MCT11 antibody for treating T cell exhaustion and enhancing cancer immunotherapy

Abstract

Monoclonal antibodies and antigen-binding fragments that specifically bind to MCT11 are provided. Nucleic acid molecules encoding the antibodies, vectors containing these nucleic acid molecules, and host cells transfected with these vectors are also disclosed. Methods of using MCT11-specific antibodies to treat cancer, inhibit or treat T-cell exhaustion, and / or enhance immunotherapy are also provided. The present invention relates to monoclonal antibodies and antigen-binding fragments that specifically bind to monocarboxylate transporter 11 (MCT11), and their use to treat cancer, reduce T-cell exhaustion, and increase response to immunotherapy in a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 223,473, filed July 19, 2021, which is incorporated by reference herein.

[0002] Field The present invention relates to monoclonal antibodies and antigen-binding fragments that specifically bind to monocarboxylate transporter 11 (MCT11), and their use to treat cancer, reduce T cell exhaustion, and increase response to immunotherapy in a subject. [Background technology]

[0003] background The programmed cell death 1 (PD-1) receptor is a checkpoint receptor that is expressed primarily on mature cytotoxic T lymphocytes. Cancer cells often express PD-1 ligands (such as PD-L1 and PD-L2), which leads to immune tolerance of cancerous cells. Certain cancer therapies target PD-1 or its ligands to reduce immune tolerance, thereby increasing T cell-mediated elimination of cancerous cells. However, only a portion of patients respond to this so-called PD-1 blockade. A potential factor limiting efficacy is the development of T cell exhaustion, another consequence of differentiation that leads to a dysfunctional state of T cells. Exhaustion limits the ability of T cells to respond to immunotherapy. Thus, therapies that increase T cell function may prove to be an effective strategy to treat cancer, improve cell therapy (e.g., adoptive cell transfer (ACT) therapy), or improve patient response to various cancer immunotherapies. Summary of the Invention [Means for solving the problem]

[0004] overview We show here that MCT11 is upregulated in terminally exhausted T cells, particularly in T cells that infiltrate tumors. We also show here that lactate uptake by exhausted T cells is blocked by treatment with anti-MCT11 antibody. Furthermore, we show that treatment with α-MCT11 mAb significantly reduces tumor growth in mouse cancer models. Thus, without being bound to any particular theory, MCT11-mediated uptake of lactate (or another MCT11 substrate) may reduce antitumor function in T cells. Based on these findings, we provide a method for treating T cell exhaustion that can enhance cancer treatments such as T cell-based immunotherapy.

[0005] Provided herein are monoclonal antibodies that specifically bind to monocarboxylate transporter 11 (MCT11). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (V H ) and the light chain variable region (V L ) (V as set forth in SEQ ID NOs: 1 and 5, respectively) H and V L In some embodiments, the antibody is a monoclonal antibody. In some examples, the monoclonal antibody comprises an antibody fragment (e.g., an antigen-binding fragment), such as a Fab fragment, a Fab' fragment, a F(ab)' fragment, a Fab ... 2 Fragment, Fv fragment, single chain variable fragment (scFV), single chain antibody dimer (scFV 2), or disulfide-stabilized variable fragment (dsFV). Antibody conjugates are also disclosed herein, for example, the disclosed monoclonal antibodies can be linked to an effector molecule or a detectable marker. In some embodiments, the antibody conjugate is an antibody-drug conjugate (where the effector molecule is a therapeutic molecule). In further embodiments, the disclosed antibody is a multispecific antibody comprising the disclosed monoclonal antibody specific for MCT11 and at least one additional antibody that binds to at least one other antigen (such as PD-1, 4-1BB / CD137, GITR, OX40, CD105, LAG3, TIM-3 / HAVCR2, NRP1, or FAS). Nucleic acid molecules encoding the monoclonal antibodies disclosed herein, vectors comprising these nucleic acid molecules, and host cells transformed with these nucleic acids or vectors are also disclosed.

[0006] Methods are provided for reducing T cell exhaustion, increasing effector function of T cells, or both. In some embodiments, the T cells are adoptive cell transfer (ACT) therapeutic T cells (e.g., tumor infiltrating lymphocytes (TILs), chimeric antigen receptor T cells (CAR-T), or engineered T cell receptor (TCR) T cells). Methods are also provided for treating cancer (or tumors), treating T cell exhaustion, and / or increasing response to cancer immunotherapy in a subject. In some examples, the subject has cancer and / or is undergoing immunotherapy. In some embodiments, the disclosed methods increase effector T cell function or reduce T cell exhaustion in a subject.

[0007] The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows typical phenotypes of "precursor-like" or "terminally exhausted" T cells. [Figure 2A-B]Figures 2A-2C show that MCT11 is upregulated in exhausted T cells in mice and humans. Figure 2A shows FACS sorting of LN CD8+ and TIL CD8+ cells for RNA-seq (left) and that MCT11 is upregulated in exhausted T cells (PD1hiTim3+) (right). Figure 2B shows MCT11 staining of human PBMC, PD1 / TIM3- and PD1 / TIM3+ cells from melanoma (MEL) or head and neck cancer (HNSCC) patients. Human tumor biopsy samples were stained with antibodies against CD8, PD-1, Tim-3, and MCT11 and analyzed by flow cytometry. Figure 2B shows MCT11 staining as a function of progression to exhaustion (PD-1+Tim3+). FIG. 2C shows MCT11 surface expression on exhausted or non-exhausted tumor-infiltrating lymphocytes (TILs) from MC38 (adenocarcinoma) or MEER (head and neck cancer) models in C56 / BL6J mice. [Figure 2C] Same as above. [Diagram 3] Figures 3A-3B show Slc16a11 transcripts per million (TPM) derived from RNA-seq for the indicated cell types. MCT11 is expressed (upregulated) on the surface of exhausted T cells, particularly those infiltrating tumors (TILs) (Figure 3A). Figure 3B was generated from published data and confirms that Slc16a11 is specific to tumor-infiltrating exhausted T cells. [Figure 4] Figure 4 shows that exhausted T cells specifically take up lactate. A schematic of the experimental design is shown above, and the graph below shows lactate uptake after 30 minutes of incubation with lactate for each of the indicated cell types. [Diagram 5] FIG. 5 shows that lactate (LA) uptake is specifically blocked when exhausted tumor infiltrating lymphocytes (TILs) are treated with a polyclonal anti-MCT antibody (labeled "αMCT11" in this figure). [Figure 6] FIG. 6 shows that lactate (LA) uptake is blocked when exhausted tumor infiltrating lymphocytes (TILs) are treated with a monoclonal anti-MCT antibody (labeled "MCT11 mAb"). [Figure 7A-B]Figures 7A-7C show that α-MCT11 mAb has activity in B16 melanoma and MEER HNSCC models. Figure 7A shows a diagram of the experimental setup. Figure 7B shows tumor growth inhibition in B6 mice bearing B16 melanoma treated with IgG2a, α-PD1, or α-MCT11 mAb (labeled "αMCT11" in this figure). Figure 7C shows tumor growth inhibition in B6 mice bearing MEER tumors treated with isotype control or α-MCT11 mAb (labeled "anti-MCT11" in this figure). [Figure 7C] Same as above. [Figure 8A-B] Figures 8A-8C show that the therapeutic effect of α-MCT11 mAb blockade is at least partially dependent on adaptive immunity. Figure 8A shows a diagram of the experimental setup. Figure 8B shows tumor growth in RAGKO mice bearing B16 melanoma treated with the indicated treatments. Figure 8C shows tumor growth in RAGKO mice bearing MEER tumors treated with the indicated treatments. [Figure 8C] Same as above. [Figure 9A-B] Figures 9A-9C show that α-MCT11 mAb works by blocking rather than depleting MCT11 expressing cells. Figure 9A shows a diagram of the experimental setup. Figure 9B shows the tumor area after treatment with IgG2a, α-MCT11 mAb (labeled "anti-MCT11" in this figure), or the FC mutant of α-MCT11 mAb (labeled "FC mut anti-MCT11). Mice whose tumors had been removed in response to MCT11 blockade (anti-MCT11 CR) were re-inoculated with MEER tumor cells. Figure 9C shows that mice from which MEER tumors had previously been removed show immunological memory against the tumor. [Figure 9C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Nucleic acid and amino acid sequences are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.

[0010] The sequence listing is submitted as an XML file ("Sequence.xml", created July 17, 2022, 20,480 bytes), which is incorporated herein by reference. In the attached sequence listing:

[0011] SEQ ID NO: 1 is MCT11 V H is the amino acid sequence.

[0012] SEQ ID NOs: 2, 3, and 4 are sequences of MCT11 V H are the amino acid sequences of CDR1, CDR2, and CDR3, respectively.

[0013] SEQ ID NO:5 is MCT11 V L is the amino acid sequence.

[0014] SEQ ID NOs: 6, 7, and 8 are sequences of MCT11 V L are the amino acid sequences of CDR1, CDR2, and CDR3, respectively.

[0015] SEQ ID NO:9 is an exemplary amino acid sequence of human MCT11. [ka]

[0016] SEQ ID NO:10 is an exemplary nucleic acid sequence encoding human SLC16A11 mRNA. [ka] [ka]

[0017] SEQ ID NO:11 is an exemplary immunizing peptide of MCT11. [ka]

[0018] SEQ ID NO:12 is an exemplary amino acid sequence of mouse MCT11. [ka]

[0019] SEQ ID NO:13 is an exemplary nucleic acid sequence encoding mouse Slc16a11 mRNA. [ka] [ka]

[0020] Detailed Description I. Terminology Overview Unless otherwise stated, technical terms are used according to conventional usage.Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, published by Jones & Bartlett Learning, 2017; The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

[0021] As used herein, the singular forms "a", "an" and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. As used herein, the term "comprises" means "includes". Thus, "comprising an antibody" means "including an antibody" and does not exclude other elements. It should be further understood that any and all base sizes given for nucleic acids are approximate and are provided for illustration purposes unless otherwise indicated. Although many methods and materials similar or equivalent to the methods and materials described herein can be used, particularly suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will take precedence. Furthermore, the materials, methods, and examples are for illustrative purposes only and are not intended to limit the present invention.

[0022] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided below:

[0023] About: Unless the context indicates otherwise, "about" refers to plus or minus 5% of the reference value. For example, "about" 100 refers to 95 to 105.

[0024] Administration: Providing or giving a drug (such as MCT11 monoclonal antibody) to a subject by any effective route. Administration can be local or systemic. Exemplary administration routes include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, intraprostatic, intrathecal, intraarterial, intraosseous, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. In some examples, monoclonal antibody is administered by intravenous injection.

[0025] Adoptive cell transfer (ACT) therapy: A type of immunotherapy that administers modified (e.g., modified to recognize tumor antigens) and / or expanded T cells to patients in need of it. The T cells for ACT therapy can be the patient's own T cells (e.g., modified and / or expanded ex vivo) or donor-derived T cells. ACT therapy includes, for example, treatment with chimeric antigen receptor T cells (CAR-T), engineered T cell receptors (TCR), or tumor infiltrating lymphocytes (TIL). ACT therapy is sometimes also referred to as adoptive cell therapy, cellular adoptive immunotherapy, or T-cell transfer therapy.

[0026] Antibody and antigen-binding fragment: An immunoglobulin, antigen-binding fragment thereof, or derivative that specifically binds to and recognizes an analyte (antigen) (such as MCT11, such as human MCT11). The term "antibody" is used herein in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, so long as they exhibit the desired antigen-binding activity.

[0027] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity to an antigen. Examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2 Antibody fragments include, but are not limited to, antigen-binding fragments produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methods (see, for example, Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2002). nd ed., Springer-Verlag, 2010).

[0028] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies).

[0029] An antibody may have one or more binding sites. When there is more than one binding site, the binding sites may be identical to each other or may be different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0030] Typically, naturally occurring immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0031] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). The combination of the heavy and light chain variable regions specifically binds to an antigen.

[0032] "V H References to "VH" or "VH" refer to the variable region of an antibody heavy chain, including the variable region of an antigen-binding fragment, such as an Fv, scFv, dsFv, or Fab. L References to " or "VL" refer to the variable domain of an antibody light chain, including the variable domain of an Fv, scFv, dsFv or Fab.

[0033] V H and V LEach of these sequences contains a "framework" region interrupted by three hypervariable regions known as "complementarity determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 th (See, e.g., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, USDepartment of Health and Human Services, 1991). The sequences of framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combination of the framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0034] CDRs are primarily responsible for binding to an epitope of an antigen. The boundaries of the amino acid sequence of a given CDR can be determined according to several well-known schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5 thed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, USDepartment of Health and Human Services, 1991; "Kabat" numbering scheme), the scheme described in Al-Lazikani et al. ("Standard conformations for the canonical structures of immunoglobulins," J. Mol. Bio., 273(4):927-948, 1997; "Chothia" numbering scheme), and the scheme described in Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27(1):55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (in the N-terminal to C-terminal direction) and are also typically identified by the chain in which the particular CDR is located. H CDR3 is the V of the antibody in which it is found. H CDR3 from V L CDR1 is the V domain of the antibody in which it is found. L The light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. The heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0035] In some embodiments, the disclosed antibodies comprise a heterologous constant domain, for example, an antibody comprises a constant domain that is different from the native constant domain, such as a constant domain that comprises one or more modifications to increase half-life.

[0036] A "single-chain antibody" (scFv) is a recombinant antibody that is a genetically fused single-chain molecule comprising one or more VFv fragments of an antibody linked by a suitable polypeptide linker. H Domains and V L The V domain in scFv is a genetically engineered molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). H -Domain and V L The intramolecular orientation of the -domains is typically not critical for scFvs. Therefore, it is possible to provide scFvs with both possible configurations (V H - domain - linker domain - V L -Domain;V L - domain - linker domain - V H In dsFv, the V H and V L has been mutated to introduce a disulfide bond to stabilize the association of the chains. H Domains and V L Also included are diabodies, which are bivalent, bispecific antibodies in which the domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain, thereby creating two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0037] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising said population are identical and / or bind to the same epitope, except for possible variant antibodies, including, for example, naturally occurring mutations or arising during the production of a monoclonal antibody preparation (such variants are generally present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci (such methods and other exemplary methods of producing monoclonal antibodies are described herein). In some instances, monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions (see, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2001). nd (See, e.g., J. Med. Soc. 1999, ed. New York: Cold Spring Harbor Laboratory Press, 2014.) Monoclonal antibodies include humanized monoclonal antibodies.

[0038] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs derived from a non-human (mouse, rat, synthetic, etc.) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor" and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from a donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if present, can be substantially identical to human immunoglobulin constant regions (e.g., at least about 85-90% identical, e.g., about 95% or more identical). Thus, all portions of a humanized antibody or antigen-binding fragment, except possibly the CDRs, are substantially identical to the corresponding portions of a natural human antibody sequence.

[0039] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and the CDRs and / or framework regions from another human antibody.

[0040] A "fully human antibody" or "human antibody" is an antibody that contains sequences derived from (or obtained from) the human genome and does not contain sequences derived from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) Fc regions derived from (or obtained from) the human genome. Human antibodies can be identified and isolated using technologies that generate antibodies based on sequences derived from the human genome (e.g., by phage display or using transgenic animals) (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1999). stEd. New York: Cold Spring Harbor Laboratory Press, 2004; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0041] Antibodies also include genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See, for example, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0042] Antibody-drug conjugate (ADC): A molecule that includes an antibody (or an antigen-binding fragment of an antibody) conjugated with a drug. ADCs can be used to specifically target a drug to a particular cell (such as a cancer cell or exhausted T cell) by specific binding of the antibody to a target antigen expressed on the cell surface. Exemplary drugs used with ADCs include antiviral agents (e.g., remdesivir, galidesivir, arbidol, favipiravir, baricitinib, or lopinavir / ritonavir), anti-microtubule agents (e.g., maytansinoids, auristatin E, and auristatin F), interchain crosslinkers (e.g., pyrrolobenzodiazepines; PBDs), the calicheamicin family (e.g., ozogamicin), topoisomerase inhibitors (e.g., govitecan / exetecan), PD-1 inhibitors, T cell agonists, or bacterial toxins (e.g., PE38). In some cases, the ADC is a bispecific ADC composed of two monoclonal antibodies or antigen-binding fragments thereof, each directed to a different antigen or epitope, conjugated with a drug. In one example, the ADC comprises an anti-mammalian MCT11 antibody or antigen-binding fragment, e.g., an anti-human MCT11, e.g., a mAb specific for human MCT11.

