WO2019183375A1

WO2019183375A1 - Antibody agents specifically recognizing monocarboxylate transporter 4 and uses thereof

Info

Publication number: WO2019183375A1
Application number: PCT/US2019/023402
Authority: WO
Inventors: Yoko Nakano; Yiyang XU; Pei Wang; Hong Liu; James J. HSIEH
Original assignee: Eureka Therapeutics, Inc.; Memorial Sloan Kettering Cancer Center
Priority date: 2018-03-22
Filing date: 2019-03-21
Publication date: 2019-09-26

Abstract

The present application provides antibody agents specifically recognizing monocarboxylate transporter 4 (MCT4), such as a cell surface-bound MCT4. Also provided are methods of making and using these antibody agents.

Description

ANTIBODY AGENTS SPECIFICALLY RECOGNIZING MONOCARBOXYLATE

TRANSPORTER 4 AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 62/646,819, filed on March 22, 2018, the contents of which are hereby incorporated by reference in their entireties.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEST FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 75004200l340SEQLIST.TXT, date recorded: March 14, 2019, size: 21 KB).

FIELD OF THE INVENTION

[0003] This invention pertains to antibodies that specifically recognize monocarboxyl ate transporter 4 (MCT4) and antibody agents comprising anti-MCT 4 antibody moieties, and methods of manufacture and uses thereof, including treating cancer.

BACKGROUND OF THE INVENTION

[0004] Cancer cells utilize blood glucose to produce chemical intermediates required for the generation of new cancer cells, a biochemical process called“aerobic glycolysis” (also referred to as the Warburg Effect) (Vander Heiden et al. (2009), Science , 324(5930): 1029-1033). This process results in accumulation of lactate, the end waste product of aerobic glycolysis, in the cancer microenvironment, which can further impair local anti-cancer immune responses (Scott and Cleveland (2016), Cell Metabolism, 24(5): 649-650). Normal cells utilize protein monocarboxylate transporters (MCTs) to shuttle lactate in and out of cells (Halestrap and Wilson (2012), IUBMB Life 64(2): 109-119). Among MCTs, MCT4 is highly expressed in cancer cells and has the highest Km for pyruvate, making it the primary carrier for the export of

overproduced lactate resulting from overconsumption of glucose. Genetic evidence suggests that MCT4 is a novel anti-cancer target in several major cancer types including breast cancer (Baenke et al. (2015), J Pathology, 237(2): 152-165), prostate cancer (Choi et al. (2016), Cell reports, 14(7): 1590-1601), colon cancer (Gotanda et al. (2013), Anticancer research, 33(7): 2941-2947), brain cancer (Lim et al. (2014), Oncogene, 33(35): 4433), pancreatic cancer (Baek et al. (2014), Cell reports, 9(6): 2233-2249), and kidney cancer (Gerlinger et al. (2012), J.

Pathology, 227(2): 146-156). Given the importance of MCT4 as an anti-cancer target, it would be useful to identify novel blocking antibodies and antibody agents specific for MCT4 to provide new therapies for the treatment of cancers characterized by high glucose

overconsumpti on .

[0005] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

[0006] The present application in one aspect provides anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g, full-length anti-MCT4 antibodies) specifically recognizing a cell surface-bound MCT4 (referred to herein as a“native format MCT4,” or“native MCT4

(nMCT4)”). The anti-MCT4 antibodies and antibody agents specifically bind to MCT4 and antagonize its ability to transport lactate.

[0007] In some embodiments, there is provided an isolated anti-monocarboxylate transporter 4 (MCT4) antibody agent that specifically recognizes a cell surface-bound MCT4 and antagonizes its lactate transport function. In some embodiments, the isolated anti-MCT4 antibody agent comprises: i) a heavy chain variable domain (V_H) comprising a heavy chain complementarity determining region (HC-CDR) 1, an HC-CDR2, and an HC-CDR3 of the heavy chain variable domain of any one of SEQ ID NOs: 2-4; and ii) a light chain variable domain (V_L) comprising a light chain complementarity determining region (LC-CDR) 1, an LC-CDR2, and an LC-CDR3 of the light chain variable domain of any one of SEQ ID NOs: 5-7. In some embodiments, the isolated anti-MCT4 antibody agent comprises: i) a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 2, and a VL comprising a LC-CDR1, an LC-CDR2, and an LC-CDR3 of the light chain variable domain of SEQ ID NO: 5; ii) a V_H comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 3, and a VL comprising a LC-CDR1, an LC-CDR2, and an LC-CDR3 of the light chain variable domain of SEQ ID NO: 6; or iii) a V_H comprising an HC-CDR1, an HC- CDR2, and an HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 4, and a VL comprising a LC-CDR1, an LC-CDR2, and an LC-CDR3 of the light chain variable domain of SEQ ID NO: 7.

[0008] In some embodiments, according to any of the anti-MCT4 antibody agents described above, the isolated anti-MCT4 antibody agent comprises: i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 amino acid substitutions; and ii) a V_L comprising a LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, or a variant thereof comprising up to about 5 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 amino acid substitution.

[0009] In some embodiments, according to any of the anti-MCT4 antibody agents described above, the anti-MCT4 antibody agent comprises: i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16; and ii) a V_L comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25.

[0010] In some embodiments, according to any of the anti-MCT4 antibody agents described above, the anti-MCT4 antibody agent comprises: i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NOs: 14; and ii) a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

[0011] In some embodiments, according to any of the anti-MCT4 antibody agents described above, the anti-MCT4 antibody agent comprises: i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NOs: 15; and ii) a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[0012] In some embodiments, according to any of the anti-MCT4 antibody agents described above, the anti-MCT4 antibody agent comprises: i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NOs: 16; and ii) a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22 and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0013] In some embodiments, according to any of the anti-MCT4 antibody agents described above, the anti-MCT4 antibody agent comprises: i) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% sequence identify to any one of SEQ ID NOs: 2-4; and ii) a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity to any one of SEQ ID NOs: 5-7. In some embodiments, the anti-MCT4 antibody agent comprises: i) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 2-4; and ii) a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the anti-MCT4 antibody agent comprises: i) a V_H comprising the amino acid sequence of SEQ ID NO: 2; and ii) a V_L comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti- MCT4 antibody agent comprises: i) a V_H comprising the amino acid sequence of SEQ ID NO: 3; and ii) a V_L comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-MCT4 antibody agent comprises: i) a V_H comprising the amino acid sequence of SEQ ID NO: 4; and ii) a V_L comprising the amino acid sequence of SEQ ID NO: 7.

[0014] In some embodiments, according to any of the anti-MCT4 antibody agents described above, the isolated anti-MCT4 antibody agent comprises: i) a V_H comprising sequences of SEQ ID NOs: 8, 11 and 14, and ii) a V_L comprising sequences of SEQ ID NOs: 17, 20 and 23. In some embodiments, the anti-MCT4 antibody comprises: i) a V_H comprising sequences of SEQ ID NOs: 9, 12 and 15, and ii) a V_L comprising sequences of SEQ ID NOs: 18, 21 and 24. In some embodiments, the anti-MCT4 antibody comprises: i) a V_H comprising sequences of SEQ ID NOs: 10, 13 and 16, and ii) a V_L comprising sequences of SEQ ID NOs: 19, 22 and 25.

[0015] In some embodiments, according to any of the anti-MCT4 antibody agents described above, the anti-MCT4 antibody agent is a full-length IgG antibody. In some embodiments, the anti-MCT4 antibody agent is a full-length IgGl antibody. In some embodiments, the anti-MCT4 antibody agent comprises a heavy chain constant region comprising the amino acid of SEQ ID NO: 27 and a light chain constant region comprising the amino acid sequence of SEQ ID NO:

29. In some embodiments, the anti-MCT4 antibody agent is a full-length IgG4 antibody. In some embodiments, the anti-MCT4 antibody agent comprises a heavy chain constant region comprising the amino acid of SEQ ID NO: 28 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 29.

[0016] In some embodiments, there is provided an isolated anti-MCT4 antibody agent that specifically binds to MCT4 competitively with an isolated anti-MCT4 antibody agent according to any of the embodiments described above and antagonizes MCT4 lactate transport function.

[0017] In some embodiments, there is provided an isolated nucleic acid encoding an isolated anti-MCT4 antibody agent according to any of the embodiments described above.

[0018] In some embodiments, there is provided a vector comprising an isolated nucleic acid according to any of the embodiments described above.

[0019] In some embodiments, there is provided an isolated host cell comprising an isolated anti-MCT4 antibody agent according to any of the embodiments described above, an isolated nucleic acid according to any of the embodiments described above, or a vector according to any of the embodiments described above.

[0020] In some embodiments, there is provided a pharmaceutical composition comprising an isolated anti-MCT4 antibody agent according to any of the embodiments described above, and a pharmaceutically acceptable carrier.

[0021] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 and/or high aerobic glycolysis, comprising administering to the individual an effective amount of a pharmaceutical composition according to any of the embodiments described above. In some embodiments, the administration is via intravenous or intratumoral route. In some embodiments, the method further comprises administering to the individual an additional therapy.

[0022] In some embodiments, according to any of the methods described above, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC.

[0023] In some embodiments, there is provided a method of producing an isolated anti-MCT4 antibody agent, comprising: a) culturing i) a host cell comprising an isolated nucleic acid according to any of the embodiments described above or a vector according to any of the embodiments described above, or ii) an isolated host cell according to any of the embodiments described above, under conditions effective to express the anti-MCT4 antibody agent; and b) obtaining the expressed anti-MCT4 antibody agent from said host cell. In some embodiments, step a) further comprises producing the host cell comprising the isolated nucleic acid or the vector.

[0024] In some embodiments, there is provided a kit comprising an isolated anti-MCT4 antibody agent according to any of the embodiments described above, an isolated nucleic acid according to any of the embodiments described above, a vector according to any of the embodiments described above, or an isolated host cell according to any of the embodiments described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows FACS analysis of the binding of 3 anti-MCT4 phage clones to parental 293 cells and 293 cells transformed to express MCT4 (293-MCT4). The binding of helper phages to the cells was included as a negative control (NC phage).

[0026] FIG. 2 shows FACS analysis of the binding of 3 anti-MCT4 phage clones to MCT4⁺ cell line RCC4. The binding of helper phages to the cells was included as a negative control (NC phage).

[0027] FIG. 3 shows FACS analysis of the binding of anti-MCT4 phage clone 2 to MCT4⁺ cell lines A498 and 786-0. Cell only and no phage conditions were included as negative controls.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present application in one aspect provides anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti-MCT4 antibodies) that specifically recognize a cell surface-bound MCT4 (referred to herein as a“native format MCT4,” or“native MCT4

(nMCT4)”), and antagonize its lactate transport function.

[0029] Using phage display technology, we identified multiple scFvs that are specific for cell surface-bound human MCT4. Flow cytometry assays demonstrated that these antibodies recognize MCT4-expressing cancer cell lines.

[0030] The present application thus provides anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti-MCT4 antibodies) comprising an antibody moiety specifically recognizing cell surface-bound MCT4. The anti-MCT4 antibody agents include, for example, full-length anti-MCT4 antibodies, anti-MCT4 scFvs, anti-MCT4 Fc fusion proteins, multi- specific (such as bispecific) anti-MCT4 antibodies, anti-MCT4 immunoconjugates, and the like.

[0031] In another aspect, there are provided nucleic acids encoding the anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti-MCT4 antibodies). [0032] In another aspect, there are provided compositions (such as pharmaceutical

compositions) comprising an anti-MCT4 antibody agent (such as an anti-MCT4 antibody, e.g., a full-length anti-MCT4 antibody).

[0033] Also provided are methods of making and using the anti-MCT4 antibody agents and antibodies, such as for treating cancer, as well as kits and articles of manufacture useful for such methods.

Definitions

[0034] As used herein,“treatment” or“treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g, metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of cancer (such as, for example, tumor volume). The methods of the invention contemplate any one or more of these aspects of treatment.

[0035] The term“antibody” includes full-length antibodies and antigen-binding fragments thereof. A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity

determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC- CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Rabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Rabat 1987; Rabat 1991). The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, d, e, g, and m heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgGl (gΐ heavy chain), IgG2 (g2 heavy chain), IgG3 (g3 heavy chain), IgG4 (g4 heavy chain), IgAl (al heavy chain), or IgA2 (a2 heavy chain).

[0036] The term“antigen-binding fragment” as used herein refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment ( e.g ., a parent scFv) binds. In some embodiments, an antigen binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0037] The term“epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.

[0038] As used herein, a first antibody agent (e.g., antibody or antibody moiety)“competes” for binding to a target MCT4 (e.g., nMCT4) with a second antibody agent (e.g., antibody or antibody moiety) when the first antibody agent inhibits target MCT4 binding of the second antibody agent by at least about 50% (such as at least about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolar concentration of the first antibody agent, or vice versa. A high throughput process for“binning” antibodies based upon their cross-competition is described in PCT Publication No. WO 03/48731.

[0039] As use herein, the term“specifically binds,”“specifically recognizing,” or“is specific for” refers to measurable and reproducible interactions, such as binding between a target and an antibody agent (e.g., antibody or antibody moiety), that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody agent that specifically recognizes a target (which can be an epitope) is an antibody agent that binds this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other targets. In some embodiments, an antibody agent that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen (such as native format MCT4) with a binding affinity that is at least about 10 times its binding affinity for other targets (such as soluble MCT4).

[0040] An“isolated” anti-MCT4 antibody agent or antibody as used herein refers to an anti- MCT4 antibody agent or antibody that (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, (3) is expressed by a cell from a different species, or, (4) does not occur in nature.

[0041] The term“isolated nucleic acid” as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the“isolated nucleic acid” (1) is not associated with all or a portion of a polynucleotide in which the“isolated nucleic acid” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

[0042] As used herein, the term“CDR” or“complementarity determining region” is intended to mean the non-conti guous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al ., J. Biol. Chem. 252:6609-6616 (1977); Kabat et ah, U.S. Dept of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al. , J. Mol. Biol. 196:901- 917 (1987); Al-Lazikani B. et al. , J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008);

Lefranc M.P. et al, Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Pliickthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al, Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al, Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entireties for use in the present invention and for possible inclusion in one or more claims herein.

