Dysfunctional antigen-specific CD8+ T cells in the tumor microenvironment

Provided herein are compositions and methods for detecting and/or targeting dysfunctional tumor antigen-specific CD8⁺ T cells in the tumor microenvironment for diagnostic, therapeutic and/or research applications. In particular, dysfunctional tumor antigen-specific CD8⁺ T cells are detected and/or targeted via their expression of cell surface receptors described herein, such as 4-1BB, LAG-3, or additional markers that correlate with 4-1BB and LAG-3 expression, such as markers differentially expressed on the surface of the T cells.

1. A method of treating dysfunctional tumor antigen-specific CD8⁺ T cells in a subject in need thereof with a solid tumor cancer comprising identifying dysfunctional T cells by testing said cells for expression of GPNMB; and administering to the subject an antibody or antibody fragment that specifically targets dysfunctional tumor antigen-specific CD8⁺ T cells, wherein the antibody or antibody fragment targets GPNMB expressed on the surface of the T cells, and wherein the dysfunctional tumor antigen-specific CD8+ T cells are within a tumor microenvironment.

2. The method of claim 1, wherein the tumor allows T cell infiltration, but is resistant to immunotherapies.

3. The method of claim 1, further comprising contacting the dysfunctional tumor antigen-specific CD8+ T cells with an anti-4-1BB and/or anti-LAG3 agent.

4. The method of claim 3, wherein the anti-4-1BB and/or anti-LAG3 agent is an antibody, antibody fragment, or antibody mimetic molecule.

5. The method of claim 1, further comprising co-administration of an additional therapeutic agent.

6. The method of claim 5, wherein the additional therapeutic agent is a chemotherapeutic or an immunotherapeutic agent.

7. The method of claim 6, wherein the additional therapeutic agent is an immunotherapeutic agent selected from the list consisting of cell-based therapies, monoclonal antibody (mAb) therapy, cytokine therapy, and adjuvant treatment.

8. The method of claim 7, wherein the immunotherapeutic agent is a mAb therapy selected from the list consisting of anti-CTLA-4 monoclonal antibodies and/or anti-PD-L1 monoclonal antibodies.

9. The method of claim 7, wherein the immunotherapeutic agent is a cell-based therapy selected from the list consisting of dendritic-cell therapy and T-cell therapy.

10. The method of claim 5, wherein the additional therapeutic agent targets PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM and/or Sema7a.

11. The method of claim 1, wherein the antibody or antibody fragment is an anti-Nrn1 antibody, antibody fragment, or antibody mimetic molecule.

12. The method of claim 1, wherein the antibody or antibody fragment is an anti-Sema7a antibody, antibody fragment, or antibody mimetic molecule.

13. The method of claim 1, wherein the antibody or antibody fragment is an anti-CRTAM antibody, antibody fragment, or antibody mimetic molecule.

The present invention claims priority to U.S. Provisional Patent Application Ser. No. 62/447,199, filed Jan. 17, 2017, which is incorporated by reference in its entirety.

This invention was made with government support under Grant No. R01 CA161005 awarded by the National Institutes of Health. The government has certain rights in the invention.

Provided herein are compositions and methods for detecting and/or targeting dysfunctional tumor antigen-specific CD8⁺ T cells in the tumor microenvironment for diagnostic, therapeutic and/or research applications. In particular, dysfunctional tumor antigen-specific CD8⁺ T cells are detected and/or targeted via their expression of cell surface receptors described herein, such as 4-1BB, LAG-3, or additional markers that correlate with 4-1BB and LAG-3 expression, such as markers differentially expressed on the surface of the T cells.

The immune system plays a critical role in protecting the host from cancer (Vesely et al., 2011; incorporated by reference in its entirety). Innate sensing of tumors leads to an adaptive T cell response through the presentation of tumor-associated antigens (TAAs) derived from mutations and epigenetic changes that contribute to carcinogenesis (Gajewski et al., 2013; incorporated by reference in its entirety). Spontaneously-primed CD8+ T cells home to tumor sites in mouse tumor models (Harlin et al., 2009; Fuertes et al., 2011; incorporated by reference in their entireties) and in a subset of patients with advanced cancer (Harlin et al., 2006; incorporated by reference in its entirety). These tumor-infiltrating lymphocytes (TIL) have the ability to recognize tumor antigens and are believed to contribute to tumor control in cancer patients, based on the correlation between activated CD8+ T cell infiltration with improved prognosis and response to immunotherapy (Fridman et al., 2012; Tumeh et al., 2014; incorporated by reference in their entireties). However, without additional manipulation, this endogenous anti-tumor response is usually not sufficient to mediate complete rejection of an established tumor (Gajewski, 2007b; Pardoll, 2012; Baitsch et al., 2011; Gajewski et al., 2006; Larkin et al., 2015). Data accumulated over the past several years have indicated that tumors with spontaneous anti-tumor T cell responses have high expression of immune-inhibitory pathways that subvert the effector phase of the response. These include PD-L1/PD-1 interactions (Pardoll, 2012; incorporated by reference in its entirety), recruitment of CD4+Foxp3+ regulatory T (Treg) cells (Gajewski, 2007a; incorporated by reference in its entirety), and metabolic dysregulation by indoleamine-2,3-dioxygenase (IDO) (Spranger et al., 2013; incorporated by reference in its entirety). However, even when CD8+ T cells specific for tumor antigens are isolated from tumors, away from these extrinsic immune inhibitory factors, they still show altered functional properties ex vivo (Harlin et al., 2006; Baitsch et al., 2011; incorporated by reference in their entireties).

Expression of PD-1 has been described to identify tumor-specific exhausted T cells (Ahmadzadeh et al., 2009; Fourcade et al., 2012; Wu et al., 2014; Gros et al., 2014; incorporated by reference in their entireties). However, T cells expressing PD-1 in the context of chronic infection can still retain effector function (Wherry and Kurachi, 2015; incorporated by reference in its entirety), and PD-1 is not required for the induction of T cell exhaustion (Odorizzi et al., 2015; incorporated by reference in its entirety). In addition to PD-1, several additional co-inhibitory receptors, including CD223 (LAG-3), CD244 (2B4), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), hepatitis A virus cellular receptor 2 (TIM-3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), are also be expressed on dysfunctional T cells and expression of a greater number of inhibitory receptors has been correlated with diminished cytokine secretion (in particular IFN-g and TNF-α) as well as proliferative capacity (Blackburn et al., 2009; incorporated by reference in its entirety). Expression of these receptors has been observed in both viral and cancer models, however, a complete analysis of both co-inhibitory and co-stimulatory receptors on the same population is lacking in the tumor setting.

Provided herein are compositions and methods for detecting and/or targeting dysfunctional tumor antigen-specific CD8⁺ T cells in the tumor microenvironment for diagnostic, therapeutic and/or research applications. In particular, dysfunctional tumor antigen-specific CD8⁺ T cells are detected and/or targeted via their expression of cell surface receptors described herein, such as 4-1BB, LAG-3, or additional markers that correlate with 4-1BB and LAG-3 expression, such as markers differentially expressed on the surface of the T cells (e.g., PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, CRTAM and Sema7a).

In some embodiments, provided herein are methods of treating a subject with cancer comprising administering an agent that specifically targets dysfunctional tumor antigen-specific CD8⁺ T cells. In some embodiments, the subject suffers from a solid tumor cancer. In some embodiments, the tumor allows T cell infiltration, but is resistant to immunotherapies. In some embodiments, the tumor environment comprises dysfunctional tumor antigen-specific CD8⁺ T cells. In some embodiments, contacting the dysfunctional tumor antigen-specific CD8⁺ T cells with an anti-4-1BB and/or anti-LAG3 agent. In some embodiments, the anti-4-1BB and/or anti-LAG3 agent is an antibody, antibody fragment, or antibody mimetic molecule. In some embodiments, methods further comprise co-administration of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic or an immunotherapeutic agent. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent selected from the list consisting of cell-based therapies, monoclonal antibody (mAb) therapy, cytokine therapy, and adjuvant treatment. In some embodiments, the immunotherapeutic agent is a mAb therapy selected from the list consisting of anti-CTLA-4 monoclonal antibodies and/or anti-PD-L1 monoclonal antibodies. In some embodiments, the immunotherapeutic agent is a cell-based therapy selected from the list consisting of dendritic-cell therapy and T-cell therapy. In some embodiments, the additional therapeutic agent targets one of the markers/receptors listed in Table 2. In some embodiments, the additional therapeutic targets a marker/receptor expressed on the surface of the T cells. In some embodiments, the additional therapeutic targets PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, CRTAM or Sema7a. In some embodiments, the additional therapeutic agent targets Nrn1, Sema7a, or CRTAM.

In some embodiments, provided herein are methods of treating a subject with cancer comprising administering a therapeutic agent that specifically targets dysfunctional tumor antigen-specific CD8⁺ T cells, wherein the agent targets one of the receptors listed in Table 2. In some embodiments, the therapeutic targets a marker/receptor expressed on the surface of the T cells. In some embodiments, the therapeutic targets PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, CRTAM or Sema7a. In some embodiments, the therapeutic agent targets Nrn1, Sema7a, or CRTAM. In some embodiments, the therapeutic agent is an antibody, antibody fragment, or antibody mimetic molecule that binds the target marker/receptor. In some embodiments, the therapeutic agent is an anti-Nrn antibody, antibody fragment, or antibody mimetic molecule. In some embodiments, the therapeutic agent is an anti-Sema7a antibody, antibody fragment, or antibody mimetic molecule. In some embodiments, the therapeutic agent is an anti-CRTAM antibody, antibody fragment, or antibody mimetic molecule.

In some embodiments, provided herein are compositions comprising: (a) one or more of an anti-4-1BB agent, an anti-LAG-3 agent, an anti-Nrn1 agent, an anti-Sema7a agent, and an anti-CRTAM agent; and (b) an immunotherapeutic agent, said composition formulated for therapeutic delivery to a subject. In some embodiments, the anti-4-1BB agent, anti-LAG-3 agent, anti-Nrn1 agent, anti-Sema7a agent, and/or anti-CRTAM agent is an antibody, antibody fragment, or antibody mimetic molecule.

In some embodiments, provided herein are compositions comprising: (a) an agent that targets and/or binds one of PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A; and (b) an immunotherapeutic agent, said composition formulated for therapeutic delivery to a subject.

In some embodiments, provided herein are methods comprising: (a) testing CD8⁺ T cells from a cell population to determine whether the CD8+ T Cells co-express LAG-3 and 4-1BB; and (b) administering one or more agents that target and/or bind one of PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A. In some embodiments, the agent is an anti-Nrn1 agent, an anti-Sema7a agent, and an anti-CRTAM agent. In some embodiments, the anti-Nrn1 agent, anti-Sema7a agent, and/or anti-CRTAM agent is an antibody, antibody fragment, or antibody mimetic molecule. In some embodiments, testing is performed in vitro.