[0043] Binding affinity: the affinity of an antibody for an antigen. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by competitive radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In some embodiments, binding affinity is measured using an Octet system (Creative Biolabs) based on biolayer interferometry (BLI) technology. In other embodiments, Kd is measured using a surface plasmon resonance assay, for example, using a BIACORES-2000 or BIACORES-3000 (BIAcore, Inc., Piscataway, NJ). In other embodiments, antibody affinity is measured by flow cytometry or surface plasmon resonance. Other exemplary methods are described in Harlow & Lane, Antibodies, A Laboratory Manual, 2003 (2003), pp. 2171-2175, 2003. nd ed., Cold Spring Harbor Publications, New York (2013).

[0044] An antibody that "specifically binds" to an antigen (e.g., MCT11, e.g., human MCT11) is an antibody that binds to an antigen with high affinity but does not bind significantly to other unrelated antigens. In one example, an antibody that specifically binds to MCT11 is an antibody that substantially binds to the MCT11 substrate to which the protein (i.e., protein in a biological sample) binds. A certain degree of non-specific interaction between the antibody and the non-target may occur. Specific binding typically results in more than a two-fold increase (e.g., more than a five-fold, more than a ten-fold, or more than a hundred-fold increase) in the amount of antibody bound (per unit time) to a protein, including an epitope or a cell or tissue expressing the target epitope, compared to a protein or a cell or tissue lacking the epitope. Specific binding of a protein under such conditions requires an antibody that is selected for its antibody specificity to a particular protein. A variety of immunoassay formats are suitable for the selection of antibodies or other ligands that are specifically immunoreactive with a particular protein. For example, a solid-phase ELISA immunoassay can be used to select monoclonal antibodies that are specifically immunoreactive with a protein. In some examples, a monoclonal antibody (such as an MCT11 mAb) specifically binds to a target (such as human MCT11) with an equilibrium constant (Kd) of 50 nM or less (such as 45 nM or less, 40 nM or less, 35 nM or less, 30 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, or 5 nM or less).

[0045] Bispecific antibody: A recombinant protein that contains antigen-binding fragments of two different monoclonal antibodies, and can thereby bind to two different antigens or two different epitopes of the same antigen. Similarly, a multispecific antibody is a recombinant protein that contains antigen-binding fragments of at least two different monoclonal antibodies (such as two, three, or four different monoclonal antibodies). In one example, a bispecific antibody includes an anti-MCT11 antibody, such as an anti-human MCT11, such as a mAb specific for human MCT11.

[0046] Cancer: A malignant tumor characterized by abnormal or unregulated cell growth. Other features often associated with cancer include metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretory products, and suppressed or exacerbated inflammatory or immunological responses, infiltration of surrounding or distant tissues or organs (such as lymph nodes). "Metastatic disease" refers to cancer cells that remain at the original tumor site and travel to other parts of the body, for example, via the bloodstream or lymphatic system.

[0047] Checkpoint inhibitors (or checkpoint blockade): therapeutic agents that target checkpoint proteins. Checkpoints help prevent overly active immune or autoimmune responses and can sometimes limit the ability of T cells to eliminate cancerous cells. When checkpoints are blocked (e.g., PD-1 blockade), T cells can better target and kill cancerous cells. Examples of checkpoint proteins found on T cells or cancerous cells include PD-1 / PD-L1 / PD-L2, and CTLA-4 / B7-1 / B7-2.

[0048] Exemplary checkpoint inhibitors include ipilimumab (Yervoy®), nivolumab (Opdivo®), pembrolizumab (Keytruda®), atezolizumab (Tencentriq®), avelumab (Bavencio®), durvalumab (Imfinzi®), cemiplimab (Libtayo®), palbociclib (Ibrance®), ribociclib (Kisquali®), and abemaciclib (Verzenio®). Further examples are provided in Qiu et al., Journal of the European Society for Therapeutic Radiology and Oncology, 126(3):450-464, 2018; Visconti et al., J Exp Clin Cancer Res. 35(1):153, 2016; and Mills et al. Cancer Res. 77(23):6489-6498, 2017.

[0049] Chimeric antigen receptor (CAR): an artificial engineered T cell receptor that grafts any specificity onto immune effector cells. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto T cells (e.g., CAR-T); the coding sequence of said antibody is easily transferred by a vector. Thus, a CAR that "specifically binds" or is "specific" to an antigen is a CAR that binds to the antigen with high affinity and does not significantly bind to other unrelated antigens. The present disclosure includes CARs specific to MCT11, for example, CARs that include MCT11-specific antigen-binding fragments (e.g., MCT11-specific scFvs disclosed herein).

[0050] In ACT therapy, CARs can be useful for the treatment of cancer. For example, T cells (obtained from a patient or donor) are modified so that the T cells express a receptor specific to the patient's particular cancer. The modified T cells (which can then recognize and kill cancer cells) are introduced into the patient. First-generation CARs typically contained intracellular domains from the CD3ζ chain (which is the main transmitter of signals from endogenous TCRs). Second-generation CARs have added intracellular signaling domains from various co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to T cells. Third-generation CARs combine multiple signaling domains (such as CD3z-CD28-41BB or CD3z-CD28-OX40) to enhance efficacy.

[0051] A multispecific CAR is a single CAR molecule composed of at least two antigen binding domains (such as scFvs and / or single domain antibodies), each binding to a different antigen or different epitopes on the same antigen (see, e.g., US2018 / 0230225). For example, a bispecific CAR refers to a single CAR molecule with two antigen binding domains, each binding to a different antigen. A bicistronic CAR refers to two complete CAR molecules, each containing an antigen binding moiety that binds to a different antigen. In some cases, a bicistronic CAR construct expresses two complete CAR molecules linked by a cleavable linker. T cells expressing a bispecific or bicistronic CAR can bind to cells expressing both antigens to which the binding moieties are directed (see, e.g., Qin et al., Blood 130:810, 2017; and WO / 2018 / 213337). Any of these CARs can be used with the methods described herein.

[0052] Complementarity determining region (CDR): A region of hypervariable amino acid sequence that defines the binding affinity and specificity of an antibody. The light and heavy chains of a mammalian immunoglobulin each contain a light chain CDR1 (sometimes called V L- CDR1 or LCDR1), CDR2 (sometimes referred to as V L - CDR2 or LCDR2), and CDR3 (sometimes called V L -CDR3 or LCDR3); and heavy chain CDR1 (sometimes referred to as V H -CDR1 or HCDR1), CDR2 (sometimes called V H - CDR2 or HCDR2), and CDR3 (sometimes called V H -CDR3 or HCDR3.

[0053] Conditions sufficient to form an immune complex: Conditions that allow an antibody or antigen-binding fragment to bind to its cognate epitope to a degree detectably greater than and / or to a degree that substantially excludes binding to all other epitopes. Conditions sufficient to form an immune complex depend on the format of the binding reaction and are typically conditions utilized in immunoassay protocols or conditions encountered in vivo. For a description of immunoassay formats and conditions, see Greenfield (Ed.), Antibodies: A Laboratory Manual, 2001, pp. 211-215. nd ed. New York: Cold Spring Harbor Laboratory Press, 2014. The conditions used in the above methods are "physiological conditions," including reference to conditions (e.g., temperature, osmolality, pH) typical inside a living mammal or mammalian cell. While it is recognized that some organs are subject to extreme conditions, the environment within an organism and cell is typically at approximately pH 7 (e.g., pH 6.0-pH 8.0, more typically pH 6.5-7.5), contains water as the primary solvent, and exists at a temperature above 0° C. and below 50° C. The osmolality is within a range that supports cell viability and growth.

[0054] Immune complex formation can be detected by conventional methods (e.g., immunohistochemistry (IHC), immunoprecipitation (IP), flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blot), magnetic resonance imaging (MRI), computed tomography (CT) scanning, radiography, and affinity chromatography).

[0055] Conjugate: A complex of two molecules linked together (e.g., linked together by a covalent bond). In one embodiment, an antibody or antibody fragment (e.g., an antigen-binding fragment) is linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. An antibody conjugate is often referred to as an "immunoconjugate." When the conjugate comprises an antibody linked to a therapeutic agent, the conjugate is often referred to as an "antibody-drug conjugate" or "ADC." Other antibody conjugates include, for example, multispecific (such as bispecific or trispecific) antibodies and chimeric antigen receptors (CARs). In one example, the conjugate comprises a monoclonal antibody specific for MCT11 disclosed herein.

[0056] A peptide linker (short peptide sequence) can be included between the antibody and the effector molecule or second protein, if desired. Because conjugates can be prepared from two molecules (such as an antibody and an effector molecule) with distinct functionalities and / or origins, conjugates are sometimes also referred to as "chimeras."

[0057] Conservative variant: A "conservative" amino acid substitution is one that does not substantially affect or reduce the function of a protein (such as the ability of the protein to interact with a target protein). For example, an MCT11-specific antibody can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity to MCT11 (e.g., human MCT11). The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0058] Individual substitutions, deletions, or additions that change, add, or delete a single amino acid or a small percentage of amino acids (e.g., less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations in which the alteration replaces an amino acid with a chemically similar amino acid.

[0059] The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0060] A non-conservative substitution is a substitution that reduces the activity or function of an antibody (such as the ability to specifically bind to MCT11). For example, if an amino acid residue is essential for the function of a protein, even a conservative substitution may destroy the activity. Thus, a conservative substitution does not change the basic function of the protein of interest.

[0061] Contacting: putting into direct physical association; including contact in both solid and liquid form, and contacting can occur either in vivo or in vitro. Contacting can include contact between one molecule and another molecule (e.g., contact of amino acids on the surface of one polypeptide, such as an antigen, with another polypeptide, such as an antibody). Contacting can also include contact of cells (e.g., T cells) by putting an antibody into direct physical association with the cell.

[0062] Control: Reference standard. In some embodiments, the control is a negative control. In other embodiments, the control is a positive control. In still other embodiments, the control is a historical control or a standard reference value or range of values ​​(e.g., a previously tested control sample, e.g., a patient group with a known prognosis or outcome, or a sample group showing baseline or normal values). In some examples, the control can be a subject that has not been treated with the agent of interest (e.g., a monoclonal antibody specific for MCT11 of the disclosure) or an alternative treatment, or a baseline reading of the subject before treatment with the agent.

[0063] The difference between the test sample and the control can be increased or, conversely, decreased. The difference can be a qualitative difference or a quantitative difference (e.g., statistically significant difference). In some examples, the difference is an increase compared to the control (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500% increase). In other examples, the difference is a reduction compared to the control (e.g., a reduction of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100%).

[0064] Degenerate variant: In the context of this disclosure, a "degenerate variant" refers to a polynucleotide encoding a polypeptide (such as an antibody heavy or light chain) that contains a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences encoding a peptide are included, so long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0065] Detectable marker: a detectable molecule (also known as a label) that is directly or indirectly conjugated to a second molecule, such as an antibody (such as an MCT11 antibody) to facilitate detection of the second molecule. For example, detectable markers can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (such as CT scan, MRI, ultrasound, fiber optic testing, and laparoscopic testing). Non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme-linked, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). Guidance on the use of detectable markers and the selection of appropriate detectable markers for various purposes can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4 th (eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons (with supplement), 2017).

[0066] Effective amount: The amount of an agent (such as the MCT11 monoclonal antibody disclosed herein) sufficient to produce a beneficial or desired result. The effective amount (also referred to as a therapeutically effective amount) may vary depending on one or more of the following: the subject and condition being treated, the weight and age of the subject, the severity of the condition, and the mode of administration, which can be determined by the clinician. Beneficial therapeutic effects can include being able to make a diagnostic determination; improving a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally alleviating a disease, symptom, disorder, or pathological condition.

[0067] In one embodiment, an "effective amount" of a therapeutic agent (e.g., an MCT11 monoclonal antibody disclosed herein) is an amount sufficient to treat cancer (or tumor) in a subject (e.g., an amount that reduces tumor volume / size, tumor weight, number of metastases, reduces metastasis volume / size, metastasis weight by at least 10%, at least 20%, at least 25%, at least 50%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 600%, or a combination thereof). In another embodiment, an "effective amount" is an amount (e.g., of an MCT11 monoclonal antibody disclosed herein) sufficient to increase T cell effector function or activity, e.g., by at least 10%, at least 20%, at least 25%, at least 50%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 600%. In some embodiments, an "effective amount" is an amount (e.g., of an MCT11 monoclonal antibody disclosed herein) sufficient to reduce the activity of a target protein (e.g., MCT11), for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100%. In further embodiments, an "effective amount" (e.g., of an MCT11 monoclonal antibody disclosed herein) is an amount sufficient to reduce T cell exhaustion, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99%. In some instances, a combination of these effects is achieved.The increase or decrease can be determined in comparison to a suitable control (eg, no administration of the therapeutic agent of interest (eg, MCT11 antibody) or other suitable control).

[0068] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic (meaning that they induce a specific immune response). An antibody specifically binds to a specific antigen epitope on a polypeptide. In some examples, the disclosed antibody or antigen-binding fragment specifically binds to an MCT11 epitope. In one example, the epitope is specific to a portion of the continuous sequence of SEQ ID NO: 9, 11, or 12.

[0069] Fc region: The constant region of an antibody excluding the first heavy chain constant domain. The Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. The Fc region may also include some or all of the flexible hinge N-terminal to these domains. For IgA and IgM, the Fc region may or may not include the tail, and may or may not be bound by a J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains Cγ2 and Cγ3, and optionally the lower part of the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include the residues following C226 or P230 relative to the Fc carboxyl terminus, where numbering is according to Kabat. For IgA, the Fc region includes immunoglobulin domains Cα2 and Cα3, and optionally includes the lower part of the hinge between Cα1 and Cα2. In some examples, the antibody specific for MCT11 includes an Fc (e.g., human IgG1 Fc or human IgG4 Fc), a fucosylated Fc, or a non-Fcr-linked Fc.

[0070] Heterologous: Derived from a different genetic source. A nucleic acid molecule heterologous to a cell from which it is derived, other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein (such as an scFv) is expressed in a cell, such as a mammalian cell. Methods for introducing heterologous nucleic acid molecules into a cell or organism are known (e.g., transformation methods including electroporation, lipofection, particle gun acceleration, or homologous recombination).

[0071] Host cell: A cell that can propagate a vector and express its DNA. The cell can be a prokaryotic or eukaryotic cell. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell, since there may be mutations that occur during replication. However, when the term "host cell" is used, such progeny are included. In some examples, the host cell is a human cell, such as a human T cell (e.g., exhausted T cell) or PBMC.

[0072] IgG: A polypeptide belonging to a class or isotype of antibody substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class is IgG 1 , IgG 2 , IgG 3 , and IgG 4 Includes.

[0073] Immune complex: An immune complex is formed by the binding of an antibody or an antigen-binding fragment (such as scFv) to a soluble antigen. The formation of the immune complex can be detected by conventional methods (e.g., immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blot), magnetic resonance imaging, CT scan, radiography, and affinity chromatography).

[0074] Immunotherapy: Therapy that uses drugs to stimulate or suppress the immune system to treat diseases such as cancer. Some examples of cancer immunotherapy include immune checkpoint inhibitors, adoptive cell therapy, vaccines, and immune system modulators. Immunotherapy can be antibodies, viruses, nucleic acids, proteins, Fc-fusion proteins, or cells (e.g., T cells or NK cells). Non-limiting examples include abemaciclib, atezolizumab, avelumab, axicabtagene ciloreucel, brinotumumab, semipilimab, durvalumab, yelamirimab, ipilimumab, nivolumab, palbociclib, pembrolizumab, pidilizumab, relatolimab, ribociclib, urelemab, outlimumab, adoptive cell transfer (ACT) therapy (e.g., chimeric antigen receptors (CARs) (e.g., tisagenlecleucel)), or engineered TCRs or tumor infiltrating lymphocytes (TILs)), and oncolytic viruses (e.g., talimogene laherparepvec (T-VEC)).

[0075] Increase or decrease: a positive or negative change in amount, respectively, from a control value (such as a value indicating no therapeutic agent, such as no MCT11 antibody). An increase is a positive change compared to the control value (such as an increase of at least 25%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%). A decrease is a negative change compared to the control value (such as a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%). In some examples, the increase or decrease is statistically significant compared to a suitable control.

[0076] For example, in some embodiments, administration of an agent (e.g., an MCT11-specific antibody disclosed herein) reduces the activity of a protein target (e.g., MCT11) by reducing (including eliminating or inhibiting) one or more activities of the target (e.g., lactate transport). In some embodiments, the activity of MCT11 is reduced by at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 99%, at least 99.9%, or even 100% compared to a suitable control (e.g., the amount of MCT11 activity observed in the absence of an agent (e.g., an MCT11 antibody)).