TABLE 1 : CDR DEFINITIONS Rabat¹ Chothia² MacCallum IMGT AHo

V_H CDR1 31-35 26-32 30-35 27-38 25-40 V_H CDR2 50-65 53-55 47-58 56-65 58-77 V_H CDR3 95-102 96-101 93-101 105-117 109-137 V_L CDR1 24-34 26-32 30-36 27-38 25-40 V_L CDR2 50-56 50-52 46-55 56-65 58-77 V_L CDR3 89-97 91-96 89-96 105-117 109-137

Residue numbering follows the nomenclature of Kabat et al. , supra

2Residue numbering follows the nomenclature of Chothia et al. , supra

3Residue numbering follows the nomenclature of MacCallum et al. , supra

4Residue numbering follows the nomenclature of Lefranc et al. , supra

5Residue numbering follows the nomenclature of Honegger and Pluckthun, supra

[0043] The term“chimeric antibodies” refer to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a biological activity of this invention ( see U.S. Patent No. 4,816,567; and Morrison et al. , Proc. Natl. Acad. Sci. USA,

81 :6851-6855 (1984)).

[0044] The term“semi-synthetic” in reference to an antibody agent (e.g., antibody or antibody moiety) means that the antibody agent has one or more naturally occurring sequences and one or more non-naturally occurring ( i.e ., synthetic) sequences.

[0045] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0046] “Single-chain Fv,” also abbreviated as“sFv” or“scFv,” are antibody fragments that comprise the V_H and V_L antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the V_H and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0047] The term“diabodies” refers to small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) typically with short linkers (such as about 5 to about 10 residues) between the V_H and V_L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e ., fragment having two antigen binding sites. Bispecific diabodies are heterodimers of two“crossover” scFv fragments in which the V_H and V_L domains of the two antibodies are present on different polypeptide chains.

Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA , 90:6444-6448 (1993).

[0048] “Humanized” forms of non-human ( e.g. , rodent) antibody agents (e.g., antibodies) are chimeric antibody agents that contain minimal sequence derived from the non-human antibody agent. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. , Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op.

Struct. Biol. 2:593-596 (1992).

[0049] “Percent (%) amino acid sequence identity” or“homology” with respect to the polypeptide and antibody agent (e.g., antibody) sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5): 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1): 113, 2004).

[0050] The terms“Fc receptor” or“FcR” are used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR of this invention is one that binds an IgG antibody (a g receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRII A (an“activating receptor”) and Fey RUB (an“inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.

Activating receptor FcyRII A contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain. Inhibiting receptor Fey RUB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). The term includes allotypes, such as FcyRIIIA allotypes: FcyRTTTA-Phel 58 FcyRTTTA-Val 1 58 FcyRIIA-Rl3 l and/ or F cyRII A-H 131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al,

Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term“FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer el al, J. Immunol. 117:587 (1976) and Kim el al, J. Immunol. 24:249 (1994)).

[0051] The term“FcRn” refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an a-chain noncovalently bound to P2-microglobulin. The multiple functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays a role in the passive delivery of immunoglobulin IgGs from mother to young and the regulation of serum IgG levels. FcRn can act as a salvage receptor, binding and transporting pinocytosed IgGs in intact form both within and across cells, and rescuing them from a default degradative pathway. [0052] The“CH1 domain” of a human IgG Fc region (also referred to as“Cl” of“Hl” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0053] “Hinge region” is generally defined as stretching from Glu2l6 to Pro230 of human IgGl (Burton, Molec. Immunol.22: 161 -206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter heavy chain S-S bonds in the same positions.

[0054] The“CH2 domain” of a human IgG Fc region (also referred to as“C2” of“H2” domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain- domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22: 161-206 (1985).

[0055] The“CH3 domain” (also referred to as“C2” or“H3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).

[0056] A“functional Fc fragment” possesses an“effector function” of a native sequence Fc region. Exemplary“effector functions” include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody variable domain) and can be assessed using various assays known in the art.

[0057] An antibody agent (e.g., antibody) with a variant IgG Fc with“altered” FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity (e.g, FcyR or FcRn) and/or ADCC activity compared to a parent polypeptide or to a polypeptide comprising a native sequence Fc region. The variant Fc which“exhibits increased binding” to an FcR binds at least one FcR with higher affinity (e.g, lower apparent K_d or IC₅₀ value) than the parent polypeptide or a native sequence IgG Fc. According to some embodiments, the improvement in binding compared to a parent polypeptide is about 3 fold, such as about any of 5, 10, 25, 50, 60, 100, 150, 200, or up to 500 fold, or about 25% to 1000% improvement in binding. The polypeptide variant which“exhibits decreased binding” to an FcR, binds at least one FcR with lower affinity ( e.g ., higher apparent ¾ or higher IC50 value) than a parent polypeptide. The decrease in binding compared to a parent polypeptide may be about 40% or more decrease in binding.

[0058] “Antibody-dependent cell-mediated cytotoxicity” or“ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies“arm” the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No.

5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo , e.g, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0059] The polypeptide comprising a variant Fc region which“exhibits increased ADCC” or mediates ADCC in the presence of human effector cells more effectively than a polypeptide having wild type IgG Fc or a parent polypeptide is one which in vitro or in vivo is substantially more effective at mediating ADCC, when the amounts of polypeptide with variant Fc region and the polypeptide with wild type Fc region (or the parent polypeptide) in the assay are essentially the same. Generally, such variants will be identified using any in vitro ADCC assay known in the art, such as assays or methods for determining ADCC activity, e.g, in an animal model etc. In some embodiments, the variant is from about 5 fold to about 100 fold, e.g. from about 25 to about 50 fold, more effective at mediating ADCC than the wild type Fc (or parent polypeptide) .

[0060] “Complement dependent cytotoxicity” or“CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described in US patent No. 6, 194,551B1 and W099/51642. The contents of those patent publications are specifically incorporated herein by reference. See also, Idusogie et al. J Immunol. 164: 4178-4184 (2000).

[0061] Unless otherwise specified, a“nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0062] The term“operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0063] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g ., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0064] An“effective amount” of an anti-MCT4 antibody agent (e.g., antibody) or composition as disclosed herein, is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and by known methods relating to the stated purpose.

[0065] The term“therapeutically effective amount” refers to an amount of an anti-MCT4 antibody agent (e.g., antibody) or composition as disclosed herein, effective to“treat” a disease or disorder in an individual. In the case of cancer, the therapeutically effective amount of the anti-MCT4 antibody agent or composition as disclosed herein can reduce the number of cancer cells; reduce the tumor size or weight; inhibit {i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the anti-MCT4 antibody agent or composition as disclosed herein can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In some embodiments, the therapeutically effective amount is an amount that extends the survival of a patient. In some embodiments, the therapeutically effective amount is an amount that improves progression free survival of a patient.

[0066] As used herein, by“pharmaceutically acceptable” or“pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and

manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

[0067] It is understood that embodiments of the invention described herein include “consisting” and/or“consisting essentially of’ embodiments.

[0068] Reference to“about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to“about X” includes description of“X”.

[0069] As used herein, reference to“not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0070] As used herein and in the appended claims, the singular forms“a,”“or,” and“the” include plural referents unless the context clearly dictates otherwise.

Anti-MCT4 antibody agents

[0071] In one aspect, the present invention provides anti-MCT4 antibody agents that specifically bind to MCT4. In some embodiments, the anti-MCT4 antibody agent is an anti- MCT4 antibody (e.g., full-length anti-MCT4 antibody). In some embodiments, the anti-MCT4 antibody agent is a full-length antibody (e.g., full-length IgG4) or antigen-binding fragment thereof, which specifically binds to MCT4 and antagonizes its ability to transport lactate. MCT4 lactate transport function can be evaluated by methods known in the art, such as intracellular pH and lactate levels, extracellular acidification rate (ECAR), and 0₂ consumption rate (OCR).

ECAR and OCR can be measured, e.g., using an XF-24 Extracellular Flux Analyzer (Seahorse Bioscience). In some embodiments, binding of the anti-MCT4 antibody agent to an MCT4- expressing cell (e.g., an MCT4-expressing cancer cell) reduces its intracellular pH by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody agent to an MCT4-expressing cell (e.g., an MCT4- expressing cancer cell) increases its intracellular lactate concentration by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody agent to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) reduces the ECAR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody agent to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) increases the OCR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, reference to an antibody agent that specifically binds to MCT4 means that the antibody agent binds to MCT4 with an affinity that is at least about 10 times (including for example at least about any of 10, 10², 10³, 10⁴, 10⁵, 10⁶, or 10⁷ times) its binding affinity for non-target. In some embodiments, the non-target is an antigen that is not MCT4. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). ¾ can be determined by methods known in the art, such as surface plasmon resonance (SPR) assay utilizing, for example, Biacore instruments, or kinetic exclusion assay (KinExA) utilizing, for example, Sapidyne instruments.

[0072] Contemplated anti-MCT4 antibody agents include, for example, full-length anti-MCT4 antibodies (e.g., full-length IgG4), anti-MCT4 scFvs, anti-MCT4 Fc fusion proteins, multi specific (such as bispecific) anti-MCT4 antibodies, anti-MCT4 immunoconjugates, and the like.

[0073] Although anti-MCT4 antibody agents containing human sequences (e.g., human heavy and light chain variable domain sequences comprising human CDR sequences) are extensively discussed herein, non-human anti-MCT4 antibody agents are also contemplated. In some embodiments, non-human anti-MCT4 antibody agents comprise human CDR sequences from an anti-MCT4 antibody agent as described herein and non-human framework sequences. Non human framework sequences include, in some embodiments, any sequence that can be used for generating synthetic heavy and/or light chain variable domains using one or more human CDR sequences as described herein, including, e.g., mammals, e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey), etc. In some embodiments, a non-human anti-MCT4 antibody agent includes an anti-MCT4 antibody agent generated by grafting one or more human CDR sequences as described herein onto a non-human framework sequence (e.g., a mouse or chicken framework sequence).

[0074] The complete amino acid sequence of an exemplary human MCT4 comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-MCT4 antibody agent described herein specifically recognizes an epitope within human MCT4. In some embodiments, the anti-MCT4 antibody agent may cross-react with MCT4 from species other than human. In some embodiments, the anti-MCT4 antibody agent may be completely specific for human MCT4 and may not exhibit species or other types of non-human cross reactivity.

[0075] In some embodiments, the anti-MCT4 antibody agent cross-reacts with at least one allelic variant of the MCT4 protein (or fragments thereof). In some embodiments, the allelic variant has up to about 30 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) amino acid substitutions (such as a conservative substitution) when compared to the naturally occurring MCT4 (or fragments thereof). In some embodiments, the anti-MCT4 antibody agent does not cross-react with any allelic variant of the MCT4 protein (or fragments thereof).

[0076] In some embodiments, the anti-MCT4 antibody agent cross-reacts with at least one interspecies variant of the MCT4 protein. In some embodiments, for example, the MCT4 protein (or fragments thereof) is human MCT4 and the interspecies variant of the MCT4 protein (or fragments thereof) is a mouse or rat variant thereof. In some embodiments, the anti-MCT4 antibody agent does not cross-react with any interspecies variant of the MCT4 protein.

[0077] In some embodiments, the anti-MCT4 antibody agent specifically recognizes MCT4 expressed on the cell surface of a cancer cell (such as solid tumor). In some embodiments, the anti-MCT4 antibody agent specifically recognizes MCT4 expressed on the cell surface of one or more of breast cancer cells prostate cancer cells, colon cancer cells, brain cancer cells, pancreatic cancer cells, and kidney cancer cells. In some embodiments, the anti-MCT4 antibody agent specifically recognizes MCT4 expressed on the cell surface of one or more of colon cancer cell line LS174T, kidney cancer cell lines A498, RCC4, and UOK262, and pancreatic cancer cell lines PL45 and Panel.

[0078] In some embodiments, according to any of the anti-MCT4 antibody agents described herein, the anti-MCT4 antibody agent comprises an anti-MCT4 antibody moiety that specifically binds to MCT4. In some embodiments, the anti-MCT4 antibody moiety comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the anti-MCT4 antibody moiety comprises an IgGl heavy chain constant region. In some embodiments, the anti-MCT4 antibody moiety comprises an IgG2 heavy chain constant region.

In some embodiments, the anti-MCT4 antibody moiety comprises an IgG3 heavy chain constant region. In some embodiments, the anti-MCT4 antibody moiety comprises an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some

embodiments, the anti-MCT4 antibody moiety comprises a lambda light chain constant region.

In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 antibody moiety comprises a kappa light chain constant region. In some embodiments, the anti-MCT4 antibody moiety comprises an antibody heavy chain variable domain and an antibody light chain variable domain.

[0079] In some embodiments, the anti-MCT4 antibody moiety comprises a heavy chain variable domain comprising one, two or three HC-CDRs of SEQ ID NO: 2. In some

embodiments, the anti-MCT4 antibody moiety comprises a heavy chain variable domain comprising one, two or three HC-CDRs of SEQ ID NO: 3. In some embodiments, the anti- MCT4 antibody moiety comprises a heavy chain variable domain comprising one, two or three HC-CDRs of SEQ ID NO: 4. In some embodiments, the anti-MCT4 antibody moiety comprises a light chain variable domain comprising one, two or three LC-CDRs of SEQ ID NO: 5. In some embodiments, the anti-MCT4 antibody moiety comprises a light chain variable domain comprising one, two or three LC-CDRs of SEQ ID NO: 6. In some embodiments, the anti- MCT4 antibody moiety comprises a light chain variable domain comprising one, two or three LC-CDRs of SEQ ID NO: 7. In some embodiments, the anti-MCT4 antibody moiety comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 2, and a light chain variable domain comprising LC- CDR1, LC-CDR2 and LC-CDR3 of the light chain variable domain of SEQ ID NO: 5. In some embodiments, the anti-MCT4 antibody moiety comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 3, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain variable domain of SEQ ID NO: 6. In some embodiments, the anti-MCT4 antibody moiety comprises a heavy chain variable domain comprising HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain variable domain of SEQ ID NO: 4, and a light chain variable domain comprising LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain variable domain of SEQ ID NO: 7.

