MIC antibodies and binding agents and methods of use thereof

Abstract

Provided are MIC antibodies, antigen-binding portions thereof, and MIC binding agents thereof for the treatment of cancer.SOLUTION: (i) a heavy chain variable (VH) region having an amino acid sequence represented by a specific sequence, and (ii) a light chain variable (VL) region having an amino acid sequence represented by a specific sequence, wherein the framework regions of the heavy chain and the light chain are optionally modified by substitution, deletion or insertion of 1 to 8 amino acids in the framework regions.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 049,012, filed July 7, 2020, which is incorporated herein by reference in its entirety for all purposes.

[0002] Description of support funds This invention was made with government support under Small Business Technology Transfer Grant 1R41CA206688-01A1 awarded by the U.S. Small Business Administration. The government has certain rights in this invention. [Background technology]

[0003] Major histocompatibility complex class I chain-related molecules A and B (MICA and MICB, respectively, commonly referred to as MIC) are a family of proteins that bind to NKG2D, an activating immune receptor expressed by natural killer (NK) cells, NKT cells, a subset of gamma delta T cells, and human CD8+ T cells. Binding of MIC to NKG2D on NK cells or T cells activates NK cells and costimulates CD8+ T cells and gamma delta T cells in vitro, among other effects.

[0004] It has been reported that MIC family molecules are expressed on tumor cells and are thought to be involved in suppressing immune responses against tumor cells. MICA is expressed more frequently and in greater amounts on the tumor cell surface than MICB. MIC proteins are found in both membrane-bound and soluble forms (sMIC), the latter of which is shed from tumor cells. Elevated serum levels of sMIC have been associated with many types of cancer, including solid tumors such as melanoma, prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, renal cancer, gastrointestinal (GI) cancer, and head and neck cancer, as well as hematological cancers such as lymphoma and multiple myeloma, and cancers of bone and soft tissue origin such as sarcoma. While membrane-restricted MIC(B) has been reported to maintain NKG2D-mediated protective antitumor immunity in mice, the soluble form correlated with tumor progression, which is caused by decreased NKG2D expression on NK cells and CD8 T cells and impaired peripheral maintenance of NK cells. Therefore, drugs that bind to sMIC may offer a therapeutic approach for patients. However, there remains a need for optimized drugs for the treatment of MIC+ cancers in humans. Summary of the Invention

[0005] The inventions disclosed herein are based, in part, on MIC-binding antibodies, antigen-binding portions thereof, and related binding agents that specifically bind to soluble MIC (sMIC) and / or cell membrane-bound MIC (also referred to as membrane-bound MIC) and exhibit improved therapeutic properties. MIC, and particularly sMIC, are important and advantageous therapeutic targets for the treatment of certain cancers. These MIC-binding antibodies, antigen-binding portions thereof, and binding agents provide compositions and methods based on the use of such antibodies, antigen-binding portions, and related binding agents in the treatment of MIC+ cancers. Accordingly, the present invention provides methods, compositions, kits, and articles of manufacture related to MIC-binding agents.

[0006] In some embodiments, a binding agent is provided comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the framework regions of the heavy and light chains are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids in the framework regions. The binding agent specifically binds to MIC. In some embodiments, the binding agent specifically binds to MIC with a higher binding affinity than antibody B10G5. In some embodiments, the binding agent comprises (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2. The binding agent specifically binds to MIC. In some embodiments, the binding agent specifically binds to MIC with a higher binding affinity than antibody B10G5.

[0007] In some embodiments, a binding agent is provided that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1 having the amino acid sequence set forth in SEQ ID NO: 11, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 13, and the VL region comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 16, and wherein the VH and VL regions each comprise a humanized framework region. In some embodiments, the humanized VH framework regions are derived from human germline genes having the amino acid sequences set forth in IMGT IGHV4-59*11 (SEQ ID NO: 29) and IGHJ4*01 (SEQ ID NO: 30) or IGHV4-30-4*01 (SEQ ID NO: 31) and IGHJ4*01 (SEQ ID NO: 30). In some embodiments, the humanized VL framework regions are derived from human germline genes having the amino acid sequences set forth in IMGT IGKV1-NL1*01 (SEQ ID NO: 32) and IMGT IGKJ1*01 (SEQ ID NO: 33), IMGT IGKV1-33*01 (SEQ ID NO: 34) and IMGT IGKJ1*01 (SEQ ID NO: 33), or IMGT IGKV1-5*01 (SEQ ID NO: 35) and IMGT IGKJ1*01 (SEQ ID NO: 33). The binding agent specifically binds to MIC. In some embodiments, the binding agent specifically binds to MIC with a higher binding affinity than antibody B10G5.