[0077] In some embodiments, target cells (e.g., exhausted T cells) are increased or decreased in the sample or in vivo. For example, in some embodiments, the number of exhausted T cells (including terminal exhausted T cells) in the sample is decreased by contacting the sample with an effective amount of an antibody specific for MCT11 and removing cells bound to the antibody (e.g., the method depletes the exhausted T cell sample). In some examples, the method decreases the number of exhausted T cells (such as terminal exhausted T cells) in the PBMC population, for example, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9%. In some embodiments, target cells (e.g., exhausted T cells, including terminal exhausted T cells) are decreased in vivo. For example, the antibody is administered to a subject to facilitate the elimination of target cells in vivo, for example, by delivering a cytotoxic agent to the target cells or otherwise inducing cell death. In some examples, the method reduces the number of target cells in vivo (such as exhausted T cells, including terminal exhausted T cells) by, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9%, or more.

[0078] Isolated: An "isolated" biological component (such as a monoclonal antibody) is substantially separated, produced separately, or purified away from other biological components (such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins) in the cells or tissues of the organism in which it occurs. "Isolated" nucleic acids and proteins include those purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids and proteins. For example, T cells isolated from a subject (including a tumor or other sample from a subject) can be at least 50% pure, e.g., at least 60%, e.g., at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more pure.

[0079] Linker: A bifunctional molecule that can be used to link two molecules into one continuous molecule, for example, to link a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine-serine (GS) linkers.

[0080] The terms "conjugating," "joining," "binding," or "linking" can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalent attachment of an effector molecule or detectable marker radionuclide or other molecule to a polypeptide (such as the disclosed monoclonal antibodies). Linking can be by either chemical or recombinant means. "Chemical means" refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0081] Monocarboxylate transporter 11 (MCT11): MCT11 is a recently characterized transport protein that can transport monocarboxylates such as lactate. MCT11 protein is encoded by Slc16a11 (SLC16A11) gene. The sequence of MCT11 / Slc16a11 is publicly available, see, for example, GenBank Accession Nos. KJ900348.1, NM_153081.3, NM_153357.3, and NM_001370549.1 (providing exemplary Slc16a11 nucleic acid sequences), and UniProt Accession No. Q8NCK7, GenBank Accession Nos. NP_001357478.1, NP_694721.2, and NP_001099267.2 (providing exemplary MCT11 protein sequences). Examples are also provided in SEQ ID NOs: 9, 10, 12, and 13.

[0082] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence.For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence (e.g., a promoter driving the expression of a heterologous nucleic acid sequence disclosed herein).Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, are in the same reading frame.

[0083] Programmed cell death protein 1 (PD-1): A cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds to two ligands, PD-L1 and PD-L2. The human form is a type 1 transmembrane protein of 268 amino acids. PD-1 is an inhibitory receptor that mediates T cell exhaustion. PD-1 sequences are publicly available, for example, from the GenBank® sequence database (e.g., accession numbers NP_005009.2 (mature peptide is aa 21-288), CAA48113.1, NP_001301026.1 (mature peptide is aa 25-288), and CAA48113.1 (mature peptide is aa 21-288) provide exemplary PD-1 protein sequences, while accession numbers L27440.1, NM_005018.2, X67914.1, AB898677.1, and EU295528.2 provide exemplary PD-1 nucleic acid sequences).

[0084] Pharmaceutically acceptable carriers: The pharma- ceutically acceptable carriers useful in the present invention are conventional carriers. Remington's Pharmaceutical Sciences, 23rd Edition, Academic Press, Elsevier, (2020) describes compositions and formulations suitable for pharmaceutical delivery of therapeutic agents (such as the MCT11 monoclonal antibodies disclosed herein).

[0085] Generally, the nature of the carrier depends on the particular administration form used.For example, parenteral formulations usually contain injection fluids that contain pharma- ceutically and physiologically acceptable fluids as vehicles (such as water, physiological saline, balanced salt solutions, aqueous dextrose, 5% human serum albumin, or glycerol).In addition to biologically natural carriers, the pharmaceutical composition to be administered can contain small amounts of non-toxic auxiliary substances (such as wetting agents or emulsifiers, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate).Additionally, supplementary active compounds can be incorporated into the composition.

[0086] Promoter: A set of nucleic acid regulatory sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription (such as a TATA element in the case of a polymerase II type promoter). A promoter also includes, as needed, distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0087] Examples of promoters include, but are not limited to, SV40 promoter, CMV enhancer-promoter, CMV enhancer / β-actin promoter, EF1a, and PGK. Both constitutive and inducible promoters are included (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). Also included are sufficient promoter elements that allow promoter-dependent gene expression to be regulated by external signals or agents in a cell type-specific, tissue-specific, or inducible manner; such elements can be located in the 5' or 3' region of the gene. Also, promoters produced by recombinant DNA or synthetic techniques can be used to transcribe nucleic acid sequences.

[0088] Recombinant: A recombinant nucleic acid or amino acid is one that has a sequence that does not occur in nature or that has a sequence that is created by the artificial combination of two sequence segments that are naturally separated.

[0089] Sequence identity: The similarity between amino acid or nucleotide sequences is expressed in terms of the similarity between the sequences, or is otherwise referred to as sequence identity. Sequence identity is often measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Polypeptide (or nucleotide sequence) homologs have a relatively high degree of sequence identity when aligned using standard methods.

[0090] Methods for alignment of sequences for comparison have been described. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0091] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), and on the Internet. A description of how to determine sequence identity using this program is available on the Internet at the NCBI website.

[0092] Subject: A vertebrate (such as a mammal, e.g., a human). Mammals include, but are not limited to, mice, apes, humans, farm animals, sport animals, and pets. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human mammal (such as a monkey or other non-human primate, mouse, rat, rabbit, pig, goat, sheep, dog, cat, horse, or cow). In some examples, the subject has cancer (or a tumor) or is undergoing cancer immunotherapy (such as adoptive cell transfer (ACT) therapy). In some examples, the subject is a laboratory animal / organism (such as a mouse, rabbit, or rat).

[0093] T cell agonist: An immunotherapy that activates T cells to promote anti-tumor function. Non-limiting examples include urelumab and utomilumab.

[0094] T cell: A white blood cell (lymphocyte) that is an important mediator of the immune response. T cells include CD3 + T cells, CD4 + T cells, and CD8 + These include, but are not limited to, CD4 + T cells are immune cells that carry a marker on their surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help coordinate immune responses, including antibody responses and killer T cell responses. CD8 + T cells carry the "cluster of differentiation 8" (CD8) marker. In some instances, CD8 + T cells are cytotoxic T lymphocytes (CTLs). CD3+ T cells bear the "cluster of differentiation 3" (CD3) marker, a multimeric protein complex historically known as the T3 complex.

[0095] Activated T cells can be detected by cell proliferation and / or increased expression or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IFN-γ, or TNFα). +T cell activation can be detected by increased cytolytic activity in response to antigen. Exhausted T cells are dysfunctional T cells (hyporesponsive) commonly found in the cancer environment. T cell exhaustion is characterized by progressive loss of effector function (e.g., loss of production of IL-2, TNF-α, and IFN-γ) and persistent expression of inhibitory receptors (such as PD-1, T cell immunoglobulin domain- and mucin domain-containing protein 3 (Tim-3), CTLA-4, lymphocyte activation gene 3 (LAG-3), and CD160). In some instances, exhausted T cells are characterized by increased expression of CD3 + T cells or CD8 + In some instances, the exhausted T cells are terminally exhausted T cells (exhausted, terminally differentiated T cells). Terminally exhausted T cells express Tim3 and maintain high PD-1 expression compared to other T cells (Tim3 + PD-1 hi T cells). Tim3 + and / or PD-1 hi The T cells that are exhausted can be determined by FACs analysis, for example, FACs analysis of a population of T cells. In some instances, terminally exhausted T cells express MCT11. A possible cause of T cell exhaustion is chronic activation or prolonged antigen stimulation.

[0096] T cell receptor (TCR): A receptor found on the surface of T lymphocytes (or T cells) responsible for the recognition of antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is composed of two different protein chains. In humans, in 95% of T cells the TCR consists of alpha (α) and beta (β) chains, whereas in 5% of T cells the TCR consists of gamma and delta (γ / δ) chains. This ratio varies during ontogeny and in disease states as well as in different species. When the TCR engages with an antigen peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors. In one example, the TCR is a recombinant TCR (such as those used in TCR-engineered T cells for ACT therapy).

[0097] Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including transduction with a viral vector, transformation with a plasmid vector, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0098] Exemplary transduction methods include chemical methods (e.g., calcium phosphate transfection), physical methods (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), lipofection, nucleofection, receptor-mediated endocytosis (e.g., DNA-protein complexes, viral envelope / capsid-DNA complexes), particle gun acceleration methods (gene guns), and biological infection with viruses such as recombinant viruses (Wolff, JA, ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)). In the case of infection with retroviruses, infectious retroviral particles are absorbed by target cells, which reverse transcribes retroviral RNA genomes and integrates the resulting provirus into cellular DNA.

[0099] Treating, treatment, and therapy: Any success or sign of success in attenuating or ameliorating an injury, pathology, or condition, including any objective or subjective parameter, such as alleviation, remission, reduction in symptoms, or making the condition more tolerable to the patient, slowing the rate of degeneration or decline, making the end point of degeneration less debilitating, improving the physical or mental health of the subject, or extending life, etc. Treatment may be assessed by objective or subjective parameters, including the results of physical exams, blood tests, and other clinical tests, etc. In some examples, treatment with the disclosed methods reduces the number, volume, and / or weight of tumors and / or metastases. In some examples, treatment with the disclosed methods reduces T cell exhaustion, such as reducing exhausted T cells (e.g., Tim3) in a subject. + PD-1 hi In some examples, treatment with the disclosed methods reduces T cell exhaustion (e.g., a reduction in the number of T cells (exhausted T cells (e.g., Tim3 + PD-1 hi T cells). In some instances, a combination of these effects is achieved.

[0100] Tumor-infiltrating lymphocyte (TIL): Lymphocyte that invades tumor tissue. For example, T cells found with tumor samples. In ACT therapy, TIL therapy generally involves isolating TIL from a patient's tumor, activating and expanding TIL in culture, and then reinfusing it into the patient. In some examples, the T cells are TIL.

[0101] Tumor, Neoplasia, or Malignancy: A neoplasm is an abnormal growth of tissue or cells resulting from excessive cell division. As a neoplasm grows, it can produce a tumor. The tumor burden in an individual is the "tumor burden" and can be measured as the number, volume, or weight of tumors. "Non-cancerous tissue" is tissue from the same organ in which a malignant neoplasm has formed but does not possess the pathology characteristic of a neoplasm. Generally, non-cancerous tissue is considered histologically normal. "Normal tissue" is tissue from an organ, said organ not affected by cancer or another disease or disorder of the organ. A "cancer-free" subject is one in which the organ in question has not been diagnosed with cancer and does not possess detectable cancer.

[0102] Exemplary tumors (such as cancers) that can be treated using the disclosed MCT11 monoclonal antibodies include solid tumors, such as breast cancer (e.g., lobular and ductal carcinomas, e.g., triple-negative breast cancer), sarcomas, lung cancer (e.g., non-small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma), lung mesothelioma, colorectal adenocarcinoma, gastric cancer, prostate adenocarcinoma, ovarian cancer (such as serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular carcinoma and germ cell tumors, pancreatic adenocarcinoma, bile duct adenocarcinoma, hepatocellular carcinoma, bladder cancer (including, e.g., transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma), renal cell adenocarcinoma, endometrial cancer (including, e.g., adenocarcinoma and mixed Mullerian tumor (carcinosarcoma)). , endocervical, epicervical, and vaginal cancers (including adenocarcinoma and squamous cell carcinoma of each of these), skin tumors (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin adnexal tumor, Kaposi's sarcoma, cutaneous lymphoma, skin adnexal tumor, and various types of sarcoma and Merkel cell carcinoma), esophageal cancer, nasopharyngeal and oropharyngeal cancers (including squamous cell carcinoma and adenocarcinoma thereof), salivary gland cancer, brain and central nervous system tumors (including tumors of glial, neuronal, and meningeal origin), peripheral nerve tumors, soft tissue sarcomas and sarcomas of bone and cartilage, head and neck squamous cell carcinoma, and lymphatic tumors (including B-cell and T-cell malignant lymphoma). In one example, the tumor is a melanoma. In one example, the tumor is a head and neck squamous cell carcinoma (HNSCC) (such as HPV-positive HNSCC).

[0103] The disclosed MCT11 monoclonal antibody can also be used to treat liquid tumors, such as lymphatic, leukocyte, or other leukemia types. In a specific example, the tumor to be treated is a hematological tumor, such as leukemia (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia), lymphoma (such as Hodgkin's lymphoma or non-Hodgkin's lymphoma), or myeloma.

[0104] Vector: A nucleic acid molecule that can be introduced (e.g., by transfection or transduction) into a host cell, thereby producing a transformed host cell (such as a transformed exhausted T cell). A recombinant DNA vector is a vector that has recombinant DNA. A vector can contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector can also contain one or more selectable marker genes and other genetic elements. A viral vector (such as AVV) is a recombinant nucleic acid vector that has at least some nucleic acid sequences from one or more viruses. A replication-deficient viral vector is a vector that requires complementation of one or more viral genomic regions required for replication due to a deficiency in at least one replication-essential gene function.

[0105] II. Description of Several Embodiments Provided herein is a monoclonal antibody that specifically binds to monocarboxylate transporter 11 (MCT11). In some examples, the antibody specifically binds to human MCT11. In some embodiments, the monoclonal antibody is an antibody fragment (e.g., an antigen-binding fragment). Also disclosed is a multispecific antibody (e.g., a bispecific antibody) that comprises a monoclonal antibody or an antibody fragment (e.g., an antigen-binding fragment) that specifically binds to MCT11. The multispecific antibody recognizes at least one antigen in addition to MCT11. The antibody is useful, for example, for treating cancer, reducing T cell exhaustion or increasing the effector function of T cells, or increasing the response of a subject to cancer immunotherapy.

[0106] A. Monoclonal antibodies and fragments thereof that specifically bind to MCT11 Disclosed herein are isolated monoclonal antibodies that specifically bind to mammalian MCT11, such as MCT11, e.g., SEQ ID NO: 9 or 12. In some examples, the monoclonal antibodies specifically bind to human MCT11 or a portion thereof, such as SEQ ID NO: 9 or 11. The disclosed antibodies each have a V domain that includes CDR1, CDR2, and CDR3. HDomains and V L In some embodiments, the antibody comprises a V domain comprising the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO:1. H domain and / or a V domain comprising the light chain CDR1, CDR2, and CDR3 of SEQ ID NO:5 L The CDRs include domains. Various CDR numbering schemes (such as the Kabat, Chothia, or IMGT numbering schemes) can be used to determine the location of the CDRs. In some examples, the numbering scheme used to determine the CDRs is the Chothia numbering scheme (see, for example, Table 1). In a non-limiting example, the monoclonal antibody includes the heavy and light chain amino acid sequences and CDRs provided in Table 1. [Table 1-1] [Table 1-2]

[0107] In some embodiments, the antibody comprises a V sequence comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth in SEQ ID NO:1. H In some examples, the antibody comprises a VFGV comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth in SEQ ID NO:5. L In a further example, the antibody comprises V independently comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth in SEQ ID NOs: 1 and 5, respectively. H and V L Includes.

[0108] In some embodiments, the antibody comprises a V sequence comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth in SEQ ID NO:1. H and a V that comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 2, 3, and 4, respectively, and an amino acid sequence that is at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth in SEQ ID NO: 5. L and comprises light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 6, 7, and 8, respectively. Thus, in this particular embodiment, variation due to sequence identity is outside the CDRs.

[0109] In some examples, the antibody comprises a V H and / or SEQ ID NO:5 L In some embodiments, the antibody comprises the V sequence consisting of SEQ ID NO:1. H and / or V consisting of SEQ ID NO:5 L Includes.

