exists as a separate molecule independent of other sequences (e.g., cDNA). The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term encompasses single-stranded and double-stranded forms of DNA.

[0082] Polypeptide: any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). A polypeptide can be between 3 and 30 amino acids in length. In one embodiment, a polypeptide is from about 7 to about 25 amino acids in length. In yet another embodiment, a polypeptide is from about 8 to about 10 amino acids in length. In yet another embodiment, a peptide is about 9 amino acids in length. With respect to a polypeptide, "comprising" indicates that additional amino acid sequences or other molecules may be included in the molecule, "consisting essentially of" indicates that additional amino acid sequences are not included in the molecule, but other agents (e.g., labels or chemical compounds) may be included, and "consisting of" indicates that additional amino acid sequences and additional agents are not included in the molecule.

[0083] Preventing, Treating, or Ameliorating a Disease: "Preventing" a disease refers to inhibiting the full development of a disease, such as a tumor. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or condition after it has begun to develop, such as reducing tumor burden or the number or size of metastases. "Ameliorating" refers to a reduction in the number or severity of signs or symptoms of a disease, such as cancer.

[0084] Programmed cell death protein (PD)-1: The PD-1 molecule is a member of the immunoglobulin gene superfamily. Human PD-1 has an extracellular region that contains an immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) ((Ishida et al., EMBO J. 11:3887, 1992; Shinohara et al., Genomics 23:704, 1994; U.S. Patent No. 5,698,520, incorporated herein by reference). These features also define a larger family of molecules termed immunoinhibitory receptors, which also include gp49B, PIR-B, and killer inhibitory receptors (KIR) (Vivier and Daeron (1997) Immunol. Today 18:286). Without being bound by theory, it is believed that the tyrosyl phosphorylated ITIM motifs of these receptors interact with phosphatases containing S112 domains, thereby resulting in inhibitory signals. A subset of these immunosuppressive receptors bind to major histocompatibility complex (MHC) molecules such as KIR, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) binds to B7-1 and B7-2. In humans, PD-1 is a 50-55 kDa type I transmembrane receptor that was first identified in T-cell lines that underwent activation-induced apoptosis. PD-1 is expressed on T cells, B cells, and macrophages. The ligands for PD-1 are the B7 family members PD-ligand 1 (PD-L1, also known as B7-H1) and PD-L2 (also known as B7-DC).

[0085] In vivo, PD-1 is expressed in activated T cells, B cells, and monocytes. Experimental data implicates the interaction of PD-1 with its ligand in the downregulation of central and peripheral immune responses. Specifically, in the presence of PD-L1, the proliferation of wild-type T cells is inhibited, but the proliferation of PD-1-deficient T cells is not inhibited. Furthermore, PD-1-deficient mice exhibit an autoimmune phenotype. Exemplary amino acid sequences of human PD-1 are described in Ishida et al., EMBO J. 11:3887, 1992;Shinohara et al. Genomics 23:704,1994;U.S. Patent No. 5,698,520).

[0086] Assembly of PD-1 (e.g., by cross-linking or by aggregation) results in the transmission of inhibitory signals in immune cells, resulting in increased immune cell anergy and concomitant decreased immune responses. PD-1 binds to two ligands, PD-L1 and PD-L2, both of which are human PD-1 ligand polypeptides that are members of the B7 family of polypeptides.

[0087] PD-1 antagonists include agents that reduce the expression or activity of PD ligand 1 (PD-L1) or PD ligand 2 (PD-L2) or reduce the interaction of PD-1 with PD-L1 or PD-L2. Exemplary compounds include antibodies (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies), RNAi molecules (e.g., anti-PD-1 RNAi molecules, anti-PD-L1 RNAi, and anti-PD-L2 RNAi, etc.), antisense molecules (e.g., anti-PD-1 antisense RNA, anti-PD-L1 antisense RNA, and anti-PD-L2 antisense RNA), dominant negative proteins (e.g., dominant negative PD-1 proteins, dominant negative PD-L1 proteins, and dominant negative PD-L2 proteins). See, e.g., PCT Publication No. 2008 / 083174, which is incorporated herein by reference.

[0088] Proliferation: The division of cells to produce progeny and can be measured in a number of ways known in the art, including but not limited to, assays that count the total number of cells, assays that count the number of cells of a particular cell type, Ki-67 assays, thymidine incorporation, and bromodeoxyuridine assays.

[0089] Purified: The term purified does not require absolute purity and is intended as a relative term. Thus, for example, a purified peptide preparation is one in which a peptide or protein is enriched more than the peptide or protein is in its natural environment in a cell. In one embodiment, the preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. Substantial purification refers to purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free from other proteins and cellular components.

[0090] Sample (Biological Sample): Includes biological samples containing fluids, tissues, cells, and their subcomponents such as DNA, RNA, and proteins. For example, common samples include tumor biopsies, bone marrow, spleen, lymph nodes, blood, e.g., peripheral blood (but also CD8, CD9, CD10, CD11, CD12, CD13, CD24, CD25, CD30, CD46, CD47, CD50, CD60, CD89, CD91, CD14, CD15, CD26, CD16, CD27, CD38, CD49, CD51, CD17, CD28, CD49, CD19, CD29, CD30, CD49, CD18, CD29, CD31, CD41, CD42, CD19, CD25, CD43, CD19, CD26, CD19, CD27, CD18, CD28, CD31, CD44, CD19, CD29, CD32, CD45, CD19, CD29, CD46, CD19, CD28, CD19, CD29, CD31, CD45, CD19, CD29, CD32, CD46, CD19, CD29, CD47, CD19, CD29, CD31, CD48, CD19, CD29, CD49, CD19, CD29, CD31, CD45, CD19, CD29, CD46, CD19, CD29, CD31 ... + CD39 + CD103 + (It may also include any other source from which T cells can be isolated).

[0091] Specific binding agent: An agent that binds only to a substantially defined target. In one embodiment, a specific binding agent is a monoclonal or polyclonal antibody that specifically binds to a PD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG3 or 4-1BB polypeptide.

[0092] The term "specifically binds" refers to, in relation to an antigen, such as a PD-1, PD-L1, CTLA-4, BTLA, TIM-3, LAG3 or 4-1BB polypeptide, that an antibody or other ligand, in whole or in part, preferentially associates with cells or tissues bearing the antigen and not with cells or tissues lacking the antigen. Of course, it is recognized that a certain degree of non-specific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, specific binding can be distinguished because it is mediated by specific recognition of the antigen. A selectively reactive antibody binds to an antigen, but may do so with low affinity. Specific binding results in a much stronger association between the antibody (or other ligand) and a cell bearing the antigen than between the antibody (or other ligand) and a cell lacking the antigen. Specific binding generally results in a greater than 2-fold, e.g., greater than 5-fold, greater than 10-fold, or greater than 100-fold increase (per unit time) in the amount of antibody or other ligand bound to a cell or tissue bearing the polypeptide, compared to a cell or tissue lacking the polypeptide. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats are suitable for selecting antibodies or other ligands that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988).

[0093] Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.

[0094] T cell: white blood cell important to the immune response. T cells include, but are not limited to, CD4 + T cells and CD8 + T cells include CD4 + T lymphocytes are immune cells that have a marker on their surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help coordinate immune responses, including antibody responses as well as killer T cell responses. CD8 + T cells bear the "cluster of differentiation 8" (CD8) marker. In one embodiment, CD8+ T cells are cytotoxic T lymphocytes. In another embodiment, CD8+ cells are suppressor T cells. A T cell is "activated" if it can respond to a specific antigen of interest presented on an antigen-presenting cell.

[0095] T cell immunoglobulin and mucin domain-containing-3 (TIM-3): a protein encoded by the HAVCR2 gene in humans. TIM3 is a Th1-specific cell surface protein immune checkpoint that regulates macrophage activation and enhances the severity of experimental autoimmune encephalomyelitis in mice. The Tim-3 pathway may interact with the PD-1 pathway in exhausted CD8+ T cells in cancer. Exemplary mRNA and protein sequences for human TIM-3 are provided in GENBANK® Accession No. NM_032782.4, April 30, 2017, which is incorporated herein by reference.

[0096] Therapeutically effective amount: A quantity of a particular substance that is sufficient to achieve a desired effect in a treated subject. For example, a therapeutically effective amount may be the amount required to inhibit or suppress the growth of a tumor. In one embodiment, a therapeutically effective amount is the amount required to eliminate a tumor, reduce the size of a tumor, or prevent the metastasis of a tumor. When administered to a subject, a dosage is generally used that achieves a concentration in a target tissue (e.g., a concentration in a tumor) that has been shown to achieve a desired effect in vitro.

[0097] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Thus, "comprising A or B" means including A or B, or including A and B. It should be further understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. All GENBANK® accession numbers are incorporated by reference herein as they are in the database on June 1, 2017. In case of conflict, the present specification, including explanations of terms, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting. Treatment method: Adoptive immunotherapy

[0098] Disclosed herein are methods for treating a subject of interest, such as a subject having a tumor. The methods include administering a therapeutically effective amount of CD8 + CD39 + CD103 + The present invention relates to a method for enhancing an immune response in a subject, for example, for enhancing the immune system, comprising administering purified CD8 T cells to a subject. + CD39 + CD103 + Administration of T cells increases the subject's ability to mount an immune response, such as against a tumor. Thus, purified selected CD8 T cells from a subject can be used to + CD39 + CD103 +By purifying and generating a population of T cells ex vivo and introducing a therapeutic amount of these cells, the immune response of the recipient subject is enhanced. As discussed below, additional agents can also be administered to the subject. The subject can be a human or veterinary subject.

[0099] In one embodiment, the method comprises the steps of: + CD39 + CD103 + The method includes isolating a population of donor cells that includes T cells (e.g., peripheral blood mononuclear cells or T cells from a tumor biopsy) and, optionally, expanding the cells. + CD39 + CD103 + The T cells are administered to the recipient, thereby generating an immune response in the recipient against the tumor. + CD39 + CD103 + The T cells can kill cells containing tumor-associated antigens or assist other immune cells. In some embodiments, an additional cancer therapeutic agent, such as a chemotherapeutic agent, can be administered.

[0100] In some embodiments, the method also includes administering to the subject a therapeutically effective amount of a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist, a LAG3 antagonist, and / or a 4-1BB agonist. Administration of the PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist, a LAG3 antagonist, and / or a 4-1BB agonist is described in more detail below. A therapeutic amount of a CD8 + CD39 + CD103 +Administration of T cells and a therapeutically effective amount of a PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, BTLA antagonist, TIM-3 antagonist, LAG3 antagonist and / or 4-1BB agonist can be used prophylactically to prevent tumor recurrence or to treat tumor relapse in the recipient.

[0101] In general, CD8 + CD39 + CD103 + T cells are self. But CD8 + CD39 + CD103 + T cells may also react with matching MHC from an allogeneic donor. In general, T cells are positive for expression of CD3, CD8, CD39, and CD103. For example, by using differentially stained anti-CD3, anti-CD8, anti-CD39, and anti-CD103 antibodies, fluorescence-activated cell sorting (FACS) can be used to identify (and, if desired, sort) a population of cells positive for CD3, CD8, CD39, and CD103. Briefly, a population of cells, such as peripheral blood mononuclear cells or T cells from a tumor biopsy, is incubated in the presence of anti-CD103, anti-CD8, anti-CD39, and optionally anti-CD3 antibodies (each conjugated to a different fluorophore) for a time sufficient for the antibodies to bind to the cells. After removing unbound antibodies, the cells are analyzed by FACS using routine methods. In a particular example, the resulting CD8 + CD39 + CD103 +The population of T cells is at least 30% pure compared to the total population of CD8+ positive cells, for example, at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, or at least about 96%, about 97%, about 98% or about 99% pure compared to the total population of CD8 positive cells.Therefore, only a limited number of xenogeneic cells are administered.Cells can be processed through more than one round of cell sorting.The population of T cells can be tested for mycoplasma, sterility, endotoxin, and quality control for function and purity before cryopreservation or before infusion into recipient.

[0102] In one embodiment, labeled antibodies specifically directed against one or more cell surface markers are used to detect CD8 + CD39 + CD103 + T cells and populations of these cells expressing additional markers are identified, quantified, and / or isolated. Antibodies can be conjugated to other compounds, including but not limited to enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, or drugs. Enzymes that can be conjugated to antibodies include but are not limited to alkaline phosphatase, peroxidase, urease, and β-galactosidase. Fluorescent dyes that can be conjugated to antibodies include but are not limited to fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanin, and Texas Red. For additional fluorescent dyes that can be conjugated to antibodies, see Haugland, RP, Handbook of Fluorescent Probes and Research Products, published by Molecular Probes, 9 thEdition (2002). Metal compounds that can be conjugated to antibodies include, but are not limited to, ferritin, colloidal gold, and especially colloidal superparamagnetic beads. Haptens that can be conjugated to antibodies include, but are not limited to, biotin, digoxigenin, oxazalone, and nitrophenol. Radioactive compounds that can be conjugated to or incorporated into antibodies are known in the art and include, but are not limited to, technetium 99 ( 99 Tc), 125 I, as well as, but not limited to, 14 C. 3 H and 35 Any radionuclide-containing amino acid, including S, may be used.

[0103] In some examples, CD8 cells from a biological sample, such as a peripheral blood sample or a tumor sample, are contacted with an appropriately labeled antibody. + CD39 + CD103 + Isolate T cells. However, other techniques with different effectiveness can be used to purify and isolate the desired cell population. The separation technique used should maximize the retention of viability of the fraction of cells collected. The specific technique used will naturally depend on the efficiency of separation, the cytotoxicity of the method, the ease and speed of separation, and what equipment and / or technical skills are required.

[0104] Data generated by a flow cytometer can be plotted in one dimension to produce a histogram, or in a two-dimensional dot plot or even in three dimensions. Regions on these plots can be sequentially separated by a series of subset extractions, called "gates", based on the fluorescence intensity. Specific gating protocols are known in the art. Plots are generally made in log scale. Since the emission spectra of different fluorochromes overlap, the signals at the detector are electronically and computer-corrected. Data accumulated using a flow cytometer can be analyzed using software such as FLOWJO® or BD FACSDiva®. The analysis is most often performed on a separate computer. The principles of gating that allow the identification of cells expressing high or low levels of a protein of interest are well known in the art. Tutorials on learning to establish gates are provided, for example, on the FLOWJO® website. In general, one skilled in the art can easily use any FACS machine and a computer program for data analysis to establish gates to separate cells expressing a particular marker. By way of example, one skilled in the art can easily use any FACS machine and a computer program for data analysis to establish gates to separate cells expressing a particular marker. By way of example, one skilled in the art can easily use any FACS machine and a computer program for data analysis to establish gates to separate cells expressing a particular marker. - ) cells, with expression of CD8 (CD8 + ) cells can be easily identified.

[0105] Additional separation procedures may include magnetic separation using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents that are bound to monoclonal antibodies or used in conjunction with complement, and "panning" utilizing monoclonal antibodies bound to solid matrices, or other conventional techniques. Magnetic beads and antibodies bound to other solid matrices such as agarose beads, polystyrene beads, hollow fiber membranes, and plastic petri dishes allow for direct separation. Antibody-bound cells can be removed from the cell suspension simply by physically separating the solid support from the cell suspension. The exact conditions and duration of incubation of the cells with the antibody linked to the solid phase depend on several system-specific factors. However, the selection of appropriate conditions is well known in the art.

[0106] Then, after a sufficient time to allow cells expressing the marker of interest (for example, but not limited to, CD8, CD39, CD103, and optionally CD3) to bind to the antibody linked to the solid phase, the unbound cells can be eluted or washed away with a physiological buffer. The bound cells are then separated from the solid phase by any suitable method, depending mainly on the nature of the solid phase and the antibody used, and quantified using methods well known in the art. In one specific, non-limiting example, the bound cells separated from the solid phase are quantified by FACS (see above).

[0107] The antibodies can be conjugated to biotin, which can then be removed using avidin or streptavidin bound to a support, or to fluorescent dyes, allowing their use in FACS to separate and quantitate cells, as is known in the art.

[0108] In another embodiment, cells can be collected from a subject using an apheresis procedure using an automated apheresis instrument (e.g., a CS-3000 blood cell separator, Baxter Health Care, Deerfield, IL, or equivalent machine). In a specific, non-limiting example, labeled antibodies specifically directed against one or more cell surface markers can be used to isolate CD8 cells, such as those disclosed herein. + CD39 + CD103 + T cells are identified and quantified.

[0109] In some embodiments, CD8 + CD39 + CD103 + The T cells are expanded in vitro prior to administration to a subject. Expansion methods are disclosed below.

[0110] The present disclosure relates to enriched (e.g., purified) CD8 + CD39 + CD103 + Therapeutic compositions comprising T cells and optionally a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist, a LAG3 antagonist, and / or a 4-1BB agonist are also provided. The compositions are useful in the methods disclosed herein, including, but not limited to, treating a subject with a tumor. In certain embodiments, the ... + CD39 + CD103 + The population of T cells is in a therapeutic dosage form for administration to a subject in need thereof. The PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, BTLA antagonist, TIM-3 antagonist, LAG3 antagonist and / or 4-1BB agonist are also provided in a therapeutic dosage form for administration to a subject in need of treatment.

[0111] Therapeutic dose of CD8 + CD39 + CD103+ T cells are administered to the subject in a therapeutically effective amount of CD8 + CD39 + CD103 + Specific, non-limiting examples of T cells include approximately 1×10 cells per kilogram of subject. 5 Approximately 1 x 10 cells per kilogram of subject 9 For example, about 1 x 10 cells per kilogram 6 Approximately 1 x 10 cells per kilogram 8 For example, about 5 x 10 cells per kilogram 6 Approximately 75 x 10 cells per kilogram 6 For example, about 25 x 10 cells per kilogram 6 Approximately 50 x 10 cells per kilogram 6 Purified CD8 administered in doses + CD39 + CD103 + T cells are one example.

[0112] Purified CD8 + CD39 + CD103 + The T cells can be administered in a single dose or multiple doses as determined by the clinician. For example, the cells can be administered at intervals of approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, or monthly, depending on the desired response and the response obtained. In some examples, once the desired response is obtained, additional CD8 + CD39 + CD103 + No T cells are administered. However, if the recipient exhibits one or more symptoms associated with the tumor, a therapeutically effective amount of CD8 + CD39 + CD103 + T cells can be administered.

[0113] Administration can be local or systemic.In some embodiments, the subject is treated with chemotherapy, and then the cells are administered intravenously.In other embodiments, the subject is also administered cytokines such as IL-2 and / or IL-15 to support the growth of the administered cells.

[0114] The purified CD8 disclosed herein + CD39 + CD103 + The T cells can be administered with a pharma- ceutically acceptable carrier, such as saline. The PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, BTLA antagonist, TIM-3 antagonist, LAG3 antagonist, and / or 4-1BB agonist can also be formulated in a pharma- ceutically acceptable carrier, as described below. They can be formulated as a single composition or as two separate compositions. In some examples, other therapeutic agents are administered with the T cells. The other therapeutic agents can be used to inhibit CD8 + CD39 + CD103 + It can be administered before, during, or after administration of the T cells. Exemplary therapeutic agents include, but are not limited to, antibacterial agents, immunostimulants such as interferon-alpha, chemotherapeutic agents, or peptide vaccines used to stimulate T cells in vitro. In a particular example, CD8 + CD39 + CD103 + The composition containing the T cells also includes one or more therapeutic agents. + CD39 + CD103 + The use of T cells can reduce tumor volume, tumor metastasis, and tumor recurrence.

[0115] In general, the method includes the steps of selecting a subject having a tumor, such as a benign or malignant tumor, administering to the subject a therapeutically effective amount of (1) CD8 + CD39 + CD103 +and (2) optionally administering a checkpoint inhibitor antagonist, such as a PD-1 antagonist, a PD-L1 antagonist, a BTLA antagonist, a TIM-3 antagonist, a LAG3 antagonist, or a CTLA-4 antagonist, or a 4-1BB agonist. The PD-1 antagonist, PD-L1 antagonist, BTLA antagonist, TIM-3 antagonist, LAG3 antagonist, or CTLA-4 antagonist, or a 4-1BB agonist, in some non-limiting examples, can be an antibody (or an antigen-binding fragment thereof) that specifically binds to PD-1, PD-L1, PD-L1, PD-L2, TIM-3, LAG3, BTLA, CTLA-4, or 4-1BB, respectively. CD8 + CD39 + CD103 + T cells are useful for treating tumors, for example, for reducing tumor volume, reducing or preventing metastasis, preventing the transformation of benign tumors into malignant tumors, and / or preventing or inhibiting tumor recurrence. Administration can be local or systemic. Appropriate administration methods are known to clinicians.