[0080] In some embodiments, the antibody heavy chain variable domain comprises an HC- CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the antibody heavy chain variable domain comprises an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16. In some embodiments, the light chain variable domain comprises an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the light chain variable domain comprises an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25. In some embodiments, a) the heavy chain variable domain comprises an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and b) the light chain variable domain comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

In some embodiments, a) the heavy chain variable domain comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) the light chain variable domain comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, a) the heavy chain variable domain comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) the light chain variable domain comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0081] In some embodiments, the anti-MCT4 antibody moiety comprises a heavy chain variable domain comprising the sequences of SEQ ID NOs: 8, 11 and 14; and a light chain variable domain comprising the sequences of SEQ ID NOs: 17, 20 and 23. In some

embodiments, the anti-MCT4 antibody moiety comprises a heavy chain variable domain comprising the sequences of SEQ ID NOs: 9, 12 and 15; and a light chain variable domain comprising the sequences of SEQ ID NOs: 18, 21 and 24. In some embodiments, the anti-MCT4 antibody moiety comprises a heavy chain variable domain comprising the sequences of SEQ ID NOs: 10, 13 and 16; and a light chain variable domain comprising the sequences of SEQ ID NOs: 19, 22 and 25.

[0082] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, and the light chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 2 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 4 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 7. [0083] Exemplary antibody sequences are shown in Tables 2-3 below. The exemplary CDR sequences in Table 2 are predicted using the IgBLAST algorithm. See , for example, Ye J. et al. Nucleic Acids Research, 4EW34-W40 (2013), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that many algorithms are known for prediction of CDR positions in antibody heavy chain and light chain variable regions, and antibody agents comprising CDRs from antibodies described herein, but based on prediction algorithms other than IgBLAST, are within the scope of this invention.

[0084] The exemplary antibody heavy chain and light chain variable region sequences are delimited according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM^® (IMGT). See , for example, Lefranc, M.-P. et ah, Nucleic Acids Res., 43 :D4l3-422 (2015), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that antibody agents comprising V_H or V_L sequences from antibodies described herein, but based on algorithms other than IMGT, are within the scope of this invention.

Table 2. Exemplary anti-MCT4 antibody CDR sequences.

Table 3. Exemplary sequences.

Full-length anti-MCT4 antibody

[0085] The anti-MCT4 antibody agent in some embodiments is a full-length anti-MCT4 antibody. In some embodiments, the full-length anti-MCT4 antibody is an IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length anti-MCT4 antibody comprises IgG constant domains, such as constant domains of any of IgGl, IgG2, IgG3, and IgG4 including variants thereof. In some embodiments, the full-length anti-MCT4 antibody comprises a lambda light chain constant region. In some embodiments, the full-length anti-MCT4 antibody comprises a kappa light chain constant region. In some embodiments, the full-length anti-MCT4 antibody is a full-length human anti-MCT4 antibody. In some embodiments, the full-length anti-MCT4 antibody comprises an Fc sequence of a mouse immunoglobulin. In some embodiments, the full- length anti-MCT4 antibody comprises an Fc sequence that has been altered or otherwise changed so that it has enhanced antibody dependent cellular cytotoxicity (ADCC) or

complement dependent cytotoxicity (CDC) effector function.

[0086] Thus, for example, in some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody specifically binds to MCT4 and antagonizes its ability to transport lactate. In some embodiments, the IgGl is human IgGl . In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) reduces its intracellular pH by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) increases its intracellular lactate concentration by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) reduces the ECAR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) increases the OCR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more).

[0087] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG2 constant domains, wherein the anti-MCT4 antibody specifically binds to MCT4 and antagonizes its ability to transport lactate. In some embodiments, the IgG2 is human IgG2. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) reduces its intracellular pH by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) increases its intracellular lactate concentration by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) reduces the ECAR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4- expressing cell (e.g., an MCT4-expressing cancer cell) increases the OCR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more).

[0088] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG3 constant domains, wherein the anti-MCT4 antibody specifically binds to MCT4 and antagonizes its ability to transport lactate. In some embodiments, the IgG3 is human IgG3. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) reduces its intracellular pH by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) increases its intracellular lactate concentration by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) reduces the ECAR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4- expressing cell (e.g., an MCT4-expressing cancer cell) increases the OCR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more).

[0089] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody specifically binds to MCT4 and antagonizes its ability to transport lactate. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) reduces its intracellular pH by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4- expressing cell (e.g., an MCT4-expressing cancer cell) increases its intracellular lactate concentration by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) reduces the ECAR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more). In some embodiments, binding of the anti-MCT4 antibody to an MCT4-expressing cell (e.g., an MCT4-expressing cancer cell) increases the OCR by at least about 5% (such as at least about any of 6, 7, 8, 9, 10, 15, 20%, or more).

[0090] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC- CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the IgGl is human IgGl . In some embodiments, the anti- MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0091] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG2 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3,

4, or 5) amino acid substitutions; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC- CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the IgG2 is human IgG2. In some embodiments, the anti- MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. [0092] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG3 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3,

4, or 5) amino acid substitutions; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC- CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the IgG3 is human IgG3. In some embodiments, the anti- MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0093] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3,

4, or 5) amino acid substitutions; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC- CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti- MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0094] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11- 13, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequences; and b) a light chain variable domain comprising an LC- CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in the LC-CDR sequences. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:

27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0095] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11- 13, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequences; and b) a light chain variable domain comprising an LC- CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in the LC-CDR sequences. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:

28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0096] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11- 13, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0097] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11- 13, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0098] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

[0099] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

[0100] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

[0101] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

[0102] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

[0103] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

[0104] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity, and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0105] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG2 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity, and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity. In some embodiments, the IgG2 is human IgG2. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0106] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG3 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity, and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity. In some embodiments, the IgG3 is human IgG3. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0107] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity, and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. [0108] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 2-4, and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0109] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 2-4, and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0110] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0111] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0112] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0113] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0114] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0115] In some embodiments, there is provided a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29.

Chimeric anti-MCT4 constructs

[0116] In some embodiments, the anti-MCT4 antibody agent is an anti-MCT4 chimeric antigen receptor (CAR) or variant thereof that specifically binds to MCT4. In some

embodiments, the anti-MCT4 antibody agent is an anti-MCT4 CAR. CARs are well known in the art, and the anti-MCT4 antibody agent can be a CAR according to any CAR known in the art, such as described in Sadelain et al. (2017) Nature 545: 423- 431, the disclosure of which is explicitly incorporated herein for use in the present invention and for possible inclusion in one or more claims herein. In some embodiments, the anti-MCT4 CAR comprises an anti-MCT4 antibody moiety according to any of the anti-MCT4 antibody moieties described herein. For example, in some embodiments, there is provided an anti-MCT4 CAR comprising an anti-MCT4 antibody moiety. In some embodiments, the anti-MCT4 antibody moiety comprises an antibody heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17- 19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity.

[0117] In some embodiments, the anti-MCT4 antibody agent is an anti-MCT4 chimeric receptor comprising T cell receptor (TCR) transmembrane domains. For example, in some embodiments, the anti-MCT4 antibody agent is an antibody-T cell receptor (abTCR) as described in PCT Patent Application Publication No. WO2017070608, the disclosure of which is explicitly incorporated herein for use in the present invention and for possible inclusion in one or more claims herein. In some embodiments, the anti-MCT4 abTCR comprises an anti-MCT4 antibody moiety according to any of the anti-MCT4 antibody moieties described herein. For example, in some embodiments, there is provided an anti-MCT4 abTCR comprising an anti- MCT4 antibody moiety. In some embodiments, the anti-MCT4 antibody moiety comprises an antibody heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity.

[0118] In some embodiments, the anti-MCT4 antibody agent is a chimeric co-stimulatory receptor comprising an anti-MCT4 antibody moiety that specifically binds to MCT4 and a co stimulatory signaling domain. In some embodiments, the anti-MCT4 chimeric co- stimulatory receptor is capable of stimulating an immune cell on the surface of which it is functionally expressed upon binding MCT4. In some embodiments, the anti-MCT4 chimeric co- stimulatory receptor lacks a functional primary immune cell signaling sequence. In some embodiments, the anti-MCT4 chimeric co-stimulatory receptor lacks any primary immune cell signaling sequence. In some embodiments, the anti-MCT4 chimeric co-stimulatory receptor comprises a single polypeptide chain comprising the anti-MCT4 antibody moiety, a transmembrane domain, and the co-stimulatory signaling domain. In some embodiments, the anti-MCT4 chimeric co stimulatory receptor comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains together form the anti-MCT4 antibody moiety, a transmembrane module, and co-stimulatory signaling module comprising the co-stimulatory signaling domain. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and the anti-MCT4 chimeric co-stimulatory receptor is a multimer, such as a dimer. In some embodiments, the first and second polypeptide chains are covalently linked, such as by a peptide linkage, or by another chemical linkage, such as a disulfide linkage. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked by at least one disulfide bond. In some embodiments, the anti-MCT4 antibody moiety is a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv).

[0119] Examples of co-stimulatory immune cell signaling domains for use in the anti-MCT4 chimeric co-stimulatory receptors of the invention include the cytoplasmic sequences of co receptors of the T cell receptor (TCR), which can act in concert with a chimeric receptor (e.g., a CAR or abTCR) to initiate signal transduction following chimeric receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0120] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequence: those that initiate antigen-dependent primary activation through the TCR (referred to herein as“primary immune cell signaling sequences”) and those that act in an antigen- independent manner to provide a secondary or co-stimulatory signal (referred to herein as“co stimulatory immune cell signaling sequences”).

[0121] Primary immune cell signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing primary immune cell signaling sequences include those derived from TOIz, FcRy, FcRp, CD3y, CD35, CD3e, CD5, CD22, CD79a, CD79b, and CD66d. A“functional” primary immune cell signaling sequence is a sequence that is capable of transducing an immune cell activation signal when operably coupled to an appropriate receptor. “Non-functional” primary immune cell signaling sequences, which may comprises fragments or variants of primary immune cell signaling sequences, are unable to transduce an immune cell activation signal. The anti-MCT4 chimeric co- stimulatory receptors described herein lack a functional primary immune cell signaling sequence, such as a functional signaling sequence comprising an IT AM. In some embodiments, the anti-MCT4 chimeric co-stimulatory receptors lack any primary immune cell signaling sequence.

[0122] The co-stimulatory immune cell signaling sequence can be a portion of the intracellular domain of a co-stimulatory molecule including, for example, CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.

[0123] In some embodiments, the anti-MCT4 antibody moiety of an anti-MCT4 chimeric co stimulatory receptor comprises an antibody heavy chain variable domain comprising an HC- CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC- CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% (for example at least about any of 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity.

[0124] In some embodiments, the anti-MCT4 chimeric co-stimulatory receptor is expressed in an immune cell. In some embodiments, the anti-MCT4 chimeric co-stimulatory receptor is expressed in an immune cell that expresses another chimeric receptor. In some embodiments, the other chimeric receptor is a CAR or an abTCR. In some embodiments, the other chimeric receptor binds to MCT4. In some embodiments, the other chimeric receptor does not bind to MCT4. In some embodiments, the other chimeric receptor binds to an antigen associated with a cancer characterized by high expression of MCT4 and/or high aerobic glycolysis. In some embodiments, the other chimeric receptor binds to an antigen associated with any of the cancers described herein (such as kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, or pancreatic cancer). In some embodiments, the other chimeric receptor binds to an antigen associated with kidney cancer. In some embodiments, the kidney cancer is renal cell carcinoma (RCC). In some embodiments, the RCC is metastatic RCC. In some embodiments, the immune cell is a T cell. In some embodiments, expression of the anti-MCT4 chimeric co-stimulatory receptor in the immune cell is inducible. In some embodiments, the expression of the anti-MCT4 chimeric co-stimulatory receptor in the immune cell is inducible upon signaling through the other chimeric receptor.

Binding affinity

[0125] Binding affinity can be indicated by K_d, K_off, K_on, or K_a. The term

“K_0ff”, as used herein, is intended to refer to the off-rate constant for dissociation of an antibody agent from the antibody agent/antigen complex, as determined from a kinetic selection set up. The term“K_on”, as used herein, is intended to refer to the on-rate constant for association of an antibody agent to the antigen to form the antibody agent/antigen complex. The term equilibrium dissociation constant“K_d”, as used herein, refers to the dissociation constant of a particular antibody agent-antigen interaction, and describes the concentration of antigen required to occupy one half of all of the antibody -binding domains present in a solution of antibody agent molecules at equilibrium, and is equal to K_off/K_on. The measurement of K_d presupposes that all binding agents are in solution. In the case where the antibody agent is tethered to a cell wall, e.g ., in a yeast expression system, the corresponding equilibrium rate constant is

expressed as EC50, which gives a good approximation of K_d. The affinity constant, K_a, is the inverse of the dissociation constant, K_d.

[0126] The dissociation constant (K_d) is used as an indicator showing affinity of antibody moieties to antigens. For example, easy analysis is possible by the Scatchard method using antibody agents marked with a variety of marker agents, as well as by using Biacore (made by Amersham Biosciences), analysis of biomolecular interactions by surface plasmon resonance, according to the user's manual and attached kit. The K_d value that can be derived using these methods is expressed in units of M (Mols). An antibody agent that specifically binds to a target may have a K_d of, for example, < 10⁷ M, < 10⁸ M, < 10⁹ M, < 10¹⁰ M, < 10¹¹ M, < 10¹² M, or < 10¹³ M.

[0127] Binding specificity of the antibody agent can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore-tests and peptide scans. In some embodiments, the binding affinity of the anti-MCT4 antibody agent is measured by testing the binding affinity of the anti- MCT4 antibody agent to cells expressing MCT4 on the surface (e.g., HepG2 cells).