[0110] In some embodiments, the monoclonal antibody is an antigen-binding fragment. An antigen-binding fragment is an antibody fragment that retains the ability to selectively bind to an antigen (e.g., MCT11 antigen, such as human MCT11). Non-limiting examples of such fragments include: (1) Fab, the fragment that contains a monovalent antigen-binding fragment of an antibody molecule that can be produced by digestion of a whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction to yield an intact light chain and a portion of the heavy chain; (3) (Fab') 2 (The fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction); F(ab') 2is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv (V expressed as two chains) H and V L a genetically engineered fragment comprising (5) Single-chain antibodies (such as scFv) (V linked by a suitable polypeptide linker as a genetically fused single-chain molecule) H and V L (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry and Snavely, IDrugs, 13(8):543-549, 2010). H -Domain and V L The intramolecular orientation of the V domain is not critical for the provided antibodies (e.g., for the provided multispecific antibodies). Thus, scFvs (V H - domain - linker domain - V L -Domain;V L - domain - linker domain - V H -domain) can be used. (6) Single-chain antibody dimer (scFV 2 ) (defined as a dimer of scFV). These are also called "mini-antibodies". (7) Disulfide-stabilized variable fragment (dsFv)

[0111] In some embodiments, the monoclonal antibody is a Fab fragment, a Fab' fragment, a F(ab)' 2 Fragment, Fv, single chain variable fragment (scFV), single chain antibody dimer (scFV 2 ), and disulfide-stabilized variable fragment (dsFv). In some examples, the antigen-binding fragment is an scFv that specifically binds to MCT11.

[0112] Antigen-binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as E. coli cells) of DNA encoding the fragment. Antigen-binding fragments can also be obtained by digestion of whole antibodies with pepsin or papain by conventional methods. For example, antigen-binding fragments can be obtained by enzymatic cleavage of antibodies with pepsin to produce F(ab') 2 This fragment can be further cleaved using a thiol reducing agent, and optionally protecting the sulfhydryl groups resulting from cleavage of disulfide bonds, to produce 3.5S Fab' monovalent fragments.

[0113] Other antibody cleavage methods (such as separation of the heavy chain to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques) can also be used, so long as the fragments bind to the antigen recognized by the intact antibody. Other suitable methods for preparing antigen-binding fragments are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, 2001, 14:1311-1315. nd , Cold Spring Harbor Laboratory, New York, 2013.

[0114] In some embodiments, the antibody is a non-human mammalian or avian antibody (e.g., mouse, rat, guinea pig, hamster, rabbit, chicken, dog, cat, sheep, pig, goat, or horse). In specific, non-limiting examples, the antibody is a murine antibody. In some examples, the antibody is a humanized antibody. In some examples, the antibody is a human antibody. In further examples, the antibody is a chimeric antibody (e.g., an antibody having a variable region from one species (e.g., mouse) and a constant region from another species (e.g., human). In some embodiments, the antibody comprises a constant region. The constant region may comprise at least one modification to increase the half-life, stability, and / or function of the monoclonal antibody.

[0115] The antibody can include any suitable framework region, such as, but not limited to, a human framework region, or an optimized or humanized framework region. Alternatively, a heterologous framework region, such as, but not limited to, a mouse or monkey framework region, can be included in the heavy or light chain of the antibody.

[0116] The antibody can be of any isotype. The antibody can be, for example, an IgA, IgM, or IgG antibody (IgG 1 , IgG 2 , IgG 3 , or IgG 4 The class of an antibody that specifically binds to MCT11 can be switched to another class, for example, an antibody that specifically binds to MCT11 that was originally an IgG can be class switched to IgA. The class can be switched, for example, by changing the constant region portion of the antibody to a different class but keeping the variable region the same. Class switching can be used to convert one IgG subclass to another subclass (IgG 1 From IgG 2 , IgG 3 , or IgG 4 It can be converted to (e.g., to ).

[0117] The antibody can be derivatized or linked to another molecule (such as another peptide or protein). In some examples, the antibody is linked (such as by chemical coupling, genetic fusion, or non-covalent association) to one or more other molecules (such as another antibody (e.g., bispecific antibody or diabody), detectable marker, effector molecule, or protein or peptide) that can mediate the association of the antibody or antibody part with another molecule (such as streptavidin core region or polyhistidine tag). In general, the antibody or antigen-binding fragment is derivatized so that binding to MCT11 is not adversely affected by derivatization or labeling.

[0118] In some examples, the disclosed antibodies are oligomers (such as dimers, trimers, tetramers, pentamers, hexamers, septamers, and octomers).

[0119] In some embodiments, the antibody is at a concentration of 1.0×10 -8 M or less, 5.0×10 -8 M or less, 1.0×10 -9 M or less, 5.0×10 -9 M or less, 1.0×10 -10 M or less, 5.0×10 -10 M or less, 1.0×10 -11 M or less, or 5.0 x 10 -11 Affinity below M (e.g., K D (measured by the equilibrium dissociation constant between the antibody and its antigen). In some instances, D The value is about 10 -4 ~10 -6 M, 10 -7 ~10 -9 M, 10 -10 ~10 -12 M, 10 -13 ~10 -15 M, 10 -6 ~10 -9 M, 10 -5 ~10 -6 M, 10 -5 ~10 -7 M, or 10 -5 ~10 -9 It is M. D can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed using the Fab version of the antibody of interest and its antigen. Other methods include ELISA-based methods, microscale thermophoresis (MST), surface plasmon resonance (SPR), and biolayer interferometry (BLI). In some examples, K Dis measured using SPR, e.g., on a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ). See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). In some examples, K D , using two or more methods (e.g., K by both SPR and BLI) D (by determining

[0120] I. Multispecific antibodies Also disclosed are multispecific antibodies (e.g., bispecific antibodies) that include the antibodies that specifically bind to MCT11 disclosed herein. In some examples, the multispecific antibodies include at least one other antibody that targets an antigen that is not an MCT11 antigen (e.g., an antibody that targets PD-1, 4-1BB / CD137, GITR, OX40, CD105, LAG3, TIM-3 / HAVCR2, NRP1, or FAS). In some examples, the multispecific antibodies include at least one other antibody that targets a T cell antigen, for example, for the multispecific antibodies to target T cells. In further examples, the multispecific antibodies include the MCT11-specific monoclonal antibodies disclosed herein and at least one other antibody that targets a different antigen of MCT11.

[0121] Any suitable method can be used to design and produce multispecific antibodies, such as crosslinking two or more antibodies of the same or different types (e.g., crosslinking a monoclonal antibody disclosed herein and an antibody that binds to another antigen) or crosslinking antigen-binding fragments (such as scFvs). Exemplary methods for making multispecific antibodies include the methods described in PCT Publication No. WO2013 / 163427. Non-limiting examples of suitable crosslinkers include heterobifunctional crosslinkers (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional crosslinkers (such as disuccinimidyl suberate) that have two separate reactive groups separated by a suitable spacer. Multispecific antibodies can have any suitable format that allows binding to MCT11 by the antibodies provided herein.

[0122] Bispecific single chain antibodies can be encoded by a single nucleic acid molecule.Non-limiting examples of bispecific single chain antibodies and methods of constructing such antibodies are provided in U.S. Patent Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, and 6,723,538. Further examples of bispecific single chain antibodies can be found in PCT Application No. WO 99 / 54440; Mack et al., J. Immunol., 158(8):3965-3970, 1997; Mack et al., Proc. Natl. Acad. Sci. USA, 92(15):7021-7025, 1995; Kufer et al., Cancer Immunol. Immunother., 45(3-4):193-197, 1997; Loeffler et al., Blood, 95(6):2098-2103, 2000; and Bruhl et al., J. Immunol., 166(4):2420-2426, 2001. The production of bispecific Fab-scFv ("bibody") molecules has been described, for example, by Schoonjans et al. (J. Immunol., 165(12):7050-7057, 2000) and Willems et al. (J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 786(1-2):161-176, 2003). For bibodies, scFv molecules can be fused to one of the VL-CL(L) or VH-CH1 chains, for example, to produce a bibody in which one scFv is fused to the C-terminus of a Fab chain.

[0123] The outermost or N-terminal variable domain is designated VD1 and the innermost variable domain is designated VD2; VD2 is proximal to the C-terminal CH1 or CL. DVD-immunoglobulin molecules can be produced in large quantities and purified to homogeneity, and are distinct from conventional IgG1 and demonstrates in vivo efficacy. Any of the disclosed monoclonal antibodies can be included in a DVD-immunoglobulin format.

[0124] II. Antibody Conjugates The antibodies, antigen-binding fragments, or multispecific antibodies (e.g., bispecific antibodies) disclosed herein can be conjugated to an agent, such as an effector molecule or detectable marker. The effector molecule or detectable marker can be covalently or non-covalently attached to the disclosed antibodies, antigen-binding fragments, or multispecific antibodies. A variety of effector molecules and detectable markers can be used, and the choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell, and the desired biological effect.

[0125] In some examples, the effector molecule is a receptor or receptor fragment (including an artificial receptor). In a non-limiting example, the antibody conjugate is a chimeric antigen receptor (CAR) comprising a MCT11-specific antigen-binding fragment (e.g., an scFv specific for MCT11 disclosed herein).

[0126] In some examples, the effector molecule is a drug (e.g., an antibody-drug conjugate). Exemplary drugs include antiviral agents (e.g., remdesivir, galidesivir, arbidol, favipiravir, baricitinib, or lopinavir / ritonavir), anti-microtubule agents (e.g., maytansinoids, auristatin E, and auristatin F), interchain crosslinkers (e.g., pyrrolobenzodiazepines; PBDs), the calicheamicin family (e.g., ozogamicin), topoisomerase inhibitors (e.g., govitecan / exetecan), PD-1 inhibitors, T cell agonists, or bacterial toxins (e.g., PE38). In some cases, the ADC is a bispecific ADC composed of two monoclonal antibodies or antigenic fragments thereof (each directed against a different antigen or epitope) conjugated with a drug.

[0127] Detectable markers are, for example, markers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (such as CT, computerized axial tomography (CAT), MRI, magnetic resonance tomography (MTR), ultrasound, fiber optic examination, and laparoscopic examination). Non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, and heavy metals or compounds (such as superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds (such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalene sulfonyl chloride, phycoerythrin, and lanthanide phosphors). Bioluminescent markers (such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP)) are also useful. The antibody, antigen-binding fragment, or multispecific antibody can also be conjugated with enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When the antibody is conjugated to a detectable enzyme, the antibody can be detected by adding an additional reagent that the enzyme uses to produce a reaction product that can be identified. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a colored reaction product that can be visually detected. The antibody, antigen-binding fragment, or multispecific antibody can also be conjugated with biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated with an enzyme or fluorescent label.

[0128] The antibody, antigen-binding fragment, or multispecific antibody may be conjugated with a paramagnetic agent, such as gadolinium. Paramagnetic agents, such as superparamagnetic iron oxide, are also useful as labels. The antibody may be conjugated with lanthanides, such as europium and dysprosium, and manganese. The antibody, antigen-binding fragment, or multispecific antibody may also be conjugated with a predetermined polypeptide epitope recognized by a secondary reporter, such as a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag, or the like.

[0129] The antibody, antigen-binding fragment, or multispecific antibody can also be conjugated with a radioactively labeled amino acid, for example, for diagnostic purposes. For example, the radioactive label can be used to detect exhausted T cells by radiography, emission spectroscopy, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes: 3 H, 14 C. 35 S, 90 Y, 99m Tc, 111 In, 125 I, 131 I. Radiolabels may be detected, for example, using photographic film or a scintillation counter, and fluorescent markers may be detected by irradiating light using a photodetector. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0130] The average number of detectable marker moieties per antibody, antigen-binding fragment, or multispecific antibody in the conjugate can range, for example, from 1 to 20 moieties per antibody or antigen-binding fragment. In some embodiments, the average number of effector molecule moieties or detectable marker moieties per antibody or antigen-binding fragment in the conjugate ranges from about 1 to about 2, about 1 to about 3, about 1 to about 8; about 2 to about 6; about 3 to about 5; or about 3 to about 4. The loading of the conjugate (e.g., effector molecule ratio per antibody) may be adjusted in different ways, for example, by (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent compared to the antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) some limited or limited reducing conditions for modification of cysteine ​​thiols, (iv) engineering the amino acid sequence of the antibody by recombinant techniques such that the number and position of cysteine ​​residues are modified to adjust the number or position of linker-effector molecule bonds.

[0131] The procedure for attaching an effector molecule or detectable marker to an antibody, antigen-binding fragment, or multispecific antibody varies depending on the chemical structure of the effector or detectable marker. Polypeptides typically have a variety of functional groups (carboxyl groups (-COOH), free amine groups (-NH 2), or sulfhydryl groups (-SH). Alternatively, the antibody, antigen-binding fragment, or multispecific antibody is derivatized to expose or attach additional reactive functional groups. Derivatization may include attachment of any suitable linker molecule. The linker may form a covalent bond with both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody, antigen-binding fragment, or multispecific antibody and the effector molecule or detectable marker are polypeptides, the linker may be linked to the constituent amino acids through their side chains (such as via a disulfide bond to cysteine) or alpha carbons, or through the amino and / or carboxyl groups of the terminal amino acids.

[0132] Considering the numerous methods that have been reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies, a suitable method for attaching a given agent to an antibody, antigen-binding fragment, or multispecific antibody (e.g., bispecific antibody) can be determined.

[0133] III. Variants In some embodiments, amino acid sequence variants of the antibodies, antigen-binding fragments, and multispecific antibodies (e.g., bispecific antibodies) disclosed herein are provided. For example, it may be desirable to improve binding affinity and / or other biological properties. Amino acid sequence variants may be included in the V and VF of an antibody. H Domain and / or V LThe domain may be prepared by introducing suitable modifications into the nucleotide sequence encoding the domain or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct retains the desired characteristics (e.g., specific binding to MCT11, such as human MCT11).

[0134] In some embodiments, variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDR and framework regions. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity (e.g., retained / improved antigen binding, reduced MCT11 activity, or improved T-cell specificity). Variants typically have the correct folding and V H Area and V L Retain the amino acid residues necessary for stabilization between the V domains and the charge characteristics of the residues to maintain the low pI and low toxicity of the molecule. H Area and V L Amino acids in the region can be substituted.

[0135] In some embodiments, the V of the antibody H contains up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs: 1. In some embodiments, the V L contains up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs: 5. In some examples, the amino acid substitutions are not within the CDR sequences (are outside the CDR regions). In such embodiments, the variant antibody retains specific binding of MCT11.

[0136] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the antigen-binding ability of the antibody. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. In some examples, substitutions, insertions, or deletions in the CDRs increase binding affinity to MCT11. Variant V as provided above H Array and V L In some embodiments of the sequences, each CDR contains no more than one, no more than two, or no more than three amino acid substitutions. In some examples, the CDRs contain no substitutions. Variant V H Array and V L In some embodiments of the sequences, only framework residues are altered, thereby leaving the CDRs unchanged.

[0137] To increase the binding affinity of the antibody, V L Segment and V H Segments can be randomly mutated, such as within the heavy or light chain CDR3 regions, in a process similar to the in vivo somatic mutation process responsible for affinity maturation of antibodies during natural immune responses. Thus, in vitro affinity maturation can be performed by PCR primers complementary to the heavy or light chain CDR3, respectively, to the V H Area and V L This can be done by amplifying a region of the V H and / or V L Random mutations have been introduced into the CDR3 region of V H Segment and V L Certain positions are "spiked" with random mixtures of the four nucleotide bases to encode the V H Segment and V L The segments can be tested to determine their binding affinity to MCT11. H The amino acid sequence of V is SEQ ID NO:1. LThe amino acid sequence of is SEQ ID NO:5.

[0138] In some embodiments, the antibody, antigen-binding fragment, or multispecific antibody is altered to increase or decrease the extent to which the antibody or antigen-binding fragment is glycosylated. Glycosylation sites may be added or deleted by altering the amino acid sequence such that one or more glycosylation sites are created or removed. If the antibody contains an Fc region, the carbohydrate attached to this region may be altered. Native antibodies produced by mammalian cells typically have a CH of the Fc region. 2 The antibody comprises a biantennary branched oligosaccharide that is generally linked by an N-linkage to Asn297 of the domain. In some examples, the antibody (e.g., an antibody specific for MCT11) comprises an Fc (e.g., human IgG1 Fc or human IgG4 fc), an afucosylated Fc, or a non-FcR-binding Fc. See, e.g., Wright et al. Trends Biotechnol. 15(1):26-32, 1997. The oligosaccharide may comprise various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibody may be modified to create an antibody variant with certain improved properties.

[0139] Further provided are antibody variants having bisected oligosaccharides (e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc). Such antibody variants may be reduced in fucosylation. In some examples, the antibody specific for MCT11 comprises an afucosylated Fc. Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region.

[0140] In some embodiments, the constant region of the disclosed antibody comprises one or more amino acid substitutions to optimize the in vivo half-life of the antibody. The serum half-life of IgG Abs is controlled by the neonatal Fc receptor (FcRn). Thus, in some embodiments, the antibody comprises amino acid substitutions that increase binding to FcRn. Non-limiting examples of such substitutions include the substitutions T250Q and M428L in the IgG constant region (see, e.g., Hinton et al., J Immunol., 176(1):346-356, 2006); M428L and N434S ("LS" mutations, see, e.g., Zalevsky, et al., Nature Biotechnol., 28(2):157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18(12):1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18(12):1759-1769, 2006). al., Int. Immunol., 18(12):1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall'Acqua et al., J. Biol. Chem., 281(33):23514-23524, 2006). The disclosed antibodies and antigen-binding fragments can be linked to or include an Fc polypeptide that includes any of the substitutions listed above (e.g., an Fc polypeptide can include an M428L substitution and an N434S substitution).