[0008] In some embodiments, the binding agent is an antibody or antigen-binding portion thereof, hi some embodiments, the binding agent is a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody.

[0009] In some embodiments, the binding agent comprises a heavy chain variable region linked to a heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG isotype. In some embodiments, the heavy chain constant region is an IgG1 constant region. In some embodiments, the heavy chain constant region is an IgG4 constant region. In some embodiments, the heavy chain variable region and heavy chain constant region have the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the binding agent comprises a light chain variable region linked to a light chain constant region. In some embodiments, the light chain constant region is a kappa isotype. In some embodiments, the light chain variable region and light chain constant region have the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the heavy chain constant region further comprises an amino acid modification that increases binding affinity to at least human Fc gamma RIII. In some embodiments, the heavy chain constant region further comprises an amino acid modification that increases at least antibody-dependent cellular cytotoxicity (ADCC) activity. In some embodiments, the heavy chain constant region further comprises at least one amino acid modification that increases CDC activity.

[0010] In some embodiments, the binding agent is monospecific. In some embodiments, the binding agent is monospecific and bivalent. In some embodiments, the binding agent is bivalent. In some embodiments, the binding agent is bivalent and bispecific or multivalent and multispecific.

[0011] In some embodiments, a pharmaceutical composition is provided that includes the binding agent of any of the embodiments described herein and a pharmaceutically acceptable carrier.

[0012] In some embodiments, a nucleic acid is provided that encodes a heavy chain variable region of a binding agent having an amino acid sequence set forth in SEQ ID NO: 1, optionally having a nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a nucleic acid is provided that encodes a light chain variable region of a binding agent having an amino acid sequence set forth in SEQ ID NO: 2, optionally having a nucleic acid sequence set forth in SEQ ID NO: 22. In some embodiments, a nucleic acid is provided that encodes a binding agent of any of the embodiments described herein, optionally having a nucleic acid sequence set forth in SEQ ID NO: 21 and SEQ ID NO: 22. In some embodiments, a vector is provided that includes any of the nucleic acids encoding the MIC-binding agent polypeptides described herein. In some embodiments, a cell is provided that includes a nucleic acid encoding any of the binding agent polypeptides described herein, or a vector that includes such nucleic acid(s).

[0013] In some embodiments, methods of treating MIC+ cancer are provided, comprising administering to a subject in need of treatment a therapeutically effective amount of a binding agent comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the framework regions of the heavy and light chains are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids in the framework regions, and wherein the binding agent specifically binds to sMIC and / or membrane-bound MIC. In some embodiments, the binding agent specifically binds to MIC with a higher binding affinity than antibody B10G5.

[0014] In some embodiments, methods are provided for treating MIC+ cancer, comprising administering to a subject in need of treatment a therapeutically effective amount of a binding agent comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the binding agent specifically binds to MIC. In some embodiments, the binding agent specifically binds to MIC with a higher binding affinity than antibody B10G5.

[0015] In some embodiments, methods of treating MIC+ cancer are provided, comprising administering to a subject in need thereof a therapeutically effective amount of any of the binding agent embodiments described herein. In some embodiments, the method comprises administering the binding agent as a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0016] In some embodiments, the cancer is a carcinoma, a sarcoma, a neuroendocrine tumor, or a hematological malignancy. In some embodiments, the carcinoma is optionally a solid tumor selected from melanoma, prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, renal cancer, and head and neck cancer. In some embodiments, the hematological malignancy is lymphoma, leukemia, or multiple myeloma.