[0116] In some embodiments, the advantages of the methods provided herein include CD8 + CD39 + CD103 + Combining T cells with checkpoint inhibitors, such as PD-1 antagonists, PD-L1 antagonists, BTLA antagonists, TIM-3 antagonists, LAG3 antagonists and / or CTLA-4 antagonists, or 4-1BB agonists, allows for a reduction in the dosage of active agents for cancer treatment, while also reducing any corresponding undesirable side effects of the treatment (e.g., cytotoxicity). In a further embodiment, another advantage of the methods provided herein is the ability to selectively inhibit CD8 + CD39 + CD103 +Combining T cells with checkpoint inhibitors, such as PD-1 antagonists, PD-L1 antagonists, BTLA antagonists, TIM-3 antagonists, LAG3 antagonists and / or CTLA-4 antagonists, or 4-1BB agonists, can treat tumors, e.g., reduce tumor volume, reduce or prevent metastasis, prevent conversion of benign tumors to malignant tumors, and / or prevent or inhibit tumor recurrence. + CD39 + CD103 + Combining T cells with checkpoint inhibitors such as PD-1 antagonists, PD-L1 antagonists, BTLA antagonists, TIM-3 antagonists, LAG3 antagonists and / or CTLA-4 antagonists, or 4-1BB agonists can increase survival.

[0117] For example, but not limited to, an additional agent such as a cancer therapeutic agent may be administered to the subject of interest. For example, but not limited to, an additional procedure such as surgical removal of a tumor may be performed on the subject.

[0118] The subject can be selected for treatment. For example, a diagnostic assay (e.g., immunohistochemical (IHC) assay) can be performed on the tumor (or a sample of the tumor) to identify the subject as a subject that may respond to the disclosed treatment method. The method of selection is disclosed below.

[0119] In a further embodiment, the subject is selected for treatment if the tumor tests positive for PD-L1 expression or PD-L2 expression by IHC assay. Exemplary assays for detecting tumors testing positive for PD-L1 expression are described in Topalian et al. 2012. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 366:2443-2454;Wolchok et al. 2013. Nivolumab plus ipilimumab in advanced melanoma. N. Engl. J. Med. 369:122-133;Herbst et al. 2014. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 515:563-567;Garon et al. 2015. Pembrolizumab for the treatment of non-small-cell lung cancer. N. Engl. J. Med. 369:122-133; 372:2018-2028; and Reck et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N. Engl. J. Med. 375:1823-1833.

[0120] The tumor may be benign or malignant. The tumor may be any tumor of interest, including, but not limited to, head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma, or rectal cancer. The lung cancer may be small cell lung cancer or non-small cell lung cancer. The liver cancer may be hepatoma. The breast cancer may be triple negative breast cancer. In some embodiments, the tumor is a head and neck tumor, such as tumor of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary gland, and paraganglioma. Further examples are skin tumor, brain tumor, cervical cancer, testicular cancer, gastrointestinal tumor, genitourinary system tumor, gynecological tumor, endocrine tumor, soft tissue and bone sarcoma, mesothelioma, melanoma, central nervous system neoplasm, or leukemia. In other embodiments, the tumor is a lung tumor, such as non-small cell lung cancer or small cell lung cancer. In further embodiments, the tumor can be a tumor of the gastrointestinal tract, such as cancer of the esophagus, stomach, pancreas, liver, biliary tree, small intestine, colon, rectum, and anal region. In still other embodiments, the tumor can be a tumor of the genitourinary system, such as cancer of the kidney, urethra, bladder, prostate, urethra, penis, and testicle. In some embodiments, the tumor is a gynecological tumor, such as cancer of the cervix, vagina, vulva, uterus, gestational trophoblastic disease, ovary, fallopian tube, peritoneum, or breast. In other embodiments, the tumor is an endocrine tumor, such as a thyroid tumor, a parathyroid tumor, an adrenal cortical tumor, a pancreatic endocrine tumor, a carcinoid tumor, and a carcinoid syndrome. The tumor may be soft tissue and bone sarcoma, mesothelioma, skin cancer, melanoma including cutaneous melanoma and intraocular melanoma, central nervous system neoplasm, childhood cancer including retinoblastoma, Wilms tumor, neurofibromatosis, neuroblastoma, Ewing sarcoma family tumor, rhabdomyosarcoma. The tumor may be lymphoma including non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, primary central nervous system lymphoma, and Hodgkin's disease. The tumor may be leukemia such as acute leukemia, chronic myeloid leukemia, and lymphocytic leukemia. The tumor may be plasma cell neoplasm, cancer of unknown primary site, peritoneal carcinomatosis, Kaposi's sarcoma, AIDS-related lymphoma, AIDS-related primary central nervous system lymphoma, AIDS-related Hodgkin's disease, and AIDS-related anogenital cancer, metastatic cancer to liver, metastatic cancer to bone, malignant pleural and pericardial effusion, and malignant ascites.

[0121] In some embodiments, tumor treatment is initiated after tumor diagnosis or after the onset of a precursor condition (e.g., development of dysplasia or benign tumor). Treatment can be initiated early in cancer, for example, before symptoms of the condition are manifest in the subject, for example, during diagnosis of stage I or when dysplasia or an in situ proliferative condition is diagnosed. However, treatment can be initiated at any stage of the disease, for example, but not limited to, stage I, stage II, stage III, and stage IV cancer. In some examples, treatment is administered to those subjects with benign tumors that may transform into malignant tumors or even metastatic tumors.

[0122] Treatment before the onset of a condition, for example, treatment when dysplasia or an early (benign) precursor condition is detected, is referred to herein as treatment of a subject "at risk" of developing a condition. In some embodiments, administration of the composition can be performed during or after the onset of a condition described herein. The composition can be administered to a subject at risk of developing a tumor.

[0123] The presence of a tumor can be determined by methods known in the art, and generally includes cytological and morphological evaluation. The tumor can be an established tumor. The cells can be in vivo or ex vivo, including cells obtained from a biopsy.

[0124] Treatment initiated after the onset of a condition, such as malignant cancer, can result in a reduction in the severity of one of the symptoms of the condition, or a complete elimination of the symptoms, or a reduction in metastases, tumor volume or number of tumors. In some examples, tumors are undetectable after treatment.

[0125] In one embodiment of the present disclosure, the formation of tumors, such as metastases, is delayed, prevented or reduced. In another embodiment, the size of the primary tumor is reduced. In a further embodiment, the symptoms of tumors are reduced. In yet another embodiment, the tumor volume is reduced. In yet another embodiment, the recurrence of tumors is delayed or prevented, for example, for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 2 months, 22 months, 23 months, or 24 months, or for 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years.

[0126] In some embodiments, the immune response can be measured, the tumor volume can be measured, the number of metastatic lesions can be measured, and / or the symptoms of the tumor can be measured. A therapeutically effective dose can increase the immune response, decrease the tumor volume, reduce the number and / or size of metastases, and / or reduce one or more symptoms of the tumor.

[0127] The disclosed methods and compositions are generally used to treat human subjects, but can also be used to treat similar or identical diseases in other vertebrates, such as other primates, dogs, cats, horses, and cattle. The appropriate mode of administration will best be determined by a medical practitioner for each subject individually. Various pharmacologic acceptable carriers and their formulations are described in standard formulation treatises, such as Remington's Pharmaceutical Sciences, by EW Martin. See also Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988. The dosage form of the pharmaceutical composition is determined by the mode of administration selected.

[0128] CD8 + CD39+ CD103 + T cells can be administered locally or systemically by any route. For example, CD8 + CD39 + CD103 + T cells can be administered intratumorally, intraperitoneally, or intravenously. In one non-limiting example, CD8 + CD39 + CD103 + T cells can be administered intravenously. PD-1, PD-L1, PD-L2, BTLA, TIM-3, LAG3, or CTLA-4 antagonists (or 4-1BB agonists) can also be administered by any route, including parenteral administration, e.g., intravenous, intraperitoneal, intramuscular, intraperitoneal, intrasternal, or intraarticular injection or infusion, or by sublingual, oral, topical, intranasal, or transmucosal administration, or by pulmonary inhalation. In some embodiments, CD8 + CD39 + CD103 + The T cells and / or PD-1, PD-L1, PD-L2, BTLA, TIM-3, LAG3, or CTLA-4 antagonists are administered to the tissue in which the tumor is located or directly to the tumor (intramyocellular). When providing a parenteral composition, for example for injection or infusion, the active agent is generally suspended in an aqueous carrier, for example, an isotonic buffer solution having a pH of about 3.0 to about 8.0, preferably about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffer solutions include sodium citrate-citric acid and sodium phosphate-phosphate, and sodium acetate-acetic acid buffers. A repository or "depot" type of slow release preparation form can be used such that a therapeutically effective amount of the preparation is delivered into the bloodstream for hours or days after transdermal injection or delivery.

[0129] In certain embodiments, a PD-1, PD-L1, PD-L2, BTLA, TIM-3, LAG3, or CTLA-4 antagonist (such as, but not limited to, an antibody or antigen-binding fragment), or a 4-1BB agonist may be administered at a dose ranging from about 0.01-10 mg / kg, 0.01-5 mg / kg, 0.01-1 mg / kg, 0.01-0.1 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-3 mg / kg, 0.5-1.0 mg / kg, 0.05-0.5 mg / kg, according to a dosing schedule including, but not limited to, administration daily, two or three times per week, weekly, every two weeks, every three weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician. As part of a combination therapy, a CD8 + CD39 + CD103 + The T cells can be administered to the subject before, after, or simultaneously with the additional agent.

[0130] In some embodiments, a PD-1 or PD-L1 antagonist (e.g., an antibody or antigen-binding fragment that specifically binds PD-1 or PD-L1) can be administered at a dose ranging from about 0.01-10 mg / kg, 0.01-5 mg / kg, 0.01-1 mg / kg, 0.01-0.1 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-3 mg / kg, 0.5-1.0 mg / kg, 0.05-0.5 mg / kg, according to a dosing schedule including, but not limited to, administration daily, two or three times per week, weekly, every two weeks, every three weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician, as part of a combination therapy, so long as the dosing schedule provides sufficient physiological concentrations of the agent to provide a therapeutic benefit. + CD39 + CD103 +T cells can be administered to the subject before, after, or simultaneously with a PD-1 or PD-L1 antagonist. In certain embodiments, a CTLA-4 antagonist (e.g., an antibody or antigen-binding fragment that specifically binds to CTLA-4) can be administered at a dose ranging from about 0.01-10 mg / kg, 0.01-5 mg / kg, 0.01-1 mg / kg, 0.01-0.1 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-3 mg / kg, 0.5-1.0 mg / kg, 0.05-0.5 mg / kg, according to a dosing schedule including, but not limited to, daily, two or three times per week, weekly, every two weeks, every three weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician, as part of a combination therapy, so long as the dosing schedule provides sufficient physiological concentrations of the agents to provide a therapeutic benefit. + CD39 + CD103 + The T cells can be administered to the subject before, after, or simultaneously with the CTLA-4 antagonist. In further embodiments, the BTLA antagonist (e.g., an antibody or antigen-binding fragment that specifically binds to BTLA) can be administered at a dose ranging from about 0.01-10 mg / kg, 0.01-5 mg / kg, 0.01-1 mg / kg, 0.01-0.1 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-3 mg / kg, 0.5-1.0 mg / kg, 0.05-0.5 mg / kg, according to a dosing schedule including, but not limited to, daily, two or three times per week, weekly, every two weeks, every three weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician. CD8 T cells can be administered as part of a combination therapy, so long as the dosing schedule provides sufficient physiological concentrations of the agents to provide a therapeutic benefit. + CD39 + CD103 +T cells can be administered to a subject before, after, or simultaneously with a BTLA antagonist. In additional embodiments, a LAG3 or TIM-3 antagonist (e.g., an antibody or antigen-binding fragment that specifically binds to LAG3 or TIM-3) can be administered at a dose ranging from about 0.01-10 mg / kg, 0.01-5 mg / kg, 0.01-1 mg / kg, 0.01-0.1 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-3 mg / kg, 0.5-1.0 mg / kg, 0.05-0.5 mg / kg, according to a dosing schedule including, but not limited to, daily, two or three times per week, weekly, every two weeks, every three weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician. CD8 antagonists can be administered as part of a combination therapy, so long as the dosing schedule provides sufficient physiological concentrations of the agents to provide a therapeutic benefit. + CD39 + CD103 + T cells can be administered to the subject before, after, or simultaneously with the LAG3 or TIM-3 antagonist. In yet other embodiments, a 4-1BB agonist (e.g., an antibody or antigen-binding fragment that specifically binds to 4-1BB) can be administered at a dose ranging from about 0.01-10 mg / kg, 0.01-5 mg / kg, 0.01-1 mg / kg, 0.01-0.1 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-3 mg / kg, 0.5-1.0 mg / kg, 0.05-0.5 mg / kg, according to a dosing schedule including, but not limited to, daily, two or three times per week, weekly, every two weeks, every three weeks, monthly, etc., or other dose and dosing schedule deemed appropriate by the treating physician, as part of a combination therapy, so long as the dosing schedule provides sufficient physiological concentrations of the agent to provide therapeutic benefit. + CD39 + CD103 + The T cells can be administered to the subject before, after, or simultaneously with the 4-1BB agonist.

[0131] A sustained release composition can be utilized. Suitable examples of sustained release compositions include suitable polymeric materials (e.g., semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules), suitable hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, and sparingly soluble derivatives (e.g., sparingly soluble salts). Depending on the location of the tumor, sustained release formulations can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by dust, ointment, gel, drops, or transdermal patch), bucally, or as an oral or nasal spray.

[0132] The pharma- ceutically acceptable carriers and excipients useful in the disclosed method are conventional.For example, parenteral formulations usually contain injectable fluids that are pharma- ceutical and physiologically acceptable fluid vehicles, such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol, etc. The excipients that can be included are, for example, proteins, such as human serum albumin or plasma preparations.If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.The actual preparation method of such dosage forms is known or will be apparent to those skilled in the art.

[0133] Kits are also available. + CD39 + CD103 +T cells and / or PD-1, PD-L1, PD-L2, LAG3, TIM-3, BTLA, or CTLA-4 antagonists can be formulated into unit dosage forms suitable for individual administration of precise dosages. The amount of active compound(s) administered depends on the subject being treated, the severity of the affliction, and the mode of administration, and is best left to the discretion of the prescribing clinician. Within these constraints, the formulation administered contains a quantity of active ingredient(s) in an amount effective to achieve the desired effect in the subject being treated. For example, multiple treatments are envisioned over a defined time interval, such as daily, biweekly, weekly, bimonthly, or monthly, to achieve chronic administration.

[0134] Additional agents such as cytokines, chemokines, or chemotherapeutic agents can be administered. These can be included in the disclosed pharmaceutical compositions. Cytokines such as interleukin 2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), or interferons such as interferon (IFN) beta can be administered. In one embodiment, a surgical procedure can be administered to the subject to prevent and treat cancer. In one embodiment, the administration is sequential. In other embodiments, the administration is simultaneous.

[0135] Examples of chemotherapeutic agents are alkylating agents, antimetabolites, natural products, or hormones and their antagonists.Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin or dacarbazine).Examples of antimetabolites include folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-FU or cytarabine), and purine analogs such as mercaptopurine or thioguanine. Examples of natural products include vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (e.g., L-asparaginase). Examples of miscellaneous agents include platinum coordination complexes (e.g., cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenal cortical suppressants (e.g., mitotane and aminoglutethimide). Examples of hormones and antagonists include corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., tamoxifen), and androgens (e.g., testosterone propionate and fluoxymesterone).Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes such as Docetaxel), Velban, Vincristine, VP-16, and some newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan, STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevalin, and Calcitriol. Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech). In some embodiments, the subject is administered sorafenib.

[0136] The additional chemotherapeutic agent may be an antibody. Antibodies may be provided in lyophilized form and rehydrated with sterile water prior to administration, but are also provided as sterile solutions of known concentrations. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and is generally administered at a dose of 0.5-15 mg per kg of body weight. Considerable experience is available in the art in administering antibody drugs, which have been marketed since the approval of RITUXAN® in the United States in 1997. The antibody may specifically bind to programmed death (PD)-1 or programmed death ligand (PD-L1) (see below). The antibody may be administered by slow infusion, rather than an intravenous push or bolus. In one example, a higher loading dose is administered, followed by maintenance doses at lower levels. For example, an initial loading dose of 4 mg / kg is infused over approximately 90 minutes, followed by weekly maintenance doses at 2 mg / kg infused over a 30 minute period for 4-8 weeks, if the previous dose was well tolerated.

[0137] Treatment regimens may also include combinations with surgery, chemotherapy, radiation, or other immunoablative agents such as CAMPATH, anti-CD3 antibody or other antibody therapy, cytotoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation, peptide vaccines such as those described in Izumoto et al. 2008 J Neurosurg 108:963-971. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), Included are antimetabolites (including, for example, folate antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin or bortezomib), immunomodulators such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0138] Common chemotherapeutic agents considered for use in combination therapy include anastrozole (ARIMIDEX®), bicalutamide (CASODEX®), bleomycin sulfate (BLENOXANE®), busulfan (MYLERAN®), busulfan injection (BUSULFEX®), capecitabine (XELODA®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (PARAPLATIN®), carmustine (BICNU®), and rifampicin (RIRI). ), chlorambucil (LEUKERAN®), cisplatin (PLATINOL®), cladribine (LEUSTATIN®), cyclophosphamide (CYTOXAN® or NEOSAR®), cytarabine, cytosine arabinoside (CYTOSAR-U®), cytarabine liposome injection (DEPOCYT®), dacarbazine (DTIC-DOME®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride ( CERUBIDINE®), Daunorubicin Citrate Liposomal Injection (DAUNOXOME®), Dexamethasone, Docetaxel (TAXOTERE®), Doxorubicin Hydrochloride (ADRIAMYCIN®, RUBEX®), Etoposide (VEPESID®), Fludarabine Phosphate (FLUDARA®), 5-Fluorouracil (ADRUCIL®, EFUDEX®), Flutamide (EULEXIN®), Tezacitibine, Gemcitabine (Diazepam), fluorodeoxycytidine), hydroxyurea (HYDREA®), idarubicin (IDAMYCIN®), ifosfamide (IFEX®), irinotecan (CAMPTOSAR®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (ALKERAN®), 6-mercaptopurine (PURINETHOL®), methotrexate (FOLEX®), mitoxantrone (NOVANTRONE®), Mylotarg,These include paclitaxel (TAXOL®), phoenix (yttrium 90 / MX-DTPA), pentostatin, polipheprosan 20 with carmustine implant (GLIADEL®), tamoxifen citrate (NOLVADEX®), teniposide (VUMON®), 6-thioguanine, thiotepa, tirapazamine (TIRAZONE®), injectable topotecan hydrochloride (HYCAMPTIN®), vinblastine (VELBAN®), vincristine (ONCOVIN®), and vinorelbine (NAVELBINE®). Exemplary alkylating agents include, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: uracil mustard (AMINOURACIL MUSTARD®, CHLORETHAMINACIL®, DEMETHYLDOPAN®, DESMETHYLDOPAN®, HAEMANTHAMINE®, NORDOPAN®, URACIL NITROGEN MUSTARD®, URACILLOST®, URACILMOSTAZA®, URAMUSTIN®, URAMUSTINE®), chlormethine (MUSTARGEN®), cyclophosphamide (CYTOXAN®, NEOSAR®, CLAFEN®, ENDOXAN®, PROCYTOX®, REVIMMUNE™), ifosfamide (MITOXANA®), melphalan (ALKEK®), urethra, isoflurane (URETHA), urethra, ... RAN®), chlorambucil (LEUKERAN®), pipobroman (AMEDEL®, VERCYTE®), triethylenemelamine (HEMEL®, HEXYLEN®, HEXASTAT®), triethylenethiophosphoramine, temozolomide (TEMODAR®), thiotepa (THIOPLEX®), busulfan (BUSILVEX®, MYLERAN®), carmustine (BiCNU®),These include lomustine (CEENU®), streptozocin (ZANOSAR®), and dacarbazine (DTIC-DOME®). Additional exemplary alkylating agents include, without limitation, oxaliplatin (ELOXATIN®); temozolomide (TEMODAR® and TEMODAL®); dactinomycin (actinomycin-D, also known as COSMEGEN®); melphalan (L-PAM, also known as L-sarcolysin, and phenylalanine mustard, ALKERAN®); altretamine (hexamethylmelamine (HMM), also known as HEXYLEN®); carmustine (BICNU®); bendamustine (TREANDA®); busulfan (BUSULFEX® and MYLERAN®); carboplatin (PARAPLATIN®); lomustine (CCNU, also known as CEENU®); cisplatin (CDDP, as PLATINOL® and PLATINOL®-AQ) chlorambucil (LEUKERAN®); cyclophosphamide (CYTOXAN® and NEOSAR®); dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-DOME®); altretamine (also known as hexamethylmelamine (HMM), HEXYLEN®); ifosfamide (IFEX®); prednumustine; procarbazine mechlorethamine (also known as nitrogen mustard, mustine, and mechlorethamine hydrochloride, MUSTARGEN®); streptozocin (ZANOSAR®); thiotepa (thiophosphatamide, also known as TESPA and TSPA, THIOPLEX®); cyclophosphamide (ENDOXAN®, CYTOXAN®, NEOSAR®, PROCYTOX®,Exemplary mTOR inhibitors include, for example, temsirolimus; ridaforolimus (formerly deforolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0, 4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication WO 03 / 064383; everolimus (AFINITOR® or RAD001); rapamycin (AY22989, SIROLIMUS®); simapimod (CAS 16430 1-51-3; emsirolimus, (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS1013101-36-4); and N 2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-, inner salt (SF1126, CAS 936487-67-1), and XL765. Exemplary immunomodulators include, for example, afutuzumab (available from ROCHE®); pegfilgrastim (NEULASTA®); lenalidomide (CC-5013, REVLIMID®); thalidomide (THALOMID®), actimid (CC4047); and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2, and interferon gamma, CAS951209-71-5, available from IRX Therapeutics). Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and RUBEX®); bleomycin (LENOXANE®); daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, CERUBIDINE®); daunorubicin liposome (daunorubicin citrate liposome, DAUNOXOME®); mitoxantrone (DHAD, NOVANTRONE®); epirubicin (ELLENCE™); idarubicin (IDAMYCIN®, IDAMYCIN PFS®); mitomycin C (MUTAMYCIN®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin. Exemplary vinca alkaloids include, for example, vinorelbine tartrate (NAVELBINE®), vincristine (ONCOVIN®), and vindesine (ELDISINE®); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, ALKABAN-AQ® and VELBAN®); and vinorelbine (NAVELBINE®).Exemplary proteosome inhibitors include bortezomib (VELCADE®); carfilzomib (PX-171-007, (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido). 2-methyl-5-thiazolyl)-pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912). Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), e.g., U.S. Pat. No. 6,111,090, European Patent No. 09. GITR fusion proteins as described in, for example, U.S. Patent No. 7,025,962, European Patent No. 1947183 B1, U.S. Patent No. 7,812,135, U.S. Patent No. 8,388,967, U.S. Patent No. 8,591,886, European Patent No. EP1866339, PCT Publication No. WO2011 / 028683, PCT Publication No. WO2013 / 03995 4, PCT Publication No. WO2005 / 007190, PCT Publication No. WO2007 / 133822, PCT Publication No. WO2005 / 055808, PCT Publication No. WO1999 / 40196, PCT Publication No. WO2001 / 03720, PCT Publication No. WO1999 / 20758, PCT Publication No. WO2006 / 083289, PCT Publication No. WO2005 / 115451, U.S. Patent No. 7,618,632, and PCT Publication No. WO2011 / 051726. How to increase CD39+CD103+CD8+T cells