[0141] In some embodiments, the antibodies disclosed herein may be further modified to include additional non-proteinaceous moieties. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing because it is stable in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and when more than one polymer is attached, the polymers can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations, including but not limited to, the particular property or function of the antibody to be improved, such as whether the antibody derivative will be used in an application under defined conditions.

[0142] IV. Additional Explanation Antibodies and antigen-binding fragments that specifically bind to the same epitope (e.g., the same MCT11 epitope bound by the disclosed monoclonal antibodies) can be identified and isolated, for example, by screening combinatorial libraries for antibodies with the desired binding characteristics. In some examples, phage display libraries are generated and screened for antibodies possessing the desired binding characteristics (e.g., binding of MCT11). Such methods are described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001); McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al. al.,J.Mol.Biol.222:581-597(1992);Marks and Bradbury,in Methods in Molecular Biology 248:161-175(Lo,ed.,Human Press,Totowa,NJ,2003);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al. al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0143] In certain phage display methods, V H Genes and V LThe repertoire of genes can be individually cloned by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages, for example, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immune sources provide antibodies that exhibit high affinity to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self antigens as well as self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be synthesized by cloning unrearranged V-gene segments from stem cells and using PCR primers that code for highly variable CDR3 regions and contain random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications that describe human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360. Competitive binding assays similar to those disclosed in the Examples section below can be used to select antibodies with desired binding characteristics.

[0144] In some examples, an antibody that binds to an epitope of interest (e.g., an MCT11 epitope) can be identified based on the ability of the antibody to cross-compete with (e.g., competitively inhibit the binding of in a statistically significant manner) an antibody provided herein in a binding assay.

[0145] Human antibodies that bind to the same epitope of MCT11 (e.g., the same MCT11 epitope that the disclosed monoclonal antibody binds) can be produced using any suitable method. Such antibodies can be prepared, for example, by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces the endogenous immunoglobulin locus or is extrachromosomal or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). (See, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology, and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology.) The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combination with a different human constant region.

[0146] In addition, additional human antibodies that bind to the same epitope can be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for human monoclonal antibody production have been described (see, for example, Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147:86 (1991)). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Further methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains.

[0147] B. Polynucleotides and Expression Nucleic acid molecules (e.g., DNA, cDNA, or RNA (e.g., mRNA)) encoding the amino acid sequences of the antibodies, antigen-binding fragments, multispecific antibodies (e.g., bispecific antibodies), or conjugates that specifically bind to MCT11 as described herein are also provided. Nucleic acids encoding these molecules can be prepared by the methods described herein using the amino acid sequences provided herein (V-VIII listed in Table 1). H Array and V L The antibody sequences can be easily generated using sequences available in the art (such as framework or constant region sequences and each CDR sequence), and the genetic code. Using the genetic code, various functionally equivalent nucleic acid sequences (which may differ in sequence but encode the same antibody sequence, or may be a combination of V and V sequences) can be generated. L and / or V H A conjugate or fusion protein comprising the nucleic acid sequence can be constructed, such as a nucleic acid encoding the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 2, 3, and 4, respectively, and the light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 6, 7, and 8, respectively. In some embodiments, the nucleic acid molecule encodes the V and V sequences of SEQ ID NOs: 1 and 5, respectively. H , V L , or V H and V L Code both.

[0148] Also disclosed are degenerate variants of the disclosed nucleic acid sequences. Silent mutations of coding sequences are due to the degeneracy (i.e. redundancy) of genetic code, whereby one or more codons can code for the same amino acid residue. Thus, for example, leucine can be coded for by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be coded for by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be coded for by AAT or AAC; aspartic acid can be coded for by GAT or GAC; cysteine ​​can be coded for by TGT or TGC; alanine can be coded for by GCT, GCC, GCA, or GCG; glutamine can be coded for by CAA or CAG; tyrosine can be coded for by TAT or TAC; isoleucine can be coded for by ATT, ATC, or ATA.

[0149] The disclosed nucleic acid sequences can be codon-optimized for expression in a particular host cell (such as human, mouse, or bacteria). By using a table of codon preferences and codon usage frequencies for a particular species, the codon usage preferences of the particular species can be utilized to engineer isolated nucleic acid molecules that code for protein products (such as the disclosed monoclonal antibodies). For example, nucleic acids can be designed to have codons that are preferentially used by a particular organism of interest (e.g., an organism for expressing a monoclonal antibody). In some examples, nucleic acids are codon-optimized for expression in humans. In some examples, nucleic acids are codon-optimized for a particular protein expression system (such as a bacterial, yeast, insect, or CHO expression system).

[0150] The disclosed nucleic acids can be prepared by any suitable method, including, for example, cloning of appropriate sequences or direct chemical synthesis by standard methods. In some examples, the disclosed nucleic acids are prepared by cloning techniques. Examples of suitable cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4 thed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, with supplements). Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), self-sustained sequence replication system (3SR), and Qβ replicase amplification system (QB). A wide variety of cloning and in vitro amplification methodologies have been previously described. In some examples, the disclosed nucleic acids are prepared by direct chemical synthesis. Numerous chemical synthesis methods have been described (e.g., the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethyl phosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid-phase phosphoramidite triester method of Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, using an automated synthesizer, e.g., as described in Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and the solid-support method of U.S. Pat. No. 4,458,066). Chemical synthesis produces single-stranded oligonucleotides. This can be converted to double-stranded DNA by hybridization with a complementary sequence, or by polymerization with DNA polymerase using the single strand as a template. Chemical synthesis of DNA is generally limited to sequences of about 100 bases, although longer sequences may be obtained by ligation of shorter sequences.

[0151] In some embodiments, the disclosed nucleic acids are included in an expression vector (e.g., a viral vector, a plasmid, or other vehicle) for expression in a host cell (e.g., a human cell, a T cell, an exhausted T cell, a protein-expressing cell (e.g., bacteria, insect, yeast, Chinese hamster ovary (CHO), human embryonic kidney (HEK293)). In some examples, the vector includes a selection marker (an antibiotic resistance gene (e.g., puromycin) or a reporter gene (e.g., green fluorescent protein (GFP) and the like). In other examples, the selection marker and / or reporter is not included in the vector. In some examples, the expression vector includes a promoter operably linked to the disclosed nucleic acid molecule. For example, the promoter can be used to express a monoclonal antibody, antigen-binding fragment, multispecific antibody, or antibody specific for MCT11 disclosed herein. It can be operably linked to the nucleic acid encoding the conjugate. The promoter can be constitutive or inducible. The promoter can be any promoter of interest, including the cytomegalovirus promoter. A typical expression vector can include sequences useful for controlling the expression of DNA encoding a protein (e.g., the disclosed MCT11-specific antibody), such as a suitable promoter, enhancer, transcription and translation terminator, initiation sequence, a start codon (i.e., ATG) in front of the protein-coding gene, splicing signals of introns, sequences for maintaining the correct reading frame of the gene to allow proper translation of mRNA, and a stop codon. The vector can encode a selection marker, such as a marker encoding drug resistance (e.g., ampicillin resistance or tetracycline resistance).

[0152] In some embodiments, the disclosed nucleic acid is contained in a viral vector. Exemplary viral vectors that can be used include, but are not limited to, polyoma, SV40, adenovirus, vaccinia virus, adeno-associated virus (AAV), herpesvirus (including HSV and EBV), Sindbis virus, alphavirus, and retrovirus of avian, murine, and human origin. Baculovirus (Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors can also be used. Other suitable vectors include orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, lentivirus vectors, alphavirus vectors, and poliovirus vectors. Specific examples of vectors are poxvirus vectors, such as vaccinia virus, fowlpox virus, and highly attenuated vaccinia virus (MVA), adenovirus, and baculovirus. Useful poxviruses include orthopox, suipox, avipox, and capripox viruses. Orthopox include vaccinia, ectromelia, and raccoon pox. One example of a useful orthopox is vaccinia. Avipox includes fowlpox, canarypox, and pigeonpox. Capripox includes goatpox and sheeppox. In one example, suipox is swinepox. Other viral vectors that can be used include other DNA viruses (such as herpesviruses and adenoviruses), and RNA viruses (such as retroviruses and polio). Biologically functional viral and plasmid DNA vectors capable of expression and replication in cells (e.g., T cells, exhausted T cells, bacteria, yeast, insect, CHO, HEK293, human) are known, and those skilled in the art can identify suitable vectors.

[0153] The disclosed nucleic acids or vectors encoding the disclosed nucleic acids can be expressed in a host cell in vitro or in a cell of a host organism in vivo by DNA transfer into the cell. In some examples, the nucleic acids or vectors disclosed herein are expressed in vitro. The host cell can be a prokaryotic or eukaryotic host cell. Numerous expression systems are available for protein expression, including E. coli, other bacterial hosts, yeast, insect, or various eukaryotic cells (COS, Chinese Hamster Ovary (CHO), HeLa, myeloma cells, human embryonic kidney (HEK293) lines, etc.). Any of these cell lines can be used to express the antibodies, antigen-binding fragments, multispecific antibodies, or conjugates disclosed herein. In some embodiments, the host cell is a T cell or an exhausted T cell. Stable transfer methods (meaning that the foreign DNA is continuously maintained in the host) can be used.

[0154] For optimal expression of the disclosed nucleic acids, the expression cassette or vector can contain (can use), for example, a strong promoter to direct transcription, a ribosome binding site for translation initiation (e.g., internal ribosome binding sequence), and a transcription / translation terminator. For expression in bacterial cells (such as E. coli), a promoter (such as T7, trp, lac, or lambda promoter), a ribosome binding site, and preferably a transcription termination signal can be used. For eukaryotic cells, such as humans, the regulatory sequences can include, for example, promoters and / or enhancers from immunoglobulin genes, HTLV, SV40, or cytomegalovirus, and polyadenylation sequences, and can further include splice donor and / or splice acceptor sequences (e.g., CMV and / or HTLV splice acceptor and donor sequences). Additional operational elements include, but are not limited to, leader sequences, termination codons, polyadenylation signals, and any other sequences necessary for proper transcription and subsequent translation of the nucleic acid sequence.

[0155] The disclosed nucleic acid or vector can be introduced into a host cell by any suitable method (e.g., transformation). Numerous transformation methods are known, such as: chemical methods (e.g., calcium phosphate transfection), physical methods (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), lipofection, nucleofection, receptor-mediated endocytosis (e.g., DNA-protein complexes, viral envelope / capsid-DNA complexes), particle gun acceleration methods (gene guns), or biological infection with viruses, such as recombinant viruses (Wolff, JA, ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)). In the case of infection with retroviruses, infectious retroviral particles are absorbed by target cells, which reverse transcribes the retroviral RNA genome, and the resulting provirus is integrated into cellular DNA. Successfully transformed cells can be selected by antibiotic resistance conferred by genes (such as amp, gpt, neo, and hyg genes) contained in the vector.

[0156] The disclosed nucleic acids can be modified without reducing the biological activity of the encoded product. For example, they can be modified to facilitate the cloning, expression, or incorporation into fusion proteins of target molecules. Such modifications include, for example, termination codons, sequences for creating conveniently located restriction sites, and sequences for adding methionine to the amino terminus to provide an initiation site, or additional amino acids (such as polyHis) to aid in purification steps.

[0157] Antibodies, antigen-binding fragments, multispecific antibodies, and conjugates can be expressed as separate proteins comprising a VH and / or a VL (optionally linked to an effector molecule or detectable marker) or can be expressed as fusion proteins. Immunoadhesins can also be expressed. Thus, in some instances, the V H and VL (For example, V comprising the CDR sequences of SEQ ID NOs: 2, 3, and 4) H and V comprising the CDR sequences of SEQ ID NOs: 6, 7, and 8. L ) as well as a nucleic acid encoding the immunoadhesin. The nucleic acid sequence can optionally encode a leader sequence.

[0158] To generate scFv, V H and V L The DNA fragment encoding V H Array and V L The sequences are joined by a flexible linker L Domains and V H A flexible linker is encoded (e.g., the amino acid sequence (Gly)) so that the protein can be expressed as a continuous single chain protein having the domains. 4 -Ser) 3 (encoding a polypeptide) can be operably linked to another fragment encoding a polypeptide (e.g., Bird et al., Science, 242(4877):423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA, 85(16):5879-5883, 1988; McCafferty et al., Nature, 348:552-554, 1990; Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2050, 2051, 2052, 2053, 2054, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080 nd ed., Springer-Verlag, 2010; Greenfield (Ed.), Antibodies: A Laboratory Manual, 2 nd ed. New York: Cold Spring Harbor Laboratory Press, 2014). Optionally, a cleavage site (such as a furin cleavage site) can be included in the linker. scFvs are monovalent (e.g., a single V H and V L ), bivalent (only when using two V H and V L ), or multivalent (more than two V H and VL Multispecific or multivalent antibodies can be generated that specifically bind MCT11 and another antigen. H and V L If necessary, V H Domain and V L A furin cleavage site can be included between the domains. A linker can also be encoded, such as in the case where the nucleic acid molecule encodes a bispecific antibody in the DVD-Ig™ format.

[0159] Any suitable antibody and antigen-binding fragment expression and isolation method can be used; including ammonium sulfate precipitation, affinity columns, and column chromatography (see generally Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). Further non-limiting examples are provided in Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011. An isolated antibody, antigen-binding fragment, multispecific antibody, or conjugate need not be 100% pure. In some examples, an isolated antibody is substantially free of harmful contaminants (e.g., compositions that are harmful when administered to humans).

[0160] Methods for the expression of antibodies, antigen-binding fragments, multispecific antibodies, and conjugates from mammalian cells and bacteria (such as E. coli), and / or for refolding into appropriate active forms have been described and are applicable to the antibodies disclosed herein. See, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2001, 144:1311-1325, 1999. nded. New York: Cold Spring Harbor Laboratory Press, 2014, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009, and Ward et al., Nature 341(6242):544-546, 1989.

[0161] C. Compositions and Dosages Compositions are provided that include one or more of the disclosed monoclonal antibodies, antigen-binding fragments, multispecific antibodies, conjugates, nucleic acid molecules or vectors encoding such molecules in a pharma- ceutically acceptable carrier. In some embodiments, the compositions include a monoclonal antibody specific for MCT11, or an antigen-binding fragment thereof. The compositions are useful, for example, for treating cancer (or tumor), increasing T cell effector function, increasing resistance to T cell exhaustion (or conversely decreasing T cell exhaustion), increasing a subject's response to immunotherapy, or a combination of these effects. In some examples, the compositions are useful for increasing a subject's response to immunotherapy, for example, increasing the response to checkpoint inhibitors, oncolytic viruses (e.g., T-VEC), or adoptive cell transfer (ACT) therapy. In some examples, the checkpoint inhibitor is an inhibitor that targets PD-1, CTLA-4, CDK4, CDK6, and / or its ligand. In specific, non-limiting examples, checkpoint inhibitors are ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, palbociclib, ribociclib, and abemaciclib.

[0162] The composition can be prepared in a unit dosage form, such as in a kit for administration to a subject.The amount and timing of administration are left to the discretion of the administering physician to achieve the desired purpose.The composition can be formulated for systemic or local administration.In one example, the antigen-binding fragment, multispecific antibody, conjugate, or nucleic acid molecule encoding such molecule is formulated for parenteral administration (such as intravenous administration).

[0163] In some embodiments, the composition is at least 70% pure (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 99.99%). In some embodiments, the composition contains less than 10% (such as less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less) of contaminants (such as other proteins or macromolecules (e.g., human, bacterial, yeast)).

[0164] The composition may be a solution of MCT11 antibody, antigen-binding fragment, multispecific antibody (e.g., bispecific antibody), conjugate, or nucleic acid molecule or vector encoding such molecule dissolved in a pharma- ceutically acceptable carrier (such as an aqueous carrier). A variety of aqueous carriers (e.g., buffered saline, etc.) may be used. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by any suitable technique. The composition may include pharma- ceutically acceptable auxiliary agents (e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.) necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, etc. The concentration of the antibody in these formulations may vary widely and is selected primarily based on fluid volume, viscosity, and body weight, etc., according to the particular mode of administration selected and the needs of the subject.