In some embodiments, provided herein are methods of identifying dysfunctional T cells by testing said cells for co-expression of 4-1BB and LAG-3. In some embodiments, provided herein are methods of identifying dysfunctional T cells by testing said cells for expression of one or more of the markers/receptors of Table 2 (e.g., a T-cell surface marker/receptor (e.g., PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, TMEM126A).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.

As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition (e.g., cancer, solid tumor cancer, etc.).

As used herein, an “immune response” refers to the action of a cell of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a subject of invading pathogens, cells or tissues infected with pathogens, or cancerous or other abnormal cells.

As used herein, the term “immunoregulator” refers to a substance, an agent, a signaling pathway or a component thereof that regulates an immune response. “Regulating,” “modifying” or “modulating” an immune response refers to any alteration in a cell of the immune system or in the activity of such cell. Such regulation includes stimulation or suppression of the immune system which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes which can occur within the immune system. Both inhibitory and stimulatory immunoregulators have been identified, some of which may have enhanced function in the cancer microenvironment.

As used herein, the term “immunotherapy” refers to the treatment or prevention of a disease or condition by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.

As used herein, “potentiating an endogenous immune response” means increasing the effectiveness or potency of an existing immune response in a subject. This increase in effectiveness and potency may be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

As used herein, the term “antibody” refers to a whole antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab′, and F(ab′)₂), unless otherwise specified (e.g., “whole antibody,” “antibody fragment”). An antibody may be a polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc.

A native antibody typically has a tetrameric structure. A tetramer typically comprises two identical pairs of polypeptide chains, each pair having one light chain (in certain embodiments, about 25 kDa) and one heavy chain (in certain embodiments, about 50-70 kDa). In a native antibody, a heavy chain comprises a variable region, V_H, and three constant regions, C_H1, C_H2, and C_H3. The V_Hdomain is at the amino-terminus of the heavy chain, and the C_H3domain is at the carboxy-terminus. In a native antibody, a light chain comprises a variable region, V_L, and a constant region, C_L. The variable region of the light chain is at the amino-terminus of the light chain. In a native antibody, the variable regions of each light/heavy chain pair typically form the antigen binding site. The constant regions are typically responsible for effector function.

In a native antibody, the variable regions typically exhibit the same general structure in which relatively conserved framework regions (FRs) are joined by three hypervariable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDRs on the heavy chain are referred to as H1, H2, and H3, while the CDRs on the light chain are referred to as L1, L2, and L3. Typically, CDR3 is the greatest source of molecular diversity within the antigen-binding site. H3, for example, in certain instances, can be as short as two amino acid residues or greater than 26. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat et al. (1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Publication No. 91-3242, vols. 1-3, Bethesda, Md.); Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196:901-917; or Chothia, C. et al. Nature 342:878-883 (1989). In the present application, the term “CDR” refers to a CDR from either the light or heavy chain, unless otherwise specified.

As used herein, the term “heavy chain” refers to a polypeptide comprising sufficient heavy chain variable region sequence to confer antigen specificity either alone or in combination with a light chain.

As used herein, the term “light chain” refers to a polypeptide comprising sufficient light chain variable region sequence to confer antigen specificity either alone or in combination with a heavy chain.

As used herein, when an antibody or other entity “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (K_a) of at least 10⁷M⁻¹(e.g., >10⁷M⁻¹, >10⁸M⁻¹, >10⁹M⁻¹, >10¹⁰M⁻¹, >10¹¹M⁻¹, >10¹²M⁻¹, >10¹³M⁻¹, etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.

As used herein, the term “anti-4-1BB antibody” or “4-1BB antibody” refers to an antibody which specifically recognizes an antigen and/or epitope presented by 4-1BB. Similarly, the terms “anti-LAG-3 antibody” and “LAG-3 antibody” refer to an antibody which specifically recognizes an antigen and/or epitope presented by LAG-3, the terms “anti-Nrn1 antibody” and “Nrn1 antibody” refer to an antibody which specifically recognizes an antigen and/or epitope presented by Nrn1, the terms “anti-CRTAM antibody” and “CRTAM antibody” refer to an antibody which specifically recognizes an antigen and/or epitope presented by CRTAM, and the terms “anti-Sema7a antibody” and “Sema7a antibody” refer to an antibody which specifically recognizes an antigen and/or epitope presented by Sema7a. Antibodies that recognize epitopes on other molecular entities may be referred to according to a similar scheme (e.g., anti-CTLA-4, anti-PD-L1, etc.).

As used herein, the term “monoclonal antibody” refers to an antibody which is a member of a substantially homogeneous population of antibodies that specifically bind to the same epitope. In certain embodiments, a monoclonal antibody is secreted by a hybridoma. In certain such embodiments, a hybridoma is produced according to certain methods known to those skilled in the art. See, e.g., Kohler and Milstein (1975) Nature 256: 495-499; herein incorporated by reference in its entirety. In certain embodiments, a monoclonal antibody is produced using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In certain embodiments, a monoclonal antibody refers to an antibody fragment isolated from a phage display library. See, e.g., Clackson et al. (1991) Nature 352: 624-628; and Marks et al. (1991) J. Mol. Biol. 222: 581-597; herein incorporated by reference in their entireties. The modifying word “monoclonal” indicates properties of antibodies obtained from a substantially-homogeneous population of antibodies, and does not limit a method of producing antibodies to a specific method. For various other monoclonal antibody production techniques, see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); herein incorporated by reference in its entirety.

As used herein, the term “antibody fragment” refers to a portion of a full-length antibody, including at least a portion antigen binding region or a variable region. Antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, Fv, scFv, Fd, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; herein incorporated by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies (e.g., papain digestion and pepsin digestion of antibody) produced by recombinant DNA techniques, or chemical polypeptide synthesis.

For example, a “Fab” fragment comprises one light chain and the C_H1and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab′” fragment comprises one light chain and one heavy chain that comprises additional constant region, extending between the C_H1and C_H2domains. An interchain disulfide bond can be formed between two heavy chains of a Fab′ fragment to form a “F(ab′)₂” molecule.

An “Fv” fragment comprises the variable regions from both the heavy and light chains, but lacks the constant regions. A single-chain Fv (scFv) fragment comprises heavy and light chain variable regions connected by a flexible linker to form a single polypeptide chain with an antigen-binding region. Exemplary single chain antibodies are discussed in detail in WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203; herein incorporated by reference in their entireties. In certain instances, a single variable region (e.g., a heavy chain variable region or a light chain variable region) may have the ability to recognize and bind antigen.

Other antibody fragments will be understood by skilled artisans.

As used herein, the term “chimeric antibody” refers to an antibody made up of components from at least two different sources. In certain embodiments, a chimeric antibody comprises a portion of an antibody derived from a first species fused to another molecule, e.g., a portion of an antibody derived from a second species. In certain such embodiments, a chimeric antibody comprises a portion of an antibody derived from a non-human animal fused to a portion of an antibody derived from a human. In certain such embodiments, a chimeric antibody comprises all or a portion of a variable region of an antibody derived from a non-human animal fused to a constant region of an antibody derived from a human.

A “humanized” antibody refers to a non-human antibody that has been modified so that it more closely matches (in amino acid sequence) a human antibody. A humanized antibody is thus a type of chimeric antibody. In certain embodiments, amino acid residues outside of the antigen binding residues of the variable region of the non-human antibody are modified. In certain embodiments, a humanized antibody is constructed by replacing all or a portion of a complementarity determining region (CDR) of a human antibody with all or a portion of a CDR from another antibody, such as a non-human antibody, having the desired antigen binding specificity. In certain embodiments, a humanized antibody comprises variable regions in which all or substantially all of the CDRs correspond to CDRs of a non-human antibody and all or substantially all of the framework regions (FRs) correspond to FRs of a human antibody. In certain such embodiments, a humanized antibody further comprises a constant region (Fc) of a human antibody.

The term “human antibody” refers to a monoclonal antibody that contains human antibody sequences and does not contain antibody sequences from a non-human animal. In certain embodiments, a human antibody may contain synthetic sequences not found in native antibodies. The term is not limited by the manner in which the antibodies are made. For example, in various embodiments, a human antibody may be made in a transgenic mouse, by phage display, by human B-lymphocytes, or by recombinant methods.

As used herein, the term “natural antibody” refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multicellular organism. For example, the antibodies produced by the antibody-producing cells isolated from a first animal immunized with an antigen are natural antibodies. Natural antibodies contain naturally-paired heavy and light chains. The term “natural human antibody” refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a human subject.

Native human light chains are typically classified as kappa and lambda light chains. Native human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA has subclasses including, but not limited to, IgA1 and IgA2. Within native human light and heavy chains, the variable and constant regions are typically joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., Fundamental Immunology (1989) Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.); herein incorporated by reference in its entirety.

The term “neutralizing antibody” or “antibody that neutralizes” refers to an antibody that reduces at least one activity of a polypeptide comprising the epitope to which the antibody specifically binds. In certain embodiments, a neutralizing antibody reduces an activity in vitro and/or In vivo. In some embodiments, by neutralizing the polypeptide comprising the epitope, the neutralizing antibody inhibits the capacity of the cell displaying the epitope.

As used herein, the term “glycoengineered”, as used herein, includes any manipulation of the glycosylation pattern of a naturally occurring or recombinant protein, polypeptide or a fragment thereof.

The term “antigen-binding site” refers to a portion of an antibody capable of specifically binding an antigen. In certain embodiments, an antigen-binding site is provided by one or more antibody variable regions.

The term “epitope” refers to any polypeptide determinant capable of specifically binding to an immunoglobulin or a T-cell or B-cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. In certain embodiments, an epitope may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope may have specific three dimensional structural characteristics (e.g., a “conformational” epitope) and/or specific charge characteristics.

An epitope is defined as “the same” as another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides having different primary amino acid sequences may comprise epitopes that are the same. In certain embodiments, epitopes that are the same may have different primary amino acid sequences. Different antibodies are said to bind to the same epitope if they compete for specific binding to that epitope.

A “conservative” amino acid substitution refers to the substitution of an amino acid in a polypeptide with another amino acid having similar properties, such as size or charge. In certain embodiments, a polypeptide comprising a conservative amino acid substitution maintains at least one activity of the unsubstituted polypeptide. A conservative amino acid substitution may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Naturally occurring residues may be divided into classes based on common side chain properties, for example: hydrophobic: norleucine, Met, Ala, Val, Leu, and Ile; neutral hydrophilic: Cys, Ser, Thr, Asn, and Gln; acidic: Asp and Glu; basic: His, Lys, and Arg; residues that influence chain orientation: Gly and Pro; and aromatic: Trp, Tyr, and Phe. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class; whereas conservative substitutions may involve the exchange of a member of one of these classes for another member of that same class.

As used herein, the term “sequence identity” refers to the degree to which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into the families (see above). The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.

The term “effective dose” or “effective amount” refers to an amount of an agent, e.g., an antibody, that results in the reduction of symptoms in a patient or results in a desired biological outcome. In certain embodiments, an effective dose or effective amount is sufficient to treat or reduce symptoms of a disease or condition.