[0017] In some embodiments, the method further comprises administering immunotherapy to the subject. In some embodiments, the immunotherapy is adoptive cell therapy or a checkpoint inhibitor. In some embodiments, the adoptive cell therapy is selected from autologous NK cells, allogeneic NK cells, autologous T cells, CAR-modified T cells, and CAR-modified NK cells. In some embodiments, the immunotherapy comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4. In some embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

[0018] In some embodiments, the method includes administering no chemotherapy to the subject for at least 4 weeks, at least 6 weeks, or at least 8 weeks prior to administration of the binding agent. In some embodiments, the binding agent is administered intravenously. In some embodiments, the binding agent is administered at a dose of about 0.1 mg / kg to about 100 mg / kg, or about 0.1 mg / kg to about 25 mg / kg, or about 0.1 mg / kg to about 20 mg / kg, or about 0.1 mg / kg to about 15 mg / kg, or about 0.1 mg / kg to about 10 mg / kg.

[0019] In some embodiments, methods are provided for reducing the level of circulating sMIC in a subject with cancer, comprising administering a therapeutically effective amount of a binding agent of any of the embodiments described herein or a pharmaceutical composition of any of the binding agent embodiments described herein, wherein the binding agent specifically binds to circulating sMIC. In some embodiments, the cancer is a carcinoma, a sarcoma, a neuroendocrine tumor, or a malignant hematological disease. In some embodiments, the carcinoma is selected from solid tumors, including, but not limited to, melanoma, prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, kidney cancer, and head and neck cancer. In some embodiments, the malignant hematological disease is lymphoma, leukemia, or multiple myeloma.

[0020] In some embodiments, a method of improving a subject's outcome in immunotherapy is provided, comprising administering to a subject having cancer an effective amount of the immunotherapy and administering to the subject a therapeutically effective amount of a binding agent of any of the embodiments described herein, or a pharmaceutical composition of any of the embodiments of the binding agent described herein, wherein the binding agent specifically binds to circulating sMIC and / or cell membrane-bound MIC, and the subject's outcome is improved compared to administration of the immunotherapy alone. In some embodiments, the improved outcome is an objective response selected from stable disease, partial response, or complete response. In some embodiments, the improved outcome is a reduction in tumor burden. In some embodiments, the improved outcome is progression-free survival or disease-free survival. In some embodiments, the immunotherapy is adoptive cell therapy or a checkpoint inhibitor. In some embodiments, the adoptive cell therapy is autologous NK cells, allogeneic NK cells, autologous T cells, CAR-modified T cells, and CAR-modified NK cells. In some embodiments, the checkpoint inhibitor comprises an antibody that specifically binds to human PD-1, human PD-L1, or CTLA4. In some embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

[0021] In some embodiments, the subject has not received chemotherapy for at least 4 weeks, at least 6 weeks, or at least 8 weeks prior to administration of the binding agent. In some embodiments, the binding agent is administered intravenously. In some embodiments, the binding agent is administered at a dose of about 0.01 mg / kg to about 100 mg / kg, or about 0.01 mg / kg to about 25 mg / kg, or about 0.01 mg / kg to about 20 mg / kg, or about 0.01 mg / kg to about 15 mg / kg, or about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 100 mg / kg, or about 0.1 mg / kg to about 25 mg / kg, or about 0.1 mg / kg to about 20 mg / kg, or about 0.1 mg / kg to about 15 mg / kg, or about 0.1 mg / kg to about 10 mg / kg.