[0165] Any suitable method may be used to prepare the administrable compositions; non-limiting examples include those described in Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013. In some embodiments, the composition is a liquid formulation, and one or more of the disclosed MCT11 antibodies, antigen-binding fragments, multispecific antibodies, or conjugates are administered at a concentration of about 0.1 mg / ml to about 50 mg / ml, e.g., about 0.5 mg / ml to about 50 mg / ml, or about 1 mg / ml to about 50 mg / ml, about 5 mg / ml to about 50 mg / ml, about 10 mg / ml to about 50 mg / ml, about 15 mg / ml to about 50 mg / ml, about 20 mg / ml to about 50 mg / ml, about 25 mg / ml to about 50 mg / ml, about 30 mg / ml to about 50 mg / ml, about 35 mg / ml to about 50 mg / ml, about 40 ... The concentration is from about 5 mg / ml to about 50 mg / ml, from about 0.1 mg / ml to about 25 mg / ml, from about 0.5 mg / ml to about 25 mg / ml, from about 1 mg / ml to about 25 mg / ml, from about 5 mg / ml to about 25 mg / ml, from about 0.1 mg / ml to about 15 mg / ml, from about 0.5 mg / ml to about 15 mg / ml, from about 1 mg / ml to about 15 mg / ml, from about 5 mg / ml to about 15 mg / ml, from about 10 mg / ml to about 25 mg / ml, from about 15 mg / ml to about 25 mg / ml, from about 20 mg / ml to about 25 mg / ml, from about 10 mg / ml to about 40 mg / ml, from about 10 mg / ml to about 30 mg / ml, or from about 15 mg / ml to about 30 mg / ml. In specific, non-limiting examples, the composition comprises about 10-25 mg / ml or about 50-200 mg / ml of the MCT11 monoclonal antibody, antigen-binding fragment, multispecific antibody, or conjugate disclosed herein.

[0166] In some embodiments, the composition comprises one or more of the disclosed MCT11 antibodies, antigen-binding fragments, multispecific antibodies, or conjugates at about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 2 In some examples, the composition comprises at least 1 mg, at least 10 mg, at least 25 mg, at least 50 mg, at least 100 mg, at least 200 mg, or at least 500 mg, e.g., 1 mg to 1 g, e.g., 1 mg to 500 mg, or 10 to 100 mg, of one or more of the disclosed MCT11 antibodies, antigen-binding fragments, multispecific antibodies, or conjugates.

[0167] Compositions comprising the disclosed MCT11 antibodies, antigen-binding fragments thereof, multispecific antibodies, conjugates, nucleic acids or vectors encoding such molecules can be provided in lyophilized form and rehydrated with a suitable sterile solution prior to administration. The solution can then be added to an infusion bag containing 0.9% sodium chloride (USP) and typically administered at a dosage of 0.5-15 mg / kg body weight. Since the approval of rituximab in 1997, there has been considerable experience in the art in administering antibody drugs marketed in the United States. The compositions can be administered by slow infusion rather than intravenous infusion or bolus. In one example, a higher loading dose is administered, followed by a lower level maintenance dose. For example, an initial loading dose of 4 mg / kg can be infused over approximately 90 minutes, followed by weekly infusions of 2 mg / kg over 30 minutes for 4-8 weeks if the previous dose was well tolerated.

[0168] Modified release parenteral formulations can be made as implants, oily injections, or as particulate systems. For an overview of protein delivery systems, see Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Lancaster, PA: Technomic Publishing Company, Inc., 1995. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain an active protein agent (such as a cytotoxin or drug) as a central core. In microspheres, the active protein agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively, and can be administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, Colloidal Drug Delivery Systems, J. Kreuter (Ed.), New York, NY: Marcel Dekker, Inc., pp. 219-342, 1994; and Tice and Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization, Applications, A. Kydonieus (Ed.), New York, NY: Marcel Dekker, Inc., pp. 315-339, 1992.

[0169] Polymers can be used for the ion-regulated release of the compositions disclosed herein.Any suitable polymer can be used, such as degradable or non-degradable polymer matrices designed for use in regulated drug delivery.Alternatively, hydroxyapatite is used as a microcarrier for the regulated release of proteins.In yet another embodiment, liposomes are used for the regulated release and drug targeting of lipid-encapsulated drugs.

[0170] D.How to use Disclosed herein is a method of using MCT11 antibodies (such as one or more of those provided herein) to treat T cell exhaustion or increase T cell effector function. For example, a therapeutically effective amount of one or more MCT11 antibodies (such as one or more of those provided herein) can be administered to a subject (such as a subject with a cancer, such as a cancer that can be treated with immunotherapy (e.g., ACT therapy). In some examples, a single dose of MCT11 antibody (or a fragment or conjugate thereof) is administered. In some examples, multiple doses of MCT11 antibody (or a fragment or conjugate thereof) are administered, such as at least two, at least three, at least four, at least five, or more separate doses. In some examples, a nucleic acid molecule (such as a vector encoding MCT11 antibody (or a fragment or conjugate thereof)) is administered (e.g., in one or more separate doses, e.g., in at least two, at least three, at least four, at least five, or more separate doses).

[0171] In some examples, the MCT11 antibody used in the disclosed methods is one provided herein (e.g., a monoclonal MCT11 antibody or an antigen-binding fragment specific for MCT11). In a non-limiting example, the MCT11 antibody used in the disclosed methods specifically binds to monocarboxylate transporter 11 (MCT11) and has a variable heavy chain (VH) of SEQ ID NO: 1, respectively. H ) domain and the variable light chain (V L In a further example, the MCT11 antibody used in the disclosed methods specifically binds to monocarboxylate transporter 11 (MCT11) and comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and a variable heavy chain (VH) domain of SEQ ID NOs: 2, 3, and 4, respectively. H5, and a variable light chain (V) of SEQ ID NO: 6, 7, and 8, respectively; L ) CDR1, CDR2, and CDR3.

[0172] In some examples, the MCT11 antibody used in the disclosed methods is a commercially available MCT11 antibody (such as an MCT11 antagonistic antibody or an antigen-binding fragment specific for MCT11) (e.g., anti-MCT11 antibody (G-4) sc-515145 from Santa Cruz Biotechnology; anti-SLC16A11 (item number: 041802-APC.200) from USBiological Life Science; anti-MCT11 (catalog number: MBS8292652) from MyBioSource; MCT-MCT11 (ab230845) from Abcam; anti-MCT1 / monocarboxylate transporter 1 antibody (ERR13706(B)) (Ab179832) from Abcam; SLC16A11 polyclonal antibody (catalog number: PA5-98710) from Thermo; SLC16A11 antibody (catalog number: Orb376095) from Biorbyt). In some examples, the MCT11 antibody is part of an antibody-drug conjugate (ADC) or a bispecific antibody that specifically binds to MCT11 and another antigen. The other antigen can be a T cell specific antigen. Examples of other antigens include, but are not limited to: PD-1, 4-1BB / CD137, GITR, OX40, CD105, LAG3, TIM-3 / HAVCR2, NRP1, or FAS.

[0173] In some examples, the method includes contacting a T cell with an effective amount of a monoclonal antibody disclosed herein or expressing a nucleic acid molecule or vector encoding a monoclonal antibody disclosed herein in the T cell, thereby reducing T cell exhaustion or increasing effector function of the T cell. In some examples, the contacting includes administering to a subject having exhausted T cells or T cells with reduced effector function. In some examples, the subject has cancer or is receiving or will receive immunotherapy (e.g., abemaciclib, atezolizumab, avelumab, axicabtagene ciloreucel, brinotumumab, semipilimab, durvalumab, yelamirimab, ipilimumab, nivolumab, palbociclib, pembrolizumab, pidilizumab, relatolimab, ribociclib, urelemab, utolimumab, adoptive cell transfer (ACT) therapy (e.g., chimeric antigen receptor (CAR) (e.g., tisagenlecleucel)), or engineered TCR or tumor infiltrating lymphocytes (TIL)), and oncolytic viruses (e.g., talimogene laherparepvec (T-VEC)).

[0174] In some examples, the T cells are isolated from a subject (e.g., a donor subject, or a subject with exhausted T cells or T cells with reduced effector function) prior to the contacting step. In some examples, the T cells are peripheral blood mononuclear cells (PBMCs). In some examples, the T cells are tumor infiltrating lymphocytes (TILs). In some examples, the T cells are reactive to tumor-specific antigens (e.g., one or more of CD19, CD20, BCMA, MUC1, PSA, CEA, HER1, HER2, TRP-2, EpCAM, GPC3, mesothelin 1 (MSLN), or EGFR). In some examples, the T cells are exhausted T cells. In some examples, the T cells are terminally exhausted T cells. The cells (e.g., PBMCs, T cells, exhausted T cells) can be isolated from a subject (e.g., a blood sample (e.g., a venous blood sample), a biopsy (e.g., a tumor sample), other sample from the subject). Several techniques are known for isolating cells of interest (e.g., density centrifugation (Ficoll approach), isolation by cell preparation tubes (CPT), or isolation by SepMate™ tubes). In some examples, apheresis or leukapheresis is used. Flow cytometry techniques (e.g., FACS) can be used to assess the composition of a cell population (e.g., PBMCs isolated from a subject) to identify cell types such as, for example, monocytes (e.g., CD14), T cells (e.g., CD3, CD8, CD4), B cells (e.g., CD20), or NK cells (e.g., CD56). FACS techniques can also be used to enrich or deplete specific cell types from a cell population (e.g., enrich or deplete cells positive for CD14, CD3, CD8, CD4, CD28, CD20, CD56, TIM3, PD-1, or combinations thereof). In some examples, T cells are isolated from or enriched for T cells in a PBMC sample (e.g., CD3 + Cells or CD8 +In some examples, the sample is enriched by negative selection, e.g., by selecting and removing undesirable cell types (e.g., cell types other than T cells, naive or memory T cells, exhausted T cells) from the sample. In some examples, FACS is used to isolate or enrich for exhausted T cells (e.g., PD-1 hi , TIM3 + T cells) are assessed in the sample, and the sample is thereby selected to enrich for exhausted T cells or conversely, to remove exhausted T cells.

[0175] In a non-limiting example, TILs are first isolated from a tumor sample (e.g., a biopsy) from a subject with cancer or tumor, and then contacted with an effective amount of an MCT11-specific antibody, or alternatively, TILs are modified to express a nucleic acid molecule or vector encoding an MCT11-specific antibody (e.g., one or more of the MCT11-specific antibodies disclosed herein), thereby increasing the effector function of TILs. In such an example, an effective amount can be, for example, an amount that improves the in vitro expansion of TILs (e.g., TILs specific for tumor antigens of a cancer (or tumor) in a subject) and / or improves the effector activity of TILs (e.g., increased cytokine production, increased cytotoxic activity, or decreased expression of T cell exhaustion markers such as TIM3 or PD-1).

[0176] Also disclosed herein is a method for treating cancer or tumor in a subject, increasing response to immunotherapy, or increasing immune response by administering an effective amount of the MCT11 monoclonal antibody disclosed herein; or administering an effective amount of a nucleic acid or vector encoding the MCT11 monoclonal antibody disclosed herein, thereby treating cancer or tumor in the subject, or increasing response to immunotherapy. In a non-limiting specific example, the method is a method for treating cancer or tumor in a subject.

[0177] In some examples, an effective amount is an amount sufficient to prevent, treat, reduce, and / or ameliorate one or more signs or symptoms of cancer (or tumor) in a subject. For example, an amount sufficient to reduce tumor size or tumor mass in a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% as compared to baseline measurements in the same subject or a suitable control. In some examples, an effective amount is an amount sufficient to inhibit or delay metastasis in a subject. For example, by reducing tumor spread in a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% as compared to baseline measurements in the same subject or a suitable control. In some examples, an effective amount is an amount that increases the life expectancy of a subject by, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or more. In other examples, an effective amount is an amount sufficient to reduce tumor density in a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%, for example, compared to a baseline measurement of the same subject or other suitable control. Non-limiting examples of suitable controls include untreated subjects or subjects that have not been administered the monoclonal antibodies or nucleic acids or vectors encoding the monoclonal antibodies disclosed herein (e.g., subjects receiving other drugs or alternative therapies).In further examples, an effective amount is an amount sufficient to target and eliminate tumor cells (e.g., eliminate at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or even 100%, compared to a suitable control).

[0178] In some embodiments, an effective amount of MCT11 monoclonal antibody is an amount that blocks monocarboxylate transport by at least one cell expressing MCT11 (e.g., exhausted T cells, regulatory T cells, or resident memory T cells). In some embodiments, MCT11 monoclonal antibody blocks lactate, pyruvate, ketone bodies, butyrate, propionate, or succinate uptake by at least one cell expressing MCT11 (e.g., exhausted T cells, regulatory T cells, or resident memory T cells).

[0179] In some embodiments, the subject is receiving, has received, or will receive at least one immunotherapy (e.g., abemaciclib, atezolizumab, avelumab, axicabtagene ciloreucel, brinotumumab, semipilimab, durvalumab, yelamirimab, ipilimumab, nivolumab, palbociclib, pembrolizumab, pidilizumab, relatolimab, ribociclib, urelemab, outlimumab, adoptive cell transfer (ACT) therapy (e.g., a chimeric antigen receptor (CAR) (e.g., tisagenlecleucel)), or engineered TCR or tumor infiltrating lymphocytes (TIL)), and an oncolytic virus (e.g., talimogene laherparepvec (T-VEC)). In some examples, the immunotherapy includes adoptive cell transfer (ACT) therapy, such as CAR (e.g., CAR-T or CAR-NK), TCR, or TIL immunotherapy. In a specific, non-limiting example, the immunotherapy is CAR-T therapy. In some examples, the immunotherapy includes a checkpoint inhibitor, such as a checkpoint inhibitor that targets PD-1, PD-L1, CD137, CD223, CTLA-4, CDK4, and / or CDK6. Exemplary checkpoint inhibitors include ipilimumab, urelemab, yelamirimab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, palbociclib, ribociclib, and abemaciclib. In some embodiments, administering the immunotherapy increases the immune response in the subject, e.g., the subject with cancer.

[0180] In some embodiments, the increased response to immunotherapy includes preventing, treating, alleviating, and / or improving one or more signs or symptoms of cancer in a subject. In some embodiments, the effective amount of the monoclonal antibody or nucleic acid or vector encoding the monoclonal antibody disclosed herein is an amount that, when administered with immunotherapy, makes the combination more effective in treating cancer (or tumor) compared to immunotherapy alone; for example, in some examples, the combination is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or more effective (preventing, treating, alleviating, and / or improving one or more signs or symptoms of cancer or tumor) than immunotherapy alone. In some examples, an effective amount is an amount that is synergistic when administered with immunotherapy (e.g., an amount that synergistically prevents, treats, reduces, and / or ameliorates one or more signs or symptoms of cancer).

[0181] In some embodiments, the subject has cancer. In some examples, the subject has a solid tumor or cancer, such as breast cancer (e.g., lobular carcinoma and ductal carcinoma, e.g., triple-negative breast cancer), sarcoma, lung cancer (e.g., non-small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma), lung mesothelioma, colorectal adenocarcinoma, gastric carcinoma, prostate adenocarcinoma, ovarian cancer (such as serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumor, testicular carcinoma and germ cell tumor, pancreatic adenocarcinoma, bile duct adenocarcinoma, hepatocellular carcinoma, bladder cancer (including, e.g., transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma), renal cell adenocarcinoma, endometrial cancer (including, e.g., adenocarcinoma and mixed Mullerian tumor (carcinosarcoma)), endocervical, epicervical, and Cancers of the vagina (such as adenocarcinoma and squamous cell carcinoma of each of these), tumors of the skin (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin adnexal tumor, Kaposi's sarcoma, cutaneous lymphoma, skin adnexal tumor, and various types of sarcoma and Merkel cell carcinoma), esophageal cancer, cancer of the nasopharynx and oropharynx (including squamous cell carcinoma and adenocarcinoma thereof), salivary gland cancer, tumors of the brain and central nervous system (including tumors of glial, neuronal, and meningeal origin), tumors of the peripheral nerves, soft tissue sarcomas and sarcomas of bone and cartilage, head and neck squamous cell carcinoma (HNSCC), and tumors of the lymphatic system (including B-cell and T-cell malignant lymphomas).

[0182] In some examples, the subject has a liquid tumor or cancer, such as lymphatic, leukocyte, or other leukemia types. In specific examples, the tumor to be treated is a hematological tumor, such as a leukemia (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia), a lymphoma (such as Hodgkin's lymphoma or non-Hodgkin's lymphoma), or a myeloma.

[0183] In specific, non-limiting examples, the subject has leukemia, colorectal cancer, cervical cancer, lung cancer, bladder cancer, head and neck cancer, pancreatic cancer, glioblastoma, head and neck squamous cell carcinoma, ovarian cancer, uterine cancer, prostate cancer, breast cancer, melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, sarcoma, or adrenal cancer. In another non-limiting example, the subject has melanoma or head and neck squamous cell carcinoma (HNSCC).