[0139] CD8 + CD39 + CD103 + T cells can be expanded in vitro. In some embodiments, CD8 T cells isolated from a subject are + CD39 + CD103 + T cells can be cultured in tissue culture medium containing glutamine, serum, and antibiotics to form primary culture. Generally, cells are seeded in suitable culture vessels. The culture vessels used to culture cell(s) can include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multiwell plates, microslides, chamber slides, tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, as long as T cells can be cultured therein. Cells can be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. In some embodiments, the culture vessel can be a tissue culture plate, e.g., a 6-well, 24-well, or 96-well plate. In other embodiments, the culture vessel can be a bioreactor, which can refer to any ex vivo device or system that supports a biologically active environment so that cells can be propagated. The bioreactor can have a volume of at least or about 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein, and can be cultured with nutrients necessary to support the growth of a population of cells.

[0140] Generally, cells are cultured in a growth medium that includes a carbon source, a nitrogen source, and a buffer to maintain pH. The medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, pyruvate, buffering agents, and inorganic salts. An exemplary growth medium contains a minimum essential medium, such as Dulbecco's Modified Eagle Medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance T cell growth. Examples of minimum essential medium include, but are not limited to, Eagle's Minimum Essential Medium (MEM) Alpha Medium, Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI)-1640 Medium, 199 Medium, and F12 Medium. Antibiotics, such as, but not limited to, penicillin, streptomycin, or tetracycline, can be added to the medium if necessary. Glutamine can also be added to the tissue culture medium. Additives such as antibiotics and amino acids are known in the art.

[0141] Additionally, the minimal essential medium can be supplemented with additional additives such as human, fetal bovine or bovine serum. Serum can be included, for example, at a concentration of 10-15% (volume / volume), for example, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% serum. The serum can be fetal bovine serum. In other embodiments, the serum is human serum, such as human AB serum.

[0142] Alternatively, the medium may be serum-free. In other cases, the growth medium may contain a serum replacement. Exemplary serum replacements are known in the art. For example, KNOCKOUT™ serum replacement is disclosed, for example, in U.S. Patent Application Publication No. 2002 / 0076747, which is incorporated herein by reference. Replacements for serum may include materials that suitably contain albumin (e.g., lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextrans, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolgiycerol, or equivalents thereto. Replacements for serum may be prepared, for example, by methods disclosed in International Publication WO 98 / 30679.

[0143] The cultures may be "xeno-free (XF)," which refers to media or culture conditions that are essentially free of components derived from xenogeneic animals. For the culture of human cells, any protein from a non-human animal, such as mouse, would be a xeno component. Thus, in some embodiments, the disclosed conditions are xeno-free. Thus, for the culture of human cells, cultures that include human AB serum may be "xeno-free."

[0144] Other culture conditions can be defined as appropriate. For example, the culture temperature may be about 30-40°C, for example, at least or about 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, but is not particularly limited thereto. In one embodiment, the cells are cultured at 37°C. The CO2 concentration can be about 1-10%, for example, about 2%-5%, or any range derivable therein. In one non-limiting example, a CO2 concentration of about 5% is utilized.

[0145] The primary culture is stimulated with an effective amount of irradiated allogeneic feeder cells and cytokines such as interleukin (IL)-15 or IL-2 to form stimulated T cells.The feeder cells can be, for example, allogeneic irradiated peripheral blood mononuclear cells.The feeder cells, including human feeder cells, can be, for example, irradiated with 4000rad gamma irradiation.

[0146] In some embodiments, the cells are also stimulated with a polyclonal T cell stimulant such as phytohemagglutinin. In some non-limiting examples, a concentration of 1 μg / ml to 2 μg / ml is utilized. In further embodiments, both a cytokine such as IL-15 and / or IL-2 and PHA are utilized.

[0147] In some embodiments, about 1,000 to about 2,000 CD8 + CD39 + CD103 + A ratio is used such that T cells are stimulated with about 100,000 to about 300,000 allogeneic feeder cells, such as irradiated allogeneic PBMCs. In another embodiment, about 1,000 to about 2,000 CD8 + CD39 + CD103 + A ratio is used such that T cells are stimulated with approximately 200,000 allogeneic feeder cells.

[0148] A cytokine such as IL-2 or IL-15 can be included in the culture. In some embodiments, IL-15 can be used at a concentration of about 5 ng / ml to about 15 ng / ml of IL-15. In some embodiments, IL-15 is included in the culture at a concentration of about 7 ng / ml to about 12 ng / ml, for example, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, or about 12 ng / ml. In one non-limiting example, IL-15 is included at a concentration of about 10 ng / ml.

[0149] The stimulated CD39+CD103+CD8+ T cell cultures are then supplemented with fresh tissue culture medium and cytokines such as IL-15 and / or IL-2 throughout the in-vitro expansion. IL-15 can be included in the tissue culture medium at a concentration of about 5 ng / ml to about 50 ng / ml of IL-15, for example, about 10 ng / ml to about 50 ng / ml of IL-15. In some embodiments, IL-15 is included in the cultures at a concentration of about 7 ng / ml to about 12 ng / ml, for example, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, or about 12 ng / ml. In one non-limiting example, IL-15 is included at a concentration of about 10 ng / ml. In other examples, IL-15 is included at a concentration of about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, or about 50 ng / ml.

[0150] The cell culture can be maintained for any period of time, in some embodiments, expanded CD39+CD103+CD8+ T cells are harvested after 15-30 days of culture. Methods for isolating and using nucleic acids encoding T cell receptors

[0151] Methods are provided for isolating nucleic acids encoding T cell receptors (TCRs) that specifically bind to tumor cell antigens. These methods involve isolating CD39 T cells, such as CD39+CD103+CD8 T cells, from a sample from a subject with a tumor that expresses the tumor cell antigen. + CD8 + The method includes isolating T cells. In some embodiments, the tumor is a solid tumor, such as head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma, or rectal cancer. The subject may be a human subject or a veterinary subject. The sample may be any sample from the subject, including, but not limited to, a peripheral blood sample or a tumor biopsy. CD8 T cells, such as CD39+CD103+CD8 T cells. + CD39 + Methods for isolating T cells are disclosed above.

[0152] In some embodiments, the method comprises administering to a subject a CD8 T cell, such as a CD39+CD103+CD8 T cell. + CD39 + This includes increasing the number of T cells. + CD39 + Methods for expanding T cells in vitro are also disclosed above. In another embodiment, primary CD8 T cells, such as CD39+CD103+CD8 T cells, are + CD39 + T cells are utilized, where the cells are not expanded in vitro.

[0153] The method includes detecting CD8 T cells, such as CD39+CD103+CD8 T cells. + CD39 + The method further includes cloning a nucleic acid molecule encoding the TCR from the T cell. Methods for cloning TCRs are known in the art, see, for example, U.S. Patent No. 8,697,854, which is incorporated herein by reference. TCRs are members of the immunoglobulin superfamily and usually consist of an α-subunit and a β-subunit. They have an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a transmembrane / cell membrane spanning region, and a short C-terminal cytoplasmic tail. Both the TCR α-chain and β-chain variable domains have three hypervariable or complementarity determining regions (CDRs), but the β-chain variable region has an additional hypervariable region (HV4) that does not usually contact the antigen and is therefore not considered a CDR.

[0154] CDR3 is the main CDR that recognizes processed antigens, but CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides. CDR1 of the beta-chain also interacts with the C-terminal portion of peptides. CDR2 is thought to recognize MHC. CDR4 of the beta-chain is not thought to be involved in antigen recognition, but has been shown to interact with superantigens. The constant domain of the TCR domain consists of a short connecting sequence in which cysteine ​​residues form disulfide bonds that form the link between the two chains. The affinity of TCRs for specific antigens makes them therapeutically useful. For example, tumors can be effectively treated by using adoptive immunotherapy with T cells expressing specific TCRs. Methods for cloning TCRs and using adoptive immunotherapy with TCR-transfected cells are disclosed in PCT Publication No. WO2006 / 031221, U.S. Patent No. 5,906,936; PCT Publication No. WO97 / 32603; PCT Publication No. WO2007 / 065957, and PCT Publication No. WO2008 / 039818. Nucleic acid molecules encoding TCRs and methods of generating T cells (or NK cells) containing such receptors are described, for example, in Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1 -9; Till et al., 2008, Blood, 1 12:2261 -2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT Publication Nos. WO2012 / 079000, WO2013 / 126726; and U.S. Patent Publication No. 2012 / 0213783.

[0155] In some embodiments, single cell RNA sequence is used for T cell receptor or pair-seq (Adaptive). In one non-limiting example, purified T cells are isolated into single cells using fluidigm or 10x genomics instruments. T cell DNA is then amplified and sequenced. T cells do not need to be antigen-stimulated or presented with antigen for this process.

[0156] Further examples of suitable cloning and sequencing techniques, as well as sufficient instructions to orient one of skill in the art to many cloning disciplines, are known (e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4 th(ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Product information from manufacturers of biological reagents and laboratory equipment can also provide useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to those of skill in the art.

[0157] Nucleic acid sequences encoding TCRs can be prepared by any suitable method. Once the entire sequence has been cloned and known, it can be synthesized by the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the diethylphosphoramidite method of Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1982, using an automated synthesizer, e.g., as described in Needham-VanDevanter et al., Nucl. Acids Res.12:6159-6168, 1984. They can also be prepared by direct chemical synthesis, such as the solid-phase phosphoramidite triester method described in 1981; and the solid-support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces single-stranded oligonucleotides, which can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.

[0158] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification systems are well known to those skilled in the art. Modifications can be made to the nucleic acids encoding the polypeptides described herein without reducing their biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the target molecule into a fusion protein. Such modifications are well known to those skilled in the art and include, for example, stop codons, methionine added to the amino terminus to provide an initiation site, additional amino acids placed on either end to create conventionally placed restriction sites, or additional amino acids (e.g., polyHis) to aid in purification steps.

[0159] The nucleic acid molecule encoding the TCR can be operably linked to a heterologous promoter. The nucleic acid molecule encoding the TCR can be included in a vector (e.g., a lentiviral vector or a gamma retroviral vector) for expression in a host cell. Exemplary cells are mammalian cells, including T cells, such as cytotoxic T lymphocytes (CTLs), and NK cells. In certain non-limiting examples, the cells are CD3 + T cells such as T cells. CD3 + T cells are CD4 + T cells or CD8 + It may be a T cell.

[0160] The nucleic acid molecules can also be expressed in recombinantly engineered cells such as bacteria, plant, yeast, insect and mammalian cells. The TCR can also be expressed as a fusion protein. Methods for expression and purification of antibodies and antigen-binding fragments are known and further described herein (see, for example, Al-Rubeai (ed), Antibody Expression and Production, Springer Press, 2011). Those skilled in the art are knowledgeable of the numerous expression systems available for expressing proteins, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as COS, CHO, HeLa and myeloma cell lines. The term "host cell" also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell, as mutations may occur during replication. Methods for stable transfer, i.e., continuously maintaining foreign DNA in a host, are known in the art. As disclosed herein, certain embodiments of the present disclosure include T cells, such as human T cells and human NK cells, that express the TCR. These T cells are CD4 + T cells or CD8 + CD3 on T cells + It may be a T cell.

[0161] Expression of the nucleic acid encoding the TCR against a promoter (either constitutive or inducible) followed by incorporation into an expression cassette. The promoter may be any promoter of interest, including the cytomegalovirus promoter and the human T-cell lymphotropic virus promoter (HTLV)-1. Optionally, an enhancer, such as the cytomegalovirus enhancer, is included in the construct. The cassette may be suitable for replication and integration in either prokaryotes or eukaryotes. A typical expression cassette contains certain sequences useful for regulating the expression of the DNA encoding the protein. For example, the expression cassette may include a suitable promoter, enhancer, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of the gene encoding the protein, splicing signals for introns, sequences to maintain the correct reading frame of the gene to allow proper translation of the mRNA, and a stop codon. The vector may encode a selection marker, such as a marker encoding drug resistance (e.g., ampicillin resistance or tetracycline resistance).

[0162] To obtain high-level expression of cloned genes, it is desirable to construct an expression cassette that contains, at a minimum, a strong promoter to direct transcription, a ribosome binding site (internal ribosome binding sequence) for translation initiation, and a transcription / translation terminator. For eukaryotic cells, the control sequences may include promoters and / or enhancers derived from, for example, immunoglobulin genes, HTLV, SV40, or cytomegalovirus, as well as polyadenylation sequences, and may further include splice donor and / or acceptor sequences (e.g., CMV and / or HTLV splice acceptor and donor sequences).

[0163] When the host is a eukaryotic organism, methods of DNA transfection can be used, such as calcium phosphate co-precipitation, microinjection, electroporation, traditional mechanical procedures such as insertion of a plasmid or viral vector packaged in a liposome, etc. Cells transformed with the cassette can be selected by resistance to antibiotics conferred by genes contained in the cassette, such as the amp, gpt, neo and hyg genes.

[0164] Eukaryotic cells can also be co-transformed with a polynucleotide sequence encoding a TCR and a second foreign DNA molecule encoding a selectable phenotype, such as a herpes simplex thymidine kinase gene. Another method is to use eukaryotic virus vectors, such as Simian Virus 40 (SV40), lentivirus or bovine papillomavirus, to transiently infect or transform eukaryotic cells to express proteins (see, for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011). Those skilled in the art can easily use expression systems, such as plasmids and vectors, that are useful for producing proteins in cells, including higher eukaryotic cells, such as COS, CHO, HeLa and myeloma cell lines.

[0165] In some embodiments, viral vectors are utilized to express TCR. Viral vectors include, but are not limited to, retroviruses, such as Simian Virus 40, adenovirus, adeno-associated virus (AAV), lentiviral vectors, and gamma retroviruses. Retroviral vectors provide a highly efficient method for gene transfer into eukaryotic cells. Furthermore, retroviral integration occurs in a controlled manner, resulting in stable integration of one or a few copies of new genetic information per cell. Without being bound by theory, lentiviral vectors have the advantage over vectors derived from oncoretroviruses, such as mouse leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. Lentiviral vectors also have the additional advantage of being less immunogenic. The use of lentiviral vectors to express TCR is known in the art, and is disclosed, for example, in US Patent Application No. 2014 / 0050708, which is incorporated herein by reference. Transposons can be used.

[0166] In some embodiments, host cells are produced for introduction into a subject of interest. The host cells can be peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC), purified T cells, or purified NK cells. The T cells can be any T cell, such as cultured T cells, e.g., primary T cells, or T cells from a cultured T cell line, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal (e.g., a human patient to whom the TCR-T cells are subsequently administered). When obtained from a mammalian subject, such as a human subject, the T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can also be enriched or purified for purification. The T cells can be, but are not limited to, CD3 + cells, CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 cells and Th2 cells, CD8 +The T cells may be of any type and at any stage of development, including T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc. + T cells or CD4 + CD3 on T cells + The cells may be T cells. In alternative embodiments, the cells may be NK cells, such as NK cells obtained from the same subject to which the TCR-NK cells are subsequently administered. In some embodiments, the cells are human cells. In other embodiments, the cells are from a veterinary subject.

[0167] Also provided are populations of cells comprising at least one host cell as described herein. The population of cells may be a heterogeneous population comprising host cells comprising any of the recombinant expression vectors encoding a TCR, at least one other cell, as well as, for example, host cells not comprising any recombinant expression vector (e.g., T cells), or cells other than T cells, such as, for example, B cells, macrophages, neutrophils, erythrocytes, etc. Alternatively, the population of cells may be a substantially homogenous population, where the population comprises primarily (e.g., consists essentially of) host cells comprising a recombinant expression vector encoding a TCR. The population may be a clonal population of cells, where all cells of the population are clones of a single host cell comprising the recombinant expression vector, and thus all cells of the population comprise the recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector as described herein. The T cells are CD8 + T cells or CD4 + The cell may be a CD3+ T cell, such as a T cell. The cell may be a NK cell.

[0168] The cells may be autologous to the recipient. The recipient may be a recipient with or at risk of having a tumor. The recipient may have been pretreated for the tumor. The tumor may be a solid tumor, such as head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma, or rectal cancer. In some embodiments, autologous T cells or NK cells are isolated from a subject, such as a human subject, and the isolated TCR is introduced into these cells. These transformed cells are then reintroduced into the subject. In this scenario, the donor and recipient are the same subject. The subject may be a human. In some embodiments, the subject is administered a therapeutically effective amount of T cells and / or NK cells expressing the cloned TCR. In certain embodiments (see US Patent Publication No. 20140271635 A1, incorporated herein by reference), a source of T cells is obtained from the subject prior to expansion and genetic modification.

[0169] In some embodiments, the T cells and / or NK cells are autologous. In other embodiments, the T cells and / or NK cells are allogeneic. The T cells and / or NK cells are then introduced into the subject as disclosed above. In one embodiment, the cells transiently express the vector 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. In one non-limiting example, the vector is transduced into the T cells by electroporation.

[0170] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, pigs (and other veterinary subjects), and non-human primates. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In other embodiments, any number of T cell lines available in the art can be used. In some embodiments, the subject can undergo leukapheresis, in which leukocytes are harvested and enriched or depleted ex vivo to select and / or isolate cells of interest, e.g., T cells and / or NK cells. These cell isolates can be expanded by methods known in the art and treated to introduce the TCR construct, thereby generating autologous cells expressing the cloned TCR. In one aspect, viral vectors, such as, but not limited to, lentiviral viral vectors, are used to generate TCR-expressing cells. In some non-limiting examples, T cells and / or NK cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those of skill in the art, such as FICOLL™ separation, or the cells can be obtained by apheresis. The apheresis product generally contains lymphocytes, including T cells, monocytes, granulocytes, B cells, NK cells, other nucleated white blood cells, red blood cells, and platelets.

[0171] Cells harvested by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing steps. In some non-limiting examples, the cells are washed with phosphate buffered saline (PBS). In alternative examples, the washing solution lacks calcium, may lack magnesium, or may lack many, but not all, divalent cations. An initial activation step in the absence of calcium can result in extended activation. The washing step can be accomplished by methods known in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CYTOMATE®, or HAEMONETICS CELL SAVER 5®) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample can be removed and the cells can be resuspended directly in culture medium.