[0022] These and other aspects of the present invention can be more fully understood by reference to the following detailed description, non-limiting examples of specific embodiments, and the accompanying drawings. [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 shows the analysis of binding affinity by FACS of humanized variants of antibody B10G5 (linked to the Fc region of human IgG1). Two humanized variants, Antibody G and Antibody K, showed higher mean fluorescence intensity (MFI) than Antibody J (Ab-J, a B10G5 chimeric antibody constructed from the F(ab)2 of the murine B10G5 antibody and the human IgG1 Fc domain). [Figure 2A] Antibody B10G5 (Figure 2A) has a lower (weaker) binding affinity than antibody K (Ab-K, Figure 2B), showing Kd = 12.1 nM vs. 7.2 nM, respectively, as determined by biolayer interferometry using Octet Red 96 (ForteBio). [Figure 2B] Antibody B10G5 (Figure 2A) has a lower (weaker) binding affinity than antibody K (Ab-K, Figure 2B), showing Kd = 12.1 nM vs. 7.2 nM, respectively, as determined by biolayer interferometry using Octet Red 96 (ForteBio). [Figure 3A] Antibody K (Ab-K) exhibits greater activity than Ab-J (chimeric B10G5) in enhancing IL-2-activated primary NK cell killing of MIC+ thyroid oncocytoma UC1 tumor cells (Figure 3A) and pancreatic PL12 cells (Figure 3B). [Figure 3B] Antibody K (Ab-K) exhibits greater activity than Ab-J (chimeric B10G5) in enhancing IL-2-activated primary NK cell killing of MIC+ thyroid oncocytoma UC1 tumor cells (Figure 3A) and pancreatic PL12 cells (Figure 3B). [Figure 4A]The levels of antibody monomer and aggregates are shown for murine antibody B10G5 (Figure 4A), chimeric Ab-K antibody (ch-Ab-K, Ab-K variable domain and murine IgG1-FC, Figure 4B), and antibody K (Ab-K, humanized, Figure 4C), as determined by dynamic light scattering (DLS) assay. [Figure 4B] The levels of antibody monomer and aggregates are shown for murine antibody B10G5 (Figure 4A), chimeric Ab-K antibody (ch-Ab-K, Ab-K variable domain and murine IgG1-FC, Figure 4B), and antibody K (Ab-K, humanized, Figure 4C), as determined by dynamic light scattering (DLS) assay. [Figure 4C] The levels of antibody monomer and aggregates are shown for murine antibody B10G5 (Figure 4A), chimeric Ab-K antibody (ch-Ab-K, Ab-K variable domain and murine IgG1-FC, Figure 4B), and antibody K (Ab-K, humanized, Figure 4C), as determined by dynamic light scattering (DLS) assay. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition For convenience, certain terms in the specification, examples, and claims are defined here. Unless otherwise stated or implicit from context, the following terms and phrases have the meanings provided below. The definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] As used herein, unless otherwise indicated, the terms "a" and "an" shall be construed to mean "one," "at least one," or "one or more." Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0026] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise," "comprising," and the like, are to be construed in an inclusive sense, i.e., "including, but not limited to," rather than in an exclusive or exhaustive sense.

[0027] As used herein, the terms "reduce," "reduce," "reduced," "reduction," "diminish," and "inhibit" are all used generically to mean a decrease by a statistically significant amount relative to a reference.

[0028] As used herein, the terms "increased," "increase," or "enhance," or "activate" are all used generically to mean an increase by a statically significant amount relative to a baseline.

[0029] As used herein, the terms "isolated" or "partially purified" refer to a nucleic acid, polypeptide, or protein that has been separated from at least one other component (e.g., nucleic acid, polypeptide, or protein) that is present with the nucleic acid, polypeptide, or protein as found in its natural source and / or that would be present with the nucleic acid, polypeptide, or protein when expressed by a cell or, in the case of secreted polypeptides and proteins, when secreted. Chemically synthesized nucleic acids, polypeptides, or proteins, or those synthesized using in vitro transcription / translation, are considered "isolated." The terms "purified" or "substantially purified" refer to an isolated nucleic acid, polypeptide, or protein that is at least 95%, by weight, including, for example, at least 96%, at least 97%, at least 98%, at least 99%, or more, of the nucleic acid, polypeptide, or protein of interest.

[0030] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues connected to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" also refer to polymers of proteinaceous amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to encoded gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0031] Major histocompatibility complex class I chain-related (MIC) polypeptides are cell surface transmembrane proteins. MIC polypeptides include, but are not limited to, human MICA isoforms (e.g., isoform 1, NCBI Reference Sequences NP_000238.1 (SEQ ID NO: 9) and 001170990) (these sequences are incorporated herein by reference) and other MICA isoforms), and human MICB isoforms (e.g., isoform 1, NCBI Reference Sequence NP_005922.2 (SEQ ID NO: 10) (this sequence is incorporated herein by reference) and other MICB isoforms). In some embodiments, a MIC polypeptide refers to MICA. In some embodiments, a MIC polypeptide refers to MICB. In some embodiments, a MIC polypeptide refers to a shared structural feature of MICA and MICB, i.e., an epitope(s) shared by MICA and MICB.