[0128] In some embodiments, the anti-MCT4 antibody agent specifically binds to a target MCT4 (e.g, nMCT4) with a K_d of about 10⁷M to about 10¹³ M (such as about 10⁷ M to about 10¹³ M, about 10⁹ M to about 10¹³ M, or about 10¹⁰ M to about 10¹² M). Thus in some embodiments, the K_d of the binding between the anti-nMCT4 antibody agent and nMCT4, the K_d of the binding between the anti-sMCT4 antibody agent and sMCT4, or the K_d of the binding between the anti-MCT4 antibody agent and MCT4 (any format), is about lO^_7M to about lO^_13M, about lxlO^_7Mto about 5^c10^_13M, about lO^_7Mto about lO^_12M, about lO^_7Mto about lO^_11M, about lO^_7M to about lO^_10M, about lO^_7M to about lO^_9M, about lO^_8M to about lO^_13M, about lxlO^_8Mto about 5^c10^_13M, about lO^_8Mto about lO^_12M, about lO^_8M to about lO^_11M, about lO^_8M to about lO^_10M, about lO^_8M to about lO^_9M, about 5^c10^_9M to about lxlO^_13M, about 5xl0^_9Mto about 1^c10^_12M, about 5xl0^_9Mto about 1^c10^_11M, about 5 x 10⁹ M to about 1 c 1 Cf ¹⁰ M, about 10⁹ M to about 10¹³ M, about 10⁹ M to about lO^_12M, about lO^_9Mto about l0^_11M, about lO^_9Mto about lO^_10M, about 5xlO^_10Mto about 1^c10¹³M, about 5 xlO ¹⁰M to about 1^c10¹²M, about 5 ^c10¹⁰M to about 1^c10^_11M, about 10¹⁰ M to aboutl 0^-13 M, about lxl 0¹⁰ M to about 5x10¹³ M, about lxl 0¹⁰ M to about 1^c10¹²M, about 1^c10¹⁰M to about 5xlO ¹²M, about 1^c10¹⁰M to about 1^c10^_11M, about 10 ^uMto about 10¹³M, about 1x10¹¹ M to about 5x10¹³M, about 10 ^uMto about 10¹² M, or about lO^_12M to about l0^_13M. In some embodiments, the K_d of the binding between the anti- nMCT4 antibody agent and an nMCT4 is about 10⁷ M to about 10¹³ M.

[0129] In some embodiments, the K_d of the binding between the anti-MCT4 antibody agent and a non-target is more than the K_d of the binding between the anti-MCT4 antibody agent and the target, and is herein referred to in some embodiments as the binding affinity of the anti- MCT4 antibody agent to the target ( e.g ., cell surface-bound MCT4) is higher than that to a non target. In some embodiments, the non-target is an antigen that is not MCT4. In some

embodiments, the K_d of the binding between the anti-MCT4 antibody agent (against nMCT4) and a non-MCT4 target can be at least about 10 times, such as about 10-100 times, about 100- 1000 times, about 10³-10⁴ times, about 10⁴-10⁵ times, about 10⁵-10⁶ times, about 10⁶-10⁷ times, about 10⁷-10⁸ times, about 10⁸-10⁹ times, about 10⁹-10¹⁰ times, about l0¹⁰-l0^u times, or about l0^u-l0¹² times of the K_d of the binding between the anti-MCT4 antibody agent and a target MCT4.

[0130] In some embodiments, the anti-MCT4 antibody agent binds to a non-target with a K_d of about 10¹ M to about 10⁶ M (such as about 10¹ M to about 10⁶ M, about 10¹ M to about 10⁵ M, or about 10² M to about 10⁴ M). In some embodiments, the non-target is an antigen that is not MCT4. Thus in some embodiments, the K_d of the binding between the anti-MCT4 antibody agent and a non-MCT4 target is about 10¹ M to about 10⁶ M, about 1 ^c 10¹ M to about 5x 10⁶ M, about 10¹ M to about 10⁵ M, about 1 ^c 10¹ M to about 5x 10⁵ M, about 10¹ M to about 10⁴ M, about 1 x 10¹ M to about 5x 10⁴ M, about 10¹ M to about 10³ M, about 1 ^c 10¹ M to about 5x 10³ M, about l0^_1M to about 10² M, about 10²M to about 10⁶ M, about 1^c10²M to about 5xl0⁶ M, about lO²Mto about 10⁵ M, about lxlO²Mto about 5xl0⁵ M, about lO²Mto about 10⁴ M, about 1 x 1 O² M to about 5 ^c 1 O⁴ M, about 1 O² M to about 10³ M, about 10³ M to about 1 O⁶ M, about 1 x 10³ M to about 5x 10⁶ M, about 10³ M to about 10⁵ M, about 1 ^c 10³ M to about 5 x 10⁵ M, about 10³ M to about 1 O⁴ M, about 1 O⁴ M to about 1 O⁶ M, about 1 ^c 1 O⁴ M to about 5 x 1 O⁶ M, about 1 O⁴ M to about 10⁵ M, or about 10⁵ M to about 1 O⁶ M.

[0131] In some embodiments, when refering to that the anti-MCT4 antibody agent specifically recognizes a target MCT4 (e.g., cell surface-bound MCT4) at a high binding affinity, and binds to a non-target at a low binding affinity, the anti-MCT4 antibody agent will bind to the target MCT4 (e.g, cell surface-bound MCT4) with a K_d of about 10⁷M to about 10¹³ M (such as about 10⁷ M to about 10¹³ M, about 10⁹ M to about 10¹³ M, or about 10¹⁰ M to about 10¹² M), and will bind to the non-target with a K_d of about 10 ¹ M to about 10 ⁶ M (such as about 10 ¹ M to about 10 ⁶ M, about 10 ¹ M to about 10 ⁵ M, or about 10 ² M to about 10 ⁴ M).

[0132] In some embodiments, when referring to that the anti-MCT4 antibody agent specifically recognizes a cell surface-bound MCT4, the binding affinity of the anti-MCT4 antibody agent is compared to a control anti-MCT4 antibody agent. In some embodiments, the K_d of the binding between the control anti-MCT4 antibody agent and a cell surface-bound MCT4 can be at least about 2 times, such as about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 10-100 times, about 100-1000 times, about 10³-10⁴ times, about 10⁴-10⁵ times, about 10⁵-10⁶ times, about 10⁶-10⁷ times, about 10⁷-10⁸ times, about 10⁸-10⁹ times, about 10⁹-10¹⁰ times, about 10¹⁰- 10¹¹ times, or about l0^u-l0¹² times of the K of the binding between the anti-nMCT4 antibody agent described herein and a cell surface-bound MCT4.

Nucleic Acids

[0133] Nucleic acid molecules encoding the anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti-MCT4 antibodies) are also contemplated. In some embodiments, there is provided a nucleic acid (or a set of nucleic acids) encoding a full-length anti-MCT4 antibody, including any of the full-length anti-MCT4 antibodies described herein. In some embodiments, the nucleic acid (or a set of nucleic acids) encoding the anti-MCT4 antibody agent described herein may further comprises a nucleic acid sequence encoding a peptide tag (such as protein purification tag, e.g., His-tag, HA tag).

[0134] Also contemplated here are isolated host cells comprising an anti-MCT4 antibody agent, an isolated nucleic acid encoding the polypeptide components of the anti-MCT4 antibody agent, or a vector comprising a nucleic acid encoding the polypeptide components of the anti- MCT4 antibody agent described herein.

[0135] The present application also includes variants to these nucleic acid sequences. For example, the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti- MCT4 antibodies) or anti-MCT4 antibody moieties of the present application under at least moderately stringent hybridization conditions.

[0136] The present invention also provides vectors in which a nucleic acid of the present invention is inserted.

[0137] In brief summary, the expression of an anti-MCT4 antibody agent (e.g, full-length anti-MCT4 antibody) by a natural or synthetic nucleic acid encoding the anti-MCT4 antibody agent can be achieved by inserting the nucleic acid into an appropriate expression vector, such that the nucleic acid is operably linked to 5’ and 3’ regulatory elements, including for example a promoter ( e.g ., a lymphocyte-specific promoter) and a 3’ untranslated region (UTR). The vectors can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0138] The nucleic acids of the present invention may also be used for nucleic acid

immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In some embodiments, the invention provides a gene therapy vector.

[0139] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0140] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

[0141] A number of viral based systems have been developed for gene transfer into

mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0142] Additional promoter elements, e.g ., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

[0143] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human

immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0144] In some embodiments, the expression of the anti-MCT4 antibody agent is inducible. In some embodiments, a nucleic acid sequence encoding the anti-MCT4 antibody agent is operably linked to an inducible promoter, including any inducible promoter described herein.

Inducible promoters

[0145] The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Exemplary inducible promoter systems for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see, e.g., Spencer, D. M. et al 1993) Science 262: 1019- 1024) and ionizing radiation-regulated elements (e.g., see Manome, Y. et al. (1993)

Biochemistry 32: 10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1014- 10153). Further exemplary inducible promoter systems for use in in vitro or in vivo mammalian systems are reviewed in Gingrich et al. (1998) Annual Rev. Neurosci 21 :377-405. In some embodiments, the inducible promoter system for use to express the anti-MCT4 antibody agent is the Tet system. In some embodiments, the inducible promoter system for use to express the anti- MCT4 antibody agent is the lac repressor system from E. coli.

[0146] An exemplary inducible promoter system for use in the present invention is the Tet system. Such systems are based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, a polynucleotide of interest is under the control of a promoter that comprises one or more Tet operator (TetO) sites. In the inactive state, Tet repressor (TetR) will bind to the TetO sites and repress transcription from the promoter. In the active state, e.g., in the presence of an inducing agent such as tetracycline (Tc), anhydrotetracy cline, doxy cy cline (Dox), or an active analog thereof, the inducing agent causes release of TetR from TetO, thereby allowing transcription to take place. Doxycycline is a member of the tetracycline family of antibiotics having the chemical name of l-dimethylamino-2,4a,5,7,l2-pentahydroxy-l l-methyl- 4,6-dioxo-l,4a,l l,l la,l2,l2a-hexahydrotetracene-3-carboxamide.

[0147] In one embodiment, a TetR is codon-optimized for expression in mammalian cells, e.g., murine or human cells. Most amino acids are encoded by more than one codon due to the degeneracy of the genetic code, allowing for substantial variations in the nucleotide sequence of a given nucleic acid without any alteration in the amino acid sequence encoded by the nucleic acid. However, many organisms display differences in codon usage, also known as“codon bias” (i.e., bias for use of a particular codon(s) for a given amino acid). Codon bias often correlates with the presence of a predominant species of tRNA for a particular codon, which in turn increases efficiency of mRNA translation. Accordingly, a coding sequence derived from a particular organism (e.g., a prokaryote) may be tailored for improved expression in a different organism (e.g., a eukaryote) through codon optimization.

[0148] Other specific variations of the Tet system include the following“Tet-Off’ and“Tet- On” systems. In the Tet-Off system, transcription is inactive in the presence of Tc or Dox. In that system, a tetracycline-controlled transactivator protein (tTA), which is composed of TetR fused to the strong transactivating domain of VP 16 from Herpes simplex virus, regulates expression of a target nucleic acid that is under transcriptional control of a tetracycline- responsive promoter element (TRE). The TRE is made up of TetO sequence concatamers fused to a promoter (commonly the minimal promoter sequence derived from the human

cytomegalovirus (hCMV) immediate-early promoter). In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive.

[0149] Conversely, in the Tet-On system, transcription is active in the presence of Tc or Dox. The Tet-On system is based on a reverse tetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusion protein comprised of the TetR repressor and the VP 16 transactivation domain. However, a four amino acid change in the TetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of Dox. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.

[0150] Another inducible promoter system is the lac repressor system from E. coli {See Brown et al., Cell 49:603-612 (1987)). The lac repressor system functions by regulating transcription of a polynucleotide of interest operably linked to a promoter comprising the lac operator (lacO).

The lac repressor (lacR) binds to LacO, thus preventing transcription of the polynucleotide of interest. Expression of the polynucleotide of interest is induced by a suitable inducing agent, e.g., i sopropy 1 -b-D-thi ogal actopy ranosi de (IPTG).

[0151] In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Eiseful selectable markers include, for example, antibiotic- resistance genes, such as neo and the like.

[0152] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, b-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene ( e.g ., Ui-Tel et al, 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0153] In some embodiments, there is provided nucleic acid encoding a full-length anti-MCT4 antibody according to any of the full-length anti-MCT4 antibodies described herein. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the full-length anti-MCT4 antibody. In some embodiments, each of the one or more nucleic acid sequences are contained in separate vectors. In some embodiments, at least some of the nucleic acid sequences are contained in the same vector. In some embodiments, all of the nucleic acid sequences are contained in the same vector. Vectors may be selected, for example, from the group consisting of mammalian expression vectors and viral vectors (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses).

[0154] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0155] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, the introduction of a polynucleotide into a host cell is carried out by calcium phosphate transfection.

[0156] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus 1, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and

5,585,362. [0157] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome e.g ., an artificial membrane vesicle).

[0158] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell {in vitro , ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the

oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a“collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0159] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example,“molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELIS As and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

Preparation of anti-MCT4 antibody agents and anti-MCT4 antibody moieties

[0160] In some embodiments, the anti-MCT4 antibody agent is a monoclonal antibody or derived from a monoclonal antibody. In some embodiments, the anti-MCT4 antibody agent comprises V_H and V_L domains, or variants thereof, from the monoclonal antibody. In some embodiments, the anti-MCT4 antibody agent further comprises CM and C_L domains, or variants thereof, from the monoclonal antibody. Monoclonal antibodies can be prepared, e.g., using known methods in the art, including hybridoma methods, phage display methods, or using recombinant DNA methods. Additionally, exemplary phage display methods are described herein and in the Examples below.

[0161] In a hybridoma method, a hamster, mouse, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro. The immunizing agent can include a polypeptide or a fusion protein of the protein of interest. Generally, peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which prevents the growth of HGPRT-deficient cells.