[0172] In some embodiments, T cells are isolated from peripheral blood lymphocytes by negative selection. Specific subpopulations of T cells, such as CD3+, CD4+, CD8+, CD28+CD45RA+, and CD45RO+ T cells, naive and / or memory T cells, can be further isolated by positive or negative selection techniques. For example, T cells can be isolated by incubating with beads conjugated with anti-CD3 / anti-CD28, such as DYNABEADS® M-450 CD3 / CD28 T, or CD3 / IL-2, for a period of time sufficient for positive selection of the desired T cells. See, for example, US Patent Publication No. 20140271635 A1. In a non-limiting example, the period is about 24 to about 72 hours and all integer values ​​therebetween. In another non-limiting example, the period is at least 24 hours, 36 hours, 48 ​​hours, or longer. Longer incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as isolation from immunocompromised individuals. Multiple rounds of selection can also be used.

[0173] Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies directed to surface markers unique to the cells to be negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on the cells to be negatively selected. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail generally includes antibodies against CD8a, CD14, CD15, CD16, CD19, CD36, CD56, CD132, TCR gamma / delta, and CD235a (glycophorin A). To enrich CD8+ cells by negative selection, the monoclonal antibody cocktail contains antibodies against CD4, CD15, CD16, CD19, CD34, CD36, CD56, CD123, TCR gamma / delta, and CD235a. T cell populations expressing one or more cytokines can be selected. Methods for screening cellular expression are disclosed in PCT Publication WO2013 / 126712.

[0174] With regard to isolating a desired population of cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied to ensure maximum cell-bead contact. In some embodiments, a concentration of 1 million cells per ml is used. In further embodiments, more than 100 million cells per ml is used. In other embodiments, a concentration of 10, 15, 20, 25, 30, 35, 40, 45, 50, 65, 70, 75, 80, 85, 90, 95, or 100 million cells per ml is used. Without being bound by theory, the use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Lower concentrations of cells can also be used. Without being bound by theory, significant dilution of the mixture of T cells and surface (e.g., particles such as beads) minimizes particle-cell interactions. This selects for cells that express large amounts of the desired antigen that binds to the particles. In some embodiments, the concentration of cells used is 5×10 per ml. 6 In another embodiment, the concentration used is about 1×10 per ml. 5 From 1 to 1 x 10 per ml 6 The number can be up to and any integer value between.

[0175] The cells can be incubated on a rotator at varying speeds and at either 2-10°C or room temperature for various lengths of time. T cells for stimulation can also be frozen after a washing step. Without being bound by theory, the freezing and subsequent thawing step results in a more homogenous product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then freezing the cells to -80°C at a rate of 1°C per minute and storing them in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing as well as uncontrolled instant freezing at -20°C or in liquid nitrogen can be used. See U.S. Patent Application Publication No. 2014-0271635 A1.

[0176] The blood sample or apheresis product can be taken from the subject at a time period prior to when the expanded cells as described herein may be needed. In that way, the source of cells to be expanded can be collected at any time necessary, and the desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions for which T cell therapy, such as those described herein, is beneficial. In one embodiment, the blood sample or apheresis is generally obtained from a healthy subject. In certain embodiments, the blood sample or apheresis is generally obtained from a healthy subject at risk of developing a disease, such as a tumor, but not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells can be expanded, frozen, and used at a later time. In certain embodiments, the sample is collected from the patient shortly after diagnosis of a particular disease, such as a tumor, but prior to any treatment as described herein. In another embodiment, the cells are isolated from a blood sample or apheresis from the subject prior to any number of relevant treatment modalities. In certain embodiments, cryopreserved cells are thawed as described herein, washed, and left at room temperature for 1 hour before use.Blood samples or apheresis products are collected from subjects when needed and may not be frozen.In some embodiments, autologous tumor-bearing T cells are isolated from individuals for subsequent transfection and infusion.

[0177] T cells can generally be activated and expanded using methods described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded by contacting with a surface bound with an agent that stimulates signals associated with the CD3 / TCR complex and a ligand that stimulates costimulatory molecules on the surface of the T cells. T cells can also be expanded using PHA, or bulk PBMCs can be transfected with CD3 / 28 or CD3 / IL2. In some non-limiting examples, a population of T cells can be stimulated as described herein, for example, by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under appropriate conditions to stimulate proliferation of T cells. An anti-CD3 antibody and an anti-CD28 antibody to stimulate proliferation of either CD4+ T cells or CD8+ T cells.Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and can be used generally as in other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol. Meth. 227(1-2):53-63, 1999). Other methods include the use of allogeneic irradiated PBMCs with phytohemagglutinin to stimulate T cell proliferation.

[0178] Once the TCR is isolated, various assays can be used to assess the activity of the molecule in appropriate in vitro and animal models, including but not limited to, the ability to expand T cells after antigen stimulation, the ability to sustain T cell expansion in the absence of restimulation, and anti-cancer activity. In some embodiments, the upregulation of activation markers, such as but not limited to 1-1BB, is assessed, such as by flow cytometry.

[0179] The method comprises administering to a subject a therapeutically effective amount of the cells of the pharmaceutical composition, for example, the T cells and / or NK cells expressing cloned TCR.Subjects in need thereof, such as subjects with tumors, can then undergo standard treatment with chemotherapy or surgery (for cancer) or antiviral agents (for HIV infection).Administering the host cells expressing heterologous TCR can result in tumor treatment, such as reducing tumor volume, reducing metastasis, or reducing tumor signs or symptoms.

[0180] A pharmaceutical composition may include a TCR-expressing host cell, e.g., a plurality of TCR-expressing host cells as described herein, in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents or excipients. + and / or CD8 +CD3 on T cells + The composition may be a T cell, such as a T cell, and / or a NK cell. Such compositions may include, for example, a buffer solution, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol, proteins, amino acids, such as polypeptides or glycine, antioxidants, chelating agents, such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0181] For cells, various aqueous carriers can be used for the introduction of cells, such as buffered saline. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., to approximate physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., if necessary. The concentrations in these formulations can vary widely and are selected primarily based on the volume of fluid, viscosity, body weight, etc., depending on the particular mode of administration selected and the needs of the subject.

[0182] In one embodiment, the pharmaceutical composition is substantially free, e.g., absent at detectable levels, of contaminants such as endotoxins, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, anti-CD8 / anti-CD39 / anti-CD103 coated bead residues, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cells or plasmid components, bacteria and fungi.

[0183] The exact amount of the composition to be administered can be determined by the physician considering individual differences in age, weight, tumor size, extent of metastasis, and condition of the patient (subject). In general, the pharmaceutical compositions comprising the T cells (and / or NK cells) described herein are administered at a dose of 10 cells / kg body weight, including all integer values ​​within the range. 4 ~10 9For example, 10 cells per kg of body weight 5 ~10 6 It can be stated that the cells can be administered in doses of 10 cells per kg. An exemplary dose is 10 cells per kg. 6 Approximately 1 x 10 cells per 1 kg 8 up to 5 x 10 cells per kg, for example 6 Approximately 7.5 x 10 cells per kg 7 up to 10 cells, e.g., about 2.5 x 10 cells per kg 7 Approximately 5.0 x 10 cells per kg 7 There are 10 pieces.

[0184] The composition can be administered at these dosages once or multiple times, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The composition can generally be administered by using injection techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The composition can be administered daily, weekly, bimonthly, or monthly. In some non-limiting examples, the composition is formulated for intravenous administration and administered multiple times. The amount and frequency of administration are determined by factors such as the subject's condition and the type and severity of the subject's disease, but the appropriate dosage can be determined by clinical trials.

[0185] In one embodiment, the TCR is introduced into cells such as T cells or NK cells, and the subject receives an initial administration of cells and one or more subsequent administrations of cells, where the one or more subsequent administrations are administered less than 15 days after the previous administration, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, the subject (e.g., a human) is provided with more than one administration of cells per week, and for example, two, three, or four administrations of TCR-expressing cells are administered per week. In one embodiment, the subject receives more than one administration of T cells per week (e.g., two, three, or four administrations per week) (also referred to as a cycle), followed by a week without administration, and then the subject is administered one or more additional administrations of TCR-expressing cells (e.g., more than one administration of cells per week). In another embodiment, a subject (e.g., a human subject) receives more than one cycle of TCR-expressing cells, with the time between each cycle being less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 3 days. In one embodiment, the TCR-expressing cells are administered every other day, three doses per week. In another embodiment, the TCR-expressing cells are administered for at least 2, 3, 4, 5, 6, 7, 8 or more weeks. The dosage of the above treatment administered to a patient varies depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed according to art-accepted practices.

[0186] In some embodiments, TCR modified T cells can replicate in vivo and persist for long periods of time, thereby resulting in sustained tumor control. In various aspects, the T cells administered to a subject, or the progeny of these cells, persist in the subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or years after administration of the T cells to the subject. In other embodiments, the cells and their progeny persist for less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, or less than 1 month, e.g., 3 weeks, 2 weeks, 1 week, after administration of the T cells to the subject.

[0187] The subject compositions can be administered in any conventional manner, including by injection, transfusion, implantation, or transplantation. The disclosed compositions can be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In some embodiments, the compositions are administered to a patient by intradermal or subcutaneous injection. In other embodiments, the compositions of the invention are administered by iv injection. The compositions can also be injected directly into a tumor or lymph node.

[0188] In one embodiment, the composition containing the population of isolated cells may also contain one or more additional pharmaceutical agents, such as one or more antibacterial agents (e.g., antibiotics, antiviral agents, and antifungal agents), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), depleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), or nonsteroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen, or naproxen sodium, cytokines (e.g., interleukin 2), or vaccines. Many chemotherapeutic agents are currently known in the art. In one embodiment, the chemotherapeutic agent is selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvival agents, biological response modifiers, antihormonal agents, e.g., antiandrogens, and antiangiogenic agents.

[0189] For the treatment of malignancies, the methods may also include administering to the subject a therapeutically effective amount of an additional cancer therapeutic agent (including chemotherapeutic agents and radiation) or performing surgery.

[0190] The TCR-expressing cells can be administered in conjunction with surgery, radiation, chemotherapy, or immunotherapy. Suitable chemotherapeutic agents are disclosed above. The TCR-expressing cells can also be administered in conjunction with PD-1, CTLA-4, TIM-3, LAG3, BTLA antagonists and / or 4-1BB agonists, as disclosed in detail below. Methods of Detection and Treatment

[0191] CD39, such as CD39+CD103+CD8 T cells + CD8 + It is disclosed herein that administration of T cells is useful in diagnosis and treatment. In these embodiments, administration of CD39 T cells, such as CD39+CD103+CD8 T cells, in a biological sample from a subject is useful. + CD8 +T cells are measured. In some embodiments, the sample is a peripheral blood sample or a tumor biopsy. The subject can be any subject, such as a human or veterinary subject. In further embodiments, the subject has a tumor, is suspected of having a tumor, or is at risk of having a tumor. The tumor can be a solid tumor. In some non-limiting examples, the solid tumor is head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma, or rectal cancer.

[0192] In some embodiments, methods are disclosed for determining whether a subject with a tumor responds to a cancer therapeutic agent, including, but not limited to, biological response modifiers (e.g., cytokines and chemokines), cancer vaccines, chemotherapy agents, immunotherapy agents, and radiation. In some embodiments, the cancer therapeutic agent can be a checkpoint inhibitor, a 4-1BB agonist, and / or radiation. The cancer therapeutic agent can be a chemical. The method can also be used to detect whether a subject responds to surgery.

[0193] These methods involve detecting CD39 T cells, such as CD39+CD103+CD8 T cells, in a biological sample from a subject. + CD8 +The method includes detecting the presence of T cells, and the presence of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, in the biological sample indicates that a cancer therapeutic agent, such as, but not limited to, a checkpoint inhibitor, a 4-1BB agonist, and / or radiation, is effective in treating the tumor in the subject. The method may also indicate that the subject responds to a surgical procedure, such as resection. The method may also include administering a cancer therapeutic agent to the subject or performing a surgical procedure. In some non-limiting examples, the cancer therapeutic agent is a checkpoint inhibitor, which may be, but is not limited to, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist, or a LAG3 antagonist. Suitable antagonists are disclosed in detail below. Suitable 4-1BB agonists are also disclosed below.

[0194] In other embodiments, a method is disclosed for determining whether a subject having a tumor will respond to a treatment regimen, such as one that includes a cancer therapeutic agent. The cancer therapeutic agent can be a chemotherapy agent or radiation. The cancer therapeutic agent can be a checkpoint inhibitor, such as a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist, or a LAG3 antagonist, or can be a 4-1BB agonist. In some embodiments, the method includes administering to the subject a first dose of a cancer therapeutic agent and detecting CD39 T cells, such as CD39+CD103+CD8 T cells, in a biological sample from the subject. + CD8 + and determining the number of CD39+CD103+CD8 T cells in the biological sample. + CD8 + An increase in the amount of T cells compared to a control indicates that the first dose of the cancer therapeutic is effective in treating the tumor in the subject.

[0195] In further embodiments, a method is disclosed for determining whether a subject with a tumor responds to a cancer therapeutic agent. The cancer therapeutic agent can be a chemotherapy agent or radiation. The cancer therapeutic agent can be a checkpoint inhibitor, such as a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist or a LAG3 antagonist, or can be a 4-1BB agonist. These methods include administering a first dose of a cancer therapeutic agent to the subject and determining the number of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, in a biological sample from the subject, and an increase in the amount of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, in the biological sample compared to a control indicates that the first dose of the cancer therapeutic agent is effective in treating the tumor in the subject. In additional embodiments, the method further includes administering a second dose of the cancer therapeutic agent to the subject, wherein the first dose is the same as the second dose or the second dose is lower than the first dose.

[0196] In yet another embodiment, methods for treating a subject with a tumor are disclosed. These methods include administering a first dose of a cancer therapeutic agent to the subject and determining the number of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, in a biological sample from the subject. A decrease or no change in the amount of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, in the biological sample compared to a control indicates that the first dose of the cancer therapeutic agent is not effective in treating the tumor in the subject. A second dose of the cancer therapeutic agent is administered to the subject, where the second dose is higher than the first dose or the second dose is the same as the first dose.

[0197] In some embodiments, the subject has a tumor or is at risk of developing a tumor, as discussed above. These subjects can be identified by a person skilled in the art, such as a physician, by suitable standard methods. The disclosed method includes selecting a subject of interest and administering a cancer therapeutic of interest, including but not limited to a checkpoint inhibitor, such as a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist, or a LAG3 antagonist. The subject can also be administered a 4-1BB agonist. The method can detect subjects that respond to a surgical procedure.

[0198] In additional embodiments, the subject is administered a therapeutically effective amount of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, and a therapeutically effective amount of a cancer therapeutic agent, including but not limited to a checkpoint inhibitor, such as a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist, or a LAG3 antagonist. In additional embodiments, the subject can be administered a 4-1BB agonist. Administration of purified CD39+CD8+ T cells, such as the CD39+CD103+CD8 T cells disclosed herein, and a checkpoint inhibitor or a 4-1BB agonist increases the subject's ability to overcome a pathology, such as a tumor. The cells and checkpoint inhibitor can be included in a single pharmaceutical composition or in separate pharmaceutical compositions. Thus, purifying a population of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, from a subject ex vivo, producing a purified population, and introducing these cells enhances the immune response of the recipient subject.Administering a therapeutically effective amount of a checkpoint inhibitor or a 4-1BB agonist also enhances the immune response of the recipient.Thus, the method disclosed herein for determining whether a chemotherapeutic agent or radiation is effective can be used in combination with (and in) any of the above-mentioned treatments.

[0199] The method can also be used to evaluate the dose of cancer therapeutic agent that is therapeutically effective for a subject.For example, the method disclosed herein can be used to determine whether the dose administered to a subject of interest can be reduced and still be effective.The method disclosed herein can also be used to determine whether the dose administered to a subject is too low and therefore must be increased to be therapeutically effective.

[0200] Any of the disclosed methods may include measuring other cell types, such as B cells and / or T cells. The disclosed methods may also include measuring expression of markers such as PD-1, PD-L1, CTLA-4, BTLA, TIM-3, or LAG3. Evaluate expression of CD8, CD39, and / or CD103.

[0201] In some embodiments, CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, are measured. An increase in the number of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, from a biological sample compared to a control indicates that the dose of the cancer therapeutic agent is useful for treating the subject, and no significant change in the number of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, compared to a control indicates that the dose of the cancer therapeutic agent is not useful for treating the subject. The control can be a previously determined standard value, or the amount of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, in a sample from a subject before administration of a cancer therapeutic agent, or the amount of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, in a sample from a subject when the subject is administered a control substance.

[0202] Generally, measuring the number of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, includes obtaining a sample containing T cells from a subject and determining the presence or number of CD39+CD8+ T cells, such as CD39+CD103+CD8 T cells, in the sample. In some examples, the sample is a biopsy sample, a blood sample, or a sample of peripheral blood mononuclear cells. Methods include immunohistochemistry and / or flow cytometry.

[0203] The method may include immunohistochemical methods, such as those on biological samples from subjects. The sample may be a tumor sample. In some embodiments, the antibody (or antigen-binding fragment), such as the antibody that binds to CD8, CD39, or CD103, is directly labeled with a detectable label. In another embodiment, the antibody (or antigen-binding fragment) that binds to CD8, CD39, or CD103 (first antibody) is unlabeled, and a second antibody or other molecule that can bind to the antibody that binds to the first antibody is utilized. As is well known to those skilled in the art, a second antibody is selected that can specifically bind to a particular species and class of first antibody. For example, if the first antibody is a human IgG, the secondary antibody can be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, protein A and protein G, both of which are commercially available.

[0204] Suitable labels for antibodies or secondary antibodies are described above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol, a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include: 125 I, 131 I, 35 S or 3 Examples include H.

[0205] Flow cytometry can also be used to quantify cells. In additional embodiments, the sample can be purified to separate T cells, such as CD39+CD8+T cells, such as CD8 T cells or CD39+CD103+CD8 T cells. In some embodiments, the method includes measuring the amount of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells. In some examples, the amount of CD39+CD8+T cells, such as CD39+CD103+CD8 T cells, in the biological sample is compared to a control. Suitable controls are described above.

[0206] In some examples, cell suspension is made from tumor sample.In one non-limiting example, under sterile conditions, tumor is cut into small pieces and digested in RPMI-1640 containing hyaluronidase, collagenase, DNase and human serum albumin.Cells can be digested, for example, at room temperature for 1 hour with magnetic stirring.Cell suspension is filtered through cell filter.Tumor-infiltrating lymphocytes can be enriched by centrifugation using density gradient solution.

[0207] Methods for isolating, detecting, and / or quantifying T cells are known in the art, and exemplary protocols are presented herein. Methods for measuring T cell proliferation are also known in the art. These methods generally involve the use of molecular and / or biochemical techniques, and not simple visual observation. Some example cells utilize fluorescence-activated cell analysis (FACS). FACS can be used to sort (isolate) cells, such as T cells, by staining the cells with appropriately labeled antibodies. In one embodiment, several antibodies (e.g., antibodies that bind CD8, CD39, and CD103) and FACS sorting can be used to generate a population of CD39+CD8+ T cells, such as substantially purified CD39+CD103+CD8 T cells. Any FACS technique can be used. See, for example, the FACS method disclosed in U.S. Pat. No. 5,061,620.

[0208] However, other techniques of differing effectiveness can be used to purify and isolate the desired cell population. The separation technique used should maximize the retention of viability of the fraction of cells harvested. The particular technique used will naturally depend on the efficiency of separation, the cytotoxicity of the method, the ease and speed of separation, and what equipment and / or technical skill is required.

[0209] Separation procedures include magnetic separation using antibody-coated magnetic beads, cytotoxic agents that are bound to monoclonal antibodies or used in conjunction with complement, and "panning" utilizing monoclonal antibodies bound to solid matrices, or other conventional techniques. Magnetic beads and antibodies bound to other solid matrices such as agarose beads, polystyrene beads, hollow fiber membranes, and plastic petri dishes allow for direct separation. Antibody-bound cells can be removed from the cell suspension simply by physically separating the solid support from the cell suspension. The exact conditions and duration of incubation of the cells with the antibody linked to the solid phase depend on several factors specific to the system used. However, the selection of appropriate conditions is well within the skill of the art.

[0210] Then, after a sufficient time for cells expressing the marker of interest (e.g., CD39 or CD103) to bind to the antibody linked to the solid phase, the unbound cells can be eluted or washed away with a physiological buffer. The bound cells are then separated from the solid phase by any suitable method, depending primarily on the nature of the solid phase and the antibody used.

[0211] The antibodies can be conjugated to biotin, which can then be removed using avidin or streptavidin bound to a support, or to fluorescent dyes, and can be used in FACS to allow for cell separation, as is known in the art.

[0212] For example, cells expressing CD8 or CD3 are first separated from other cells by cell surface expression of CD8 or CD3. The isolated CD8 + Cells or CD3 +The purity of the cells is confirmed, if desired, for example, using a BD LSRFORTESSA® flow cytometer (Becton Dickinson, San Jose, Calif.). In one embodiment, a further purification step is performed, such as FACS sorting of the population of cells. In one example, this sorting can be performed to detect the expression of CD39, CD103, and CD8.