[0032] As used herein, "soluble MIC" or "sMIC" refers to a portion of a MIC polypeptide (MICA or MICB) that includes the alpha1 and alpha2 domains and the alpha3 domain or a portion of the alpha3 domain up to the proteolytic cleavage site, but lacks the transmembrane domain (e.g., the extracellular portion of MIC). In some embodiments, soluble MICA can comprise the amino acid sequence set forth in Genbank Accession No. CAA77031.1 (SEQ ID NO: 27), or a variant thereof, such as amino acid residues 24-297 of Genbank Accession Nos. AAU95072.1, AAO45822.1, AFR69318.1, AFR69319.1, or AAH16929.1, or reference sequence NP_000238.1, or amino acids 1-274 of Genbank Accession No. CAE45581.1, or amino acids 1-273 of Genbank Accession Nos. AAD52069.1, AAD52070.1, or QDW65494.1, the disclosures of which are incorporated herein by reference. In some embodiments, the soluble MICB can comprise an amino acid sequence set forth in Genbank Accession No. ARB08539.1 (SEQ ID NO: 28), AAB71646.1, AAB71647.1, or AAB71644.1, or a variant thereof, such as amino acid residues 24-297 of Genbank Accession No. ABO16470.1, ABB51802.1, AAB42011.1, AAB71643.1, or Q29980.1, or reference sequence NP_005922.2, or amino acids 1-273 of Genbank Accession No. AAC39848.1, AEK67483.1, AFR7773.1, AXY93666.1, CAB72098.1, or AAC39849.1, the disclosures of which are incorporated herein by reference. Unless otherwise specified, use of the term "MIC" is intended to refer to both cell membrane-bound and soluble forms of MIC.

[0033] As used herein, antibody B10G5 refers to the MIC antibody of the same name described in US Pat. No. 9,803,017, the disclosure of which is incorporated herein by reference for all purposes.

[0034] As used herein, "epitope" refers to the amino acids typically bound by an immunoglobulin VH / VL pair, such as the antibodies and binding agents described herein. Epitopes can be formed on a polypeptide from adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from adjacent amino acids typically survive exposure to denaturing solvents, whereas epitopes formed by tertiary folding typically are lost upon treatment with denaturing solvents. An epitope typically comprises at least three, more commonly at least five, about nine, or about eight to ten amino acids in a unique spatial conformation. An epitope defines the minimal binding site of an antibody or other binding agent and thus represents the target of specificity for the antibody, its antigen-binding portion, or other immunoglobulin-based binding agent. In the case of single-domain antibodies, an epitope represents the structural unit bound by an isolated variable domain.

[0035] As used herein, "specifically binds" means that a binding agent (e.g., an antibody or portion thereof) described herein specifically binds to a target molecule. -5 M (10000nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12Specific binding refers to the ability to bind to a target, such as the MIC, with a KD of M or less. Specific binding can be affected, for example, by the affinity and avidity of the binder and the concentration of the target polypeptide. One of skill in the art can determine appropriate conditions under which the antibodies and other binders described herein selectively bind to the MIC using any suitable method, such as titrating the binder in a suitable cell binding assay. A binder that specifically binds to the MIC is not displaced by a dissimilar competitor. In certain embodiments, a MIC antibody or antigen-binding portion thereof is said to specifically bind to the MIC when it preferentially recognizes its target antigen, the MIC, in a complex mixture of proteins and / or macromolecules.

[0036] In some embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent described herein is administered in a 10 -5 M (10000nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In some embodiments, the MIC antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to a MIC polypeptide with a dissociation constant (KD) of about 10 M or less. -5 M~10 -6 In some embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a MIC polypeptide with a dissociation constant (KD) of about 10 -6 M~10 -7 In some embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a MIC polypeptide with a dissociation constant (KD) of about 10 -7 M~10 -8 In some embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a MIC polypeptide with a dissociation constant (KD) of about 10 -8M~10 -9 In some embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a MIC polypeptide with a dissociation constant (KD) of about 10 -9 M~10 -10 In some embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a MIC polypeptide with a dissociation constant (KD) of about 10 -10 M~10 -11 In some embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a MIC polypeptide with a dissociation constant (KD) of about 10 -11 M~10 -12 Specifically binds to a MIC polypeptide with a dissociation constant (KD) of 10 M. In some embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent described herein specifically binds to a MIC polypeptide with a dissociation constant (KD) of 10 M. -12 Specifically binds to the MIC polypeptide with a dissociation constant (KD) less than M

[0037] As used herein, the phrase "specifically binds to MIC with a higher binding affinity than antibody B10G5" refers to binding affinity to soluble MIC.