[0162] In some embodiments, the immortalized cell lines fuse efficiently, support stable high- level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. In some embodiments, the immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies.

[0163] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide. The binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by

immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0164] After the desired hybridoma cells are identified, the clones can be sub cloned by limiting dilution procedures and grown by standard methods. Goding, supra. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0165] The monoclonal antibodies secreted by the sub clones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0166] In some embodiments, according to any of the anti-MCT4 antibody agents described herein, the anti-MCT4 antibody agent comprises sequences from a clone selected from an antibody library (such as a phage library presenting scFv or Fab fragments). The clone may be identified by screening combinatorial libraries for antibody fragments with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding

characteristics. Such methods are reviewed, e.g., in Hoogenboom el al, Methods in Molecular Biology 178: 1-37 (O'Brien et al. , ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in McCafferty et al, Nature 348:552-554; Clackson et al, Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee et 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).

[0167] In certain phage display methods, repertoires of V_H and V_L genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et aI., Ahh. Rev. Immunol.,

12: 433-455 (1994). Phage typically display antibody fragments, either as scFv fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g, from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al, EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro , as described by Hoogenboom and Winter, J. Mol. Biol ., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

[0168] The anti-MCT4 antibody agents can be prepared using phage display to screen libraries for anti-MCT4 antibody moieties specific to the target MCT4 (e.g, nMCT4). The library can be a human scFv phage display library having a diversity of at least one x 10⁹ (such as at least about any of 1 ^c 10⁹, 2.5 ^c 10⁹, 5 ^c 10⁹, 7.5 ^c 10⁹, 1 ^c 10¹⁰, 2.5 ^c 10¹⁰, 5 ^c 10¹⁰, 7.5 ^c 10¹⁰, or 1 ^c 10¹¹) unique human antibody fragments. In some embodiments, the library is a naive human library constructed from DNA extracted from human PMBCs and spleens from healthy donors, encompassing all human heavy and light chain subfamilies. In some embodiments, the library is a naive human library constructed from DNA extracted from PBMCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi -synthetic human library, wherein heavy chain CDR3 is completely randomized, with all amino acids (with the exception of cysteine) equally likely to be present at any given position (see, e.g, Hoet, R.M. et al. , Nat. Biotechnol. 23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 of the semi synthetic human library has a length from about 5 to about 24 (such as about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) amino acids. In some embodiments, the library is a fully-synthetic phage display library. In some embodiments, the library is a non human phage display library.

[0169] Phage clones that bind to the target MCT4 (e.g, nMCT4) with high affinity can be selected by iterative binding of phage to the target MCT4, which is bound to a solid support (such as, for example, beads for solution panning or mammalian cells for cell panning), followed by removal of non-bound phage and by elution of specifically bound phage. The bound phage clones are then eluted and used to infect an appropriate host cell, such as E. coli XL 1 -Blue, for expression and purification. In an example of cell panning, HEK293 cells over-expressing MCT4 on cell surface are mixed with the phage library, after which the cells are collected and the bound clones are eluted and used to infect an appropriate host cell for expression and purification (all see Examples). The panning can be performed for multiple (such as about any of 2, 3, 4, 5, 6 or more) rounds with solution panning, cell panning, or a combination of both, to enrich for phage clones binding specifically to the target MCT4. Enriched phage clones can be tested for specific binding to the target MCT4 by any methods known in the art, including for example ELISA and FACS.

[0170] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in ET.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures ( e.g by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells as described above or MCT4-specific phage clones of the invention can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains and/or framework regions in place of the homologous non-human sequences (ET.S. Patent No. 4,816,567; Morrison et al, supra ) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody agent of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody agent of the invention to create a chimeric bivalent antibody agent.

[0171] The antibodies can be monovalent antibodies. Methods for preparing monovalent antibodies are known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

[0172] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using any method known in the art.

[0173] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant-domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.

Human and Humanized Antibodies

[0174] The anti-MCT4 antibody agents (e.g, full-length anti-MCT4 antibodies) can be humanized antibody agents or human antibody agents. Humanized forms of non-human (e.g, murine) antibody moieties are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab’, F(ab’)₂, scFv, or other antigen-binding subsequences of antibodies) that typically contain minimal sequence derived from non-human immunoglobulin. Humanized antibody moieties include human immunoglobulins, immunoglobulin chains, or fragments thereof (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibody moieties can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.

[0175] Generally, a humanized antibody agent has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an“import” variable domain. According to some embodiments, humanization can be essentially performed following the method of Winter and co-workers (Jones et al. , Nature, 321 : 522-525 (1986); Riechmann et al. , Nature,

332: 323-327 (1988); Verhoeyen et ah, Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such“humanized” antibody moieties are antibody moieties (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the

corresponding sequence from a non-human species. In practice, humanized antibody moieties are typically human antibody moieties in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0176] As an alternative to humanization, human antibody moieties can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ -line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g, Jakobovits et al. , PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al. , Year in Immunol.,

7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97/17852. Alternatively, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al, Bio/Technology,

10: 779-783 (1992); Lonberg et al, Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812- 813 (1994); Fishwild et al. , Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

[0177] Human antibody agents may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al, J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al, J. Immunol., 147(1): 86-95 (1991).

Anti-MCT4 variants

[0178] In some embodiments, amino acid sequence variants of the anti-MCT4 antibody agents (e.g., full-length anti-MCT4 antibody) provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody agent. Amino acid sequence variants of an antibody agent may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody agent, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody agent. Any

combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g ., antigen-binding.

[0179] In some embodiments, anti-MCT4 antibody agent variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Amino acid substitutions may be introduced into an antibody agent of interest and the products screened for a desired activity, e.g. , retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0180] Conservative substitutions are shown in Table 4 below.

TABLE 4: CONSERVATIVE SUBSTITITIONS

[0181] Amino acids may be grouped into different classes according to common side-chain properties:

a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

c. acidic: Asp, Glu;

d. basic: His, Lys, Arg;

e. residues that influence chain orientation: Gly, Pro; f. aromatic: Trp, Tyr, Phe.

[0182] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0183] An exemplary substitutional variant is an affinity matured antibody agent, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques.

Briefly, one or more CDR residues are mutated and the variant antibody moieties displayed on phage and screened for a particular biological activity (e.g, binding affinity). Alterations (e.g, substitutions) may be made in HVRs, e.g, to improve antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g, Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or specificity determining residues (SDRs), with the resulting variant V_H or V_L being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in

Molecular Biology 178: 1-37 (O'Brien et al, ed., Human Press, Totowa, NJ, (2001).)

[0184] In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody agent variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0185] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody agent to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR“hotspots” or SDRs. In some embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0186] A useful method for identification of residues or regions of an antibody agent that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by

Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g, charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid ( e.g ., alanine or polyalanine) to determine whether the interaction of the antibody agent with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen- antibody agent complex can be determined to identify contact points between the antibody agent and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0187] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody agent with an N-terminal methionyl residue. Other insertional variants of the antibody agent molecule include the fusion to the N- or C-terminus of the antibody agent to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half- life of the antibody agent.

Fc Region Variants

[0188] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody agent (e.g., a full-length anti-MCT4 antibody or anti-MCT4 Fc fusion protein) provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC effector function, often related to binding to Fc receptors (FcRs). In some embodiments, the Fc region variant has decreased ADCC effector function. There are many examples of changes or mutations to Fc sequences that can alter effector function. For example, WO 00/42072 and Shields et al. J Biol. Chem. 9(2): 6591-6604 (2001) describe antibody variants with improved or diminished binding to FcRs. The contents of those publications are specifically incorporated herein by reference.

[0189] Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is a mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell (e.g, a cancer cell), whose membrane-surface antigens have been bound by specific antibodies (e.g, an anti-MCT4 antibody). The typical ADCC involves activation of NK cells by antibodies. An NK cell expresses CD16 which is an Fc receptor. This receptor recognizes, and binds to, the Fc portion of an antibody bound to the surface of a target cell. The most common Fc receptor on the surface of an NK cell is called CD16 or F cy R 111. Binding of the Fc receptor to the Fc region of an antibody results in NK cell activation, release of cytolytic granules and consequent target cell apoptosis. The contribution of ADCC to tumor cell killing can be measured with a specific test that uses NK-92 cells that have been transfected with a high-affinity FcR. Results are compared to wild-type NK-92 cells that do not express the FcR.

[0190] In some embodiments, the invention contemplates an anti-MCT4 antibody agent variant (such as a full-length anti-MCT4 antibody variant) comprising an Fc region that possesses some but not all effector functions, which makes it a desirable candidate for applications in which the half-life of the anti-MCT4 antibody agent in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody agent lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRI I and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457- 492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad.

Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™ non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo , e.g. , in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody agent is unable to bind Clq and hence lacks CDC activity. See, e.g. , Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano- Santoro et al. , J.

Immunol. Methods 202: 163 (1996); Cragg, M. S. et al, Blood 101 : 1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al, Int'l. Immunol. 18(12): 1759-1769 (2006)). [0191] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

[0192] Certain antibody agent variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0193] In some embodiments, there is provided an anti-MCT4 antibody agent (such as a full- length anti-MCT4 antibody) variant comprising a variant Fc region comprising one or more amino acid substitutions which improve ADCC. In some embodiments, the variant Fc region comprises one or more amino acid substitutions which improve ADCC, wherein the

substitutions are at positions 298, 333, and/or 334 of the variant Fc region (EU numbering of residues). In some embodiments, the anti-MCT4 antibody agent (e.g, full-length anti-MCT4 antibody) variant comprises the following amino acid substitution in its variant Fc region:

S298A, E333A, and K334A.

[0194] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g, as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al., J.

Immunol. 164: 4178-4184 (2000).

[0195] In some embodiments, there is provided an anti-MCT4 antibody agent (such as a full- length anti-MCT4 antibody) variant comprising a variant Fc region comprising one or more amino acid substitutions which increase half-life and/or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-lives and improved binding to FcRn are described in US2005/0014934A1 (Hinton et all). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265,

272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g, substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

[0196] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.

[0197] Anti-MCT4 antibody agents (such as full-length anti-MCT4 antibodies) comprising any of the Fc variants described herein, or combinations thereof, are contemplated. Glycosylation Variants

[0198] In some embodiments, an anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) provided herein is altered to increase or decrease the extent to which the anti-MCT4 antibody agent is glycosylated. Addition or deletion of glycosylation sites to an anti-MCT4 antibody agent may be conveniently accomplished by altering the amino acid sequence of the anti-MCT4 antibody agent or polypeptide portion thereof such that one or more glycosylation sites is created or removed.

[0199] Where the anti-MCT4 antibody agent comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al, TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g. , mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the“stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an anti-MCT4 antibody agent of the invention may be made in order to create anti-MCT4 antibody agent variants with certain improved properties.

[0200] The N-glycans attached to the CH2 domain of Fc is heterogeneous. Antibodies or Fc fusion proteins generated in CHO cells are fucosylated by fucosyltransferase activity. See Shoji- Hosaka et al. , J. Biochem. 2006, 140:777- 83. Normally, a small percentage of naturally occurring afucosylated IgGs may be detected in human serum. N-glycosylation of the Fc is important for binding to FcyR; and afucosylation of the N-glycan increases Fc's binding capacity to FcyRIIIa. Increased FcyRIIIa binding can enhance ADCC, which can be advantageous in certain antibody agent therapeutic applications in which cytotoxicity is desirable.

[0201] In some embodiments, an enhanced effector function can be detrimental when Fc- mediated cytotoxicity is undesirable. In some embodiments, the Fc fragment or CH2 domain is not glycosylated. In some embodiments, the N-glycosylation site in the CH2 domain is mutated to prevent from glycosylation.

[0202] In some embodiments, anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) variants are provided comprising an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. Specifically, anti-MCT4 antibody agents are contemplated herein that have reduced fusose relative to the amount of fucose on the same anti-MCT4 antibody agent produced in a wild-type CHO cell. That is, they are characterized by having a lower amount of fucose than they would otherwise have if produced by native CHO cells ( e.g ., a CHO cell that produce a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene). In some embodiments, the anti-MCT4 antibody agent is one wherein less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon comprise fucose. For example, the amount of fucose in such an anti-MCT4 antibody agent may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. In some embodiments, the anti-MCT4 antibody agent is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the anti-MCT4 antibody agent is completely without fucose, or has no fucose or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or“fucose-deficient” antibody agent variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al, especially at Example 11), and knockout cell lines, such asa-l,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al. , Biotechnol. Bioeng., 94(4):680-688 (2006); and W02003/085107).

[0203] Anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) variants are further provided with bisected oligosaccharides, e.g, in which a biantennary oligosaccharide attached to the Fc region of the anti-MCT4 antibody agent is bisected by GlcNAc. Such anti- MCT4 antibody agent (such as a full-length anti-MCT4 antibody) variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody agent variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), US 2005/0123546 (Umana et al. ), and Ferrara et al. , Biotechnology and Bioengineering, 93(5): 851-861 (2006). Anti-MCT4 antibody agent (such as full-length anti-MCT4 antibody) variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such anti-MCT4 antibody agent variants may have improved CDC function. Such antibody agent variants are described, e.g. , in WO 1997/30087 (Patel et al. ); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

[0204] In some embodiments, the anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) variants comprising an Fc region are capable of binding to an F cyRI II . In some embodiments, the anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) variants comprising an Fc region have ADCC activity in the presence of human effector cells (e.g, T cell) or have increased ADCC activity in the presence of human effector cells compared to the otherwise same anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) comprising a human wild-type IgGlFc region.

Cysteine Engineered Variants

[0205] In some embodiments, it may be desirable to create cysteine engineered anti-MCT4 antibody agents (such as a full-length anti-MCT4 antibody) in which one or more amino acid residues are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the anti-MCT4 antibody agent. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the anti-MCT4 antibody agent and may be used to conjugate the anti-MCT4 antibody agent to other moieties, such as drug moieties or linker-drug moieties, to create an anti-MCT4 immunoconjugate, as described further herein. Cysteine engineered anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti-MCT4 antibodies) may be generated as described, e.g, in U.S. Pat. No. 7,521,541.