[0213] The method may also include a step of measuring cell proliferation. Methods for analyzing cell proliferation, such as assessing cell proliferation, are known in the art. For example, a membrane dye dilution technique can be utilized, which involves chemically labeling cells of interest ex vivo with a fluorescent dye. Tritium-labeled nucleoside analogs (generally 3 H-thymidine deoxyribonucleoside, 3 Labeling with H-TdR or bromodeoxyuridine (BrdU) can be used. FACS analysis is available to measure BrdU incorporation. Surrogate markers of proliferation such as DNA content and cell cycle associated proteins can also be used.

[0214] In one embodiment, the measurement of Ki67 or PCNA can be used. The Ki67 antigen is a prototypic cell cycle-associated nuclear protein expressed by proliferating cells in all phases of the active cell cycle (G1, S, G2 and M phases). It is absent in quiescent (G0) cells. Ki67 antibodies are useful in establishing proliferation. Ki67 antibodies can be used to quantify proliferating and quiescent cells (Ki67 index). Ki67 is routinely used as a marker of cell cycle progression and proliferation; antibodies against Ki67 are commercially available, for example from ABCAM®, and methods are available for using these antibodies in immunohistochemistry and FACS analysis.

[0215] Other methods can be used to detect cells in an active cell cycle at the time of sampling. The proliferation of lymphocytes, such as CD39+CD8+ T cells, e.g., CD39+CD103+CD8 T cells, can also be measured by using a method that utilizes stable isotopes to label DNA in biological samples containing cells. DNA is uniformly and highly labeled by a de novo synthetic pathway. The stable isotope labels used, e.g., 2 H-glucose or heavy water ( 2 H2O or H2 18 O) is non-toxic to animals and humans and generally considered safe by the US Food and Drug Administration (FDA) (see US Patent Application Publication No. 2009 / 0155179). Measurement of incorporation of stable isotope labels into lymphocyte DNA includes the following steps: (i) release of DNA from chromatin without DNA extraction or further isolation, hydrolysis of DNA into deoxyribonucleotides, (ii) selective release of deoxyribose from purine deoxyribonucleotides, (iii) derivatization of purine deoxyribose into a volatile derivative (e.g., pentane tetraacetate, pentafluorobenzyl tetraacetyl derivative, or another suitable derivative) suitable for analysis by gas chromatography / mass spectrometry (GC / MS), (iv) GC / MS analysis of said derivative, (v) analysis of the pattern of mass isotopomer abundance of said derivative, and (vi) calculation of excess enrichment value from said pattern, which is a measure of stable isotope incorporation. Particular embodiments of each of these methods are taught (see US Pat. No. 5,910,40). PD-1, CTLA-4, TIM-3, LAG3, BTLA antagonists and 4-1BB agonists

[0216] Checkpoint inhibitors, such as PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG3 antagonists, TIM-3 antagonists and / or BTLA antagonists, can be used to inhibit, for example, CD8 + CD39+ CD103 + In combination with T cells, it is useful in the methods disclosed herein. 4-1BB agonists are also useful in the methods disclosed herein. PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG3 antagonists, TIM-3 antagonists, BTLA antagonists, and / or 4-1BB agonists can be chemical or biological compounds. The agents can be antibodies, including but not limited to chimeric, humanized, or human antibodies. Suitable antagonists and agonists also include antigen-binding fragments of these antibodies (see above for a description of antigen-binding fragments). Antagonists can be, for example, inhibitor nucleic acid molecules or small molecules, for example, molecules less than 900 daltons or less than 800 daltons.

[0217] A PD-1 antagonist can be any chemical compound or biological molecule that blocks the binding of PD-L1 or PD-L2 expressed on cells to human PD-1 expressed on immune cells (T cells, B cells, or NKT cells). Alternative names or synonyms for PD-1 and its ligands include PDCD1, PD1, CD279, and SLEB2 for PD-1; PDCD1L1, PD-L1, B7H1, B7-4, CD274, and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc, and CD273 for PD-L2. An exemplary human PD-1 amino acid sequence can be found in NCBI Accession No.: NP_005009. Exemplary human PD-L1 and PD-L2 amino acid sequences can be found in NCBI Accession Nos. NP_054862 and NP__079515, April 28, 2017, respectively, which are incorporated by reference. In vivo, PD-1 is expressed on activated T cells, B cells, and monocytes. In humans, PD-1 is a 50-55 kDa type I transmembrane receptor that was first identified in a T cell line that undergoes activation-induced apoptosis. PD-1 is expressed on T cells, B cells, and macrophages. The ligands for PD-1 are the B7 family members PD-ligand 1 (PD-L1, also known as B7-H1) and PD-L2 (also known as B7-DC). PD-L1 or PD-L2 inhibitors can be used in the methods disclosed herein.

[0218] Experimental data implicates the interaction of PD-1 and its ligand in the downregulation of central and peripheral immune responses. Specifically, in the presence of PD-L1, the proliferation of wild-type T cells is inhibited, but the proliferation of PD-1-deficient T cells is not inhibited. (See, for example, Ishida et al., EMBO J. 11:3887, 1992; Shinohara et al. Genomics 23:704,1994; U.S. Patent No. 5,698,520, which are incorporated herein by reference).

[0219] Additional PD-1 amino acid sequences are disclosed in U.S. Pat. No. 6,808,710 and U.S. Patent Application Publication Nos. 2004 / 0137577, 2003 / 0232323, 2003 / 0166531, 2003 / 0064380, 2003 / 0044768, 2003 / 0039653, 2002 / 0164600, 2002 / 0160000, 2002 / 0110836, 2002 / 0107363, and 2002 / 0106730, which are incorporated by reference herein.

[0220] PD-1 is a member of the CD28 / CTLA-4 family of molecules based on its ability to bind to PD-L1. In vivo, like CTLA-4, PD-1 is rapidly induced on the surface of T cells in response to anti-CD3 (Agata et al. Int. Immunol. 8:765, 1996). T cell exhaustion is concomitant with induction of PD-1 expression. See PCT Publication No. 2008 / 083174, incorporated herein by reference. The cytotoxicity of T cells can be increased by contacting T cells with an agent that reduces PD-1 expression or activity. An agent that reduces PD-1 expression or activity can be used to increase immune responses, such as immune responses against tumors. Without being bound by theory, reducing PD-1 expression or activity leads to increased cytotoxic T cell activity, increasing specific immune responses.

[0221] PD-1 family members bind to one or more receptors, such as PD-L1 and PD-L2, on antigen-presenting cells. An exemplary amino acid sequence of PD-L1 is provided as GENBANK® Accession No. AAG18508, which is incorporated herein by reference as available on October 4, 2000. An exemplary PD-L2 precursor amino acid sequence is provided as GENBANK® Accession No. AAK15370, which is incorporated herein by reference as available on April 8, 2002. An exemplary variant PD-L2 precursor amino acid sequence is provided as GENBANK® Accession No. Q9BQ51, which is incorporated herein by reference as available on December 12, 2006.

[0222] Antagonists useful in the methods disclosed herein include agents that reduce the expression or activity of PD ligand 1 (PD-L1) or PD ligand 2 (PD-L2), or reduce the interaction of PD-1 and PD-L1 or PD-1 and PD-L2, which are PD-antagonists. Exemplary compounds include antibodies (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies), RNAi molecules (e.g., anti-PD-1 RNAi molecules, anti-PD-L1 RNAi, and anti-PD-L2 RNAi), antisense molecules (e.g., anti-PD-1 antisense RNA, anti-PD-L1 antisense RNA, and anti-PD-L2 antisense RNA), dominant negative proteins (e.g., dominant negative PD-1 proteins, dominant negative PD-L1 proteins, and dominant negative PD-L2 proteins), and small molecule inhibitors. Any of these PD-1 antagonists are useful in the methods disclosed herein.

[0223] Other antibodies are also useful in the methods disclosed herein (e.g., anti-CTLA-4 antibodies, and anti-LAG3, anti-TIM-3 or anti-BTLA antibodies), RNAi molecules (e.g., anti-CTLA-4 RNAi molecules, anti-LAG3 RNAi, anti-TIM-3 RNAi and anti-BTLA RNAi), antisense molecules (e.g., anti-CTLA-4 antisense RNA, anti-LAG3 antisense RNA, anti-TIM-3 antisense RNA and anti-BTLA antisense RNA). Also useful dominant negative proteins are dominant negative CTLA-4 proteins, dominant negative LAG3 proteins, dominant negative LAG-3 proteins and dominant negative BTLA proteins. Any of these antagonists are useful in the methods disclosed herein. In addition, 4-1BB agonists, such as antibodies and RNA aptamers that bind to 4-1BB, are useful in the methods disclosed herein. TGF-β receptor inhibitory proteins or dominant negative proteins are also useful.

[0224] Antagonists are agents capable of reducing the expression or activity of a target in cells. In some embodiments, PD-1, PD-L1, PD-L2, LAG3, TIM-3, CTLA-4 or BTLA expression or activity is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to such expression or activity in a control. Exemplary reductions in activity are at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or the complete absence of detectable activity. In one example, the control is cells that have not been treated with a PD-1 antagonist. In another example, the control is cells that have been contacted with a standard value or an agent, such as a carrier, that is known not to affect activity. Expression or activity can be determined by any standard method in the art. In one non-limiting example, a PD-1 antagonist inhibits or reduces binding of PD-1 to PD-L1, PD-L2, or both. In one non-limiting example, a PD-L1 antagonist reduces binding of PD-L1 or PD-1.

[0225] An agonist is an agent capable of increasing the expression or activity of a target in a cell. In some embodiments, 4-1BB expression or activity is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to such expression or activity in a control. Exemplary increases in activity are at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or the complete absence of detectable activity. In one example, the control is cells that have not been treated with a 4-1BB agonist. In another example, the control is cells that have been contacted with a standard value or an agent, such as a carrier, that is known not to affect activity. Expression or activity can be determined by any standard method in the art. In one non-limiting example, the 4-1BB agonist stimulates or increases binding. A.Antibodies

[0226] In some embodiments, the antagonist is an antibody. Exemplary amino acid sequences of antibodies that bind to PD-1 are disclosed, for example, in US Patent Application Publication No. 2006 / 0210567, which is incorporated herein by reference. Antibodies that bind to PD-1 are also disclosed in US Patent Application Publication No. 2006 / 0034826, which is also incorporated herein by reference. Antibodies that bind to PD-1 are also disclosed in U.S. Patent No. 7,488,802, U.S. Patent No. 7,521,051, U.S. Patent No. 8,008,449, U.S. Patent No. 8,354,509, U.S. Patent No. 8,168,757, and PCT Publication No. WO2004 / 004771, PCT Publication No. WO2004 / 072286, PCT Publication No. WO2004 / 056875, and U.S. Patent Application Publication No. 2011 / 0271358. The antibody may be KEYTRUDA® (pembrolizumab). The antibody may be an anti-PD-1 antibody, such as nivolumab (ONO-4538 / BMS-936558) or OPDIVO® from Ono Pharmaceuticals. PD-L1 binding antagonists include YW243.55.S70, MPDL3280A, MDX-1105, and MEDI 4736. See U.S. Patent Application Publication No. 2017 / 0044256. Examples of monoclonal antibodies that specifically bind human PD-L1 and are useful in the methods and compositions of the disclosure are disclosed in PCT Publication No. WO2013 / 019906, PCT Publication No. WO2010 / 077634 A1, and U.S. Patent No. 8,383,796. Checkpoint inhibitor antibodies against PD-1 (e.g., nivolumab, pidilizumab, and pembrolizumab) or PD-L1 (e.g., durvalumab, atezolizumab, and avelumab) are useful in any of the methods disclosed herein. Antibodies that bind to PD-1, PD-L2, and PD-1 are also disclosed in Patent No. 8,552,154. In some examples, the antibody binds at least 10% to CTLA-4, BTLA, PD-1, PD-L1, or PD-L2. 7 M -1, e.g., at least 10 8 M -1 , at least 5 × 10 8 M -1 Or at least 10 9 M -1 and the like. All of these antibodies and antigen-binding fragments are useful in the methods disclosed herein.

[0227] Exemplary antibodies that specifically bind to CTLA-4 are disclosed in PCT Publication No. WO 2001 / 014424, PCT Publication No. WO 2004 / 035607, U.S. Patent Application Publication No. 2005 / 0201994, European Patent No. EP 1141028, and European Patent No. EP 1212422B1. Additional CTLA-4 antibodies are disclosed in U.S. Patent No. 5,811,097, U.S. Patent No. 5,855,887, U.S. Patent No. 6,051,227, U.S. Patent No. 6,984,720, U.S. Patent No. 6,682,736, U.S. Patent No. 6,207,156, U.S. Patent No. 5,977,318, U.S. Patent No. 6,682,736, U.S. Patent No. 7,109,003, U.S. Patent No. 7,132, 281, U.S. Patent No. 7,452,535, and U.S. Patent No. 7,605,238; PCT Publication No. WO 01 / 14424, PCT Publication No. WO 00 / 37504, PCT Publication No. WO 98 / 42752, U.S. Patent Application Publication No. 2000 / 037504, U.S. Patent Application Publication No. 2002 / 0039581, and U.S. Patent Application Publication No. 2002 / 086014. Antibodies that specifically bind to CTLA-4 are also disclosed in Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho et al., J. Clin. Oncol., 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998). In some embodiments, the CTLA-4 antagonist is Ipilmumab (also known as MDX-010 and MDX-101 and YERVOY®), see PCT Publication No. WO2001 / 014424, which is incorporated herein by reference. These antibodies and antigen-binding fragments are useful in the methods disclosed herein.

[0228] In further embodiments, BTLA antagonists are utilized in the methods disclosed herein.Antibodies that specifically bind to BTLA are disclosed, for example, in US Patent Application Publication No. 2016 / 0222114, US Patent Application Publication No. 2015 / 0147344, and US Patent Application Publication No. 2012 / 0288500, all of which are incorporated herein by reference.Biological agents that modulate BTLA activity, particularly those that use herpes virus entry mediator (HVEM) cis complexes, are disclosed in US Patent Application Publication No. 2014 / 0220051 and US Patent Application Publication No. 2010 / 0104559, both of which are incorporated herein by reference.In yet other embodiments, antibodies specifically bind to TIM-3, such as TSR-022. In further embodiments, the antibody specifically binds LAG3, such as BMS-986016, GSK2831781, or the antibodies disclosed in PCT Publication WO2015042246A1, which is incorporated herein by reference. See also Clinical Trial Number NCT01968109 for "Safety Study of Anti-LAG-3 With and Without Anti-PD-1 in the Treatment of Solid Tumors," available on the internet at clinicaltrials.gov, which is incorporated herein by reference. These antibodies and antigen-binding fragments are useful in the methods disclosed herein.

[0229] 4-1BB agonist antibodies are also useful.Suitable antibodies are disclosed, for example, in U.S. Patent No. 8,337,850 and PCT Publication WO2015179236A1, both of which are incorporated herein by reference.Antibodies useful in any of the disclosed methods include urelumab (BMS-663513) and PF-05082566 (Pfizer).

[0230] Antibodies useful in the disclosed methods include monoclonal antibodies, humanized antibodies, deimmunized antibodies (e.g., to reduce human-anti-mouse responses), chimeric antibodies, and immunoglobulin (Ig) fusion proteins. Antigen-binding fragments of these antibodies are also useful in the methods disclosed herein. Polyclonal antibodies can be prepared by one of skill in the art, such as by immunizing a suitable subject (e.g., a veterinary subject) with an immunogen. Antibody titers in immunized subjects can be monitored over time by standard techniques, such as by using an enzyme-linked immunosorbent assay (ELISA) using immobilized antigen. In one example, antibodies that specifically bind to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 (or a combination thereof) can be isolated from a mammal (e.g., from serum) and further purified by techniques known to those of skill in the art. For example, antibodies can be purified using Protein A chromatography to isolate IgG antibodies.

[0231] Antibody-producing cells can be obtained from a subject and used to prepare monoclonal antibodies by standard techniques (Kohler and Milstein Nature 256:495 49, 1995;Brown et al., J. Immunol. 127:539 46, 1981;Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96, 1985;Gefter, ML et al. (1977) Somatic Cell Genet. 3:231 36;Kenneth, RH in Monoclonal Antibodies: A New Dimension In Biological Analyses. Plenum Publishing Corp., New York, NY (1980);Kozbor et al. Immunol. Today 4:72, 1983;Lerner, EA (1981) Yale J. Biol. Med. 54:387 402; Yeh et al., Proc. Natl. Acad. Sci. 76:2927 31, 1976). In one embodiment, an immortal cell line (typically a myeloma) is fused with lymphocytes (typically splenocytes) from a mammal immunized with PD-1, PD-L1, PD-L2, TIM-3, LAG3, BTLA, or CTLA-4, and the resulting hybridoma cell culture supernatant is screened to identify hybridomas that produce a monoclonal antibody that specifically binds to the polypeptide of interest.

[0232] In one embodiment, to generate hybridomas, an immortal cell line (e.g., a myeloma cell line) is obtained from the same mammalian species as the lymphocytes. For example, mouse hybridomas can be generated by fusing lymphocytes from a mouse immunized with CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB peptides with an immortalized mouse cell line. In one example, a mouse myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin, and thymidine ("HAT medium") is utilized. Any of a number of myeloma cell lines can be used as fusion partners according to standard techniques, including, for example, P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653, or Sp2 / O-Ag14 myeloma lines, which are available from the American Type Culture Collection (ATCC), Rockville, Md. HAT-sensitive mouse myeloma cells can be fused with mouse splenocytes using polyethylene glycol ("PEG"). The hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused (and ineffectively fused) myeloma cells. Hybridoma cells producing the desired monoclonal antibody can be detected, for example, by screening the hybridoma culture supernatant for the production of an antibody that binds to PD-1, PD-L1, TIM-3, LAG3, BTLA, CTLA-4, PD-L2 or 4-1BB molecules using an immunological assay (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)).

[0233] As an alternative to preparing hybridomas that secrete monoclonal antibodies, monoclonal antibodies that specifically bind to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BB can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BB to isolate immunoglobulin library members that specifically bind to the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., but not limited to, Pharmacia and Stratagene). Examples of methods and reagents that are particularly suitable for use in generating and screening antibody display libraries can be found, for example, in U.S. Pat. No. 5,223,409; PCT Publication No. WO 90 / 02809; PCT Publication No. WO 91 / 17271; PCT Publication No. WO 92 / 18619; PCT Publication No. WO 92 / 20791; PCT Publication No. WO 92 / 15679; PCT Publication No. WO 92 / 01047; PCT Publication No. WO 93 / 01288; PCT Publication No. WO 92 / 09690; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978 7982, 1991; Hoogenboom et al., Nucleic Acids Res. 19:4133 4137, 1991.

[0234] In one embodiment, the sequence of the specificity determining region of each CDR is determined. Residues outside the SDR (non-ligand contact site) are replaced. For example, at most one, two or three amino acids can be replaced in any of the CDR sequences in the table above. The production of chimeric antibodies containing framework regions from one antibody and CDRs from a different antibody is well known in the art. For example, humanized antibodies can be routinely produced. The antibody or antibody fragment can be a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor monoclonal antibody that binds to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB, and immunoglobulin heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks. Humanized monoclonal antibodies can be produced by transferring donor complementarity determining regions (CDRs) from the variable heavy and variable light chains of a donor mouse immunoglobulin (such as a CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BB specific antibody) into a human variable domain, followed by substitution of human residues in the framework regions as necessary to retain affinity. The use of antibody components derived from a humanized monoclonal antibody obviates potential problems associated with the immunogenicity of the constant region of the donor antibody. Techniques for producing humanized monoclonal antibodies have been described, for example, by Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. USA 89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al., J. Immunol.150:2844, 1993.The antibody may be of any isotype, but in some embodiments the antibody is an IgG, including but not limited to IgG1, IgG2, IgG3 and IgG4. In some embodiments, the humanized immunoglobulin has at least 10 nucleotides to an antigen of interest (e.g., CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB). 7 M -1 , e.g., at least 10 8 M -1 , at least 5 × 10 8 M -1 Or at least 10 9 M -1 It specifically binds with an affinity constant of

[0235] In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework may be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework may be at least about 75%, at least about 85%, at least about 99% or at least about 95% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Human framework regions and the mutations that can be made in humanized antibody framework regions are known in the art (see, for example, U.S. Patent No. 5,585,089, which is incorporated herein by reference).