[0038] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0039] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not listed in the description of the embodiments.

[0040] Except in the examples, or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean + / - 1%.

[0041] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of two standard deviations (2SD) above or below a reference value.

[0042] Other terms are defined within the description of various aspects of the invention.

[0043] Provided herein are MIC-binding antibodies (also referred to as MIC antibodies or MIC-binding antibodies) and antigen-binding portions thereof that specifically bind to MIC. The MIC antibodies surprisingly exhibit improved properties compared to antibody B10G5. In some embodiments, the MIC antibodies reduce the level of circulating free sMIC in a subject. In some embodiments, the MIC-binding antibodies, or antigen-binding portions thereof, comprise (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the MIC-binding antibodies, or antigen-binding portions thereof, comprise (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2, wherein the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy and light chain variable regions are unmodified. In some embodiments, the MIC-binding antibody, or antigen-binding portion thereof, comprises (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the heavy and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In a further aspect of any of these embodiments, the MIC-binding antibody, or antigen-binding portion thereof, specifically binds to MIC with a higher binding affinity than antibody B10G5.

[0044] In some embodiments, provided herein are binding agents comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the binding agent specifically binds to MIC. In some embodiments, provided herein are binding agents comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, provided herein are binding agents comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the heavy and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. As described herein, the binding agent comprises a MIC antibody or antigen-binding portion(s) thereof, and can include other peptides or polypeptides covalently bound to the MIC antibody or antigen-binding portion thereof. In any of these embodiments, the binding agent specifically binds to MIC. In some embodiments, the binding agent specifically binds to MIC with a higher binding affinity than antibody B10G5.

[0045] In some embodiments, a binding agent is provided that comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1 having the amino acid sequence set forth in SEQ ID NO: 11, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 13, and the VL region comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 16, and each VH and VL comprises a humanized framework region. In some embodiments, the VH framework regions are derived from human germline genes having the amino acid sequences set forth in IMGT IGHV4-59*11 (SEQ ID NO: 29) and IGHJ4*01 (SEQ ID NO: 30) or IGHV4-30-4*01 (SEQ ID NO: 31) and IGHJ4*01 (SEQ ID NO: 30). In some embodiments, the VL framework regions are derived from human germline genes having the amino acid sequences set forth in IMGT IGKV1-NL1*01 (SEQ ID NO: 32) and IMGT IGKJ1*01 (SEQ ID NO: 33), IMGT IGKV1-33*01 (SEQ ID NO: 34) and IMGT IGKJ1*01 (SEQ ID NO: 33), or IMGT IGKV1-5*01 (SEQ ID NO: 35) and IMGT IGKJ1*01 (SEQ ID NO: 33).

[0046] In some embodiments, a binding agent is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1 having the amino acid sequence set forth in SEQ ID NO:11, HCDR2 having the amino acid sequence set forth in SEQ ID NO:12, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:13, and wherein the VH region comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:1.

[0047] In some embodiments, a binding agent is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL region comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 16, and wherein the VL region comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0048] In some embodiments, the binding agent comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1 having the amino acid sequence set forth in SEQ ID NO:11, HCDR2 having the amino acid sequence set forth in SEQ ID NO:12, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:13, and the VH region has an amino acid sequence identical to the amino acid sequence of SEQ ID NO:1 by at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or less. and an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 16, wherein the VL region comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0049] In some embodiments, the binding agent comprises a heavy chain comprising a heavy chain variable (VH) region and a light chain comprising a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1 having the amino acid sequence set forth in SEQ ID NO: 11, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 13; the VH region comprises the amino acid sequence of SEQ ID NO: 1; the VL region comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 16; and the VL region comprises the amino acid sequence of SEQ ID NO: 2. and a light chain comprising an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a binding agent is provided comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4. In some such embodiments, the binding agent is an antibody.