Derivatives

[0206] In some embodiments, an anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the anti-MCT4 antibody agent 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), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, proly propylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols ( e.g ., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the anti-MCT4 antibody agent may vary, and if more than one polymer are attached, they can be the same 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 properties or functions of the anti-MCT4 antibody agent to be improved, whether the anti-MCT4 antibody agent derivative will be used in a therapy under defined conditions, etc.

[0207] In some embodiments, conjugates of an anti-MCT4 antibody agent (such as a full- length anti-MCT4 antibody) and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et ah, Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the anti-MCT4 antibody agent-nonproteinaceous moiety are killed.

Pharmaceutical Compositions

[0208] Also provided herein are compositions (such as pharmaceutical compositions, also referred to herein as formulations) comprising an anti-MCT4 antibody agent (such as a full- length anti-MCT4 antibody), nucleic acid encoding the antibody agent, vector comprising the nucleic acid encoding the antibody agent, or host cell comprising the nucleic acid or vector. In some embodiments, there is provided a pharmaceutical composition comprising an anti-MCT4 antibody agent and optionally a pharmaceutically acceptable carrier.

[0209] Suitable formulations of the anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti-MCT4 antibodies) are obtained by mixing an anti-MCT4 antibody agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and/or non-ionic surfactants such as TWEEN™, PLEIRONICS™ or polyethylene glycol (PEG). Exemplary formulations are described in W098/56418, expressly incorporated herein by reference. Lyophilized formulations adapted for subcutaneous administration are described in W097/04801. Such lyophilized formulations may be

reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the individual to be treated herein.

Lipofectins or liposomes can be used to deliver the anti-MCT4 antibody agents of this invention into cells.

[0210] The formulation herein may also contain one or more active compounds in addition to the anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an anti neoplastic agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent in addition to the anti-MCT4 antibody agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of anti-MCT4 antibody agent present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein or about from 1 to 99% of the heretofore employed dosages.

[0211] The anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti- MCT4 antibodies) may also be entrapped in microcapsules prepared, for example, by

coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in macroemulsions. Sustained-release preparations may be prepared.

[0212] Sustained-release preparations of the anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti-MCT4 antibodies) can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody agent (or fragment thereof), which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate ), or poly(vinylalcohol)),

polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D (-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydro gels release proteins for shorter time periods. When encapsulated antibody agents remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in

immunogenicity. Rational strategies can be devised for stabilization of anti-MCT4 antibody agents depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0213] In some embodiments, the anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) is formulated in a buffer comprising a citrate, NaCl, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination of the foregoing. In some embodiments, the anti-MCT4 antibody agent is formulated in a buffer comprising about 100 mM to about 150 mM glycine. In some embodiments, the anti-MCT4 antibody agent is formulated in a buffer comprising about 50mM to about 100 mM NaCl. In some embodiments, the anti-MCT4 antibody agent is formulated in a buffer comprising about lOmM to about 50 mM acetate. In some embodiments, the anti-MCT4 antibody agent is formulated in a buffer comprising about lOmM to about 50 mM succinate. In some embodiments, the anti-MCT4 antibody agent is formulated in a buffer comprising about 0.005% to about 0.02% polysorbate 80. In some embodiments, the anti-MCT4 antibody agent is formulated in a buffer having a pH between about 5.1 and 5.6. In some embodiments, the anti-MCT4 antibody agent is formulated in a buffer comprising 10 mM citrate, 100 mM NaCl, lOOmM glycine, and 0.01% polysorbate 80, wherein the formulation is at pH 5.5.

[0214] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by, e.g ., filtration through sterile filtration membranes.

Methods of treatment using anti-MCT4 antibody agents

[0215] The anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti- MCT4 antibodies) and/or compositions of the invention can be administered to individuals (e.g, mammals such as humans) to treat a disease and/or disorder involving abnormally high levels of MCT4 and/or abnormally high rates of aerobic glycolysis, such as cancers characterized by high levels of MCT4 and/or high rates of aerobic glycolysis, including, for example, kidney cancer (renal cell carcinoma), cervical cancer, and prostate cancer. The present application thus in some embodiments provides a method of treating a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) in an individual comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising an anti-MCT4 antibody agent (e.g., a full- length anti-MCT4 antibody), such as any one of the anti-MCT4 antibodies (e.g., full-length anti- MCT4 antibodies) described herein.

[0216] In some embodiments, the cancer is selected, for example, from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB-deficient metastatic RCC. In some embodiments, the individual is human.

[0217] For example, in some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising an anti-MCT4 antibody agent (e.g., full-length anti-MCT4 antibody) according to any of the embodiments described herein. In some embodiments, the anti-MCT4 antibody agent specifically binds to MCT4 and antagonizes its ability to transport lactate. In some embodiments, the anti-MCT4 antibody agent is a full- length antibody. In some embodiments, the full-length anti-MCT4 antibody is an IgGl or IgG4 antibody. In some embodiments, the cancer is selected, for example, from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB-deficient metastatic RCC. In some embodiments, the individual is human.

[0218] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB-deficient metastatic RCC. In some embodiments, the individual is human.

[0219] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

[0220] In some embodiments, there is provided method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB-deficient metastatic RCC. In some embodiments, the individual is human. [0221] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB-deficient metastatic RCC. In some embodiments, the individual is human.

[0222] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

[0223] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a) a heavy chain variable domain comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some

[0224] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB -deficient metastatic RCC. In some embodiments, the individual is human.

[0225] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB -deficient metastatic RCC. In some embodiments, the individual is human.

[0226] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgGl constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the IgGl is human IgGl. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 27 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB -deficient metastatic RCC. In some embodiments, the individual is human.

[0227] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB -deficient metastatic RCC. In some embodiments, the individual is human. [0228] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB -deficient metastatic RCC. In some embodiments, the individual is human.

[0229] In some embodiments, there is provided a method of treating an individual having a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) comprising administering to the individual an effective amount of a composition comprising a full-length anti-MCT4 antibody comprising IgG4 constant domains, wherein the anti-MCT4 antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the IgG4 is human IgG4. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-MCT4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 28 and the anti-MCT4 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is metastatic RCC, such as SDHB -deficient metastatic RCC. In some embodiments, the individual is human.

[0230] In some embodiments, the individual is a mammal ( e.g ., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In some embodiments, the individual is younger than about 60 years old (including for example younger than about any of 50, 40, 30, 25, 20, 15, or 10 years old). In some embodiments, the individual is older than about 60 years old (including for example older than about any of 70, 80, 90, or 100 years old). In some embodiments, the individual is diagnosed with or genetically prone to one or more of the diseases or disorders described herein (such as kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, or pancreatic cancer). In some embodiments, the individual has one or more risk factors associated with one or more diseases or disorders described herein.

[0231] The present application in some embodiments provides a method of delivering an anti- MCT4 antibody agent (such as any one of the anti-MCT4 antibodies described herein, e.g. , an isolated anti-MCT4 antibody) to a cell expressing MCT4 on its surface in an individual, the method comprising administering to the individual a composition comprising the anti-MCT4 antibody agent.

[0232] Many diagnostic methods for cancer or any other disease exhibiting abnormal MCT4 expression and the clinical delineation of those diseases are known in the art. Such methods include, but are not limited to, e.g., immunohistochemistry, PCR, and fluorescent in situ hybridization (FISH).

[0233] In some embodiments, the anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti-MCT4 antibodies) and/or compositions of the invention are administered in combination with a second, third, or fourth agent (including, e.g, an antineoplastic agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) to treat diseases or disorders involving abnormal MCT4 expression. In some embodiments, the agent includes, for example, MCT1/2 inhibitors (e.g., AZD3965), oxidative phosphorylation inhibitors (e.g., metformin), and glutaminase inhibitors (e.g., CB-839).

[0234] Cancer treatments can be evaluated by, e.g, tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression and/or activity. Approaches to determining efficacy of the therapy can be employed, including for example, measurement of response through radiological imaging.

[0235] In some embodiments, the efficacy of treatment is measured as the percentage tumor growth inhibition (% TGI), calculated using the equation l00-(T/C x 100), where T is the mean relative tumor volume of the treated tumor, and C is the mean relative tumor volume of a non- treated tumor. In some embodiments, the %TGI is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or more than 95%.

Dosing and Method of Administering the anti-MCT4 antibody agent Compositions

[0236] The dose of the anti-MCT4 antibody agent (such as isolated anti-MCT4 antibody) compositions administered to an individual (such as a human) may vary with the particular composition, the mode of administration, and the type of disease being treated. In some embodiments, the amount of the composition (such as composition comprising isolated anti- MCT4 antibody agent) is effective to result in an objective response (such as a partial response or a complete response) in the treatment of cancer. In some embodiments, the amount of the anti-MCT4 antibody agent composition is sufficient to result in a complete response in the individual. In some embodiments, the amount of the anti-MCT4 antibody agent composition is sufficient to result in a partial response in the individual. In some embodiments, the amount of the anti-MCT4 antibody agent composition administered (for example when administered alone) is sufficient to produce an overall response rate of more than about any of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90% among a population of individuals treated with the anti-MCT4 antibody agent composition. Responses of an individual to the treatment of the methods described herein can be determined, for example, based on RECIST levels.

[0237] In some embodiments, the amount of the composition (such as composition comprising isolated anti-MCT4 antibody agent) is sufficient to prolong progress-free survival of the individual. In some embodiments, the amount of the composition is sufficient to prolong overall survival of the individual. In some embodiments, the amount of the composition (for example when administered along) is sufficient to produce clinical benefit of more than about any of 50%, 60%, 70%, or 77% among a population of individuals treated with the anti-MCT4 antibody agent composition.

[0238] In some embodiments, the amount of the composition (such as composition comprising isolated anti-MCT4 antibody agent), alone or in combination with a second, third, and/or fourth agent, is an amount sufficient to decrease the size of a tumor, decrease the number of cancer cells, or decrease the growth rate of a tumor by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding tumor size, number of cancer cells, or tumor growth rate in the same subject prior to treatment or compared to the corresponding activity in other subjects not receiving the treatment. Standard methods can be used to measure the magnitude of this effect, such as in vitro assays with purified enzyme, cell- based assays, animal models, or human testing.

[0239] In some embodiments, the amount of the anti-MCT4 antibody agent (such as a full- length anti-MCT4 antibody) in the composition is below the level that induces a toxicological effect (i.e., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the composition is administered to the individual.

[0240] In some embodiments, the amount of the composition is close to a maximum tolerated dose (MTD) of the composition following the same dosing regimen. In some embodiments, the amount of the composition is more than about any of 80%, 90%, 95%, or 98% of the MTD.

[0241] In some embodiments, the amount of an anti-MCT4 antibody agent (such as a full- length anti-MCT4 antibody) in the composition is included in a range of about 0.001 pg to about

1000 pg.

[0242] In some embodiments of any of the above aspects, the effective amount of an anti- MCT4 antibody agent (such as a full-length anti-MCT4 antibody) in the composition is in the range of about 0.1 pg/kg to about 100 mg/kg of total body weight.

[0243] The anti-MCT4 antibody agent compositions can be administered to an individual (such as human) via various routes, including, for example, intravenous, intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intra-tracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, sustained continuous release formulation of the composition may be used. In some

embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraportally. In some embodiments, the composition is

administered intraarterially. In some embodiments, the composition is administered

intraperitoneally. In some embodiments, the composition is administered intrahepatically. In some embodiments, the composition is administered by hepatic arterial infusion. In some embodiments, the administration is to an injection site distal to a first disease site. Cancers

[0244] The anti-MCT4 antibody agents (such as anti-MCT4 antibodies, e.g., full-length anti- MCT4 antibodies) in some embodiments can be useful for treating cancer characterized by high expression of MCT4 and/or high aerobic glycolysis. Cancers that may be treated using any of the methods described herein include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise solid tumors. Types of cancers to be treated with the anti-MCT4 antibody agents of the invention include, but are not limited to, carcinoma, blastoma, sarcoma, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumor s/cancers are also included. In some embodiments, the anti-MCT4 antibody agent is used for treating solid tumor.

[0245] In some embodiments, according to any of the methods described above, the cancer is kidney cancer. MCT4 is highly expressed in kidney cancer cell lines including, for example, A498, RCC4, and UOK262. In some embodiments, the kidney cancer is renal cell carcinoma (RCC), such as metastatic RCC. In some embodiments, the metastatic RCC is SDHB -deficient metastatic RCC. SDHB-deficient metastatic RCC is a very rare, aggressive form of kidney cancer that manifests with clinical evidence of severe Warburg effect. In some embodiments, the renal cell carcinoma is an adenocarcinoma. In some embodiments, the renal cell carcinoma is a clear cell renal cell carcinoma, papillary renal cell carcinoma (also called chromophilic renal cell carcinoma), chromophobe renal cell carcinoma, collecting duct renal cell carcinoma, granular renal cell carcinoma, mixed granular renal cell carcinoma, renal angiomyolipomas, or spindle renal cell carcinoma. In some embodiments, the individual may be a human who has a gene, genetic mutation, or polymorphism associated with renal cell carcinoma (e.g., VHL, TSC1, TSC2, CUL2, MSH2, MLH1, INK4a/ARF, MET, TGF- a, TGF-bI, IGF-I, IGF-IR, ART, and/or PTEN) or has one or more extra copies of a gene associated with renal cell carcinoma. In some embodiments, the renal cell carcinoma is associated with (1) von Hippel-Lindau (VHL) syndrome, (2) hereditary papillary renal carcinoma (HPRC), (3) familial renal oncocytoma (FRO) associated with Birt-Hogg-Dube syndrome (BHDS), or (4) hereditary renal carcinoma (HRC). In some embodiments, the renal cell carcinoma is at any of stage I, II, III, or IV, according to the American Joint Committee on Cancer (AJCC) staging groups. In some embodiments, the renal cell carcinoma is stage IV renal cell carcinoma.