As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

The term “treatment” encompasses both therapeutic and prophylactic/preventative measures unless otherwise indicated. Those in need of treatment include, but are not limited to, individuals already having a particular condition as well as individuals who are at risk of acquiring a particular condition or disorder (e.g., those having a genetic or epigenetic predisposition; based on age, gender, lifestyle, etc.). The term “treating” refers to administering an agent to a subject for therapeutic and/or prophylactic/preventative purposes.

A “therapeutic agent” refers to an agent that may be administered In vivo to bring about a therapeutic and/or prophylactic/preventative effect.

A “therapeutic antibody” refers to an antibody that may be administered In vivo to bring about a therapeutic and/or prophylactic/preventative effect.

As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

As used herein, the term pharmaceutical composition” refers to the combination of an active agent (e.g., binding agent) with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintigrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference in its entirety.

As used herein, a “diagnostic” or “diagnostic test” includes the detection, identification, or characterization of a disease state or condition of a subject. For example, a disease or condition may be characterized to determine the likelihood that a subject with a disease or condition will respond to a particular therapy, determine the prognosis of a subject with a disease or condition (or its likely progression or regression), determine the effect of a treatment on a subject with a disease or condition, or determine a future treatment course of action.

Provided herein are compositions and methods for detecting and/or targeting dysfunctional tumor antigen-specific CD8⁺ T cells in the tumor microenvironment for diagnostic, therapeutic and/or research applications. In particular, dysfunctional tumor antigen-specific CD8⁺ T cells are detected and/or targeted via their expression of cell surface receptors described herein, such as 4-1BB, LAG-3, or additional markers that correlate with 4-1BB and LAG-3 expression, such as markers differentially expressed on the surface of the T cells (e.g., PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, CRTAM and Sema7a).

Experiments conducted during development of embodiments herein identified markers/receptors that correlate and/or are responsible for tumor antigen-specific CD8⁺ T cell dysfunction. In some embodiments, the markers/receptors are overexpressed in dysfunctional tumor antigen-specific CD8⁺ T cells. In such embodiments, detecting the level (e.g., above a threshold) of such markers provides a diagnostic for detecting tumor antigen-specific CD8⁺ T cell dysfunction. Further, in such embodiments, targeting (e.g., inhibiting (e.g., expression and/or activity of)) such markers/receptors provides a therapeutic. In other embodiments, the markers/receptors are underexpressed in dysfunctional tumor antigen-specific CD8⁺ T cells. In such embodiments, detecting the level (e.g., below a threshold) of such markers provides a diagnostic for detecting tumor antigen-specific CD8⁺ T cell dysfunction. Further, in such embodiments, targeting (e.g., enhancing (e.g., expression and/or activity of)) such markers/receptors provides a therapeutic.

Transcription factor Egr2 is a critical regulator of the anergic state in CD4⁺ T cell clones manipulated in vitro (Zheng et al., 2013; 2012; incorporated by reference in their entireties). Egr2 has also been shown to be involved in negative regulation of T cell activation in several in vivo model systems (Sumitomo et al., 2013; incorporated by reference in its entirety). Egr2 contributes to upregulation of DGKa and -z which act to blunt TCR-mediated Ras pathway activation (Zha et al., 2006; incorporated by reference in its entirety). By comparing gene expression profiling of anergized cells along with Egr2 ChIP-Seq analysis multiple additional Egr2-driven gene targets were identified (Zheng et al., 2013; incorporated by reference in its entirety). These gene targets include 4-1BB (Tnfrsf9 or CD137), Lag3, Nrn1, Sema7a, Crtam, and Rankl, which encode cell surface proteins.

4-1BB is a co-stimulatory molecule transiently expressed after TCR engagement. Lag3 (lymphocyte-activation gene 3 or CD223) is a CD4 homologue and functions as an inhibitory receptor. Expression of 4-1BB and Lag3 is regulated following TCR engagement and continues throughout differentiation. In humans, 4-1BB and LAG-3 are expressed on CD8+ TILs from human melanoma tumors (Gros et al., 2014; Baitsch et al., 2012; incorporated by reference in their entireties). In both mice and humans, either molecule alone are expressed on populations of activated T cells. However, co-expression is more limited and is rarely observed in circulating T cells. The function of CD8+ TILs co-expressing these markers is unknown.

Experiments were conducted during development of embodiments herein to investigate the detailed characteristics of CD8+ TILs expressing 4-1BB and LAG-3 using mouse tumor models. It was found that the co-expression of 4-1BB and LAG-3 was sufficient to identify tumor antigen-specific dysfunctional CD8+ TILs enriched in the expression of Egr2 target genes. These CD8+ TILs failed to make IL-2 following in vitro stimulation, yet still produced IFN-g and Treg-recruiting chemokines and lysed target cells ex vivo, indicating they are not completely functionally inert. Combinatorial treatment with anti-LAG-3/anti-4-1BB restored the function of this population and promoted in situ acquisition of KLRG-1hi effector cells. Additional gene expression profiling provided a complete phenotyping of this T cell subset, which revealed expression of a broad panel of both inhibitory receptors and co-stimulatory receptors (e.g., receptors of Table 2 (e.g. Nrn1, Sema7a, CRTAM, etc.)). Inhibitory receptors and co-stimulatory receptors identified in this profiling that are displayed on the surface of T cells include PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A. These approaches have thus enabled the characterization of the population of tumor antigen-specific CD8+ T cells that arise specifically within the tumor microenvironment having altered functional properties. In some embodiments, this population is a target for immunotherapeutic approaches to restore desired functionality and promote tumor regression. In some embodiments, the receptors/markers identified herein (e.g., 4-1BB, LAG-3, receptors/markers of Table 2 (e.g., surface markers/receptors (e.g. Nrn1, Sema7a, CRTAM, etc.), etc.) etc.) are targeted (e.g., via immunotherapeutic approaches) to restore desired immunoresponsiveness, to promote tumor regression, and/or for the treatment of cancer.

Experiments conducted during development of embodiments herein applied knowledge of Egr2 targets to evaluate applicability of these markers toward understanding dysfunctional T cells within tumors in vivo. The data indeed confirm that co-expression of LAG-3 and 4-1BB is sufficient to identify the majority of tumor antigen-specific CD8⁺ T cells within the tumor microenvironment. Co-expression of these markers was not observed within peripheral lymphoid organs in tumor-bearing mice, indicating that a property unique to the tumor context drives 4-1BB and LAG-3 expression. In addition, acquisition of LAG-3 and 4-1BB expression was not observed within tumors that were undergoing successful rejection, indicating that the acquisition of this phenotype occurs under conditions of incomplete antigen clearance.

In some embodiments, cancer treatment methods described herein comprise administration (or co-administration with one or more additional therapies/therapeutics) of one or more anti-4-1BB and/or anti-LAG-3 agents (e.g., antibodies, antibody fragments, antibody mimetic molecules (e.g., DARPins, affibodies, aptamers, nanobodies, etc.), etc.). In some embodiments, an anti-4-1BB and/or anti-LAG-3 agents is administered to render cancer cells, tumor(s), and/or the tumor microenvironment accessible or susceptible to treatment with additional therapies/therapeutics (e.g., immunotherapeutics). Anti-4-1BB and/or anti-LAG-3 agents that find use in embodiments described herein are not limited by their mechanism of action. Agents may be small molecules, peptide, polypeptides, proteins, nucleic acids (e.g., antisense, RNAi, etc.), antibodies, antibody fragments, etc.

In some embodiments, cancer treatment methods described herein comprise enhancing the activity or expression of a marker/receptor identified herein that negatively correlates with tumor antigen-specific CD8⁺ T cell dysfunction.

Experiments conducted during development of embodiments herein identified receptors/markers that are differentially expressed in dysfunctional CD8+ TILs (See Table 2). Testing of targets of interest identified in that screen demonstrate that at least neuritin 1 (Nrn1), cytotoxic and regulatory t-cell molecule (CRTAM), and Semaphorin 7A (Sema7a) are regulators of anti-tumor immunity, with Nm1 and CRTAM blockade correlating with increased tumor area, and Sema7a blockade correlating with decreased tumor area.

In some embodiments, cancer treatment methods described herein comprise administration (or co-administration with one or more additional therapies/therapeutics) of agents (e.g., antibodies, antibody fragments, antibody mimetic molecules (e.g., DARPins, affibodies, aptamers, nanobodies, etc.), etc.) that target one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM, Sema7a, etc.). In some embodiments, an agent is administered to render cancer cells, tumor(s), and/or the tumor microenvironment accessible or susceptible to treatment with additional therapies/therapeutics (e.g., immunotherapeutics). Agents targeting one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, Nrn1, CRTAM, Sema7a, etc.) that find use in embodiments described herein are not limited by their mechanism of action. Agents may be small molecules, peptide, polypeptides, proteins, nucleic acids (e.g., antisense, RNAi, etc.), antibodies, antibody fragments, etc. In some embodiments, an antagonist of Nrn1 is administered. In some embodiments, an antagonist of CRTAM is administered. In some embodiments, an agonist of Sema7a is administered.

In some embodiments, antibodies, antibody fragments, antibody mimetic molecules (e.g., DARPins, affibodies, aptamers, nanobodies, etc.) targeting 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, CRTAM, Sema7a, etc.), or fragments thereof, are provided. Such agents may be naked, deriving their effect by target binding (e.g., neutralizing the target), or may be conjugated to a functional moiety (e.g., drug, toxin, effector moiety, etc.).

In some embodiments, a subject is treated with (i) one or more agents (e.g., antibodies, antibody fragments, antibody mimetic molecules (e.g., DARPins, affibodies, aptamers, nanobodies, etc.), etc.) that target 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, CRTAM, Sema7a, etc.), as well as (ii) one or more additional cancer therapies. Such therapies include chemotherapy, immunotherapy, radiation, surgery, etc. In some embodiments, agents targeting the receptors/markers described herein are co-administered with one or more additional agents for the treatment of cancer.