[0236] Antibodies, such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full-length molecules and fragments thereof, such as Fab, F(ab')2, and Fv, which contain heavy and light chain variable regions and are capable of binding to specific epitopic determinants. These antibody fragments retain some ability to selectively bind to their antigen or receptor. These fragments include: (1) Fab, the fragment containing a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibodies (e.g., scFv), defined as genetically engineered molecules containing the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker as a genetically fused single chain molecule. Examples include:

[0237] Methods for producing these antigen-binding fragments are known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). In some embodiments, the variable region comprises a light chain variable region and a heavy chain variable region expressed as individual polypeptides. Fv antibodies are generally about 25 kDa and contain a complete antigen-binding site with three CDRs per heavy and light chain. To generate these antibodies, V H and V L can be expressed in a host cell from two separate nucleic acid constructs. H and V LWhen expressed without adjacent chains, the chains of an Fv antibody are generally held together by non-covalent interactions. However, these chains tend to dissociate upon dilution, and therefore methods have been developed to cross-link the chains with glutaraldehyde, intermolecular disulfides, or peptide linkers. Thus, in one example, the Fv can be a disulfide-stabilized Fv (dsFv) in which the heavy and light chain variable regions are chemically linked by a disulfide bond.

[0238] In additional embodiments, the Fv fragment comprises V fragments connected by a peptide linker. H Chain and V L These single-chain antigen-binding proteins (scFv) contain V chains connected by oligonucleotides. H Domains and V L They are prepared by constructing a structural gene containing a DNA sequence encoding the domain. The structural gene is inserted into an expression vector, which is then introduced into a host cell, such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two V domains. Methods for making scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio / Technology 11:1271, 1993; and Sandhu, supra).

[0239] Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expressing DNA encoding the fragment in E. coli. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be generated by enzymatic cleavage of antibodies with pepsin to produce a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent and, if necessary, a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages to generate 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647 and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

[0240] Other methods of cleaving antibodies, such as separating heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques, can also be used so long as the fragment binds to the antigen recognized by the intact antibody.

[0241] It will be appreciated by those skilled in the art that conservative variants of antibodies can be made. Such conservative variants used in antibody fragments, such as in dsFv or scFv fragments, include the V H Area and V L The amino acid residues necessary for correct folding and stabilization between the domains are retained, as well as the charge characteristics of the residues to preserve the low pI and low toxicity of the molecule.H Area and V L Amino acid substitutions (e.g., substitutions of at most one amino acid, at most two amino acids, at most three amino acids, at most four amino acids, or at most five amino acids) can be made in regions to increase yield. Thus, one of skill in the art can readily review the amino acid sequence of an antibody of interest, locate one or more of the amino acids in the simple table above, identify conservative substitutions, and create conservative variants using well-known molecular techniques.

[0242] Effector molecules, such as therapeutic, diagnostic, or detectable moieties, can be linked to antibodies that specifically bind to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB using any number of means known to those skilled in the art. Both covalent and non-covalent attachment means can be used. The procedure for attaching effector molecules to antibodies varies depending on the chemical structure of the effector. Polypeptides generally contain a variety of functional groups that are available for reaction with appropriate functional groups on antibodies to effect attachment of effector molecules; for example, carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups. Alternatively, antibodies are derivatized to expose or attach additional reactive functional groups. Derivatization can involve attachment of any of a number of linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to attach an antibody to an effector molecule. The linker can form a covalent bond to both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and effector molecule are polypeptides, the linker can be attached to the constituent amino acids through their side chains (e.g., through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0243] Nucleic acid sequences encoding antibodies can be prepared by, for example, cloning appropriate sequences or by other methods, such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethyl phosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; or by using an automated synthesizer, e.g., as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984, Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, It can be prepared by any suitable method, including by direct chemical synthesis by the solid-phase phosphoramidite triester method described by 1981; and the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis produces single-stranded oligonucleotide.It can be converted into double-stranded DNA by hybridization with complementary sequence, or by polymerization using DNA polymerase with single strand as template.Although chemical synthesis of DNA is generally limited to about 100 bases of sequence, it is understood by those skilled in the art that longer sequences can be obtained by ligating short sequences.

[0244] Exemplary nucleic acid coding sequences encoding antibodies that specifically bind to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques, and sufficient instructions to guide one of skill in the art through many cloning exercises, can be found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to those of skill in the art.

[0245] Nucleic acids can also be prepared by amplification techniques, including polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustained sequence replication systems (3SR). A wide variety of cloning methods, host cells, and in vitro amplification systems are well known to those skilled in the art.

[0246] In one example, a useful antibody is prepared by inserting a cDNA encoding a variable region from an antibody that specifically binds to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB into a vector containing a cDNA encoding an effector molecule (EM). The insertion is made such that the variable region and the EM read in frame, thus creating one continuous polypeptide. Thus, the encoded polypeptide contains a functional Fv region and a functional EM region. In one embodiment, a cDNA encoding a detectable marker (e.g., an enzyme) is ligated to the scFv such that the marker is located at the carboxyl terminus of the scFv. In another example, the detectable marker is located at the amino terminus of the scFv. In another example, a cDNA encoding a detectable marker is ligated to the heavy chain variable region of an antibody that specifically binds to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB such that the marker is located at the carboxyl terminus of the heavy chain variable region. The heavy chain-variable region can then be ligated to the light chain variable region of an antibody that specifically binds to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB using a disulfide bond. In yet another embodiment, a cDNA encoding a marker is ligated to the light chain variable region of an antibody that specifically binds to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB such that the marker is located at the carboxyl terminus of the light chain variable region. The light chain-variable region can then be ligated to the heavy chain variable region of an antibody that specifically binds to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB using a disulfide bond.

[0247] Once the nucleic acid encoding the antibody or its functional fragment is isolated and cloned, the protein can be expressed in recombinantly engineered cells, such as bacteria, plants, yeast, insect and mammalian cells. One or more DNA sequences encoding the antibody or its functional fragment can be expressed in vitro by transferring the DNA into a suitable host cell. The cell can be a prokaryotic or eukaryotic cell. This term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell, since mutations may occur during replication. Methods for stable transfer, i.e., continuous maintenance of foreign DNA in a host, are known in the art.

[0248] The polynucleotide sequence encoding the antibody or its functional fragment can be operably linked to an expression control sequence. The expression control sequence operably linked to the coding sequence is ligated to achieve expression of the coding sequence under conditions compatible with the expression control sequence. The expression control sequence includes, but is not limited to, a suitable promoter, enhancer, transcription terminator, a start codon (i.e., ATG) in front of the gene encoding the protein, splicing signals of introns, maintaining the correct reading frame of the gene to allow proper translation of the mRNA, and a stop codon.

[0249] The polynucleotide sequence encoding antibody or its functional fragment can be manipulated to allow insertion or incorporation of sequence, and can be inserted into expression vectors, including but not limited to plasmid, virus or other vehicles that can be expressed in either prokaryotes or eukaryotes.Hosts can include microorganisms, yeast, insects and mammalian organisms.Methods for expressing DNA sequences with eukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors that can be expressed and replicated in hosts are known in the art.

[0250] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of incorporating DNA can be prepared from cells that are harvested after exponential growth phase and then treated by CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be carried out after forming protoplasts of host cells, if desired, or by electroporation.

[0251] When the host is a eukaryotic organism, conventional mechanical procedures such as calcium phosphate coprecipitation, microinjection, electroporation, insertion of a plasmid packaged in a liposome, or a method of transfection of DNA such as a viral vector can be used. Eukaryotic cells can also be co-transformed with a polynucleotide sequence encoding an antibody or a functional fragment thereof and a second foreign DNA molecule encoding a selectable phenotype such as a herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector such as Simian Virus 40 (SV40) or bovine papilloma virus to transiently infect or transform eukaryotic cells to express proteins (see, for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Those skilled in the art can readily use expression systems such as useful plasmids and vectors to produce proteins in cells, including higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0252] Isolation and purification of recombinantly expressed polypeptides can be performed by conventional means, including preparative chromatography and immunological separation. Once recombinant antibodies are expressed, they can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see generally R. Scopes, Protein Purification, Springer-Verlag, NY, 1982). Substantially pure compositions of at least about 90-95% homogeneity are disclosed herein, and compositions having 98-99% or greater homogeneity can be used for pharmaceutical purposes. For therapeutic use, once purified, either partially or to the desired homogeneity, the polypeptide should be substantially free of endotoxins.

[0253] Methods for expressing and / or refolding single-chain antibodies, including single-chain antibodies from bacteria such as E. coli, to a suitable active form have been described and are well known and are applicable to the antibodies disclosed herein. See Buchner et al., Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544, 1989, all of which are incorporated herein by reference.

[0254] In many cases, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants and subsequent refolding. During the solubilization step, a reducing agent must be present to separate disulfide bonds, as is well known in the art. An exemplary buffer with a reducing agent is 0.1 M Tris, pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of disulfide bonds can be carried out in the presence of reduced and oxidized low molecular weight thiol reagents, as described in Saxena et al., Biochemistry 9: 5015-5021, 1970, which is incorporated herein by reference, and in particular in Buchner et al., supra.

[0255] Renaturation is generally accomplished by diluting (e.g., 100-fold) the denatured and reduced protein into a refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

[0256] As a modification to the two-chain antibody purification protocol, the heavy and light chain regions are solubilized and reduced separately, and then combined in the refolding solution.Exemplary yields are obtained when these two proteins are mixed in a molar ratio such that the molar excess of one protein over the other does not exceed 5 times.After the redox-shuffling is completed, it is desirable to add an excess of oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution.

[0257] In addition to recombinant methods, the antibodies and functional fragments thereof disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of polypeptides less than about 50 amino acids in length can be achieved by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described in Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284;Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Longer proteins can also be synthesized by condensation of the amino and carboxyl termini of short fragments. Methods for forming peptide bonds by activation of the carboxyl terminus (eg, by using the coupling reagent N,N'-dicyclohexylcarbodimide) are well known in the art. B. Inhibitory nucleic acids

[0258] Inhibitory nucleic acids that reduce the expression and / or activity of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 can also be used in the methods disclosed herein. One embodiment is a small inhibitory RNA (siRNA) to interfere with or inhibit the expression of a target gene. Nucleic acid sequences encoding PD-1, PD-L1, and PD-L2 are disclosed in GENBANK® Accession Nos. NM_005018, AF344424, NP_079515, and NP_054862, available on April 28, 2017, all of which are incorporated by reference.

[0259] Generally, siRNA is generated by the cleavage of relatively long double-stranded RNA molecules by Dicer or DCL enzymes (Zamore, Science, 296:1265-1269, 2002; Bernstein et al., Nature, 409:363-366, 2001). In animals and plants, siRNA is assembled in RISC and guides the sequence-specific ribonucleolytic activity of RISC, which leads to the cleavage of mRNA or other RNA target molecules in the cytoplasm. In the nucleus, siRNA also guides heterochromatin-associated histone and DNA methylation, which leads to the transcriptional silencing of individual genes or large chromatin domains. PD-1 siRNA is commercially available from Santa Cruz Biotechnology, Inc. and others.

[0260] The present disclosure provides an RNA suitable for interfering with or inhibiting expression of a target gene, the RNA comprising a double-stranded RNA of about 15 to about 40 nucleotides containing 3' and / or 5' overhangs of 0 to 5 nucleotides on each strand. The sequence of the RNA is substantially identical to a portion of the mRNA or transcript of a target gene, such as CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2, whose expression is desired to be interfered with or inhibited. For purposes of the present disclosure, an RNA that is "substantially identical" to a particular portion of the mRNA or transcript of a target gene whose expression is desired to be interfered with or inhibited differs in sequence by no more than about 30 percent, and in some embodiments differs by no more than about 10 percent from the particular portion of the mRNA or transcript of the target gene. In certain embodiments, the sequence of the RNA is exactly identical to the particular portion of the mRNA or transcript of the target gene.

[0261] Thus, the siRNA disclosed herein comprises a double-stranded RNA of about 15 to about 40 nucleotides in length and a 3' or 5' overhang having a length of 0 to 5 nucleotides on each strand, where the sequence of the double-stranded RNA is substantially identical to a portion of an mRNA or transcript of a nucleic acid encoding CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 (see above). In certain examples, the double-stranded RNA contains about 19 to about 25 nucleotides, e.g., 20 nucleotides, 21 nucleotides, or 22 nucleotides, that are substantially identical to a nucleic acid encoding CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2. In further examples, the double-stranded RNA contains about 19 to about 25 nucleotides that are 100% identical to a nucleic acid encoding CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2. In this context, "about" should not refer solely to integer quantities. In one example, "about" 20 nucleotides refers to a length of 19-21 nucleotides.

[0262] With respect to overhangs on double-stranded RNA, the length of the overhang is independent between the two strands, since the length of one overhang does not depend on the length of the overhang of the other strand. In certain examples, the length of the 3' or 5' overhang is 0 nucleotides in at least one strand, and in some cases, 0 nucleotides in both strands (hence blunt dsRNA). In other examples, the length of the 3' or 5' overhang is 1 nucleotide to 5 nucleotides in at least one strand. More specifically, in some examples, the length of the 3' or 5' overhang is 2 nucleotides in at least one strand, or 2 nucleotides in both strands. In certain examples, the dsRNA molecule has a 2 nucleotide 3' overhang in both strands.

[0263] Thus, in one particular provided RNA embodiment, the double-stranded RNA contains 20, 21, or 22 nucleotides, and the 3' overhang is 2 nucleotides in length on both strands. In the RNA embodiments provided herein, the double-stranded RNA contains about 40-60% adenine + uracil (AU) and about 60-40% guanine + cytosine (GC). More specifically, in a particular example, the double-stranded RNA contains about 50% AU and about 50% GC.

[0264] Also described herein is an RNA that further comprises at least one modified ribonucleotide, for example in the sense strand of the double-stranded RNA. In a particular embodiment, the modified ribonucleotide is in the 3' overhang of at least one strand, more particularly in the 3' overhang of the sense strand. Examples of modified ribonucleotides are specifically intended to include ribonucleotides that contain detectable labels (e.g., fluorophores such as rhodamine or FITC), thiophosphate nucleotide analogs, deoxynucleotides (considered modified because the base molecule is a ribonucleic acid), 2'-fluorouracil, 2'-aminouracil, 2'-aminocytidine, 4-thiouracil, 5-bromouracil, 5-iodouracil, 5-(3-aminoallyl)-uracil, inosine, or 2'O-Me nucleotide analogs.

[0265] Antisense and ribozyme molecules of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 are also useful in the methods disclosed herein. Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a particular mRNA molecule (Weintraub, Scientific American 262:40, 1990). In cells, antisense nucleic acids hybridize with the corresponding mRNA to form a double-stranded molecule. Since cells do not translate mRNA that is double-stranded, antisense nucleic acids interfere with the translation of mRNA. Antisense oligomers of about 15 nucleotides are preferred because they are easily synthesized and are less likely to cause problems than larger molecules when introduced into target cells that produce CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (see, eg, Marcus-Sakura, Anal. Biochem. 172:289, 1988).

[0266] Antisense oligonucleotides can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.Antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.For example, antisense nucleic acid molecules can be chemically synthesized using naturally occurring nucleotides, or can use various modified nucleotides, such as phosphorothioate derivatives and acridine-substituted nucleotides, designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between antisense nucleic acid and sense nucleic acid. Examples of modified nucleotides that can be used to generate antisense nucleic acids include, among others, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine-e,5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine.

[0267] The use of oligonucleotides to stop transcription is known as triplex strategy, because bloomers wrap around double-helical DNA, thereby forming a triple-stranded helix.Thus, these triplex compounds can be designed to recognize unique sites on selected genes (Maher, et al., Antisense Res. and Dev. 1(3):227, 1991;Helene, C., Anticancer Drug Design 6(6):569, 1991).This type of inhibitory oligonucleotide is also useful in the methods disclosed herein.

[0268] Ribozymes, which are RNA molecules that have the ability to specifically cleave other single-stranded RNA in a manner similar to DNA restriction endonucleases, are also useful. By modifying the nucleotide sequences that code for these RNAs, it is possible to engineer molecules that recognize and cleave specific nucleotide sequences within the RNA molecule (Cech, J. Amer. Med. Assn. 260:3030, 1988). The main advantage of this approach is that it is sequence-specific, so only mRNAs with a particular sequence are inactivated.

[0269] There are two basic types of ribozymes: Tetrahymena-type (Hasselhoff, Nature 334:585, 1988) and "hammerhead"-type. Tetrahymena-type ribozymes recognize sequences that are 4 bases in length, while "hammerhead"-type ribozymes recognize sequences that are 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will be present exclusively in the target mRNA species. Thus, hammerhead-type ribozymes are preferred over Tetrahymena-type ribozymes for inactivating specific mRNA species, and 18-base recognition sequences are preferred over shorter recognition sequences.

[0270] A variety of delivery systems are known and can be used to administer siRNA and other inhibitory nucleic acid molecules as therapeutic agents. Such systems include, for example, liposomes, microparticles, microcapsules, nanoparticles, inclusion in recombinant cells capable of expressing therapeutic molecule(s) (see, for example, Wu et al., J. Biol. Chem. 262, 4429, 1987), construction of therapeutic nucleic acid as part of retroviral or other vectors, and the like. C. Small molecules

[0271] CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1 or PD-L2 antagonists and 4-1BB agonists include molecules identified according to methods known in the art from both large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries. Screening methods that detect decreased activity of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1 or PD-L2 (e.g., cell death detection for PD-1, PD-L1 and PD-L2) are useful for identifying compounds from various sources for activity. Screening methods that detect increased activity of 4-1BB are also useful for identifying compounds from such sources. Initial screening can be performed using a diverse library of compounds, various other compounds and compound libraries. Thus, molecules that bind to CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2, that inhibit the expression of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2, and that inhibit the activity of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 can be identified. Molecules that increase the expression and / or activity of 4-1BB can also be identified. These small molecules can be identified from combinatorial libraries, natural product libraries, or other small molecule libraries. Additionally, CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, and PD-L2 antagonists, and 4-1BB agonists can be identified as compounds from commercial sources as well as commercially available analogs of identified inhibitors. In some embodiments, the small molecules are less than 900 daltons, or less than 800 daltons.

[0272] The exact source of the test extract or compound is not important to the identification of antagonists. Thus, antagonists can be identified from virtually any number of chemical extracts or compounds. Examples of such extracts or compounds that can be antagonists include, but are not limited to, plant-based extracts, fungal-based extracts, prokaryotic-based extracts or animal-based extracts, fermentation broths, and synthetic compounds, as well as the modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, sugar-based compounds, lipid-based compounds, peptide-based compounds, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wis.). Agonists and antagonists can be identified from synthetic compound libraries commercially available from a number of companies, including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, Conn.). CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, and PD-L2 antagonists, or 4-1BB agonists can be identified from rare chemical libraries, such as those available from Aldrich (Milwaukee, Wis.).CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, and PD-L2 antagonists, or 4-1BB agonists, can be identified in libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts that are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, USA (Cambridge, Mass.). Natural and synthetically produced libraries and compounds are readily modified by conventional chemical, physical, and biochemical means.

[0273] Useful compounds can be found in many chemical classes, but generally useful compounds are organic compounds, including small organic compounds. Small organic compounds having a molecular weight of more than 50 Daltons but less than about 2,500 Daltons, for example, less than about 750 Daltons or less than about 350 Daltons, can be utilized in the methods disclosed herein. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. Compounds can be modified to enhance efficacy, stability, pharmaceutical suitability, and the like. In some embodiments, useful compounds have a Kd of less than 1 nM, less than 10 nm, less than 1 μM, less than 10 μM, or less than 1 mM for CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2. D. Peptide Variants

[0274] Immunoadhesins that specifically bind to human CTLA-4, human BTLA, human TIM-3, human LAG3, human PD-1, human PD-L1, or human PD-L2 can also be utilized. Immunoadhesins are fusion proteins that contain an extracellular or binding portion of a protein fused to a constant region, such as the Fc region of an immunoglobulin molecule. Examples of immunoadhesion molecules that specifically bind to PD-1 are disclosed in PCT Publication Nos. WO2010 / 027827 and WO2011 / 066342, both of which are incorporated by reference. These immunoadhesion molecules include AMP-224 (also known as B7-DCIg), a PD-L2-FC fusion protein. Additional PD-1 antagonists that are fusion proteins are disclosed, for example, in U.S. Patent Application Publication No. 2014 / 0227262, which is incorporated by reference herein.

[0275] In one embodiment, the LAG3 antagonist useful in the disclosed methods is IMP321, a soluble LAG3 that has been used to activate dendritic cells. In another embodiment, the TIM-3 antagonist useful if use in the disclosed methods is CA-327 (Curis).

[0276] The CTLA-4 antagonist can be a dominant negative protein or an immunoadhesin. For example, see US Patent Application Publication No. 2016 / 0264643, which is incorporated herein by reference. Additional anti-CTLA-4 antagonists include any inhibitors, including but not limited to, small molecules that can inhibit the ability of CTLA-4 to bind to its cognate ligand, can destroy the ability of B7 to CTLA-4, can destroy the ability of CD80 to bind to CTLA-4, and can destroy the ability of CD86 to bind to CTLA-4.

[0277] In one embodiment, variants of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 proteins that function as antagonists can be identified by screening combinatorial libraries of mutants, such as point mutants or truncation mutants, of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 proteins to identify proteins with antagonist activity. In one example, the antagonist is a soluble protein.