[0246] In some embodiments, according to any of the methods described above, the solid tumor is breast cancer. In some embodiments, the breast cancer is early stage breast cancer, non- metastatic breast cancer, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, breast cancer in remission, breast cancer in an adjuvant setting, or breast cancer in a neoadjuvant setting. In some embodiments, the breast cancer is in a neoadjuvant setting. In some embodiments, the breast cancer is at an advanced stage. In some embodiments, the breast cancer (which may be HER2 positive or HER2 negative) includes, for example, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, and metastatic breast cancer. In some embodiments, the individual may be a human who has a gene, genetic mutation, or polymorphism associated with breast cancer (e.g., BRCA1, BRCA2, ATM, CHEK2, RAD51, AR, DIRAS3, ERBB2, TP53, ART, PTEN, and/or PDK) or has one or more extra copies of a gene (e.g., one or more extra copies of the HER2 gene) associated with breast cancer. In some embodiments, the method further comprises identifying a cancer patient population (i.e. breast cancer population) based on a hormone receptor status of patients having tumor tissue not expressing both ER and PgR.

[0247] In some embodiments, according to any of the methods described above, the solid tumor is prostate cancer. In some embodiments, the prostate cancer is an adenocarcinoma. In some embodiments, the prostate cancer is a sarcoma, neuroendocrine tumor, small cell cancer, ductal cancer, or a lymphoma. In some embodiments, the prostate cancer is at any of the four stages, A, B, C, or D, according to the Jewett staging system. In some embodiments, the prostate cancer is stage A prostate cancer (e.g., the cancer cannot be felt during a rectal exam). In some embodiments, the prostate cancer is stage B prostate cancer (e.g., the tumor involves more tissue within the prostate, and can be felt during a rectal exam, or is found with a biopsy that is done because of a high PSA level). In some embodiments, the prostate cancer is stage C prostate cancer (e.g., the cancer has spread outside the prostate to nearby tissues). In some embodiments, the prostate cancer is stage D prostate cancer. In some embodiments, the prostate cancer is androgen independent prostate cancer (AIPC). In some embodiments, the prostate cancer is androgen dependent prostate cancer. In some embodiments, the prostate cancer is refractory to hormone therapy. In some embodiments, the prostate cancer is substantially refractory to hormone therapy. In some embodiments, the individual is a human who has a gene, genetic mutation, or polymorphism associated with prostate cancer (e.g., RNASEL/HPC1,

ELAC2/HPC2, SR-A/MSR1, CHEK2, BRCA2, PON1, OGG1, MIC-I, TLR4, and/or PTEN) or has one or more extra copies of a gene associated with prostate cancer.

[0248] In some embodiments, according to any of the methods described above, the solid tumor is brain cancer. In some embodiments, the brain cancer is glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, or anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive

neuroectodermal tumors, visual pathway and hypothalamic glioma, or glioblastoma. In some embodiments, the brain cancer is glioblastoma (also called glioblastoma multiforme or grade 4 astrocytoma). In some embodiments, the glioblastoma is radiation-resistant. In some

embodiments, the glioblastoma is radiation-sensitive. In some embodiments, the glioblastoma may be infratentorial. In some embodiments, the glioblastoma is supratentorial. In some embodiments, the individual may be a human who has a gene, genetic mutation, or

polymorphism associated with brain cancer (e.g., glioblastoma) (e.g., NRP/B, MAGE-E1, MMACI-E1, PTEN, LOH, p53, MDM2, DCC, TP-73, Rbl, EGFR, PDGFR-a, PMS2, MLH1, and/or DMBT1) or has one or more extra copies of a gene associated with brain cancer (e.g., glioblastoma) (e.g., MDM2, EGFR, and PDGR-a).

[0249] In some embodiments, according to any of the methods described above, the solid tumor is colon cancer. In some embodiments, the individual may be a human who has a gene, genetic mutation, or polymorphism associated with colon cancer (e.g., RAS, ART, PTEN, POK, and/or EGFR) or has one or more extra copies of a gene associated with colon cancer.

[0250] Cancer treatments can be evaluated by, e.g., tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression and/or activity. Approaches to determining efficacy of the therapy can be employed, including for example, measurement of response through radiological imaging.

Articles of Manufacture and Kits

[0251] In some embodiments of the invention, there is provided an article of manufacture containing materials useful for the treatment of a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer), or for delivering an anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) to a cell expressing MCT4 on its surface. The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. Generally, the container holds a composition which is effective for treating a disease or disorder described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-MCT4 antibody agent of the invention. The label or package insert indicates that the composition is used for treating the particular condition. The label or package insert will further comprise instructions for administering the anti-MCT4 antibody agent composition to the patient. Articles of manufacture and kits comprising combinatorial therapies described herein are also

contemplated.

[0252] Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage,

administration, contraindications and/or warnings concerning the use of such therapeutic products. In some embodiments, the package insert indicates that the composition is used for treating cancer (such as HCC, melanoma, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms’ tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, or liposarcoma).

[0253] Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0254] Kits are also provided that are useful for various purposes, e.g ., for treatment of a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer), or for delivering an anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody) to a cell expressing MCT4 on its surface, optionally in combination with the articles of manufacture. Kits of the invention include one or more containers comprising an anti-MCT4 antibody agent composition (or unit dosage form and/or article of manufacture), and in some embodiments, further comprise another agent (such as the agents described herein) and/or instructions for use in accordance with any of the methods described herein. The kit may further comprise a description of selection of individuals suitable for treatment. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g, a paper sheet included in the kit), but machine-readable instructions (e.g, instructions carried on a magnetic or optical storage disk) are also acceptable.

[0255] For example, in some embodiments, the kit comprises a composition comprising an anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody). In some embodiments, the kit comprises a) a composition comprising an anti-MCT4 antibody agent, and b) an effective amount of at least one other agent, wherein the other agent enhances the effect ( e.g ., treatment effect, detecting effect) of the anti-MCT4 antibody agent. In some embodiments, the kit comprises a) a composition comprising an anti-MCT4 antibody agent, and b) instructions for administering the anti-MCT4 antibody agent composition to an individual for treatment of a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). In some embodiments, the kit comprises a) a composition comprising an anti-MCT4 antibody agent, b) an effective amount of at least one other agent, wherein the other agent enhances the effect (e.g., treatment effect, detecting effect) of the anti-MCT4 antibody agent, and c) instructions for administering the anti-MCT4 antibody agent composition and the other agent(s) to an individual for treatment of a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). The anti-MCT4 antibody agent and the other agent(s) can be present in separate containers or in a single container. For example, the kit may comprise one distinct composition or two or more compositions wherein one composition comprises an anti-MCT4 antibody agent and another composition comprises another agent.

[0256] In some embodiments, the kit comprises a nucleic acid (or set of nucleic acids) encoding an anti-MCT4 antibody agent (such as a full-length anti-MCT4 antibody). In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding an anti- MCT4 antibody agent, and b) a host cell for expressing the nucleic acid (or set of nucleic acids). In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding an anti-MCT4 antibody agent, and b) instructions for i) expressing the anti-MCT4 antibody agent in a host cell, ii) preparing a composition comprising the anti-MCT4 antibody agent, and iii) administering the composition comprising the anti-MCT4 antibody agent to an individual for the treatment of a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). In some embodiments, the kit comprises a) a nucleic acid (or set of nucleic acids) encoding an anti-MCT4 antibody agent, b) a host cell for expressing the nucleic acid (or set of nucleic acids), and c) instructions for i) expressing the anti-MCT4 antibody agent in the host cell, ii) preparing a composition

comprising the anti-MCT4 antibody agent, and iii) administering the composition comprising the anti-MCT4 antibody agent to an individual for the treatment of a cancer characterized by high MCT4 expression and/or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). [0257] The kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging ( e.g ., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

[0258] The instructions relating to the use of the anti-MCT4 antibody agent compositions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. For example, kits may be provided that contain sufficient dosages of an anti- MCT4 antibody agent (such as a full-length anti-MCT4 antibody) as disclosed herein to provide effective treatment of an individual for an extended period, such as any of a week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more. Kits may also include multiple unit doses of the anti-MCT4 antibody agent and pharmaceutical compositions and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.

[0259] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES

Materials

[0260] Cell lines HEK293, RCC4, A498, and 786-0 were obtained from the American Type Culture Collection.

Example 1. Selection and Characterization of scFvs Specific for Human MCT4

[0261] This example demonstrates the selection and characterization of fully human scFvs specific for human MCT4 (hMCT4) from a collection of human scFv antibody phage display libraries. In particular, this example demonstrates the selection of human scFvs that specifically bind to human MCT4 in native format (cell surface-bound MCT4). The scFvs were selected based on high specificity for human MCT4 via panning against cell surface-bound human MCT4 in its native conformation. These human anti-MCT4 scFvs provide a valuable source of antibody components for the construction of anti-MCT4 antibody agents in various formats, e.g, full- length IgG, multi-specific anti-MCT4 antibodies, and the like.

[0262] A collection of 10 human scFv antibody phage display libraries (diversity = lOx lO¹⁰) constructed by Eureka Therapeutics (trademarked as E-ALPHA™ phage libraries) was used for the selection of human scFvs specific for human MCT4. E- ALPHA™ phage libraries included naive libraries comprising fully naive human heavy and light chain repertoires, and semi synthetic libraries containing fully naive human light chain repertoires and semi-synthetic heavy chain with completely randomized heavy chain CDR3 regions. The naive antibody repertoires were cloned from PBMCs and spleens of healthy donors or from PBMCs of autoimmune disease (such as systemic lupus erythematosus and rheumatoid arthritis) donors.

[0263] The E-APLPHA™ scFv phage libraries were screened (panned) against human MCT4 by co-incubation with MCT4-expressing HEK293 cells (HEK293-MCT4). After extended washing with PBS, HEK293-MCT4 cells with bound scFv antibody phage were spun down. The bound clones were then eluted and used for 2-3 additional rounds of panning to enrich for scFv phage clones that bound MCT4 specifically. The bound clones were then eluted and used to infect E. coli XLl-Blue cells. The phage clones expressed in bacteria were then purified.

[0264] 450 phage clones identified from the cell panning were then tested by FACS analysis for binding to HEK293-MCT4 cells. Briefly, 0.2 million cells (in PBS + 5% FBS + 0.05% NaN₃₎ were incubated for 2 h at 4 C with 50 mΐ of -1.0 x 10 pfu/mL page in PBS. FACS was carried out using primary antibody mouse anti-MT3 mAh (Thermo #MA 1 - 12900) and secondary antibody PE anti-mouse IgG (Vectors Lab #EI-2007) 3 unique clones (anti-MCT4 clone 1, anti-MCT4 clone 2, and anti-MCT4 clone 3) were identified that demonstrated specific binding for HEK293-MCT4 cells (FIG. 1). The 3 clones were then tested for binding to MCT4+ cancer cell lines RCC4, A498, and 786-0. As shown in FIGS. 2 and 3, anti-MCT4 clone 2 bound specifically to MCT4+ cancer cell lines RCC4 (FIG. 2) and 786-0 (FIG. 3).

Example 2. Generation of full-length human IgG anti-MCT4 antibodies

[0265] Full-length human IgGl of the selected phage clones are produced, for example, in

HEK293 and Chinese hamster ovary (CHO) cell lines, as described (Tomimatsu, K. et al. ,

Biosci. Biotechnol. Biochem. 73(7): 1465-1469, 2009). In brief, antibody variable regions from the phage clones are subcloned into mammalian expression vectors, with matching human lambda light chain constant region (SEQ ID NO: 29) and human IgGl constant region (SEQ ID NO: 27) sequences (see Table 5). Molecular weight of the purified full-length IgGl antibodies is measured, for example, under both reducing and non-reducing conditions by electrophoresis. SDS-PAGE of purified IgGl antibodies is performed to determine protein purity.

Table 5

[0266] Full-length human IgG4 of the selected phage clones are produced, for example, in HEK293 and Chinese hamster ovary (CHO) cell lines, as described (Tomimatsu, K. et al. , Biosci. Biotechnol. Biochem. 73(7): 1465-1469, 2009). In brief, antibody variable regions from the phage clones are subcloned into mammalian expression vectors, with matching human lambda light chain constant region (SEQ ID NO: 29) and human IgG4 constant region (SEQ ID NO: 28) sequences ( see Table 6). Molecular weight of the purified full-length IgG4 antibodies can be measured under both reducing and non-reducing conditions by electrophoresis. SDS- PAGE of purified IgG4 antibodies can be performed to determine protein purity.

Table 6

Example 3. Characterization of full-length human IgG anti-MCT4 antibodies

[0267] Anti-MCT4 IgG antibodies are tested for binding towards MCT4-expressing cells, such as HEK293-MCT4 cells, by flow cytometry. Dose dependence of binding is tested. Briefly, MCT4-expressing cells are incubated with varying amounts of the anti-hMCT4 IgG antibodies, for example, at 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014 or 0 pg/ml, on ice for 1 hour. The anti- MCT4 IgG antibodies are evaluated for their affinity towards the MCT4-expressing cells by EC50 of the dose dependence curve (MFI vs. antibody concentration). Furthermore, apparent K_D is determined based on EC50 value. Binding affinity of the anti-MCT4 IgG antibodies can be determined, for example, by ForteBio.

[0268] Specificity of the full-length IgG anti-MCT4 antibodies is further determined by measuring binding to LS174T colon adenocarcinoma cells, which normally express high levels of both MCT1 and MCT4, and LS174 MCT4 ^/ cells that lack MCT4 (Marchiq et al., 2015), for example by flow cytometry. Binding is evaluated at both normal and low oxygen conditions, for example, at 20% oxygen and 1% oxygen conditions. Additional MCT4-expressing cancer cell lines with deletion of MCT4, such as PANC-l (ATCC® CRL-1469™), UOK262, PL45 (ATCC® CRL-2558™), and Capan-2 (ATCC® HTB-80™), can be included for further specificity confirmation.