In some embodiments, exemplary anticancer agents suitable for use in compositions and methods described herein include, but are not limited to: 1) alkaloids, including microtubule inhibitors (e.g., vincristine, vinblastine, and vindesine, etc.), microtubule stabilizers (e.g., paclitaxel (Taxol), and docetaxel, etc.), and chromatin function inhibitors, including topoisomerase inhibitors, such as epipodophyllotoxins (e.g., etoposide (VP-16), and teniposide (VM-26), etc.), and agents that target topoisomerase I (e.g., camptothecin and isirinotecan (CPT-11), etc.); 2) covalent DNA-binding agents (alkylating agents), including nitrogen mustards (e.g., mechlorethamine, chlorambucil, cyclophosphamide, ifosphamide, and busulfan (MYLERAN), etc.), nitrosoureas (e.g., carmustine, lomustine, and semustine, etc.), and other alkylating agents (e.g., dacarbazine, hydroxymethylmelamine, thiotepa, and mitomycin, etc.); 3) noncovalent DNA-binding agents (antitumor antibiotics), including nucleic acid inhibitors (e.g., dactinomycin (actinomycin D), etc.), anthracyclines (e.g., daunorubicin (daunomycin, and cerubidine), doxorubicin (adriamycin), and idarubicin (idamycin), etc.), anthracenediones (e.g., anthracycline analogues, such as mitoxantrone, etc.), bleomycins (BLENOXANE), etc., and plicamycin (mithramycin), etc.; 4) antimetabolites, including antifolates (e.g., methotrexate, FOLEX, and MEXATE, etc.), purine antimetabolites (e.g., 6-mercaptopurine (6-MP, PURINETHOL), 6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir, chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and 2′-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (e.g., fluoropyrimidines (e.g., 5-fluorouracil (ADRUCIL), 5-fluorodeoxyuridine (FdUrd) (floxuridine)) etc.), and cytosine arabinosides (e.g., CYTOSAR (ara-C) and fludarabine, etc.); 5) enzymes, including L-asparaginase, and hydroxyurea, etc.; 6) hormones, including glucocorticoids, antiestrogens (e.g., tamoxifen, etc.), nonsteroidal antiandrogens (e.g., flutamide, etc.), and aromatase inhibitors (e.g., anastrozole (ARIMIDEX), etc.); 7) platinum compounds (e.g., cisplatin and carboplatin, etc.); 8) monoclonal antibodies (e.g., conjugated with anticancer drugs, toxins, and/or radionuclides, etc.; neutralizing antibodies; etc.); 9) biological response modifiers (e.g., interferons (e.g., IFN-.alpha., etc.) and interleukins (e.g., IL-2, etc.), etc.); 10) adoptive immunotherapy; 11) hematopoietic growth factors; 12) agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid, etc.); 13) gene therapy techniques; 14) antisense therapy techniques; 15) tumor vaccines; 16) therapies directed against tumor metastases (e.g., batimastat, etc.); 17) angiogenesis inhibitors; 18) proteosome inhibitors (e.g., VELCADE); 19) inhibitors of acetylation and/or methylation (e.g., HDAC inhibitors); 20) modulators of NF kappa B; 21) inhibitors of cell cycle regulation (e.g., CDK inhibitors); and 22) modulators of p53 protein function.

In some embodiments, agents targeting 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. Nrn1, Sema7a, CRTAM, etc.) are administered to overcome immune invasion of the cancer cells, tumor, tumor microenvironment, etc. In some embodiments, one or more additional cancer immunotherapies are employed (e.g., concurrently or serially) to make use of the immune-responsiveness of the treated cells/tumor. Suitable immunotherapies may include, but are not limited to: cell-based therapies (e.g., dendritic cell or T cell therapy, etc.), monoclonal antibody (mAb) therapy (e.g., naked mAbs, conjugated mAbs), cytokine therapy (e.g., interferons, interleukins, etc.), adjuvant treatment (e.g., polysaccharide-K), etc.

In some embodiments, agents targeting 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, CRTAM, Sema7a, etc.) are co-administered with agents (e.g., small molecules, peptides, antibodies, antibody fragments, etc.) that target one or more cancer cell or tumor) markers or components. In some embodiments, such co-administration renders the cancer cells, tumor, and/or tumor microenvironment susceptible and/or accessible to the treatment with the additional agent.

In some embodiments, agents for use in the methods and compositions described herein target and/or binds a cancer or tumor cell marker or component, selected from the group including but not limited to, epidermal growth factor receptor (EGFR, EGFR1, ErbB-1, HER1). ErbB-2 (HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF-binding proteins (IGFBPs), IGFR ligand family (IGF-1R); platelet derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGFR) family, VEGF family; HGF receptor family: TRK receptor family; ephrin (EPH) receptor family: AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR) receptor family; discoidin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; Transforming growth factor alpha (TGF-α), TGF-α receptor; Transforming growth factor-beta (TGF-β), TGF-β receptor; Interleukin β receptor alpha2 chain (IL13Ralpha2), Interleukin-6 (IL-6), 1L-6 receptor, interleukin-4, IL-4 receptor, Cytokine receptors, Class I (hematopoietin family) and Class II (interferon/1L-10 family) receptors, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily (TNTRSF), death receptor family, TRAIL-receptor; cancer-testis (CT) antigens, lineage-specific antigens, differentiation antigens, alpha-actinin-4, ARTC1, breakpoint cluster region-Abelson (Bcr-abl) fusion products, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), beta-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, Elongation factor 2 (ELF2), Ets variant gene 6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low density lipid receptor/GDP-L fucose: beta-Dgalactose 2-alpha-Lfucosyltraosferase (LDLR/FUT) fusion protein, HLA-A2, MLA-A11, heat shock protein 70-2 mutated (HSP70-2M), KIAA0205, MART2, melanoma ubiquitous mutated 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, Myosin class 1, NFYC, OGT, OS-9, pml-RARalpha fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT12, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, Triosephosphate Isomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8, GnT-V (aberrant N-acetyl glucosaminyl transferase V, MGAT5), HERV-K MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognized antigen on melanoma (CAMEL), MAGE-A1 (MAGE-1). MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10. MAGE-All, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5. MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1), MART-1/Melan-A (MLANA), gp100, gp100/Pme117 (S1LV), tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2, SAGE, Sp17. SSX-1, 2, 3, 4, TRP2-1NT2, carcino-embryonic antigen (CEA), Kallikrein 4, mammaglobin-A, OA1 prostate specific antigen (PSA), prostate specific membrane antigen, TRP-1/, 75. TRP-2 adipophilin, interferon inducible protein absent in melanoma 2 (AIM-2). BING-4, CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250intestinal carboxyl esterase (iCE), alpha-feto protein (AFP), M-CSF, mdm-2, MUCI, p53 (TP53), PBF, PRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, survivin (BIRCS), human telomerase reverse transcriptase (hTERT), telomerase, Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE, TPTE, immunoglobulin idiotypes, Bence-Jones protein, estrogen receptors (ER), androgen receptors (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3, cancer antigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), beta-human chorionic gonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96. GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4), carcinoembryogenic antigen peptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), etc.

Examples of antibodies which can be incorporated into compositions and methods disclosed herein include, but are not limited, to antibodies such as trastuzumab (anti-HER2/neu antibody); Pertuzumab (anti-HER2 mAb); cetuximab (chimeric monoclonal antibody to epidermal growth factor receptor EGFR); panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFR antibody); Zalutumumab (anti-EGFR mAb); Necitumumab (anti-EGFR mAb); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptor bispecific antibody); Rituximab (chimeric murine/human anti-CD20 mAb); Obinutuzumab (anti-CD20 mAb); Ofatumumab (anti-CD20 mAb); Tositumumab-1131 (anti-CD20 mAb); Ibritumomab tiuxetan (anti-CD20 mAb); Bevacizumab (anti-VEGF mAb); Ramucirumab (anti-VEGFR2 mAb); Ranibizumab (anti-VEGF mAb); Aflibercept (extracellular domains of VEGFR1 and VEGFR2 fused to IgG1 Fc); AMG386 (angiopoietin-1 and -2 binding peptide fused to IgG1 Fc); Dalotuzumab (anti-IGF-1R mAb); Gemtuzumab ozogamicin (anti-CD33 mAb); Alemtuzumab (anti-Campath-1/CD52 mAb); Brentuximab vedotin (anti-CD30 mAb): Catumaxomab (bispecific mAb that targets epithelial cell adhesion molecule and CD3); Naptumomab (anti-5T4 mAb); Girentuximab (anti-Carbonic anhydrase ix); or Farletuzumab (anti-folate receptor). Other examples include antibodies such as Panorex™ (17-1A) (murine monoclonal antibody); Panorex (@(17-1A)) (chimeric murine monoclonal antibody); BEC2 (ami-idiotypic mAb, mimics the GD epitope) (with BCG); Oncolym (Lym-1 monoclonal antibody); SMART M195 Ab, humanized 13′ 1 LYM-1 (Oncolym). Ovarex (B43.13, anti-idiotypic mouse mAb); 3622W94 mAb that binds to EGP40 (17-1A) pancarcinoma antigen on adenocarcinomas; Zenapax (SMART Anti-Tac (IL-2 receptor); SMART M195 Ab, humanized Ab, humanized); NovoMAb-G2 (pancarcinoma specific Ab); TNT (chimeric mAb to histone antigens); TNT (chimeric mAb to histone antigens); Gliomab-H (Monoclonals—Humanized Abs); GNI-250 Mab; EMD-72000 (chimeric-EGF antagonist); LymphoCide (humanized IL.L.2 antibody); and MDX-260 bispecific, targets GD-2, ANA Ab, SMART IDIO Ab, SMART ABL 364 Ab, or ImmuRAIT-CEA.

In some embodiments, an agent that finds use in embodiments herein specifically binds a component of a regulatory T cell, myeloid suppressor cell, or dendritic cell. In another aspect, the targeting moiety specifically binds one of the following molecules: CD4; CD25 (IL-2α receptor; IL-2αR); cytotoxic T-lymphocyte antigen-4 (CTLA-4; CD152); Interleukin-10 (IL-10); Transforming growth factor-beta receptor (TGF-βR); Transforming growth factor-beta (TGF-β); Programmed Death-1 (PD-1); Programmed death-1 ligand (PD-L1 or PD-L2); Receptor activator of nuclear factor-κB (RANK); Receptor activator of nuclear factor-κB (RANK) ligand (RANKL); LAG-3; glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR; TNFRSF18); or Interleukin-4 receptor (IL-4R). In some embodiments, the agent is an agonist that increases the function of the targeted molecule. In other embodiments, the agent is an antagonist that inhibits the function of the targeted molecule.

In some embodiments, an agent that finds use in embodiments herein binds a specific cytokine, cytokine receptor, co-stimulatory molecule, co-inhibitory molecule, or immunomodulatory receptor that modulates the immune system. In another aspect, the targeting moiety specifically binds one of the following molecules: tumor necrosis factor (TNF) superfamily; tumor necrosis factor-α (TNF-α); tumor necrosis factor receptor (TNFR) superfamily; Interleukin-12 (IL-12); IL-12 receptor; 4-1BB (CD137); 4-1BB ligand (4-1BBL; CD137L); OX40 (CD134; TNR4); OX40 ligand (OX40L; CD40; CD40 ligand (CD40L); CTLA-4; Programmed death-1 (PD-1); PD-1 ligand I (PD-L1: B7-H1); or PD-1 ligand 2 (PD-L2; B7-DC); B7 family; B7-1 (CD80); B7-2 (CD86); B7-H3; B7-H4; GITR/AITR: GITRL/AITRL; BTLA; CD70; CD27; LIGHT; HVEM: Toll-like receptor (TLR) (TLR 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, the agent is an agonist that increases the function of the targeted molecule. In other embodiments, the agent is an antagonist that inhibits the function of the targeted molecule.