[0278] In a further embodiment, variants of 4-1BB that function as agonists can be identified by screening combinatorial libraries of 4-1BB mutants, such as point mutations or truncation mutants, to identify proteins with agonist activity. The agonist can be a soluble protein.

[0279] Thus, libraries of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB variants can be generated by combinatorial mutagenesis at the nucleic acid level, which are encoded by a variegated gene library. Libraries of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB variants can be generated, for example, by enzymatically ligating a mixture of synthetic oligonucleotides into a gene sequence such that a degenerate set of potential sequences can be expressed as individual polypeptides or as a set of larger fusion proteins containing the set of sequences of interest (e.g., for phage display).

[0280] There are various methods that can be used to generate a library of potential CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BB variants from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be carried out in an automatic DNA synthesizer, and then the synthesized genes are ligated into a suitable expression vector. The use of a degenerate set of genes allows all of the sequences encoding the desired set of potential CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BB sequences to be provided as one mixture. Methods for synthesizing degenerate oligonucleotides are known in the art (e.g., see Narang, et al., Tetrahedron 39:3, 1983; Itakura et al. Annu. Rev. Biochem. 53:323, 1984; Itakura et al. Science 198:1056, 1984).

[0281] Additionally, libraries of fragments of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BB protein coding sequences can be used to generate populations of fragments for screening and subsequent selection of designated antagonist (or agonist, in the case of 4-1BB) variants. In one embodiment, a library of coding sequence fragments can be generated by treating double-stranded PCR fragments of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2 or 4-1BB coding sequences with a nuclease under conditions that result in only about one nicking event per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA that may contain sense / antisense pairs of different nicked products, removing single-stranded portions from the reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, PD-L2, or 4-1BB.

[0282] There are several techniques known in the art for screening the gene products of combinatorial libraries made by point mutation or truncation, and for screening cDNA libraries for gene products with selected properties. Such techniques can be adapted to the rapid screening of gene libraries produced by combinatorial mutagenesis of proteins. The most widely used techniques that can be applied to high-throughput analysis for screening large gene libraries generally include cloning gene libraries into replicable expression vectors, transforming suitable cells with the resulting library of vectors, and expressing combinatorial genes under conditions that allow the detection of desired activity to facilitate the isolation of the vectors that code for the genes whose products are detected. Recursive ensemble mutagenesis (REM) can be used in combination with screening assays to identify CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 antagonists, or 4-1BB agonists (Arkin and Youvan, Proc. Natl. Acad. Sci. USA 89:7811 7815, 1992; Delagrave et al., Protein Eng. 6(3):327 331, 1993).

[0283] In one embodiment, a cell-based assay can be utilized to analyze a library of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 variants. For example, a library of expression vectors can be transfected into a cell line that normally synthesizes and secretes CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2. The transfected cells are then cultured to secrete CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 and the specific CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 (respectively) variants. The effect of expression of the variants on activity in the cells or supernatant can be detected, such as, for example, by any of the functional assays. Plasmid DNA can then be recovered from cells in which endogenous activity has been inhibited, and individual clones can be further characterized.

[0284] Peptide mimetics can also be used as CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 antagonists. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. In general, peptidomimetics are structurally similar to paradigm polypeptides (e.g., polypeptides with PD-1 biological activity), but with one or more peptide linkages replaced with --CH2NH--, --CH2S--, --CH2--CH2--, --CH.=.CH-- (cis and trans), --COCH2--, --CH(OH)CH2--, and --CH2SO- linkages as necessary. These peptide linkages can be replaced by methods known in the art (see, for example, Morley, Trends Pharm. Sci. pp. 463 468, 1980; Hudson et al. Int. J. Pept. Prot. Res. 14:177 185, 1979; Spatola, Life Sci. 38:1243 1249, 1986; Holladay, et al. Tetrahedron Lett. 24:4401 4404, 1983). Peptidomimetics can be economically obtained, stable, and can have increased half-life or absorption. Labeling of peptidomimetics usually involves covalent attachment of one or more labels, either directly or through a spacer (e.g., by an amide group), to a non-interfering position(s) on the peptidomimetic predicted by quantitative structure-activity data and / or molecular modeling. Such non-interfering positions are generally positions that do not form direct contacts with the macromolecule(s) to which the peptidomimetic binds to produce a therapeutic effect. Derivatization of the peptidomimetic should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.

[0285] Dominant negative proteins or nucleic acids encoding dominant negative proteins that interfere with the biological activity of CTLA-4, BTLA, TIM-3, LAG3, PD-1, PD-L1, or PD-L2 can also be used in the methods disclosed herein. A dominant negative protein is any amino acid molecule having a sequence that has at least 50%, 70%, 80%, 90%, 95%, or even 99% sequence identity to at least 10 amino acids, 20 amino acids, 35 amino acids, 50 amino acids, 100 amino acids, or more than 150 amino acids of the wild-type protein to which the dominant negative protein corresponds. For example, a dominant negative PD-L1 has a mutation that binds to PD-1 more tightly than native (wild-type) PD-1, but does not activate any cell signaling through PD-1.

[0286] Dominant negative protein can be administered as an expression vector.Expression vector can be a non-viral vector or a viral vector (e.g., retrovirus, recombinant adeno-associated virus, or recombinant adenovirus vector).Alternatively, dominant negative protein can be administered as a recombinant protein directly systemically or to the infected area, for example, by using microinjection techniques.

[0287] Polypeptide antagonists can be produced by expressing polynucleotides encoding amino acid sequences in prokaryotic or eukaryotic host cells, often as part of a larger polypeptide (such as fusion protein with ras or enzyme). Alternatively, such peptides can be synthesized by chemical methods. Methods for the expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art (see Maniatis el al. Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, NY; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Kaiser et al., Science 243:187, 1989; Merrifield, Science 232:342, 1986; Kent, Annu. Rev. Biochem. 57:957, 1988).

[0288] Peptides can be produced, for example, by direct chemical synthesis and used as antagonists. Peptides can be produced as modified peptides with non-peptide moieties attached to the N-terminus and / or C-terminus by covalent linkages. In certain preferred embodiments, either the carboxy or amino terminus, or both, are chemically modified. The most common modifications of terminal amino and carboxyl groups are acetylation and amidation, respectively. Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal modifications such as amidation, as well as other terminal modifications including cyclization, can be incorporated into various embodiments. Certain amino- and / or carboxy-terminal modifications and / or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as, for example, enhanced stability, increased potency and / or efficacy, resistance to serum proteases, and desirable pharmacokinetic properties.

[0289] The present disclosure is illustrated by the following non-limiting examples. EXAMPLES

[0290] Example 1 material and method Healthy donor blood samples and patient blood and tissue samples: peripheral blood, uninvolved lymph nodes, metastatic lymph nodes and tumor samples were obtained from individuals with HNSCC, melanoma, colon cancer, rectal cancer, lung cancer, colorectal liver metastases and ovarian cancer. All subjects signed written informed consent approved by the institution's Institutional Review Board (Providence Portland Medical Center, IRB).

[0291] At the time of sample collection, the patient was treatment naive, and had previously undergone extensive treatment including chemotherapy, radiation therapy, surgery and immunotherapy, or none of the above.

[0292] Peripheral blood mononuclear cells were purified from whole blood on a Ficoll-Paque PLUS (GE Healthcare) gradient and stored frozen prior to analysis.

[0293] Tumor specimens were prepared as follows: Briefly, under sterile conditions, tumors were cut into small pieces and digested in RPMI-1640 supplemented with 0.5 mg / ml hyaluronidase, 1 mg / ml collagenase (both Sigma-Aldrich), 30 U / ml DNase (Roche) and a final concentration of 1.5% human serum albumin (MP Biomedicals). Cells were digested for 1 h at room temperature with stirring using a magnetic stir bar. The cell suspension was filtered through a 70 μm filter. Tumor-infiltrating lymphocytes were enriched by Ficoll-Paque PLUS density centrifugation as described above. Tumor single cell suspensions were stored frozen until further analysis.

[0294] Antibodies and flow cytometry: Fluorescently labeled antibodies were purchased from the following manufacturers: Biolegend: CD3 (UCHT1), CD4 (OKT-4 or RPA-T4), CD8 (RPA-T8), CD25 (BC96), CD38 (HIT2), CD45RA (HI100), CD69 (F N50), HLA-DR (L243), CTLA-4 (BNI3), 4-1BB (4B4-1), CCR7 (G043H7), Granzyme B (GB11), IFN-g (4S.B3), TNF-a (Mab11) BD Bioscience: CD27(M-T271), CD127(HIL-7R-M21), PD-1(EH12), Ki-67(B56), eBioscience: CD28 (CD28.2), CD39 (eBioA1), CD103 (Ber-ACT8 and B-Ly7), FOXP3 (PCH101), ICOS (ISA-3) R&D:TIM-3(344823)

[0295] Fixable live / dead dye was used to distinguish live cells (Biolegend). Cell surface staining was performed in FACS buffer (PBS supplemented with 1% FBS and 0.01% NaN3). Intracellular staining was performed using Fix / Perm kits from eBioscience according to the manufacturer's instructions. To analyze cytokine production by peripheral blood mononuclear cells and TILs ex vivo, cells were stimulated with PMA (0.2 μM) and ionomycin (1 μg / ml) for 5 h, with BFA (10 μg / ml) present for the last 2.5 h. Intracellular cytokine staining was performed using CytoFix / CytoPerm kits from BD Bioscience according to the manufacturer's instructions. Stained cells were acquired on LSRII and Fortessa flow cytometers or FACS AriaII (all BD) for cell sorting. Data were analyzed with FlowJo software (Treestar).

[0296] Cell sorting and T cell expansion: Cryopreserved PBMCs and TILs were thawed and T lymphocytes were enriched using Stemcell's T cell enrichment kit. For TIL enrichment, Epcam beads (StemCell) were added to the cocktail. The enriched fraction was then labeled and the population of interest was purified to 99% purity after cell sorting on a FACS AriaII (BD).

[0297] For TCR sequencing analysis, cell pellets were frozen until further processing.

[0298] To expand DN CD8+, SP CD8+ and DP CD8+ TILs as well as naïve and memory CD8 TILs from PBMCs, T cells were sorted and cultured in complete RPMI-1640 supplemented with 2 mM glutamine, 1% (vol / vol) non-essential amino acids, 1% (vol / vol) sodium pyruvate, penicillin (50 U / ml), streptomycin (50 ug / ml) and 10% fetal bovine serum (Hyclone). T cells (2000–5000 cells / well) were cultured on irradiated (4000 rad) allogeneic feeder cells (2 × 10 cells) in 96-well round-bottom plates (Corning / Costar). 5 T cells were polyclonally stimulated with 1 μg / ml PHA (Sigma) in the presence of 100 μg / ml rh IL-15 (Biolegend) and 10 ng / ml rh IL-15 (Biolegend). After one week, when T cell clusters had formed, the cells were split into an additional 96-well plate in complete medium with IL-15, which was repeated two days later, resulting in four identical replicates. The four replicates were then pooled into wells of a 24-well plate on day 12. T cell lines were maintained until analysis.

[0299] Acquisition of Microarray Data: Samples for microarrays were processed in a similar manner to flow analysis. Tumor digestion was completed in a 50 ml conical tube with a magnetic stir bar for 1 hour at room temperature using 1 mg / ml collagenase (Sigma, C-5138), 0.5 mg / ml hyaluronidase (Sigma, H-6254) in RPMI (Life Technologies, 11875-093) with 0.3% human albumin (MP Biomedicals 823051) and 30 u / ml DNASE (Roche 04536282001). After digestion, samples were filtered through a 70 μm filter. Samples were then diluted 1:2 with RPMI and layered over Ficoll (GE, 17-1440-02) to enrich for lymphocytes by a centrifugation step. The enriched cells were stained for CD3, CD8, CD103, CD39 and sorted using a BD FACSAria cell sorter. The sorted cells were lysed and RNA was purified using a Direct-zol RNA miniprep kit (zymo research), then the RNA was reverse transcribed into cDNA and amplified. The amplified cDNA was hybridized onto an Affymetrix Prime-View gene chip.

[0300] Microarray Data Analysis: CHP files generated by Affymetrix GeneChip Command Console (AGCC) v. 3.1.1 and Affymetrix Expression Console v.1.1 software from Affymetrix Primeview gene expression arrays were loaded using BRB ArrayTools, version 4.5.1 (available on the Internet at brb_nci.nih.gov / BRB-ArrayTools / ) software and annotated with their sample type (i.e., DN, SP, DP). The Affymetrix Quality Control module was run to determine that expression results were within specifications (applied BioConductor R module:affy / affyQCReport). Differential expression between sample types was determined using the Class Comparison / Between Groups of Arrays BRBarray module with set significance thresholds for univariate tests (reporting least squares and Kolmogorov-Smirnov tests sorted by p-values ​​for univariate tests). The diagram / visualization of samples module was then used to generate a multidimensional analysis of the three groups using the selected genes (applied BRBarray R module: MDS.R).

[0301] In-vitro T cell activation: Naive CD8 T cell subsets were isolated by magnetic CD8 T cell enrichment (Stemcell), labeled with antibodies against CD4, CD8, CD45RA and CCR7, and sorted. Naive T cells 1 × 10 5 T cells were cultured with anti-CD3 / CD28 Dynabeads (Life Technologies) at a bead:T cell ratio of 1:2 in the presence or absence of 2ng / ml rh TGFβ-1 (R&D). After 24 hours, beads were removed by magnetic capture for half of the experiments. Expression of activation and differentiation markers was assessed on days 1, 2, 3, 4, 7, and 9.

[0302] Deep TCR sequencing and clonality analysis of TCR VB genes: Deep sequencing of the variable VJ or VDJ regions of the TCRβ gene was performed on genomic DNA of sorted T cell populations. 4 ~1×10 5 DNA was extracted from circulating and tumor-resident CD8 T cell subsets spanning a range of 100-fold (DNeasy Blood and Tissue Kit, Qiagen). The TCRβ CDR3 region was sequenced and mapped (ImmunoSEQ, Adaptive Biotech). Coverage per sample was >10×. Only data from productive rearrangements were extracted from the ImmunoSEQ Analyzer platform for further analysis. Clonality of the different T cell subsets was assessed by nucleotide sequence comparison of the 500 most abundant clones within each subset.

[0303] To compare the TCR Vβ overlap (or similarity) of two given populations, we used the Morisita overlap index. The Morisita-Horn similarity index accounts for both the number of common clonotypes and the distribution of clonotype sizes, and is most sensitive with respect to the clonal size of the dominant clonotype (see Venturi et al., J Immunologi Meth, 2008).

[0304] Assessment of target cell recognition: upregulation of 4-1BB and CD25 and IFN-γ secretion: Upregulation of 4-1BB and CD25 and release of IFN-γ were used as measures to assess tumor cell recognition by expanded autologous CD8 T cells. Coculture experiments were performed 17-20 days after the first expansion. On the previous day, expanded T cells were counted and starved overnight in medium without IL-15 to downregulate the expression of remaining 4-1BB and CD25. Expanded CD8 T cells (1×10 5) were cultured alone or with tumor cells (autologous and allogeneic, T cell:target cell ratio = 10:1). In some conditions, tumor cells were pre-incubated with 30ug / ml anti-MHC class I blocking antibody (BD Bioscience, clone W6 / 32) for 3 hours before adding T cells. As a positive control, Nunc Maxisorp plates were coated with anti-CD3 antibody (OKT3) and T cells were added. All conditions were plated in triplicate. After 24 hours, supernatants were collected and analyzed by cytometric bead array (CBA) analysis. Cells were pooled for each condition and labeled with viability dye followed by CD39, CD103, CD25 and 4-1BB cell surface staining. Cells were analyzed by flow cytometry. For CBA, the manufacturer's protocol was followed. Specifically, IFN-γ and TNF-α were analyzed.

[0305] Live target cell killing assay: T cell-mediated target cell killing assays were performed in an Incucyte Zoom System housed inside a 37°C / 5% CO2 cell incubator according to the manufacturer's protocol (Essen Bioscience). To assess T cell killing of autologous tumor cells, 5000-10000 tumor cells (autologous and allogeneic tumor cell lines) were seeded in triplicate into 96-well flat-bottom plates to reach 10% confluency. Expanded autologous T cell subsets were counted and starved overnight without exogenous IL-15. T cells were then added at 1 × 10 5The cells were cultured with or without autologous and allogeneic tumor cells (T cell:tumor cell ratio = 10:1). In some conditions, anti-MHC class I antibody (BD Bioscience, clone W6 / 32) was added. In all conditions, NucView 488 caspase 3 / 7 substrate (Essen Bioscience) was added to monitor active caspase 3 / 7. Plates were incubated at 37 °C for 24 h and four images from three experimental replicates were captured every hour using a 10x objective to visualize T cell killing and caspase 3 / 7 activity (green fluorescence). The green channel acquisition time was 400 ms. Cell segmentation was achieved by applying a mask to exclude small T cells for phase contrast. An area filter was applied to capture images from 1000 μm 2 Objects below were excluded. Green background noise was subtracted by the Top-Hat method of background heterogeneity correction with a radius of 20 μm and a threshold of 2 green correction units. Fluorescence signals were quantified after applying a mask to the experiment. The amount of T cell killing / apoptosis was calculated by the provided Zoom software (Essen Bioscience).

[0306] Statistical analysis: Statistical tests were performed using Prism software (GraphPad, San Diego Calif.) Significance was determined by one-way ANOVA analysis with Tukey's correction as described in the figure legends. Example 2 result

[0307] Identification of tumor-reactive CD8 T cells is important for assessing the level and quality of antitumor responses in cancer patients, as well as for understanding the mode of action of new cancer treatment strategies, such as immunotherapy. Recently, human tumor-reactive CD8 T cells were identified by co-expression of CD103 and PD-1 in high-grade serous ovarian cancer (HGSC) and non-small cell lung cancer (NCLC). To further define their nature and function, tumor-infiltrating CD8 T cells (TILs) from two types of human ovarian tumors were sorted into CD103-positive and CD103-negative subsets, and their gene expression profiles were determined by microarray. For this analysis, we focused on differentially expressed cell surface molecules that can be easily detected by flow cytometry. One of the largest differences observed in the gene array comparison that met the criteria was ENTPD1, ​​a gene that encodes CD39 and is found on the cell surface (Table 1). To confirm the gene array results and to better understand the diversity of tumor-infiltrating CD8 T cells, CD8 T cells isolated from head and neck squamous cell carcinoma (HNSCC) patients were stained and gated for the CD103+ population. CD39, as well as additional cell surface markers associated with activation / exhaustion and developmental states, were investigated. The complexity of simultaneously analyzing all flow cytometry markers necessitated a method termed t-distributed stochastic neighbor embedding (t-SNE), which is widely used for mass cytometry data. Interestingly, the high expression of CD39 in CD103+CD8 T cells overlapped with high expression levels of PD-1 and CD69. Conversely, IL-7R was expressed at low levels in CD103+CD8 T cells (CD127) (Figures 1A and 1B), suggesting a more effector-like T cell phenotype. Furthermore, the combinatorial expression of CD103 and CD39 on CD8 T cells led to the identification of three distinct cell populations, including CD103-CD39- (double negative (DN) CD8), CD103+CD39- (single positive (SP) CD8) and CD103+CD39+ (double positive (DP) CD8).DP CD8 T cells were detected at relatively high frequencies in several human solid malignancies, including HNSCC, lung cancer, melanoma, ovarian cancer, and rectal cancer (Figure 1C), but the frequency of these cells was highest in melanoma. In contrast, DP CD8 T cells were present at very low frequencies in a subset of HNSCC patients and in the majority of patients with colon cancer and colorectal liver metastases (CRLM) (Figures 1C and 1D). Surprisingly, TILs from one patient with colon cancer contained very high frequencies of DP CD8 T cells. This patient was diagnosed with Lynch syndrome, a rare inherited disorder caused by mutations in mismatch repair genes that result in a high rate of mutations. Interestingly, tumors with a high mutation frequency are more likely to harbor multiple neoantigens, and these patients have better outcomes to checkpoint blockade immunotherapy, as exemplified by longer progression-free survival (Le DT, N Engl J Med 2015; Snyder A, N Engl J Med 2014; Van Allen EM, Science 2015; Rizvi NA, Science 2015).

[0308] We then investigated whether DP CD8 T cells are found only at sites where tumor cells are present. To address this, we analyzed several HNSCC patients for whom we had access to primary tumors, metastatic lymph nodes (LNs), uninvolved LNs and peripheral blood. Figure 1e shows results from a representative patient, where co-expression of CD39 and CD103 by CD8 T cells was found specifically in primary tumors and metastatic LNs, but was absent or present at very low frequencies in CD8 T cells in peripheral blood and uninvolved LNs. Importantly, this expression profile was confirmed in the majority of HNSCC patients analyzed (Figure 1F). Thus, the results indicate that CD39 expression in CD103+CD8 T cells identifies a population of CD8 T cells that are specifically induced within the tumor microenvironment.