Antagonism ofMCT4 lactate transport

[0269] Antagonism of the lactate transport function of MCT4 mediated by the full-length IgG anti-MCT4 antibodies is determined in cancer cell lines expressing MCT4, including, for example, kidney cancer cell lines A498, RCC4, and UOK262, and pancreatic cancer cell lines PL45 and Panel. Cells insensitive to the deficiency of MCT4, such as 293T cells, are included as a negative control. The impact of the full-length IgG anti-MCT4 antibodies on cell proliferation and cell death in these cells is determined. Cell proliferation is measured, for example, by cell number counts and MTT assays; alternatively proliferation index is assayed by BrdU incorporation or Ki-67 immunostaining. Cell death is measured, for example, by staining of annexin-V and cleaved caspase 3 as well as TUNEL assays. The effect of full-length IgG anti-MCT4 antibodies on intracellular pH and lactate levels, extracellular acidification rate (ECAR), and 0₂ consumption rate (OCR) is also determined, for example as measured by Seahorse XF Analyzers (Agilent). These studies are also carried out with combined treatment of the cells with the full-length IgG anti-MCT4 antibodies and the MCT1/2 chemical inhibitor AZD3965, the oxidative phosphorylation inhibitor metformin, or the glutaminase inhibitor CB- 839.

Pharmacokinetics and pharmacodynamics

[0270] The tissue/blood distribution and area under curve of the full-length IgG anti-MCT4 antibodies is determined in mice using standard techniques.

In vivo efficacy

[0271] The effect of the full-length IgG anti-MCT4 antibodies on tumor growth in mouse xenograft models of MCT4-dependent cancer, for example kidney cancer cell line A498, RCC4, or UOK262 xenograft mice, or pancreatic cancer cell line PL45 or Panel xenograft mice, is determined. The dosing schedule is chosen based on the results of the pharmacokinetic and pharmacodynamics studies. EXEMPLARY EMBODIMENTS

Embodiment 1. An isolated anti-monocarboxylate transporter 4 (MCT4) antibody agent that specifically recognizes a cell surface-bound MCT4 and antagonizes its lactate transport function.

Embodiment 2. The isolated anti-MCT4 antibody agent of embodiment 1, comprising:

i) a heavy chain variable domain (V_H) comprising a heavy chain complementarity determining region (HC-CDR) 1, an HC-CDR2, and an HC-CDR3 of the heavy chain variable domain of any one of SEQ ID NOs: 2-4; and

ii) a light chain variable domain (V_L) comprising a light chain complementarity determining region (LC-CDR) 1, an LC-CDR2, and an LC-CDR3 of the light chain variable domain of any one of SEQ ID NOs: 5-7.

Embodiment 3. The isolated anti-MCT4 antibody agent of embodiment 1, comprising:

i) a V_H comprising a HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, or a variant thereof comprising up to about 5 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, or a variant thereof comprising up to about 5 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16, or a variant thereof comprising up to about 5 amino acid substitutions; and

ii) a V_L comprising a LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, or a variant thereof comprising up to about 5 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25, or a variant thereof comprising up to about 5 amino acid substitution.

Embodiment 4. The isolated anti-MCT4 antibody agent of embodiment 3, wherein the antibody agent comprises:

i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16; and

ii) a V_L comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25.

Embodiment 5. The isolated anti-MCT4 antibody agent of embodiment 4, wherein the antibody agent comprises:

i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:

8, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NOs: 14; and

ii) a V_L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:

17, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23.

Embodiment 6. The isolated anti-MCT4 antibody agent of embodiment 4, wherein the antibody agent comprises:

i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:

9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NOs: 15; and

18, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 24.

Embodiment 7. The isolated anti-MCT4 antibody agent of embodiment 4, wherein the antibody agent comprises:

i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:

10, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NOs: 16; and

19, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22 and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

Embodiment 8. The isolated anti-MCT4 antibody agent of embodiment 4, wherein the antibody agent comprises:

i) a V_H comprising sequences of SEQ ID NOs: 8, 11 and 14, and a V_L comprising sequences of SEQ ID NOs: 17, 20 and 23;

ii) a V_H comprising sequences of SEQ ID NOs: 9, 12 and 15, and a V_L comprising sequences of SEQ ID NOs: 18, 21 and 24; or iii) a V_H comprising sequences of SEQ ID NOs: 10, 13 and 16, and a V_L comprising sequences of SEQ ID NOs: 19, 22 and 25.

Embodiment 9. The isolated anti-MCT4 antibody agent of any one of embodiments 2-4, wherein the antibody agent comprises:

i) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 2-4, or a variant thereof having at least about 95% sequence identify to any one of SEQ ID NOs: 2-4; and

ii) a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 5-7, or a variant thereof having at least about 95% sequence identity to any one of SEQ ID NOs: 5-7.

Embodiment 10. The isolated anti-MCT4 antibody agent of embodiment 9, wherein the antibody agent comprises:

i) a V_H comprising the amino acid sequence of any one of SEQ ID NOs: 2-4; and ii) a V_L comprising the amino acid sequence of any one of SEQ ID NOs: 5-7.

Embodiment 11. The isolated anti-MCT4 antibody agent of embodiment 10, wherein the antibody agent comprises:

i) a V_H comprising the amino acid sequence of SEQ ID NO: 2; and

ii) a V_L comprising the amino acid sequence of SEQ ID NO: 5.

Embodiment 12. The isolated anti-MCT4 antibody agent of embodiment 10, wherein the antibody agent comprises:

i) a V_H comprising the amino acid sequence of SEQ ID NO: 3; and

ii) a V_L comprising the amino acid sequence of SEQ ID NO: 6.

Embodiment 13. The isolated anti-MCT4 antibody agent of embodiment 10, wherein the antibody agent comprises:

i) a V_H comprising the amino acid sequence of SEQ ID NO: 4; and

ii) a V_L comprising the amino acid sequence of SEQ ID NO: 7.

Embodiment 14. The isolated anti-MCT4 antibody agent of any one of embodiments 1-13, wherein the anti-MCT4 antibody agent is a full-length IgG antibody.

Embodiment 15. The isolated anti-MCT4 antibody agent of embodiment 14, wherein the anti- MCT4 antibody agent is a full-length IgGl antibody.

Embodiment 16. The isolated anti-MCT4 antibody agent of embodiment 15, wherein the anti- MCT4 antibody agent comprises a heavy chain constant region comprising the amino acid of SEQ ID NO: 27 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 29.

Embodiment 17. The isolated anti-MCT4 antibody agent of embodiment 14, wherein the anti- MCT4 antibody agent is a full-length IgG4 antibody.

Embodiment 18. The isolated anti-MCT4 antibody agent of embodiment 17, wherein the anti- MCT4 antibody agent comprises a heavy chain constant region comprising the amino acid of SEQ ID NO: 28 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 29.

Embodiment 19. An isolated anti-MCT4 antibody agent that specifically binds to MCT4 competitively with the isolated anti-MCT4 antibody agent of any one of embodiments 01-18 and antagonizes MCT4 lactate transport function.

Embodiment 20. An isolated nucleic acid encoding the isolated anti-MCT4 antibody agent of any one of embodiments 1-19.

Embodiment 21. A vector comprising the isolated nucleic acid of embodiment 20.

Embodiment 22. An isolated host cell comprising the anti-MCT4 antibody agent of any one of embodiments 1-19, isolated nucleic acid of embodiment 20, or the vector of 21.

Embodiment 23. A pharmaceutical composition comprising the isolated anti-MCT4 antibody agent of any one of embodiments 1-19, and a pharmaceutically acceptable carrier.

Embodiment 24. A method of treating an individual having a cancer characterized by high MCT4 and/or high aerobic glycolysis, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 23.

Embodiment 25. The method of embodiment 24, wherein the administration is via

intravenous or intratumoral route.

Embodiment 26. The method of embodiment 24 or 25, further comprising administering to the individual an additional therapy.

Embodiment 27. The method of any one of embodiments 24-26, wherein the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer.

Embodiment 28. The method of any one of embodiments 24-27, wherein the cancer is renal cell carcinoma (RCC). Embodiment 29. The method of embodiment 28, wherein the cancer is metastatic RCC.

Embodiment 30. A method of producing an isolated anti-MCT4 antibody agent, comprising: a) culturing a host cell comprising the isolated nucleic acid of embodiment 20 or the vector of embodiment 21, or the isolated host cell of embodiment 22 under conditions effective to express the anti-MCT4 antibody agent; and

b) obtaining the expressed anti-MCT4 antibody agent from said host cell.

Embodiment 31. The method of embodiment 30, wherein step (a) further comprises producing a host cell comprising the isolated nucleic acid of embodiment 20 or the vector of embodiment 21.

Embodiment 32. A kit comprising the isolated anti-MCT4 antibody agent of any one of embodiments 1-19, the isolated nucleic acid of embodiment 20, the vector of embodiment 21, or the isolated host cell of embodiment 22.

Claims

CLAIMS What is claimed is:

1. An isolated anti-monocarboxylate transporter 4 (MCT4) antibody agent that specifically recognizes a cell surface-bound MCT4 and antagonizes its lactate transport function.

2. The isolated anti-MCT4 antibody agent of claim 1, comprising:

3. The isolated anti-MCT4 antibody agent of claim 1, comprising:

4. The isolated anti-MCT4 antibody agent of claim 3, wherein the antibody agent comprises:

i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 8-10, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11-13, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 14-16; and ii) a V_L comprising an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 17-19, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 20-22, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 23-25.

5. The isolated anti-MCT4 antibody agent of claim 4, wherein the antibody agent comprises:

i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:

6. The isolated anti-MCT4 antibody agent of claim 4, wherein the antibody agent comprises:

i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:

7. The isolated anti-MCT4 antibody agent of claim 4, wherein the antibody agent comprises:

i) a V_H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:

8. The isolated anti-MCT4 antibody agent of claim 4, wherein the antibody agent comprises:

i) a V_H comprising sequences of SEQ ID NOs: 8, 11 and 14, and a V_L comprising sequences of SEQ ID NOs: 17, 20 and 23; ii) a V_H comprising sequences of SEQ ID NOs: 9, 12 and 15, and a V_L comprising sequences of SEQ ID NOs: 18, 21 and 24; or

iii) a V_H comprising sequences of SEQ ID NOs: 10, 13 and 16, and a V_L comprising sequences of SEQ ID NOs: 19, 22 and 25.

9. The isolated anti-MCT4 antibody agent of any one of claims 2-4, wherein the antibody agent comprises:

10. The isolated anti-MCT4 antibody agent of claim 9, wherein the antibody agent comprises:

11. The isolated anti-MCT4 antibody agent of claim 10, wherein the antibody agent comprises:

i) a V_H comprising the amino acid sequence of SEQ ID NO: 2; and

ii) a V_L comprising the amino acid sequence of SEQ ID NO: 5.

12. The isolated anti-MCT4 antibody agent of claim 10, wherein the antibody agent comprises:

i) a V_H comprising the amino acid sequence of SEQ ID NO: 3; and

ii) a V_L comprising the amino acid sequence of SEQ ID NO: 6.

13. The isolated anti-MCT4 antibody agent of claim 10, wherein the antibody agent comprises:

i) a V_H comprising the amino acid sequence of SEQ ID NO: 4; and

ii) a V_L comprising the amino acid sequence of SEQ ID NO: 7.

14. The isolated anti-MCT4 antibody agent of any one of claims 1-13, wherein the anti- MCT4 antibody agent is a full-length IgG antibody.

15. The isolated anti-MCT4 antibody agent of claim 14, wherein the anti-MCT4 antibody agent is a full-length IgGl antibody.

16. The isolated anti-MCT4 antibody agent of claim 15, wherein the anti-MCT4 antibody agent comprises a heavy chain constant region comprising the amino acid of SEQ ID NO: 27 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 29.

17. The isolated anti-MCT4 antibody agent of claim 14, wherein the anti-MCT4 antibody agent is a full-length IgG4 antibody.

18. The isolated anti-MCT4 antibody agent of claim 17, wherein the anti-MCT4 antibody agent comprises a heavy chain constant region comprising the amino acid of SEQ ID NO: 28 and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 29.

19. An isolated anti-MCT4 antibody agent that specifically binds to MCT4 competitively with the isolated anti-MCT4 antibody agent of any one of claims 01-18 and antagonizes MCT4 lactate transport function.

20. An isolated nucleic acid encoding the isolated anti-MCT4 antibody agent of any one of claims 1-19.

21. A vector comprising the isolated nucleic acid of claim 20.

22. An isolated host cell comprising the anti-MCT4 antibody agent of any one of claims 1-19, isolated nucleic acid of claim 20, or the vector of 21.

23. A pharmaceutical composition comprising the isolated anti-MCT4 antibody agent of any one of claims 1-19, and a pharmaceutically acceptable carrier.

24. A method of treating an individual having a cancer characterized by high MCT4 and/or high aerobic glycolysis, comprising administering to the individual an effective amount of the pharmaceutical composition of claim 23.

25. The method of claim 24, wherein the administration is via intravenous or intratumoral route.

26. The method of claim 24 or 25, further comprising administering to the individual an additional therapy.

27. The method of any one of claims 24-26, wherein the cancer is selected from the group consisting of kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, and pancreatic cancer.

28. The method of any one of claims 24-27, wherein the cancer is renal cell carcinoma (RCC).

29. The method of claim 28, wherein the cancer is metastatic RCC.

30. A method of producing an isolated anti-MCT4 antibody agent, comprising:

a) culturing a host cell comprising the isolated nucleic acid of claim 20 or the vector of claim 21, or the isolated host cell of claim 22 under conditions effective to express the anti-MCT4 antibody agent; and

b) obtaining the expressed anti-MCT4 antibody agent from said host cell.

31. The method of claim 30, wherein step (a) further comprises producing a host cell comprising the isolated nucleic acid of claim 20 or the vector of claim 21.

32. A kit comprising the isolated anti-MCT4 antibody agent of any one of claims 1-19, the isolated nucleic acid of claim 20, the vector of claim 21, or the isolated host cell of claim 22