In some embodiments, agents (e.g., immunotherapeutics) targeting 4-1BB, LAG-3 and/or one or more receptors/markers of Table 2 (e.g. PD-1, TIM-3, OX-40ICOS, TIGIT, CD244, TNFRSF18, Nrn1, Nrp1, KLRG1, GM156, GPNMB, GPR65, TMEM205, and TMEM126A, CRTAM, Sema7a, etc.) are co-administered (e.g., serially or sequentially) with one or more adjuvants. Suitable adjuvants include, but are not limited to, one or more of: oil emulsions (e.g., Freund's adjuvant); saponin formulations; virosomes and viral-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing compositions (e.g., mineral salts, such as aluminium salts and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and/or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazene; muramyl peptides; imidazoquinolone compounds; and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

Adjuvants may also include immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-.gamma.), macrophage colony stimulating factor, and tumor necrosis factor. In addition to variant B7-DC polypeptides, other co-stimulatory molecules, including other polypeptides of the B7 family, may be administered. Proteinaceous adjuvants may be provided as the full-length polypeptide or an active fragment thereof, or in the form of DNA, such as plasmid DNA.

Pharmaceutical and immunotherapeutic compositions described herein may be delivered by any suitable route of administration (e.g., oral delivery, parenteral delivery, mucous membrane delivery, pulmonary delivery, intravenous delivery, etc.). Appropriate formulations for such delivery routes are understood in the field.

Non-limiting examples of cancers that may be treated with the compositions and methods described herein include, but are not limited to: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), pancreatic cancer (e.g., adenocarcinoma), breast cancer, colon cancer, lung cancer (e.g. non-small cell lung cancer), esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies. In some embodiments, the cancer is a solid tumor cancer.

Some embodiments described herein are particularly useful for the treatment of tumors that do not otherwise respond to immunotherapeutic approaches. In some embodiments, provided herein is the treatment of cancers that are non-responsive (or have a reduced response) to T cells or antigen presenting cells (e.g., dendritic cells (e.g., CD103⁺DCs, etc.), etc.). In some embodiments, provided herein is the treatment of cancers that are non-responsive to treatments, despite T cell infiltration. In some embodiments, compositions and methods described herein find use in the treatment of cancers in which T cells are not appropriately primed against tumor-associated antigens. In some embodiments, compositions and methods described herein find use in the treatment of cancers comprising tumors or cells that are defective in recruitment of dendritic cells (e.g., CD103⁺ DCs, etc.). In some embodiments, compositions and methods described herein find use in the treatment of cancers comprising tumors or cells that are defective in production of the chemokine CCL4.

In some embodiments, the therapeutic compositions and methods herein find use with those described in, for example WO 2016/141312; incorporated by reference in its entirety.

In some embodiments, methods are provided for testing sample (e.g., cell, tissue, population of cells, tumor, blood, urine, saliva, etc.) from a subject for one or more biomarkers (e.g., biomarkers of dysfunctional tumor antigen-specific CD8⁺ T cells). Such biomarkers may comprise nucleic acids, small molecules, proteins, peptides, etc., and may be detected using any suitable assay of technique. In some embodiments, provided herein are DNA-, RNA-, small molecule, and/or protein-based diagnostic methods that either directly or indirectly detect the biomarkers of the evasion of immune response or immunotherapy by cancer cells or tumors. The present invention also provides compositions, reagents, and kits for such diagnostic purposes.

In some embodiments, biomarkers are detected at the nucleic acid (e.g., RNA) level. For example, the presence or amount of biomarker nucleic acid (e.g., mRNA) in a sample is determined (e.g., to determine the presence or level of biomarker expression). Biomarker nucleic acid (e.g., RNA, amplified cDNA, etc.) may be detected/quantified using a variety of nucleic acid techniques known to those of ordinary skill in the art, including but not limited to nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification (e.g., by PCR, RT-PCR, qPCR, etc.), microarray, Southern and Northern blotting, sequencing, etc. Non-amplified or amplified nucleic acids can be detected by any conventional means. For example, in some embodiments, nucleic acids are detected by hybridization with a detectably labeled probe and measurement of the resulting hybrids. Nucleic acid detection reagents may be labeled (e.g., fluorescently) or unlabeled, and may by free in solution or immobilized (e.g., on a bead, well, surface, chip, etc.).

In some embodiments, biomarkers are detected at the protein level. For example, the presence or amount of biomarker protein in a sample is determined (e.g., to determine the presence or level of biomarker expression or localization). In some embodiments, reagents are provided for the detection and/or quantification of biomarker proteins. Suitable reagents include primary antibodies (e.g., that bind to the biomarkers), secondary antibodies (e.g., that bind primary antibodies), antibody fragments, aptamers, etc. Protein detection reagents may be labeled (e.g., fluorescently) or unlabeled, and may by free in solution or immobilized (e.g., on a bead, well, surface, chip, etc.).

In some embodiments, biomarker capture reagents are provided to localize, concentrate, aggregate, etc. a biomarker. For example, in some embodiments a biomarker capture reagent that interacts with the biomarker is linked to a solid support (e.g., a bead, surface, resin, column, and the like) that allows manipulation by the user on a macroscopic scale. Often, the solid support allows the use of a mechanical means to isolate and purify the biomarker from a heterogeneous solution. For example, when linked to a bead, separation is achieved by removing the bead from the heterogeneous solution, e.g., by physical movement. In embodiments in which the bead is magnetic or paramagnetic, a magnetic field is used to achieve physical separation of the capture reagent (and thus the target) from the heterogeneous solution. Magnetic beads used to isolate targets are described in the art, e.g., as described in European Patent Application No. 87309308, incorporated herein in its entirety for all purposes.

Compositions for use in the diagnostic methods or testing steps described herein include, but are not limited to, probes, amplification oligonucleotides, and antibodies. Any of the detection and/or diagnostic reagents used in embodiments described herein may be provided alone or in combination with other compositions in the form of a kit. Kits may include any and all components necessary or sufficient for assays including, but not limited to, the detection reagents, buffers, control reagents (e.g., tissue samples, positive and negative control sample, etc.), solid supports, labels, written and/or pictorial instructions and product information, inhibitors, labeling and/or detection reagents, package environmental controls (e.g., ice, desiccants, etc.), and the like. In some embodiments, the kits provide a sub-set of the required components, wherein it is expected that the user will supply the remaining components. In some embodiments, the kits comprise two or more separate containers wherein each container houses a subset of the components to be delivered.

In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of expression a biomarker) into data of predictive value for a clinician. In some embodiments, computer analysis combines various data into a single score or value that is predictive and/or diagnostic. The clinician can access the predictive data using any suitable means. Thus, in some preferred embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject. Contemplated herein are any methods capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information providers, medical personal, and subjects. For example, in some embodiments of the present invention, a sample (e.g., a biopsy, cell, or blood sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, third-party testing service, genomic profiling business, etc. to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves and directly send it to a profiling center. In some embodiments, a report is generated (e.g., by a clinician, by a testing center, by a computer or other automated analysis system, etc.). A report may contain test results, diagnoses, and/or treatment recommendations.

Materials and Methods

Mice and Tumor Inoculation

Female C57BL/6 mice ranging from 6 to 8 weeks were purchased from Taconic Farms. CD45.1 and Rag2^−/− mice on the C57BL/6 background were obtained from Taconic Farms and bred at the University of Chicago. 2C/Rag2^−/− and P14/Rag2^−/− mice have been previously described (Brown et al., 2006; incorporated by reference in its entirety). pLCK-CreERT2×ROSA-YFP mice were generated and have been described (Evaristo et al., 2016; incorporated by reference in its entirety). B16.SIY.dsRed (Kline et al., 2012; incorporated by reference in its entirety), C1498.SIY.GFP (Zhang et al., 2009; incorporated by reference in its entirety), and MC57.SIY.GFP (Spiotto et al., 2002; incorporated by reference in its entirety) tumor cells were engineered to express either dsRed or GFP in frame with the H2-K^b-restricted model antigen SIYRYYGL. The 1969.SIY.GFP cell line was engineered by retroviral transduction of the 1969 cell line (Diamond et al., 2011; incorporated by reference in its entirety) using the pLEGFP plasmid expressing cDNA for SIYRYYGL (Spiotto et al., 2002; incorporated by reference in its entirety). For experiments, mice 6 to 9 weeks of age and received 2×10⁶tumor cells subcutaneously on either the left flank or both the left and right flank. All mice were maintained according to the National Institute of Health Animal Care guidelines and studied under IACUC-approved protocols.

To generate the targeting construct for the Egr2^EGFPknock-in reporter mice, a 12.6 kb mouse genomic DNA fragment including the egr2 gene was excised with SacII and cloned into a pEasy-Flox vector adjacent to the thymidine kinase (TK) selection marker. A cassette containing IRES2-eGFP and a LoxP-flanked neomycin selection marker was inserted into an Nhe1 site between the translation stop codon (TGA) and the polyadenylation signal of the egr2 gene. ES cell clones from 129 mice were electroporated and selected for Neomycin resistance. ES cell clones were verified for homologous insertion in the endogenous locus by PCR and southern blot with 5′ and 3′ probes. Mice were backcrossed to C57BL/6 for over 8 generations.

TIL Isolation

Tumors were harvested from mice at the indicated time points. Tumors were dissociated through a 50 μm filter and washed with PBS. TILs were further enriched by layering Ficoll-Hypaque beneath the cell suspension followed by centrifugation without breaks for 30 min at 400×g. The buffy-layer was isolated and washed twice with PBS before staining. For isolating specific cell populations by FACS, tumors were pooled when indicated and the cell layer was re-purified by Ficoll-Hypaque centrifugation twice. For day 28 tumors, after Ficoll-Hypaque separation, T cells were further purified by negative bead selection according to manufacturer's instructions (MAGNISORT, eBiosciences). Cells were then washed with PBS, stained at 4° C. for 15 minutes before resuspending in complete DMEM (cDMEM: 10% FBS, 100U/mL Penicillin-Streptomycin, 1% MEM Non-Essential Amino Acids, 50 μM (3-ME, 0.01M MOPS), and were sorted into either RLT lysis buffer (QIAGEN) or cDMEM depending on the experimental assay. Cells sorted into RLT buffer were put directly on dry ice as soon as the sort was finished.