[0184] In some embodiments, the methods disclosed herein reduce T cell exhaustion. In some examples, the methods reduce T cell exhaustion in a subject (e.g., a subject with cancer or undergoing immunotherapy). In some examples, T cell exhaustion is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% compared to a suitable control (e.g., compared to before treatment with MCT11 antibody). The reduction in T cell exhaustion can be measured, for example, by measuring a reduction in lactate uptake, a reduction in expression of PD-1 or Tim3, an increase in cytokine production (e.g., INF-γ, TNFα, or IL-2), or an increase in cytotoxic activity (e.g., increased tumor-specific targeting or killing), or another indicator of T cell effector activity, compared to a suitable control (e.g., a measurement from untreated exhausted T cells, or a baseline measurement of exhausted T cells prior to contact (including administration) or expression of an antibody specific for MCT11). In some embodiments, the method reduces MCT11 activity, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, compared to a suitable control. In some embodiments, the method reduces lactate transport or uptake in T cells (such as exhausted T cells or terminally exhausted T cells) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, compared to a suitable control (e.g., compared to the amount of lactate uptake before treatment with the MCT11 antibody). In some instances, a combination of these effects is achieved.

[0185] In some embodiments, the methods disclosed herein involve the proliferation and differentiation of T cells (e.g., CD3 + or CD8 +T cells) effector function (e.g., cytokine secretion, cell proliferation, tumor targeting, elimination of cancerous cells). In some examples, the method increases the effector function of T cells in a subject. In some embodiments, the method increases the effector function of T cells by, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%. at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more, compared to a suitable control (e.g., compared to the amount of effector function before treatment with the MCT11 antibody). Increased effector function of T cells can be measured, for example, by decreased lactate uptake, decreased expression of PD-1 or Tim3, increased cytokine production (e.g., INF-γ, TNFα, or IL-2), increased cell proliferation (in vitro or in vivo expansion), or increased cytotoxic activity (e.g., increased tumor-specific targeting or killing), or other indices of effector function, compared to a suitable control (e.g., measurements from untreated T cells, or baseline measurements of T cells prior to contact (including administration) or expression of an antibody specific for MCT11). In some instances, a combination of these effects is achieved.

[0186] In some embodiments, the methods include administering to a subject an MCT11 monoclonal antibody, antigen-binding fragment, multispecific antibody, or conjugate disclosed herein and a pharma- ceutically acceptable carrier. Although the dosage of the MCT11 antibody, antigen-binding fragment, multispecific antibody, or conjugate can vary, exemplary dosages are between about 0.01 to about 50 mg per kg of subject body weight, e.g., about 0.01 mg / kg to about 20 mg / kg, about 0.5 mg / kg to about 20 mg / kg, about 1 mg / kg to about 20 mg / kg, about 5 mg / kg to about 20 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 0.01 The range is from about 1 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 5 mg / kg to about 15 mg / kg, from about 5 mg / kg to 20 mg / kg, from about 5 mg / kg to 25 mg / kg, from about 5 mg / kg to 30 mg / kg, from about 5 mg / kg to 35 mg / kg, from about 5 mg / kg to 40 mg / kg, from about 5 mg / kg to 50 mg / kg, from about 10 mg / kg to 20 mg / kg, from about 10 mg / kg to 30 mg / kg, from about 10 mg / kg to 40 mg / kg, from about 10 mg / kg to 50 mg / kg, from about 20 mg / kg to 40 mg / kg, or from about 20 mg / kg to 50 mg / kg. In some examples, administration is intravenous and comprises about 0.5 to about 3 mg / kg of the antibody, antigen-binding fragment, multispecific antibody, or conjugate, administered once every 2 to 4 weeks, in some examples, the dose is 50 mg, 100 mg, 200 mg, or 500 mg, administered once every 3 weeks.

[0187] In some embodiments, a DNA or RNA encoding an MCT11 antibody (such as a disclosed MCT11 monoclonal antibody, antigen-binding fragment, or multispecific antibody) is administered to a subject, for example, to produce the antibody in vivo using the subject's cellular machinery. In a non-limiting example, an effective amount of an mRNA encoding an scFV is administered to a subject. Methods of administering exogenous mRNA for in vivo protein expression are disclosed, for example, in Schlake et al. (2019) Molecular Therapy 27(4):773-784, which is incorporated by reference in its entirety.

[0188] Any suitable method of administration of nucleic acids may be used; non-limiting examples include U.S. Pat. Nos. 5,643,578, 5,593,972, and 5,817,637, 5,880,103, which are incorporated by reference in their entireties, describing several methods of delivery of nucleic acids encoding proteins to a subject.

[0189] In some embodiments, an effective amount of a vector encoding one or more nucleic acid molecules encoding a monoclonal antibody, antigen-binding fragment, multispecific antibody, or conjugate disclosed herein is administered to the subject. Upon administration of an effective amount of the vector, an effective amount of the monoclonal antibody, antigen-binding fragment, multispecific antibody, or conjugate is expressed in the subject. In some examples, the nucleic acid or vector is injected, for example, subcutaneously, intramuscularly, intradermally, intraperitoneally, intratumorally, intraprostatically, or intravenously. In a non-limiting specific example, the nucleic acid or vector is administered by intramuscular injection. In some examples, the dosage for intramuscular injection is about 0.5 μg / kg to about 50 mg / kg, for example, 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0190] Multiple doses of the MCT11 antibody, antigen-binding fragment, multispecific antibody, conjugate, or nucleic acid molecule or vector encoding such molecule disclosed herein may be administered daily, weekly, or monthly, according to a dosing schedule determined by, for example, a physician. In some examples, the antibody, antigen-binding fragment or multispecific antibody, conjugate, or nucleic acid molecule or vector encoding such molecule is administered weekly, every two weeks, every three weeks, every four weeks, every month, or less frequently. In some examples, treatment is administered every other day for three days. A skilled clinician can select a dosing schedule based on the subject, the condition being treated, previous treatment history, tumor burden and tumor type, clinical stage and grade of disease, general health, and other factors. Dosages can be administered once or applied periodically until the desired result is achieved or side effects require treatment to be discontinued. In general, the dose is sufficient to allow the patient to show the desired response without unacceptable toxicity.

[0191] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage is usually chosen so that it is within the range of ED with little or minimal toxicity. 50 The effective dose can be determined from cell culture assays and animal studies.

[0192] Administration includes local administration and systemic administration, such as by subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. In some embodiments, the antibody, antigen-binding fragment, multispecific antibody, or nucleic acid molecule encoding such molecule, or composition comprising such molecule, is administered once a day by a single subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. The antibody, antigen-binding fragment, multispecific antibody, conjugate, or nucleic acid molecule encoding such molecule, or composition comprising such molecule, can also be administered by direct injection at or near the disease site (e.g., tumor or cancer location). A further method of administration is by osmotic pump (e.g., Alzet pump) or minipump (e.g., Alzet miniosmotic pump) capable of delivering the antibody, antigen-binding fragment, conjugate, or nucleic acid molecule encoding such molecule, or composition comprising such molecule by controlled release, continuous release, and / or slow release over a predetermined period of time. The osmotic pump or minipump can be implanted subcutaneously or near the target site.

[0193] In some examples, the methods disclosed herein further include treating the subject with one or more of surgery, radiation, chemotherapy, biotherapy, or immunotherapy. In some examples, the immunotherapy includes one or more of checkpoint inhibitors, T cell agonist antibodies, oncolytic viruses (e.g., T-VEC), or adoptive cell transfer (ACT) immunotherapy.

[0194] Examples of chemotherapeutic agents that can be used in combination with MCT11 antibodies include alkylating agents, such as nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine); antimetabolites, such as folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or or cytarabine), and purine analogs, such as mercaptopurine or thioguanine; or natural products, such as vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase). Additional agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide); hormones and antagonists, such as corticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone).Examples of the most commonly used chemotherapy drugs include adriamycin, melphalan (Alkeran®), Ara-C (cytarabine), carmustine, busulfan, lomustine, carboplatinum, cisplatinum, cyclophosphamide (Cytoxan®), daunorubicin, dacarbazine, 5-fluorouracil, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or Other taxanes include docetaxel, vinblastine, vincristine, VP-16, while newer drugs include gemcitabine (Gemzar®), trastuzumab (Herceptin®), irinotecan (CPT-11), leustatin, navelbine, rituximab (Rituxan®) imatinib (STI-571), topotecan (Hycamtin®), capecitabine, ibritumomab (Zevalin®), and calcitriol. A skilled clinician can select additional therapies (from those listed herein or other current therapies) appropriate for a subject depending on factors such as the subject, the cancer being treated, treatment history, and other factors.

[0195] In some examples, the method further includes treating the subject with an additional therapeutic agent, such as a monoclonal antibody (e.g., anti-CTLA-4, anti-PD1, or anti-PDL1), a T cell agonist antibody (e.g., urelumab and utomilumab), an oncolytic virus, an adoptive cell transfer (ACT) therapy, or any combination of two or more of the foregoing. In some examples, the additional therapeutic agent is a cell cycle inhibitor or a checkpoint inhibitor. In some examples, the checkpoint inhibitor targets PD-1, PD-L1, CTLA-4, CDK4, and / or CDK6. Exemplary inhibitors include ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, palbociclib, ribociclib, and abemaciclib. In some examples, the subject is administered ACT treatment, e.g., chimeric antigen receptor (CAR)-expressing T cells, engineered TCR T cells, or tumor infiltrating lymphocytes (TILs).

[0196] The additional therapeutic agent may be administered substantially simultaneously with the disclosed composition. In some examples, the additional therapeutic agent is administered before administration of the composition, for example, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 12 days, at least 14 days, at least 3 weeks, at least 4 weeks, at least 1 month, or more. Multiple doses of the additional therapeutic agent can be administered to the subject, for example, twice daily, once daily, every other day, twice weekly, weekly, every other week, every 3 weeks, monthly, or less frequently. A skilled clinician can select the administration schedule based on the subject, the condition being treated, previous treatment history, tumor volume and tumor type, clinical stage and aggressiveness of the disease, and the subject's overall health, as well as other factors.

[0197] Also provided are compositions or kits that can be used with the disclosed methods. In some examples, the compositions or kits include, for example, one or more MCT11 antibodies, antigen-binding fragments, multispecific antibodies, conjugates, or nucleic acid molecules or vectors encoding such molecules in a pharma- ceutically acceptable carrier. The kits can include additional reagents, for example, one or more additional antibodies (e.g., anti-CD3, anti-CD8, anti-CD28, anti-CD44, anti-PD1, anti-TIM3), transfection reagents, vectors, culture media, antibiotics, or cytokines (e.g., IL-2, IL-15, and IL-7). The kits can include cells, for example, cells for protein expression. In some examples, the reagents are in separate containers. EXAMPLES

[0198] Example 1 Materials and Methods This example provides the materials and methods used to generate the data discussed in the examples that follow.

[0199] RNA-seq C57 / BL6 mice were implanted with B16 melanoma. When tumors reached 7 mm in any direction, LNs and tumors were harvested and processed to identify CD8 + T cells were sorted based on expression of CD44, PD-1, and Tim-3 from lymph nodes (LN) and tumor infiltrating lymphocytes (TIL). RNA-seq was performed on 1,000 cells isolated from the following compartments: LN CD44 hi , TIL PD-1 lo , TIL PD-1 mid , TIL PD-1 hi , and TIL PD-1 hi Tim3 + LN- and TIL-derived CD4 +T cells were sequenced from separate experiments. RNA was prepared from cell lysates of 1000 cells using the Clontech SMARTer® kit and sequenced on an Illumina NextSEQ®. TPM was calculated after alignment to the mouse genome (mm9 assembly). A plot of transcripts per million (TPM) of Slc16a11 (encoding MCT11) is shown (Figure 3A).

[0200] Lactate uptake C57 / BL6 mice were implanted with B16 melanoma as described above to generate TIL preparations. TILs were loaded with pHrodo® Red (a pH-sensitive dye) and incubated in Hank's Balanced Salt Solution (HBSS). Lactic acid was pulsed for 30 minutes and pH changes were measured by flow cytometry as described in Watson et al. (2021) Nature, 591:645-651 (incorporated herein by reference in its entirety). In experiments testing antibody inhibition of MCT11, preparations were incubated with 10 μg / mL polyclonal or monoclonal anti-MCT11 (see Example 3) antibodies before pulsing with LA.

[0201] In vivo experiments B16 melanoma Mouse, 1 x 10 5 Tumor cells were subcutaneously injected. Mice bearing 3 mm diameter B16 melanoma tumors were treated with 100 μg / mouse of MCT11 mAb (see Example 3), PD-1 mAb, or isotype control via intraperitoneal route for three times every other day (see FIG. 7A). Tumor growth inhibition was followed.

[0202] MEER (HPV positive head and neck squamous cell carcinoma (HNSCC) Mouse, 1 x 10 5Tumor cells were subcutaneously injected. Mice bearing tumors with a diameter of 3 mm were treated with 100 μg / mouse of MCT11 mAb (see Example 3) or isotype control via the intraperitoneal route, every other day for three times. Tumor growth inhibition was followed.

[0203] Fc variant antibody (LALAPG) Mouse, 1 x 10 5 MEER tumor cells were subcutaneously injected. Mice bearing tumors with a diameter of 3 mm were treated with 100 μg / mouse of isotype control, MCT11 mAb (see Example 3), or mutant MCT11 mAb via the intraperitoneal route. Tumor growth inhibition was followed.

[0204] Mice whose tumors were removed in response to MCT11 blockade were then treated with 1 × 10 5 MEER cells / mice were re-inoculated with MEER tumor cells. Tumor growth inhibition was followed.

[0205] Example 2 Discovery of MCT11 on exhausted T cells Using RNA-seq and metabolic profiling, we found that terminally exhausted T cells (dysfunctional T cells commonly found in cancer environments) highly express a novel nutrient transporter called MCT11 (encoded by Slc16a11) (Figures 3A and 3B). MCT11 may transport monocarboxylates (short-chain carbon sources such as lactate, pyruvate, and short-chain fatty acids). Upregulation of MCT11 in exhausted T cells in humans and mice was confirmed by flow cytometry and RNA-Seq (see Figures 2A-2C and Figures 3A-3B). Furthermore, we confirmed that terminally exhausted T cells specifically take up monocarboxylates such as lactate (Figure 4). However, MCT11 is not expressed on the surface of exhausted T cells induced by chronic viral infection. These findings indicate that MCT11 may be important in flowing nutrients to terminally exhausted T cells.

[0206] Example 3 Generation of monoclonal MCT11 antibodies Mice were immunized against MCT11 and monoclonal antibodies were generated from fusion with myeloma cells as standard practice. After single cell cloning and selection of high producing clones, they were screened for binding to the immunizing peptide (SEQ ID NO: 11). Clones were selected based on surface binding to MCT11 overexpressing cell lines but not to cell lines lacking MCT11. Sequence information of MCT11 mAbs is provided in Table 1.

[0207] Example 4 MCT11 blockade of lactate uptake Non-exhausted tumor-infiltrating T cells (TILs that do not express MCT11) and LN-derived T cells do not significantly take up lactate. In contrast, terminally exhausted tumor-infiltrating T cells (TILs that express high levels of PD-1 and Tim-3) actively take up lactate (Figure 4). We investigated whether MCT11 blockade could inhibit lactate uptake in terminally exhausted T cells.

[0208] Mice were implanted with B16 melanoma. After tumors reached 5 mm in diameter, preparations of lymph nodes (LN) and tumor infiltrating lymphocytes (TIL) were loaded with the pH indicator dye pHrodo® Red and cell surface stained for CD8, PD-1, and Tim-3. After pulsing with LA, preparations were incubated with 10 μg / mL of polyclonal anti-MCT11 antibody (Abcam, ab230845). Samples were then pulsed with 5 mM lactate (LA) for 30 min. Lactate uptake was blocked by treatment of exhausted T cells with polyclonal antibody (Figure 5).

[0209] The lactate uptake assay was repeated using a purified monoclonal antibody specific for MCT11 (mouse IgG2a(k) isotype) (described in Example 3), again showing that incubation with 10 μg / mL of anti-MCT11 was able to disrupt lactate uptake in terminally exhausted T cells ( FIG. 6 ), demonstrating that MCT11 supports lactate uptake and that an antibody-mediated approach can be used to prevent the uptake of this toxic metabolite.