[0309] To better understand the biology of DN, SP and DP CD8 T cells, the three cell populations were directly sorted ex vivo and their global gene expression profiles were determined by microarray. For this analysis, T cells were isolated from three HNSCCs and two ovarian tumors. Comparison of DP and DN CD8 T cells identified 372 differentially expressed genes between these two cell populations. Principal component analysis and unsupervised hierarchical clustering on this selected list of genes revealed that DP and DN CD8 T cells have distinct mRNA profiles, whereas SP CD8 T cells show an intermediate gene signature (Figures 2a and b). Principal component analysis and unsupervised hierarchical clustering performed on this set of selected genes revealed that the gene expression profiles are cell type specific and not segregated by patient (Figures 2A, 2B). When transcripts from DP CD8 T cells were compared to DN CD8 T cells, some of the largest increases were associated with the activation / exhaustion phenotype, such as MKI67, TNFRSF9, CTLA-4, HAVCR2, and GZMB. In contrast, the most downregulated genes in DP CD8 T cells compared to DN CD8 T cells were those involved in T cell recirculation patterns, such as KLF2, CCR7, SELL, S1PR1, and KLF2. This gene expression profile is indicative of a resident memory T signature (Figure 2C). Importantly, the expression profiles of these activation and recirculation-related mRNAs were consistent across all five tumors (Figure 2D). Thus, DP CD8 T cells display a gene expression signature of cells undergoing antigen-driven stimulation and activation in tumors, which may also result in loss of recirculation outside of tumor tissue.

[0310] To further evaluate the specific properties of tumor-infiltrating CD8 T cells, the expression of differentiation and activation markers was analyzed at the protein level by flow cytometry. DP CD8 T cells expressed higher levels of CD69 compared to SP CD8 T cells or DN CD8 T cells (Figure 3A). CD69 is an activation molecule that is upregulated after T cell stimulation and antagonizes sphingosine 1-phosphate receptor 1 (S1PR1)-mediated release from tissues (Mackay 2015; Skon 2013). DP CD8 T cells also expressed lower levels of CCR7, IL7R (CD127) and CD28, indicative of an effector memory phenotype (ref). Interestingly, even though DN CD8 T cells and SP CD8 T cells expressed PD-1, DP CD8 T cells expressed significantly higher levels of this protein (Figure 3B). Furthermore, CTLA-4, and TIM-3 were almost exclusively expressed within the DP CD8 T cell population. Thus, DP CD8 T cells displayed a highly activated effector memory phenotype, especially when compared with DN and SP CD8 T cells. Furthermore, the increased frequency of 4-1BB and Ki-67 within the DP CD8 T cell population suggests that these cells have recently been activated and expanded within the tumor, indicating recent recognition of cognate antigen.

[0311] The effector functions of DN, SP and DP CD8 T cells were directly assessed ex vivo by analyzing their cytokine production at the single cell level. DP CD8 T cells showed a lower frequency of cells capable of producing IFN-γ and / or TNF-α compared to DN and SP CD8 T cells, with similar results obtained in six HNSCC patients (Figures 3C, 3D). In contrast, DP CD8 T cells had an increased cytotoxic potential, as demonstrated by a significantly higher frequency of granzyme B-positive cells (Figure 3E).

[0312] Considering that DP CD8 T cells were found only at sites where tumor cells were present, we identified factors involved in this particular phenotype to better understand the development of these cells. It is established that TGF-β drives the expression of CD103 in T cells. TGF-β is produced within the tumor microenvironment and plays a major role in cancer progression by inducing epithelial-mesenchymal transition, thereby resulting in enhanced motility and invasion. CD39 expression was found on chronically stimulated / exhausted CD8 T cells in a mouse model of chronic LCMV infection, as well as in patients with chronic HCV and HIV infection (Gupta PK, PLOS Pathogen 2015), supporting the role of sustained TCR stimulation in its expression. Therefore, in naive CD8 T cells, we analyzed the kinetics of CD103 and CD39 upregulation in the presence or absence of TGF-β after TCR engagement (Figure 4 and supplementary data). To address the role of sustained TCR stimulation in CD39 expression, T cells were stimulated continuously with CD3 / CD28 beads for 9 days or the beads were removed 24 hours after the initiation of culture. CD39 expression was detected within 3 days of culture and increased by day 9. CD103 was upregulated when T cells were stimulated in the presence of TGF-β, with more than 80% of cells being positive after 7 days. Optimal CD103 and CD39 upregulation was found only after sustained TCR stimulation. Importantly, the lack of CD39 upregulation in the absence of sustained TCR stimulation was not due to limited T cell activation, since expression of other activation markers such as PD-1 was rapidly induced in the cells (Figure 4). In contrast to CD103 expression, TGF-β did not affect CD39 expression in the in vitro culture system. Thus, sustained TCR stimulation and TGF-β are necessary factors to promote CD103 and CD39 expression in CD8 T cells. Thus, DP CD8 T cells appear to acquire their phenotype upon repeated exposure to their cognate antigen leading to chronic TCR signaling within tumors in a TGF-β-rich environment.

[0313] This data suggested that DP CD8 T cells recognize their cognate antigens within the tumor site. If correct, this would result in selective expansion of dominant TCR clonotypes, resulting in increased clonality compared to other CD8 T cell populations. To address this, TCRβ clonotypes within DN, SP and DP CD8 T cell populations, as well as total memory CD8 T cells from paired blood and uninvolved LNs, were assessed by sequencing the highly variable CDR3 region of the TCRB gene. DP CD8 T cells were more oligoclonal compared to any of the other CD8 T cell populations analyzed (Figure 5A). The 30 most frequent clonotypes in each patient accounted for 56%, 61% and 66% of the DP CD8 T cell population in HPV+HNSCC, HPV-HNSCC and ovarian tumors, respectively, but only 26%, 23% and 38% of the DN CD8 T cells and less than 20% of the memory peripheral CD8 T cells. The 30 most expanded DP CD8 T cell clonotypes were much less frequent in the DN CD8 T cell population, accounting for less than 0.06% of the DN CD8 T cell repertoire in these three patients. Interestingly, very few TCR clonotypes were shared between DP CD8 T cells and other tumor-infiltrating CD8 T cell subsets (Figure 5B). In contrast, a greater number of TCR clonotypes were shared between DN and SP CD8 T cells. As predicted by the resident memory signature (Figure 2D), the majority of TCR clonotypes present on DN CD8 T cells were also shared with memory CD8 T cells in uninvolved LNs as well as with memory CD8 T cells in peripheral blood (R and SP, respectively). 2= 0.7710 and 0.2022), suggesting that DN CD8 T cells can recirculate and potentially sense the tumor environment without ultimately recognizing their cognate antigen (Figure 5C). In comparison, clonotypes present in DP CD8 T cells were only detected in minor amounts in uninvolved LNs or peripheral blood. Calculation of the Morisita index, an abundance-based similarity index that determines the overlap between the two populations, further supported our results and also demonstrated the consistency of this finding across five patient samples (Figure 5D). Collectively, these results suggest that DP CD8 T cells encounter their cognate antigen within the tumor site, leading to their activation and local expansion of tumor-specific T cell clones.

[0314] If this is correct, then DP CD8 TILs should be enriched for tumor reactivity. Therefore, autologous tumor cell lines were generated from four melanoma and two HNSCC tumors to determine whether DP CD8 TILs are enriched for tumor reactivity and killing. CD8 T cells were sorted based on CD103 and CD39 expression directly from tumor digests, and the three CD8 T cell populations were expanded in vitro. After expansion, DN CD8 TILs, SP CD8 TILs, and DP CD8 TILs were screened for reactivity against autologous tumor cell lines as assessed by 4-1BB upregulation and IFN-γ secretion. In all six patients tested, DP CD8 T cells were found to be highly enriched for tumor reactivity compared to DN CD8 TILs and SP CD8 TILs (Figure 6A). Enrichment of tumor-reactive CD8 TILs among DP CD8 cells was exemplified by the highest tumor-to-T cell ratio for patient 1, which reached 87% reactive T cells. The recognition of autologous tumors was specific and MHC class I restricted, since no reactivity was detected in DP TIL subsets after MHC class I blockade or when cells were cultured with allogeneic tumor cell lines (Figure 6B). To address whether tumor-reactive DP CD8 T cells could kill autologous tumor cells, expanded T cell subsets were co-cultured with autologous tumor cells and tumor-specific killing was monitored using the Incucyte live cell analysis system. This system allows caspase 3 / 7-dependent apoptosis to be visualized by microscopy in real time at 37°C. Consistent with 4-1BB upregulation, only DP CD8 T cells killed autologous tumor cells, as exemplified by increasing numbers of caspase 3 / 7+ events (Figures 6C, 6D). This killing was MHC class I dependent, since this effect was blocked by the MHC class I antibody W6 / 32. In contrast, little or no autologous tumor cell killing by DN CD8 T cells was observed.

[0315] Finally, the relationship between the frequency of DP CD8 TILs and patient survival was evaluated in surgical samples. This analysis focused on a small cohort of HNSCC patients (n=62). After surgery, the frequency of DP CD8 T cells among the total CD8 TILs in the primary tumor was determined by flow cytometry, and the patients then received standard of care treatment(s). Patients were separated into groups with high and low DP CD8 T cell frequency compared to the mean frequency of DP CD8 T cells for this cohort. Using this strategy, it was found that patients with a higher percentage of DP CD8 TILs in the tumor at the time of surgery correlated with longer overall survival (OS) (Figure 6E), which showed greater significance in the HPV-negative subgroup (Figure 6F). Collectively, co-expression of CD103 and CD39 was shown to strongly enrich tumor-reactive CD8 TILs and their frequency, which correlated with longer overall survival in HNSCC patients.

[0316] Based on these results, we addressed whether the TCR clonotypes that were over-represented in DP CD8 TILs were indeed tumor-specific. DP CD8 TILs were co-cultured with autologous tumor cells for 20 hours, after which 4-1BB+CD25+CD8 T cells were sorted. When these T cells were co-cultured with autologous tumor lines, the majority of the ex-vivo over-represented clonotypes were found within the 4-1BB+CD25+ fraction of DP CD8 TILs, demonstrating that they were tumor-reactive (Figure 7A, 7B). The data imply that utilizing TCRs from DP CD8 populations will be therapeutic in the adoptive transfer setting. Of note, in two different HNSCC samples, a strong overlap of DP CD8 TIL TCR repertoires was observed between primary tumors and metastatic LNs within the same patient (Figure 7C). This result is important as it suggests that the same DP CD8 TIL TCRs can be isolated from either metastatic LNs or primary tumors and used for TCR therapy.

[0317] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the invention and should not be construed as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. Accordingly, the inventors claim as their invention all that comes within the scope and spirit of these claims. The present invention provides, for example, the following items. (Item 1) 1. A method for isolating a nucleic acid encoding a T cell receptor (TCR) that specifically binds to a tumor cell antigen, comprising: isolating CD39+CD103+CD8+ T cells from a sample from a subject having a tumor that expresses the tumor cell antigen; The CD8 + CD39 + CD103 + cloning a nucleic acid molecule encoding a TCR from the T cell; thereby isolating a nucleic acid molecule encoding said TCR. (Item 2) 2. The method of claim 1, wherein the tumor is head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma or rectal cancer. (Item 3) 2. The method of claim 1, wherein the sample is peripheral blood or a tumor biopsy. (Item 4) Prior to cloning the T cell receptor, the CD8 + CD39 + CD103 + Steps for expanding T cells in vitro 4. The method according to any one of items 1 to 3, further comprising: (Item 5) 5. The method according to any one of items 1 to 4, wherein the subject is a human. (Item 6) A nucleic acid molecule encoding a TCR, produced by the method described in item 1. (Item 7) A TCR encoded by a nucleic acid molecule encoding a TCR produced by the method according to any one of items 1 to 5. (Item 8) 7. An isolated host T cell transfected with the nucleic acid molecule of item 6. (Item 9) The host T cell of item 8, wherein the TCR is autologous to the subject. (Item 10) 9. The isolated host T cell of item 8, wherein the TCR is allogeneic to the subject. (Item 11) 1. A method of treating a subject having a tumor, comprising administering to the subject a therapeutically effective amount of CD8 + CD39 + CD103 + administering T cells to the subject, thereby treating the subject having the tumor. (Item 12) 12. The method of claim 11, further comprising administering to the subject a therapeutically effective amount of a programmed death (PD)-1 antagonist, a programmed death-ligand (PD-L1) antagonist, a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antagonist, a B- and T-lymphocyte attenuator (BTLA) antagonist, a T-cell immunoglobulin and mucin domain-containing-3 (TIM-3) antagonist, a lymphocyte-activation gene 3 (LAG3) antagonist, or a 4-1BB agonist. (Item 13) 13. The method of claim 12, wherein a) the PD-1 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to PD-1; b) the PD-L1 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to PD-L1; c) the CTLA-4 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4; d) the BTLA antagonist is an antibody or antigen-binding fragment thereof that specifically binds to BTLA; e) the TIM-3 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to TIM-3; and e) the LAG3 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to LAG3. (Item 14) 14. The method of claim 13, wherein the antibody that specifically binds to PD-1, the antibody that specifically binds to PD-L1, the antibody that specifically binds to CTLA-4, the antibody that specifically binds to BTLA, the antibody that specifically binds to TIM-3, or the antibody that specifically binds to LAG3 is a human or humanized monoclonal antibody. (Item 15) 13. The method of claim 12, wherein the PD-1 antagonist, the PD-L1 antagonist, the CTLA-4 antagonist, the BTLA antagonist, the TIM-3 antagonist, or the LAG3 antagonist is a small inhibitory RNA, an antisense RNA, a ribozyme, a small molecule, or a dominant negative protein. (Item 16) 16. The method according to any one of items 11 to 15, wherein the tumor is a solid tumor. (Item 17) 17. The method of claim 16, wherein the solid tumor is head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma or rectal cancer. (Item 18) The CD8 + CD39 + CD103 + 18. The method of any one of items 11 to 17, wherein the T cells are autologous. (Item 19) 18. The method of any one of items 11 to 17, further comprising the step of resecting the tumor in the subject. (Item 20) 20. The method of any one of items 11 to 19, wherein the subject is a human. (Item 21) CD8 + CD39 + CD103 + A method for expanding T cells, comprising: CD8 + CD39 + CD103 + culturing the T cells in tissue culture medium containing glutamine, serum, and antibiotics to form a primary culture; stimulating the primary culture with an effective amount of irradiated allogeneic feeder cells and interleukin (IL)-15 to form stimulated T cells; culturing the stimulated T cells in tissue culture medium and an effective amount of IL-15. thereby, said CD8+ CD39 + CD103 + How to increase T cells. (Item 22) 22. The method of claim 21, comprising stimulating the primary culture in about 5 ng / ml to about 50 ng / ml of IL-15 and / or culturing the clusters of T cells in about 5 ng / ml to about 50 ng / ml of IL-15. (Item 23) 23. The method of claim 22, comprising stimulating the primary culture in about 10 ng / ml of IL-15 and / or culturing the clusters of T cells in about 10 ng / ml of IL-15. (Item 24) The step of stimulating the primary culture with an effective amount of irradiated allogeneic feeder cells comprises stimulating the primary culture with about 1,000 to about 2,000 CD8 + CD39 + CD103 + 24. The method of any one of items 21 to 23, comprising stimulating the T cells with about 100,000 to about 300,000 allogeneic feeder cells. (Item 25) 25. The method according to item 24, wherein about 1,000 to about 2,000 CD39+CD103+CD8+ cells are stimulated with about 200,000 allogeneic feeder cells. (Item 26) 1. A method for determining whether a subject having a tumor will respond to a checkpoint inhibitor or radiation, comprising: CD8 in a biological sample from the subject + CD39 + CD103 + Detecting the presence of T cells Including, The CD8 + CD39 + CD103 + The method, wherein the presence of T cells indicates that the checkpoint inhibitor or radiation is effective in treating the tumor in the subject. (Item 27) 27. The method of claim 26, further comprising administering the checkpoint inhibitor or radiation to the subject. (Item 28) 1. A method for determining whether a subject having a tumor will respond to a cancer therapeutic agent, comprising: administering to the subject a first dose of the cancer therapeutic agent; CD8 in a biological sample from the subject + CD39 + CD103 + Steps for determining the number of T cells Including, CD8 in the biological sample + CD39 + CD103 + An increase in the number of T cells compared to a control indicates that the first dose of the cancer therapeutic agent is effective in treating the tumor in the subject. (Item 29) 1. A method for treating a subject having a tumor, comprising: administering to the subject a first dose of a cancer therapeutic agent; CD8 in a biological sample from the subject + CD39 + CD103 + Determining the number of T cells, CD8 in the biological sample + CD39 + CD103 + an increase in the amount of T cells relative to a control indicates that the first dose of the cancer therapeutic agent is effective in treating the tumor in the subject; and administering a second dose of the cancer therapeutic agent, wherein the first dose is the same as the second dose or the second dose is lower than the first dose; The method includes: (Item 30) 1. A method for treating a subject having a tumor, comprising: administering to the subject a first dose of a cancer therapeutic agent; CD8 in a biological sample from the subject + CD39 + CD103 + Determining the number of T cells, CD8 in the biological sample + CD39 + CD103 + a decrease or no change in the amount of T cells compared to a control indicates that the first dose of the cancer therapeutic agent is not effective in treating the tumor in the subject; administering a second dose of the cancer therapeutic agent, wherein the second dose is higher than the first dose or the second dose is the same as the first dose; and The method includes: (Item 31) The control is a CD8 + CD39 + CD103 + 31. The method according to item 29 or 30, wherein the control is the number of T cells or is a standard value. (Item 32) 32. The method of any one of items 27 to 31, wherein the cancer therapeutic agent is a checkpoint inhibitor. (Item 33) 33. The method of any one of items 27 to 32, wherein the cancer therapeutic agent is a chemotherapeutic agent. (Item 34) The method of claim 28 or claim 33, wherein the checkpoint inhibitor is a programmed death (PD)-1 antagonist, a programmed death-ligand (PD-L) 1 antagonist, a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antagonist, a B- and T-lymphocyte attenuator (BTLA) antagonist, a T-cell immunoglobulin and mucin domain-containing-3 (TIM-3) antagonist, a lymphocyte-activation gene 3 (LAG3) antagonist, or a 4-1BB agonist to the subject. (Item 35) 35. The method of any one of items 27 to 34, wherein the sample is a peripheral blood sample or a tumor biopsy. (Item 36) 36. The method according to any one of items 37 to 35, wherein the subject is a human. (Item 37) 37. The method of any one of items 27 to 36, wherein the tumor is a solid tumor. (Item 38) 38. The method of claim 37, wherein the solid tumor is head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma or rectal cancer.

Claims

1. A composition for treating a tumor in a subject having a tumor, comprising a CD8 ＋ CD39 ＋ CD103 ＋ A composition comprising T cells.

2. 2. The composition of claim 1, wherein the composition is administered to the subject in combination with a programmed death (PD)-1 antagonist, a programmed death-ligand (PD-L1) antagonist, a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antagonist, a B- and T-lymphocyte attenuator (BTLA) antagonist, a T-cell immunoglobulin and mucin domain-containing-3 (TIM-3) antagonist, a lymphocyte-activation gene 3 (LAG3) antagonist, or a 4-1BB agonist.

3. 3. The composition of claim 2, wherein a) the PD-1 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to PD-1; b) the PD-L1 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to PD-L1; c) the CTLA-4 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4; d) the BTLA antagonist is an antibody or antigen-binding fragment thereof that specifically binds to BTLA; e) the TIM-3 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to TIM-3; and e) the LAG3 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to LAG3.

4. 4. The composition of claim 3, wherein the antibody that specifically binds to PD-1, the antibody that specifically binds to PD-L1, the antibody that specifically binds to CTLA-4, the antibody that specifically binds to BTLA, the antibody that specifically binds to TIM-3, or the antibody that specifically binds to LAG3 is a human or humanized monoclonal antibody.

5. 3. The composition of claim 2, wherein the PD-1 antagonist, the PD-L1 antagonist, the CTLA-4 antagonist, the BTLA antagonist, the TIM-3 antagonist, or the LAG3 antagonist is a small inhibitory RNA, an antisense RNA, a ribozyme, a small molecule, or a dominant negative protein.

6. The composition of claim 1 , wherein the tumor is a solid tumor.

7. 7. The composition of claim 6, wherein the solid tumor is head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma or rectal cancer.

8. The CD8 ＋ CD39 ＋ CD103 ＋ The composition of any one of claims 1 to 7, wherein the T cells are autologous.

9. The composition of claim 1 , wherein the subject has had a tumor resection.

10. The composition of any one of claims 1 to 9, wherein the subject is a human.

11. 2. The composition of claim 1, wherein the tumor is head and neck squamous cell carcinoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, bladder cancer, cervical cancer, liver cancer, prostate cancer, breast cancer, glioblastoma or rectal cancer.

Images