Flow Cytometry and Antibodies

Cell suspensions were washed twice in PBS before staining an FACS buffer (10% FBS, 2 mM EDTA, 0.001% NaN₃). Cells were stained for 30 min on ice and fixed in 1% PFA. Antibodies against the following molecules were used: CD3 (17A2, AX700), 2B4 (2B4, FITC), CD127 (A7R34, PE), OX-40 (OX-86, PE), 4-1BB (17B5, Biotin, APC), CD160 (7H1, PE-Cy7), LAG-3 (C9B7W, PerCPeFluor710), PD-1 (RMP1-30, PE-Cy7), NRP1 (3E12, BV421), GITR (DTA-1, FITC), ICOS (7E.17G9, BV421), KLRG-1 (2F1, eF450, BV605), TIGIT (1G9, APC), TIM-3 (RMT3-23, PE), CD4 (RM4-5, BV605), CD45.1 (A20, FITC), CD45.2 (104, PE), CD8a (53-6.7, BV711). Fixable Viability Dye 506 (eBioscience) was used for live/dead discrimination. Staining of SIY-specific T cells was performed utilizing the SIYRYYGL-Pentamer (PE) (Proimmune); a SIINFEKL-pentamer (PE) was used as a non-specific control. All flow cytometric analysis was conducted on an LSRFortessa (BD) and analyzed using FlowJo software (Tree Star).

Quantitative Real-Time PCR

Total RNA was extracted from sorted cell populations using the RNEasy Micro Kit (QIAGEN) following the manufacturer's protocol. cDNA was synthesized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to manufacturer's instructions. Transcript levels were determined using primer-probe sets (Tables 1a and 1b) developed through the online ProbeFinder Software and the Universal Probe Library (Roche) with the exception of IL-2 (Mm00434256_m1) and 18S (Hs99999901_s1). To minimize batch effect, when possible, all samples probed for a gene were run on the same 96-well qRT-PCR plate. All primer-probe sets either contained a primer spanning an exon-exon boundary or primers spanning an intron. Expression levels of transcripts were normalized to 18S expression.

In vivo proliferation was measured by a BrdU pulse 24 hours prior to flow cytometric analysis. Each mouse received 0.8 mg BrdU injected i.p. (intraperitoneal) on day 12 after tumor inoculation. TILs were isolated and surface stain was performed as described above. Following surface staining, cells were fixed and permeabilized using the Foxp3 staining kit (BD), according to manufacturer's protocol, and incubated with 100 μl PBS/DNase solution (300 μg/ml) for 30 minutes at 37° C. Cells were washed and incubated for 30 minutes at room temperature with anti-BrdU (FITC, Bu20a) and then washed with and resuspended in PBS.

In Vitro Stimulation Assays

Tissue culture-treated 96-well round bottom plates were coated with anti-CD3E (1 μg/ml; 2C11) in DPBS overnight at 4° C. or for 2 hours at 37° C. Cells were sorted into cold cDMEM media and put on ice as soon as the sort was finished. Cells were then pelleted, resuspended in 50 μl cDMEM and incubated with soluble anti-CD28 (2 μg/ml; PV-1) for 10-12 hours for a final volume of 100 μl. After stimulation supernatants were removed for ELISA or bead-based immunoassay (LegendPlex), and cells were washed once with DPBS and resuspended in 15 μl of RNAlater Stabilization Solution (QIAGEN) or 300 μl of RLT buffer. Cells were stored at −80° C. until RNA isolation was performed.

Protein Quantification

Measurement of protein concentration was determined either by a standard ELISA or bead-based immunoassay (LEGENDplex, BioLegend). ELISAs were performed according to manufacturer's protocol (Ready-SET-Go ELISA; eBioscience) on supernatants from in vitro stimulations. Absorbance values were obtained at 450 nm using an Emax microplate reader (Molecular Devices) and IL-2 concentration was determined by standard curve. Protein concentration values were normalized to the number of sorted cells plated. LEGENDplex assays were performed according to manufacturer's protocols. IL-2 concentration (FIG. 4B) was confirmed by both methods in separate experiments with no significant difference in IL-2 concentration between the two methods.

Spectratype Analysis and Sequencing Mice were injected with 2×10⁶B16.SIY.dsRed tumor cells. 14 days later, tumors were harvested and specific CD8⁺ TIL subpopulations were sorted into RLT buffer (QIAGEN) and immediately frozen. cDNA was synthesized from sorted cell populations and CDR3 regions were amplified by PCR with 21 different Vβ-5′ primers paired with a FAM-Cβ1.1 primer (Table 1). Three Vβ PCR reactions did not reach significant amplification for analysis and were removed from the analysis. For sequencing, Cβ-Vβ PCR products were purified using the QIAquick PCR purification kit (QIAGEN) and sequenced at the University of Chicago Genomics Core Facility. Cβ-Vβ PCR products were analyzed by capillary electrophoresis at the University of Chicago Genomics core and CDR3 peaks were aligned using the Liz500 ladder. Spectratype graphs were displayed using the GeneiousR9 software (Kearse et al., 2012). To generate the frequency profile for each Vβ spectratype, the area under each peak was measured using peak studio. The Hamming Distance (Currier and Robinson, 2001; incorporated by reference in its entirety) was calculated between each Vβ spectratype from each CD8⁺ spleen and TIL population within a given mouse. To determine significance between the HD from each comparison the HDs for each Vβ from mice were averaged and a One-Way ANOVA with Dunn's correction for multiple comparisons was performed.
TCR Transgenic T Cell Transfer Experiments

Cell suspensions were generated from spleens and lymph nodes from congenic 2C/Rag2^−/−/CD45.1/2 and/or P14/Rag2^−/−/CD45.2 mice and T cells were purified by CD8⁺ negative selection (Miltenyi Biotechnologies) over magnetic columns according to the manufacturer's protocol. TCR Transgenic (Tg) T cells were washed with PBS, resuspended at a concentration of 10×10⁶/ml and 1×10⁶TCR Tg cells were adoptively transferred into CD45.1 tumor bearing mice by tail vein transfer in a volume of 0.1 mL. After indicated times, 2C T cells and corresponding host CD8⁺ T cells were sorted and stimulated as described above.

In Vitro Cytotoxicity Assay

Per individual experiment, 10 C57BL/6 mice were injected s.c. (subcutaneous) with 2×10⁶B16.SIY cells on both left and right flanks. On day 14, all 20 tumors were pooled and dissociated using the Tumor Dissociation Kit (Miltenyi Biotec) following the manufacturer's protocol. Tumor cell suspensions were washed 3-5 times with PBS and TILs were enriched for by Ficoll-Hypaque gradient centrifugation. TILs were stained, sorted and put directly on ice. TILs were titrated and added directly to a 96-well plate containing 50,000 P815 mastocytoma cells and 1 μg/mL anti-CD3. For a positive control, OT-I cells were isolated from OT-I/Rag2^−/− mice and stimulated with plate-bound anti-CD3 (0.25 μg/mL), anti-CD28 (2 μg/mL) and 100 U/mL IL-2 for 2-3 days. For a negative control, P815 cells were cultured alone or cultured with naïve CD8⁺ T cells isolated from lymph nodes. After 12 hours of incubation, cells were stained for Thy1, CD45, CD8α, Fixable Viability Dye 450 (eBioscience) and/or propidium Iodide.

Gene Expression Analysis

Total RNA for the CD8⁺ TIL subpopulations was isolated following the manufacturer's protocol (RNEasy Micro Kit: QIAGEN) from sorted cells pooled from 10 mice. Samples were analyzed by the University of Chicago Genomics Facility using Illumina MouseRef8 microarray chips. Two experimental replicates were performed, and the results were log₂transformed and averaged. Probe sets that revealed a 1.5-fold difference abs(log₂(ratio)>1.5)) relative to CD8⁺4-1BB⁻LAG-3⁻PD-1⁻ cells were identified and used for subsequent analysis. The microarray data are available in the Gene Expression Omnibus database under accession number GSE79919. For cross-study comparisons, log 2-fold change values were extracted using the GEO2R online software from the hypofunctional CD8⁺ TIL data set, GSE79858 ((GSM2107353, GSM2107353 and GSM2107355) versus (GSM2107350, GSM2107351, GSM210732)) and the CD8⁺ T cell exhausted data set, GSE41870 ((GSM1026819, GSM1026820, GSM1026821) versus (GSM1026786, GSM1026787, GSM1026788, GSM1026789)). Upregulated genes showing a 2-fold difference were used for analysis. Multiple genes names with from the GEO2R extracted data were identified and matched to gene names from the Illumina data set. The rank-rank hypergeometric overlap (RRHO) analysis (Plaisier et al., 2010; incorporated by reference in its entirety) was conducted (Rosenblatt and Stein, 2014; incorporated by reference in its entirety).

Gene Ontology Enrichment Analysis

In a pair-wise fashion, shared upregulated genes were used as the input for the ClueGO software with the Cytoscape application (Shannon et al., 2003; incorporated by reference in its entirety). Both the Biological Process and Immune System Process Gene Ontology Annotations were used for analysis. Only pathways with a Bonferroni step down correction p-value>0.01 were considered when generating pathway nodes. Non-redundant pathways with the greatest number of genes found within each node were used as examples in FIG. 6A.

Antibody and FTY720 Treatments

Mice were treated i.p. with 100 μg/mouse of anti-4-1BB (Bio-X-Cell; LOB12.3) antibody and/or 100 μg/mouse anti-LAG-3 (Bio-X-Cell; C9B7W). For tumor outgrowth experiments, mice were treated on day 7, 10, 13 and 16 after tumor inoculation. For ex vivo functional experiments mice were treated on day 7, 10 and 13 and cells were sorted on day 14. For experiments blocking lymph node egress, 25 μg of FTY720 was given by gavage one day prior to first antibody treatment (day 6) and continued every day until endpoint on day 14.

Results

4-1BB and LAG-3 Identify a Major Population of CD8⁺ TILs

To determine whether 4-1BB and LAG-3 could identify dysfunctional CD8⁺ TILs, the expression pattern of LAG-3 and 4-1BB was examined using the well-characterized B16.SIY model of melanoma. On day 7 following tumor inoculation, the 4-1BB⁺LAG-3⁺ population comprised 15.8% of all CD8⁺ TILs. The frequency of this population significantly increased to 44% by day 21. The frequency of 4-1BB⁻LAG-3⁺ (4⁻L⁺) population also increased 1.9-fold from day 7 to day 14 to comprise 25% of the CD8⁺ TIL compartment. In contrast, the frequency of the 4-1BB⁻LAG-3⁻ (4⁻L⁻) population decreased by 2.7-fold by day 21. There was no significant increase in the proportion or number of 4-1BB⁺LAG-3⁻CD8⁺ TILs within the time frame of the experiment (FIGS. 1A and B). Similar patterns were seen when analyzing absolute numbers of cell subsets (FIGS. 1C and D). Acquisition of these phenotypes was specific for the tumor microenvironment, as they were not observed in the spleen or tumor-draining lymph node (TdLN) (FIG. 1A). These data indicate that the tumor microenvironment preferentially supports the induced co-expression of LAG-3 and 4-1BB.