[0210] Example 5 MCT11 blockade in vitro To test the functional significance of MCT11 blockade, TIL preparations are restimulated with PMA / ionomycin in the presence of 5 mM lactate for 5 hours in vitro with or without MCT11-specific antibodies. The ability of TILs to produce cytokines and granzyme B is measured, as is lactate uptake.

[0211] Example 6 Functional modulation of antitumor responses in vivo We investigated whether mAb-mediated blockade of MCT11 could functionally modulate antitumor immune responses in vivo by treatment of a mouse model bearing B16 melanoma tumors. MCT11 blockade with MCT11 mAb (see Example 3) demonstrated a similar functional effect of PD-1 blockade in the B16 model of aggressive melanoma, significantly reducing tumor growth (Figure 7B). Similarly, mAb-mediated blockade of MCT11 in a mouse model bearing MEER tumors (HPV-positive HNSCC) was also tested. MCT11 blockade with MCT11 mAb significantly reduced tumor growth in the MEER model (Figure 7C). Also, mice whose tumors were cleared after α-MCT11 mAb treatment showed immunological memory when re-inoculated with MEER tumor cells (Figure 9C). Notably, the mice were healthy and did not lose weight, indicating that MCT11 blockade was not toxic. This is consistent with the fact that MCT11 germline knockout mice have no obvious phenotype. Thus, MCT11 blockade can be used to block lactate uptake on terminally exhausted T cells and rescue cell function.

[0212] Example 7 MCT11 blocking mechanism To determine whether MCT11 blockade acts through adaptive immunity, experiments similar to those described in Example 6 were performed in RAG1-deficient mice (RAGKO), which lack B and T cells. Little difference was observed between isotype control (IgG2a) and α-MCT11 mAb (see Example 3) treatments, indicating that α-MCT11 acts through the immune system (see Figures 8A-8C). Furthermore, an Fc mutant of α-MCT11 mAb (LALAPG) was created to determine whether α-MCT11 mAb functions as a blocking antibody or whether α-MCT11 mAb depletes MCT11-expressing cells. B6 mice inoculated with MEER tumor cells were treated with isotype control, α-MCT11 mAb, or mutant α-MCT11 mAb (Fc mut anti-MCT11) (Figures 9A and 9B). The results indicate that α-MCT11 mAb functions as a blocking antibody rather than by depletion of MCT11-expressing cells.

[0213] Example 8 Administration of monoclonal α-MCT11 to treat cancer In this example, an effective amount (e.g., 200 mg intravenously once every 3 weeks) of a monoclonal antibody that specifically binds to MCT11 (e.g., a commercially available MCT11 antibody or the MCT11 mAb of Example 3) is intravenously administered to a patient in need of cancer treatment. The antibody can be administered alone or in combination with other immunotherapy regimens, such as checkpoint blockade antibodies (PD-1, CTLA4, LAG3), T cell agonist antibodies (41BB, OX40, GITR), oncolytic viruses (such as T-VEC), or ACT (CAR-T, TCR-T, TIL) therapy.

[0214] In specific examples, the MCT11 specific antibody is administered before, after, or substantially simultaneously with immunotherapy, such as abemaciclib, atezolizumab, avelumab, axicabtagene ciloreucel, brinotumumab, semipilimab, durvalumab, yelamirimab, ipilimumab, nivolumab, palbociclib, pembrolizumab, pidilizumab, relatorimab, ribociclib, urelemab, utrimumab, adoptive cell transfer (ACT) therapy (e.g., chimeric antigen receptor (CAR) (e.g., tisagenlecleucel)), or engineered TCR or tumor infiltrating lymphocyte (TIL)), or oncolytic virus (e.g., talimogene laherparepvec (T-VEC)). In some examples, the MCT11 specific antibody is administered substantially simultaneously with immunotherapy. One or more signs or symptoms of the cancer (or tumor) (e.g., tumor size, tumor burden, tumor density, clinical grade, presence of metastases, number of metastases, morbidity, mortality, or other measurements (qualitative or quantitative)) are measured periodically. The measurements can be compared, for example, to measurements obtained before administration of the MCT11-specific antibody to the subject, or can be compared to a control group, for example, subjects not administered the MCT11-specific antibody. In this example, administration of the MCT11-specific antibody ameliorates one or more signs or symptoms of the cancer (or tumor).

[0215] Example 9 Use of α-MCT11 to alleviate exhausted T cells In this example, an effective amount of an MCT11 antibody (such as a monoclonal antibody (e.g., the MCT11 mAb of Example 3 or a commercially available MCT11 antibody) is administered to remove exhausted T cells from the sample. In some embodiments, the method includes contacting the sample with an effective amount of an antibody specific for MCT11 and removing cells bound to the antibody from the sample, thereby generating a sample depleted of exhausted T cells.

[0216] The sample can be a cell sample, for example, a PBMC sample or a population of T cells. The method can include, for example, obtaining a PBMC sample from a subject, such as a subject with cancer. The population of T cells can include a cell population for ACT therapy, such as CAR-T cells, TCR cells, and TIL cells for use in ACT therapy. Before or after (or both before and after) contacting the cell sample with an antibody specific for MCT11, the cell sample can be cultured, for example, to expand cells (such as expanding T cells).

[0217] In a non-limiting example, the sample is a PBMC sample. PBMC can be cultured ex vivo, for example, to expand cells (such as T cells). Exhausted T cells (such as terminally exhausted T cells) can be removed from a PBMC population (either PBMC obtained directly from a subject or PBMC expanded ex vivo afterward) by contacting PBMC with MCT11 antibody (such as monoclonal antibody). Cells in the PBMC population that bind to the MCT11 antibody (which are exhausted T cells) can be separated from other cells in the PBMC population, and thus the PBMC population can be enriched for non-exhausted or non-terminally exhausted T cells. In some examples, such methods remove at least 20% of the exhausted T cells (such as terminally exhausted T cells) in a PBMC population, e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the exhausted T cells (such as terminally exhausted T cells). In some examples, such methods produce a population of PBMC cells that is substantially free of exhausted T cells (such as terminally exhausted T cells).

[0218] Exhausted T cells (including terminal exhausted T cells) can be removed from the sample, for example, by flow cytometry, magnetic separation, or panning. In one example, a PBMC sample or a population of T cells is incubated with MCT11 antibody and a suitable labeled secondary antibody (such as one that contains a fluorophore), and the labeled cells are separated from the unlabeled cells (for example, by flow cytometry). In some examples, the MCT11 antibody is directly labeled with a fluorophore instead of using a secondary antibody. In one example, magnetic separation is used (for example, by using paramagnetic particles coated with MCT11 antibody incubated with PBMC, and cells that do not adhere to the particles are separated, for example, by using centrifugation and / or washing (wherein the supernatant is collected)). In one example, panning is used (for example, by using a solid support coated with MCT11 antibody incubated with PBMC, and cells that do not adhere to the support are collected, for example, by washing).

[0219] In some examples, the methods provide samples depleted of exhausted T cells (such as terminally exhausted T cells) for use in anti-cancer immunotherapy (such as the methods provided herein).

[0220] In another example, an effective amount of an MCT11 antibody (such as a monoclonal antibody (e.g., the MCT11 mAb of Example 3 or a commercially available MCT11 antibody) is administered to deplete exhausted T cells from the subject. In some embodiments, the method includes administering an effective amount of an antibody specific for MCT11 to the subject, thereby depleting exhausted T cells in the subject. In some examples, subjects with cancer or undergoing immunotherapy are selected for treatment.

[0221] In view of the numerous possible embodiments to which the inventive principles of this disclosure may be applied, it should be recognized that the described embodiments are merely illustrative of the invention and should not be construed as limiting the scope of the invention, which is rather defined by the following claims, and the inventors therefore claim as their invention all that comes within the scope and spirit of these claims.

Claims

1. Variable heavy chain (V Ｈ ) domain and variable light chain (V Ｌ ) domain, a monoclonal antibody that specifically binds to monocarboxylate transporter 11 (MCT11), Said V Ｈ domain contains the heavy chain complementarity determining regions (CDRs) 1, 2, and 3 of SEQ ID NO: 1, Said V Ｌ domain-containing monoclonal antibody comprising complementarity-determining regions (CDRs) 1, 2, and 3 of the light chain of SEQ ID NO:

5.

2. The monoclonal antibody according to claim 1, wherein the CDR sequences are defined using the Kabat, IMGT, or Chothia numbering scheme.

3. The heavy chain CDR1, CDR2, and CDR3 each contain the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, The monoclonal antibody according to claim 1, wherein the light chain CDR1, CDR2, and CDR3 each contain the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8.

4. Said V Ｈ The amino acid sequence of the domain is at least 90% identical to SEQ ID NO: 1 and contains the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 1, The above-mentioned V Ｌ The monoclonal antibody according to claim 1, wherein the amino acid sequence of the domain is at least 90% identical to SEQ ID NO: 5 and includes the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:

5.

5. Said V Ｈ domain contains or consists of SEQ ID NO: 1, Said V Ｌ The monoclonal antibody according to claim 1, wherein the domain comprises or consists of SEQ ID NO:

5.

6. The antibody is an antigen-binding fragment selected from Fab fragments, Fab' fragments, F(ab)' ２ fragments, Fv, single-chain variable fragments (scFv), dimers of single-chain antibodies (scFv ２ ), and disulfide-stabilized variable fragments (dsFv), the monoclonal antibody according to claim 1.

7. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a mouse antibody.

8. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a humanized antibody.

9. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a human antibody.

10. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a chimeric antibody.

11. The monoclonal antibody according to claim 1, comprising a constant region.

12. The monoclonal antibody according to claim 11, wherein the constant region comprises at least one modification for increasing the half-life, stability, and / or function of the monoclonal antibody.

13. An antibody conjugate comprising the monoclonal antibody according to claim 1 linked to an effector molecule or a detectable marker.

14. An antibody-drug conjugate comprising the monoclonal antibody according to claim 1 linked to a therapeutic agent.

15. A multispecific antibody comprising the monoclonal antibody according to claim 1 and at least one antibody that specifically binds to an additional antigen.

16. The multispecific antibody according to claim 15, wherein the additional antigen is PD-1, 4-1BB / CD137, GITR, OX40, CD105, LAG3, TIM-3 / HAVCR2, NRP1, or FAS.

17. An isolated nucleic acid molecule encoding the monoclonal antibody according to claim 1.

18. The nucleotide sequence of SEQ ID NO: 1, or a degenerate variant thereof; The nucleotide sequence of SEQ ID NO: 5, or a degenerate variant thereof; or The nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 5, or a degenerate variant thereof The isolated nucleic acid molecule according to claim 17, comprising.

19. An isolated nucleic acid molecule according to claim 17, operably linked to a promoter.

20. A vector comprising the nucleic acid molecule according to claim 17.

21. A host cell comprising the nucleic acid molecule according to claim 17 or a vector comprising said nucleic acid molecule.

22. A composition comprising a monoclonal antibody according to any one of claims 1 to 12, a conjugate according to claim 13, an antibody-drug conjugate according to claim 14, a multispecific antibody according to claim 15 or claim 16, a nucleic acid molecule according to any one of claims 17 to 19, or a vector according to claim 20, and a pharmaceutically acceptable carrier.

23. The composition according to claim 22, for use in the treatment of cancer or tumor in a subject.

24. An in vitro method for reducing T cell exhaustion, reducing lactate uptake by exhausted T cells, increasing the effector function of T cells, or combinations thereof, (a) contacting said exhausted T cells or T cells in vitro with an antibody specific for monocarboxylate transporter 11 (MCT11) in an effective amount; or (b) expressing in vitro a nucleic acid molecule or vector encoding an antibody specific for MCT11 in said exhausted T cells or T cells; comprising thereby reducing T cell exhaustion, reducing lactate uptake by exhausted T cells, increasing the effector function of T cells, or combinations thereof.

25. wherein the antibody specific for MCT11 comprises a monoclonal antibody according to any one of claims 1 to 12, a conjugate according to claim 13, an antibody-drug conjugate according to claim 14, a multispecific antibody according to claim 15 or claim 16; or the nucleic acid molecule or vector encoding the antibody specific for MCT11 comprises a nucleic acid molecule according to any one of claims 17 to 19 or a vector according to claim 20, the method according to claim 24.

26. The method according to claim 24, wherein said exhausted T cells or T cells are adoptive cell transfer (ACT) -treated T cells.

27. The method according to claim 24, wherein said exhausted T cells or T cells are isolated from a subject having cancer.

28. The method according to claim 24, wherein the exhausted T cells are terminal exhausted T cells.

29. A composition for use in the treatment of cancer or tumor in a subject, the composition comprising a therapeutically effective amount of the following: An antibody specific for monocarboxylate transporter 11 (MCT11), The monoclonal antibody according to any one of claims 1 to 12, The conjugate according to claim 13, The antibody-drug conjugate according to claim 14, The multispecific antibody according to claim 15 or claim 16, The nucleic acid molecule according to any one of claims 17 to 19, or The vector according to claim 20, A composition comprising the same.

30. The composition according to claim 29, wherein the subject is further administered with immunotherapy.

31. The composition according to claim 30, wherein the immunotherapy comprises one or more of adoptive cell transfer (ACT) therapy, atezolizumab, avelumab, axicabtagene ciloleucel, blinatumomab, cemiplimab, durvalumab, yerramlimab, ipilimumab, nivolumab, pembrolizumab, pidilizumab, relatlimab, urelumab, and utomilumab.

32. The composition according to claim 31, wherein the ACT therapy comprises tumor-infiltrating lymphocyte (TIL) therapy, chimeric antigen receptor T cell (CAR-T) therapy, or engineered T cell receptor (TCR) therapy.

33. The composition according to claim 29, wherein the therapeutically effective amount increases effector T cell function, reduces T cell exhaustion, reduces lactate turnover by exhausted T cells, or a combination thereof in the subject.

34. The composition according to claim 30, wherein the therapeutically effective amount of the composition increases the subject's response to the immunotherapy.

35. A method for removing exhausted T cells from a sample, comprising: (1) contacting the sample with an effective amount of an antibody specific for MCT11, and (2) removing the cells bound to the antibody, thereby generating a sample depleted of exhausted T cells. A method comprising the same.

36. (a) the antibody specific for MCT11 binds to SEQ ID NO: 11, (b) the exhausted T cells are terminal exhausted T cells, (c) the sample is a PBMC sample isolated from a subject or a population of T cells, and / or The method according to claim 35, wherein removing the cells bound to the antibody comprises removing the cells by flow cytometry, magnetic separation, or panning. **Claim 37**: The method according to claim 36, wherein the population of T cells of (c) comprises tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T), or engineered T cell receptor (TCR) T cells. **Claim 38** The method according to claim 36, wherein the sample depleted of exhausted T cells is for treating cancer in a subject. **Claim 39** A composition for use in a method of increasing an immune response in a subject, the composition comprising an antibody specific for monocarboxylate transporter 11 (MCT11), and optionally, increasing the immune response comprises increasing the immune response against cancer in the subject. **Claim 40** The composition according to claim 39, wherein the subject has previously received immunotherapy or immunotherapy is to be further administered. **Claim 41** The composition according to claim 40, wherein the immunotherapy comprises an antibody, a virus, a nucleic acid, a protein, an Fc-fusion protein, a cell, a T cell, or an NK cell. **Claim 42** The composition according to claim 41, wherein the immunotherapy comprises at least one of abemaciclib, atezolizumab, avelumab, axicabtagene ciloleucel, blinatumomab, cemiplimab, durvalumab, yerramlimab, ipilimumab, nivolumab, palbociclib, pembrolizumab, pidilizumab, relatlimab, ribociclib, urelumab, utomumab, adoptive cell transfer (ACT) therapy, or talimogene laherparepvec (T-VEC) vaccine. **Claim 43** The composition according to claim 39, wherein the antibody specific for MCT11 blocks the uptake of lactate, pyruvate, ketone bodies, butyrate, propionate, or succinate by at least one cell expressing MCT11, and optionally, the at least one cell comprises at least one of exhausted T cells, regulatory T cells, or resident memory T cells. **Claim 44** The composition according to claim 39, wherein the antibody specific for MCT11 is a human antibody or a humanized antibody. **Claim 45** The composition according to claim 39, wherein the antibody specific for MCT11 comprises Fc, optionally human IgG1 Fc, human IgG4 Fc, fucosylated Fc, or non-FcR-binding Fc.

46. The composition according to claim 39, wherein MCT11 comprises SEQ ID NO:

9.

47. The composition according to claim 39, wherein the MCT11 antibody binds to SEQ ID NO:

11.

48. The composition according to claim 39, wherein the subject is human.

49. The composition according to claim 29, wherein the cancer is a solid cancer.

50. The composition according to claim 49, wherein the cancer is melanoma.

51. The composition according to claim 29, wherein the subject is human.

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