The selective increase in cell numbers and proportional shift towards the 4-1BB⁻ LAG-3⁺ and 4-1BB⁺LAG-3⁺ populations during tumor progression indicated that expansion of these populations was occurring within the tumor microenvironment. CD8⁺ TILs were stained for Ki67 at day 14 after tumor inoculation and analyzed by flow cytometry. 81% of 4-1BB⁻LAG-3⁺ cells and 85% of 4-1BB⁺LAG-3⁺ cells were Ki67⁺ compared to only 32% of the 4-1BB⁻LAG-3⁻ TILs (FIG. 1E). Mice were pulsed with BrdU on day 12, and 24 hours later the CD8⁺ TIL subpopulations were analyzed for BrdU incorporation. Indeed, the 4-1BB⁻LAG-3⁺ and 4-1BB⁺LAG-3⁺ populations incorporated more BrdU compared to the 4-1BB⁻LAG-3⁻ population (FIG. 1F). These data indicate that once CD8⁺ T cells arrive at the tumor site, a fraction of TILs expands within the tumor, and that these expanding TILs are identified by increased expression of 4-1BB and LAG-3.

To determine if upregulation of LAG-3 and 4-1BB was simply a product of the B16.SIY tumor model or if it is a more general feature of CD8⁺ T cells within tumors, T cells from three additional progressively growing tumor models, C1498.SIY, MC38.SIY, EL4.SIY and B16F10 parental were analyzed. TILs were analyzed for expression of 4-1BB and LAG-3 at day 14. A pattern of expression was found that is similar to that seen in CD8⁺ TILs isolated from B16.SIY tumors (FIGS. 1G and I). The results from the B16F10 parental tumor confirm that presence of SIY is not required to see co-expression of 4-1BB and LAG-3. In order to determine whether the 4-1BB⁺LAG-3⁺ TIL subset was generated only in progressing tumors or also in tumors that were rejected, T cell phenotypes in the 1969.SIY and MC57.SIY fibrosarcoma tumor models were analyzed, which are more immunogenic and undergo spontaneous rejection. Distinctly fewer 4-1BB⁺LAG-3⁺ cells were found among the CD8⁺ TIL compartment in the 1969.SIY and MC57.SIY tumors (Figure H and I). Over time, co-expression of 4-1BB and LAG-3 was maintained in B16.SIY tumors but not MC57.SIY tumors (FIG. 1J). These data indicate that the acquisition of the LAG-3⁺4-1BB⁺ TIL phenotype preferentially occurs within the tumor microenvironment and only upon conditions of tumor progression rather than regression.

CD8⁺ 4-1BB⁺LAG-3⁺ TILs Express Egr2 and Multiple Egr2 Gene Targets

Experiments conducted during development of embodiments herein to determine whether Egr2 expression itself was also characteristic of T cells within the CD8⁺ TIL compartment; an Egr2-IRES-GFP (Egr2^GFP) knock-in reporter mouse was utilized. Approximately 14% of all CD8⁺ TILs were GFP⁺ on both day 7 and day 14 (FIG. 2A). To confirm that Egr2 is faithfully reported, CD8⁺ TILs expressing high and low levels of EGFP were sorted and screened for Egr2 and several Egr2 targets by qRT-PCR. The Egr2-GFP^hipopulation expressed greater levels of Egr2 and many Egr2-target genes previously defined using in vitro anergy models. These include Tnfrsf9, Lag3, Ngn, Sema7a, Crtam, Ccl1 and Nrn1 (FIG. 2B). Expression of 4-1BB and LAG-3 in the Egr2-GFP^hiCD8⁺ TILs was confirmed by flow cytometry. The majority of Egr2-GFP^hicells expressed LAG-3 and/or 4-1BB. The Egr2GFP^locells also showed expression of 4-1BB and LAG-3 on a subpopulation at day 14 (FIG. 2C). This result indicates either that CD8⁺ TILs expressing Egr2 encompass only a subset of the TILs expressing LAG-3 and/or 4-1BB, or that Egr2 is transiently expressed and is subsequently downregulated after the induction of LAG-3 and 4-1BB.

Using Egr2 target genes from in vitro anergic CD4⁺ T cell clones (Zheng et al., 2013; incorporated by reference in its entirety), the Egr2-driven transcriptional program was examined in sorted 4-1BB⁻LAG-3⁻ and 4-1BB⁺LAG-3⁺ cells by qRT-PCR. Of the 43 Egr2 target genes examined, 10 showed detectably increased expression in 4-1BB⁺LAG-3⁺ population, while expression of a similar subset of genes was increased in the 4-1BB⁻LAG-3⁺ population (FIG. 2D). Collectively, these data demonstrate that Egr2 is expressed in a subpopulation of CD8⁺ TILs expressing LAG-3 and/or 4-1BB, and that a subset of known Egr2 targets was detected in these larger T cell populations as a whole.

It was next examined whether Egr2 was required for expression of LAG-3 and 4-1BB among CD8⁺ TIL in vivo. To this end Egr2^f1ox/flox×pLCK-CreERT2×ROSA-YFP mice were utilized, in which oral tamoxifen administration results in a fraction of the CD8⁺ T cells deleting Egr2 and expressing YFP (FIG. 2E). This allowed comparison of both Egr2-sufficient (YFP⁻) and Egr2-deficient (YFP⁺) CD8⁺ within the same tumor. To determine that Egr2 was in fact deleted from the YFP⁺ fraction, both YFP⁺ and YFP⁻CD8⁺ TILs were sorted and Egr2 transcripts were measured directly ex vivo and upon ex vivo stimulation. The YFP⁺CD8⁺ TILs expressed substantially less Egr2 transcripts compared to the YFP⁻ counterparts (FIG. 2E). To determine if Egr2 is required for 4-1BB and LAG-3 expression, CD8⁺ TILs were analyzed at day 7 and 14 after tumor inoculation and compared the YFP⁺ and YFP⁻ populations to mice not treated with tamoxifen. At day 7, the YFP⁺ fraction expressed less 4-1BB and LAG-3 compared to the YFP⁻ population and the WT CD8⁺ TILs. However, expression of 4-1BB and LAG-3 was not significantly different at day 14 (FIG. 2F). This indicates that other transcriptional regulators compensate and contribute to the expression of LAG-3 and 4-1BB, especially at later time points.

Egr3 has been shown to have overlapping function with Egr2 (Safford et al., 2005; incorporated by reference in its entirety) and HIF1α can contribute to 4-1BB expression (Palazón et al., 2012). To investigate whether these transcription factors may compensate for 4-1BB and/or LAG-3 expression we sorted Egr2GFP^hiand Egr2GFP^loCD8⁺ TILs expressing 4-1BB and LAG-3 on day 7 and analyzed expression of Egr3 and HIF1α by qRT-PCR. Egr3 and HIF1α were indeed expressed in both the Egr2GFP^hiand Egr2GFP^lopopulations. It was confirmed differential expression of Egr2 and CCL1 to between the Egr2GFP^hiand Egr2GFP^lopopulations to assure sort purity (FIG. 2G). Together, these data indicate that Egr2 contributes to upregulation of 4-1BB and LAG-3 expression at early time points, but that other transcriptional regulators compensate and drive expression of LAG-3 and 4-1BB as the T cell-tumor interaction progresses.

CD8⁺ 4-1BB⁺LAG-3⁺ TILs are Oligoclonal and Enriched for Tumor Antigen Specificity

Not all T cells in the tumor microenvironment are specific for tumor-associated antigens, as memory T cells specific for irrelevant antigens are often found among TIL, and non-specific T cell trafficking has been documented in vivo (Harlin et al., 2006; incorporated by reference in its entirety). Experiments conducted during development of embodiments herein to determine whether 4-1BB⁺LAG-3⁺CD8⁺ TILs are tumor-antigen specific. LAG-3, 4-1BB and Egr2 are upregulated after TCR stimulation and experiments indicate that this population expands within the tumor microenvironment in situ. Three complementary techniques were employed. First, the CD8⁺ TILs were isolated based on LAG-3 and 4-1BB expression by cell sorting and performed TCRβ spectratype analysis. Compared to the 4-1BB⁻LAG-3⁻ TILs and CD8⁺ splenocytes, the 4-1BB⁺LAG-3⁺ TILs had a non-Gaussian distribution and shared one or two dominant peaks (FIG. 3A). Analysis of several Vβs displaying one dominant peak revealed that Vβ7 contained a single CDR3β sequence shared between the 4-1BB⁻LAG-3⁺ and 4-1BB⁺LAG-3⁺ populations, indicating a clonal relationship (FIG. 3A). To measure the oligoclonality of the CDR3β repertoires the Hamming Distance (HD) was calculated for each Vβ between the CD8⁺ TIL subpopulations and the splenic CD8⁺ population within three separate mice (FIG. 8). By transforming each spectratype into area under the curve frequency profiles the Hamming Distance computes the changes in frequency and reports a value of comparison between 0 and 1, with 0 indicating a completely identical frequency profile and 1 signifying a completely discordant profile. As a control, the HD of the splenic CD8⁺ populations between different mice was calculated (FIG. 3B, black bar). Since the splenic CD8⁺ spectratypes are largely Gaussian this value represents the HD between two similar distributions. Analysis of the HD between the CD8⁺ TIL subpopulations revealed that the 4-1BB⁺LAG-3⁺ and 4-1BB⁻LAG-3⁺ but not the 4-1BB⁻LAG-3⁻CDR3β distributions are significantly different (less Gaussian) compared to the splenic CD8⁺ population (FIG. 3B). These data indicate that the 4-1BB⁺LAG-3⁺ and 4-1BB⁻LAG-3⁺ populations are oligoclonal expanded subsets of TILs, indicating antigen specificity in these subpopulations.

As a second approach, the B16.SIY melanoma and MC38.SIY adenocarcinoma models were utilized. CD8⁺ T cells specific for the H-2K^b-restricted SIY epitope (SIYRYYGL) were monitored. SIYRYYGL/K^bpentamer⁺ (H-2K^b/SIY) cells were found in expanded numbers within B16.SIY and MC38.SIY tumors at day 14 after tumor inoculation (FIG. 3C). Nearly 47% of the H-2K^b/SIY⁺ cells expressed both 4-1BB and LAG-3, in contrast to 32% of the H-2K^b/SIY⁻ population (FIGS. 3C and E). This enrichment of antigen-specific CD8⁺ TILs in the 4-1BB⁺LAG-3⁺ populations indicates that these markers identify tumor antigen-specific TILs. The H-2K^b/SIY⁻ cells also contained significant numbers of 4-1BB⁺LAG-3⁺ cells, which is consistent with the notion that tumor antigens other than SIY are also recognized by subsets of CD8⁺ TILs in vivo (FIG. 3C). H-2K^b/SIY⁺ cells in the spleen or TdLN did not co-express 4-1BB and LAG-3, indicating that this phenotype is acquired within the tumor microenvironment.

These features were also analyzed in the context of tumor-antigen specific CD8⁺ TILs in two spontaneously rejected tumor models. To this end, H-2K^b/SIY-specific CD8⁺ TILs cells were evaluated from MC57.SIY and 1969.SIY tumors. At day 14 after tumor inoculation, approximately 5% of the H-2K^b/SIY-specific CD8⁺ TILs were found in the 4-1BB⁺LAG-3⁺ fraction. As with the B16.SIY tumors, no H-2K^b/SIY-specific CD8 T cells co-expressed 4-1BB and LAG-3 in the TdLN or spleen (not shown) (FIG. 3D). Unlike the B16.SIY and MC38.SIY tumors, no significant enrichment of 4-1BB⁺LAG-3⁺ H-2K^b/SIY-specific CD8⁺ TILs was observed (FIGS. 3D and E). These data indicate that tumor antigen specificity per se does not determine dysfunctionality, and that this is a feature unique to the microenvironment of progressing tumors.

As a third measure to determine if tumor-antigen specific CD8⁺ T cells acquire the 4-1BB⁺LAG-3⁺ phenotype, congenically marked 2C and P14 transgenic (Tg) T cells, isolated from 2C/Rag2^−/− and P14/Rag2^−/− mice, were transferred into tumor-bearing hosts. The 2C TCR is specific for the SIY model antigen expressed by B16.SIY tumor cells, while P14 is an irrelevant TCR specific for the LCMV-derived gp_33-41epitope; both TCRs are H-2K^b-restricted. 2C and P14 Tg CD8⁺ T cells were transferred via tail vein 7 days after tumor inoculation. Seven days after transfer, tumors and TdLNs were extracted and the phenotypic profile of the transferred populations was analyzed. This system allowed for the analysis of two T cell populations with defined antigen specificities within the same tumor microenvironment, as well as the polyclonal host CD8⁺ T cells. The 2C T cells were more efficiently recruited and expanded within the tumor microenvironment compared to the P14 T cells and encompassed a large fraction of the total CD8⁺ TIL population (FIG. 3F). Of the 2C T cells, nearly all expressed LAG-3 and or 4-1BB while this was true for only a small percentage of the P14 cells (FIGS. 3G and H). Consistent with the SIY-K^bpentamer analysis, the co-expression of LAG-3 and 4-1BB on 2C T cells was not observed in the TdLN. Together, these results demonstrate that the 4-1BB⁺LAG-3⁺ phenotype is a property of tumor antigen-specific TIL under conditions of tumor progression.

CD8⁺ TILs Expressing LAG-3 and 4-1BB Exhibit Defective IL-2 Production Yet Produce IFN-γ and Treg-Recruiting Chemokines

Based on the characteristics of the in vitro T cell anergy model that led to the identification of Egr2 as an important regulator, experiments conducted during development of embodiments herein to determine whether the tumor-antigen specific 4-1BB⁺LAG-3⁺CD8⁺ TIL population is dysfunctional in their capacity to produce IL-2. To this end each subpopulation was sorted and stimulated with anti-CD3 and anti-CD28 mAb and analyzed IL-2 production by qRT-PCR and ELISA. Since nearly all CD8⁺ TILs displayed an activated phenotype, CD8⁺CD44⁺ splenocytes were used as a positive control. Indeed, the 4-1BB⁺LAG-3⁺ cells showed a 100-fold reduction in Il-2 mRNA and as much as a 40-fold reduction in IL-2 protein levels compared to the 4-1BB⁻LAG-3⁻ population (FIGS. 4A and 4B). As a second approach, Egr2^hiTIL (which are also largely 4-1BB⁺LAG-3⁺) was examined by utilizing the Egr2-GFP reporter mice. Indeed, ex vivo stimulated Egr2-GFP^hiCD8⁺ TILs also exhibited reduced Il-2 transcript compared to Egr2-GFP′° cells (FIG. 4C). As a final approach, congenically marked 2C T cells were adoptively transferred intravenously into tumor-bearing hosts and recovered the 2C T cells 7 days later from the tumor and TdLN. 2C T cells isolated from tumors exhibited a reduced capacity to produce Il-2 transcripts, at a level equivalent to 4-1BB⁺LAG-3⁺ TILs, compared to 2C CD44⁺ T cells isolated from the TdLN (FIG. 4D). In chronic infection models, expression of PD-1 has been suggested to identify intrinsically dysfunctional or “exhausted” CD8⁺ T cells. To determine if PD-1 alone might be sufficient to identify cells that lack the capacity to produce IL-2, CD8⁺ TILs that lacked expression of LAG-3 and 4-1BB were isolated and tested for the ability of the PD-1⁺ fraction to produce IL-2. Approximately ˜10% of CD8⁺ TILs were 4-1BB⁻LAG-3⁻PD-1⁺ on day 14 and 21 (FIGS. 4E and F). Upon ex vivo stimulation, this population retained the capacity to produce ll-2 mRNA at a level comparable to the 4-1BB⁻LAG-3⁻ cells (FIG. 4G). These results indicate that PD-1 expression alone is not sufficient to identify dysfunctional TIL in the tumor microenvironment.

To further examine functional alterations during tumor progression protein levels of IL-2, IFN-γ and TNF-α were tested after TCR stimulation. As the loss of the ability of CD8⁺ TILs to produce cytokines is suggested to be a temporal process reported initiated following entry into the tumor microenvironment (Waugh et al., 2016; Schietinger et al., 2016; incorporated by reference in their entireties) or progressively after 30 days in the chronic LCMV model (Wherry et al., 2007; incorporated by reference in its entirety), cytokine production was tested on day 7, 14, 21 and 28. The 4-1BB⁺LAG-3⁺ population lost the capacity to produce IL-2 as early as day 7 while the 4-1BB⁻LAG-3⁺ population lost IL-2 production between day 7 and day 14 (FIG. 5A). The 4-1BB⁻LAG-3⁻ population did not lose the ability to produce IL-2 at any time point tested (FIG. 5A), supporting the notion that this population is not tumor antigen specific and that differentiation into the dysfunctional state is an antigen-dependent process (Schietinger et al., 2016; incorporated by reference in its entirety). The 4-1BB⁺LAG-3⁺ population produced more IFN-γ at all time points after day 7 compared to their negative counterparts, albeit with a slight decrease in IFN-γ production over time. While the increase in IFN-γ was maintained until later time points, TNF-α production was lost by day 28 (FIG. 5A).

Experiments were conducted during development of embodiments herein to evaluate production of cytokines directly in the tumor without in vitro restimulation, which may more closely reflect which T cells were receiving TCR stimulation in situ. Each T cell population was sorted directly ex vivo without any culturing and mRNA levels were measured by qRT-PCR. Elevated Ifn-γ and Gzmb transcripts were observed from the 4-1BB⁺LAG-3⁺ subpopulation, along with a slight decrease in Tnf-α levels, compared to the 4-1BB⁻LAG-3⁻ cells (FIG. 5B). Production of IFN-γ in primary TILs was confirmed by injecting tumors with Brefeldin A prior to analysis by intracellular cytokine staining. Consistent with the mRNA expression, the 4-1BB⁺LAG-3⁺ population produced significantly greater amounts of IFN-γ protein (FIG. 5C). Thus, the 4-1BB⁺LAG-3⁺ TIL are not completely devoid of functionality, as they continue to produce IFN-γ despite defective production of IL-2. This phenotype is consistent with in vitro T cell anergy models (Jenkins et al., 1987; incorporated by reference in its entirety).

To test whether the 4-1BB⁺LAG-3⁺ population still retains cytotoxic capacity, re-directed lysis was performed by co-culturing anti-CD3 bound P815 mastocytoma target cells with the different CD8⁺ TIL subpopulations directly after sorting. 4-1BB⁺LAG-3⁺CD8⁺ TILs isolated from day 14 tumors were able to lyse target cells at a comparable efficacy to in vitro primed OT-I cells. 4-1BB⁺LAG-3⁺ TILs isolated from day 21 tumors were still able to lyse target cells, albeit to a lesser extent compared to primed OT-I cells (FIG. 5D).

CD8⁺ T cells in the tumor can be the source of the chemokine CCL22 that recruits FoxP3⁺ regulatory T cells (Tregs) to the tumor microenvironment (Spranger et al., 2013; incorporated by reference in its entirety). In addition, the chemokine Ccl1 was an Egr2 target in anergic T cells (Zheng et al., 2013; incorporated by reference in its entirety), and it has been suggested that CCL1 also contribute to Treg recruitment in the tumor context in vivo (Hoelzinger et al., 2010; incorporated by reference in its entirety). However, whether all CD8⁺ T cells in the tumor produce these chemokines or if they are only produced by subpopulations of T cells had not been determined. To address this the CD8⁺ TIL phenotypic subpopulations were analyzed for Ccl1 and Ccl22 mRNA expression directly ex vivo by qRT-PCR. Indeed, the 4-1BB⁺LAG-3⁺ TIL population produced substantially greater Ccl1 and Ccl22 compared to their negative counterparts or to splenic CD8⁺CD44⁺ T cells (FIG. 4K). As a control, expression of a distinct chemokine Ccl5 was found not to be differentially expressed.

Together, these data show that co-expression of 4-1BB and LAG-3 delineates tumor antigen-specific CD8⁺ TIL that lack the ability to produce IL-2 yet retain the ability to produce IFN-γ, kill target cells in vitro, and secrete chemokines capable of Treg recruitment. Given the fact that IFN-γ is responsible for the upregulation of PD-L1 and IDO in the tumor microenvironment, and that chemokines produced by CD8⁺ TIL contribute to Treg recruitment (Spranger et al., 2013; incorporated by reference in its entirety), these data indicate that the 4-1BB⁺LAG-3⁺ population contributes to the network of immune suppressive mechanisms within the tumor microenvironment that limit the efficacy of anti-tumor immunity.

Gene Expression Profiling Reveals that CD8⁺ 4-1BB⁺LAG-3⁺ TILs Express an Extensive Array of Additional Co-Stimulatory and Co-Inhibitory Receptors

Having in hand surface markers that define tumor antigen-specific dysfunctional CD8⁺ TILs, experiments conducted during development of embodiments herein to compare the gene expression profile of this population to other published profiles of dysfunctional CD8⁺ T cells to determine genes that regulate or are differentially expressed in cells in this dysfunctional state. To this end, a cross-study comparison was conducted of the transcriptional profiles of the “dysfunctional” 4-1BB⁺LAG-3⁺CD8⁺ TILs, “hypofunctional” CD8⁺ TILs from a study utilizing the murine CT26 tumor model (Waugh et al., 2016; incorporated by reference in its entirety) and LCMV “exhausted” GP33 specific CD8⁺ T cells (Doering et al., 2012; incorporated by reference in its entirety). The results are depicted in Table 2. Only genes with a 2-fold increase over controls from each study independently were considered. Over a 2-fold greater number of genes was found to be shared between the dysfunctional TIL dataset and the previously published hypofunctional CD8⁺ TIL data, than with the exhausted T cell profile (FIG. 6A). In addition, a rank-rank hypergeometric overlap (RRHO) analysis indicated a greater statistically significant overlap (FIG. 10A) and a greater correlation (FIG. 10B) between the current dysfunctional TIL and the published hypofunctional CD8⁺ TIL gene expression profiles compared to the virally-induced exhausted CD8⁺ T cell profile, indicating a more similar molecular program between CD8⁺ T cells isolate from tumors compared to chronic viral infection.