[0050] In some embodiments, binding agents provided herein, such as MIC antibodies, exhibit good thermal stability and / or low levels of aggregation (e.g., high molecular weight (HMW) aggregates). The thermal stability of MIC antibodies can be assessed, for example, by Tm and Tag, which can be obtained by intrinsic protein fluorescence (IPF) (266 nm excitation, 280-450 nm emission scan) and static light scattering (SLS) at 473 nm using the Uncle system (Unchained Labs), respectively. The level of aggregation of MIC antibodies can be measured, for example, by using HPLC-SEC or by using dynamic light scattering (DLS). In some embodiments, the level of HMW aggregates of the MIC antibody can be less than 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, or 0.5% based on the peak area of ​​antibody monomers and HMW aggregates as a percentage of the total amount of MIC antibody, i.e., at least 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the MIC antibody when in solution is in monomeric form. In some embodiments, the polydispersity index (PDI) of the MIC antibody may be less than 0.1, as determined by DLS.

[0051] In certain embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent specifically binds to a conformational epitope on MICA and MICB located within approximately amino acid positions 66-77, 136-144, and 247-258 of the amino acid sequence set forth in SEQ ID NO:27 or 28. In certain embodiments, the MIC antibody or antigen-binding portion thereof or other binding agent has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2, with optional framework region substitutions described herein, and competes for specific binding with antibody B10G5, which specifically binds to a conformational epitope on MICA and MICB located within approximately amino acid positions 66-77, 136-144, and 247-258 of the amino acid sequence set forth in SEQ ID NO:27 and 28.

[0052] In some embodiments, the compositions and methods described herein relate to the suppression of the immunosuppressive effects of sMIC (e.g., reducing the level and / or activity of sMIC available for interaction with the cellular receptor NKG2D) by an MIC antibody, antigen-binding portion thereof, or other binding agent in vivo. In some embodiments, the suppression of sMIC can be a reduction in serum levels of unbound MIC and restoration of cell surface NKG2D expression on NK and CD8 T cells. In some embodiments, the suppression of sMIC can be a reduction in the level of MIC (e.g., the level of sMIC in the circulation).

[0053] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds to an antigen. The term generally refers to antibodies consisting of two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions, including full-length antibodies (having heavy and light chain constant regions) and their antigen-binding portions. For example, intact monoclonal antibodies, Fab, Fab', F(ab') 2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAbs), diabodies, multispecific antibodies, dual specific antibodies, diabodies, and single chain (see, e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference).

[0054] Each heavy chain is composed of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may include three domains, CH1, CH2, and CH3, and optionally a fourth domain, CH4. Each light chain is composed of a variable region (abbreviated as VL) and a constant region. The light chain constant region is a CL domain. The VH and VL regions may be further divided into hypervariable regions called complementarity-determining regions (CDRs), and may incorporate conserved regions called framework regions (FRs). Thus, each VH and VL region consists of three CDRs and four FRs arranged from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure is well known to those skilled in the art.

[0055] The amino acid sequence of the VH CDR of the MIC antibody is set forth in SEQ ID NO: 1, consisting of amino acids 26-34 (GYSITSDYA, HCDR1, SEQ ID NO: 11), 50-58 (GYISYSGST, HCDR2, SEQ ID NO: 12), and 97-105 (ARGGTYFDY, HCDR3, SEQ ID NO: 13). The amino acid sequence of the VL CDR of the MIC antibody is set forth in SEQ ID NO: 2, consisting of amino acids 24-32 (RASAHINNW, LCDR1, SEQ ID NO: 14), 50-56 (DATSLES, LCDR2, SEQ ID NO: 15), and 98-107 (QHYWSTPWT, LCDR3, SEQ ID NO: 16). The phrase "the CDRs of the heavy chain or light chain variable region are unaltered" refers to these VH and VL CDRs (SEQ ID NOs: 11-16), which have no amino acid substitutions, deletions, or insertions.

[0056] As used herein, the "antigen-binding portion" of an MIC antibody refers to a portion of an MIC antibody described herein having the VH and VL sequences of the MIC antibody (represented by SEQ ID NO: 1 and SEQ ID NO: 2, optionally modified as described herein). According to the term "antigen-binding portion" of an antibody, examples of antigen-binding portions include Fab, Fab', F(ab') and Fab'. 2 , Fv, disulfide-linked Fv, scFv, single-domain antibodies (dAbs), diabodies, and single chains. As used herein, the terms Fab, F(ab'), and Fv refer to the following: (i) a Fab fragment, i.e., a monovalent fragment composed of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked together at the hinge region via a disulfide bridge; and (iii) an Fv fragment composed of the VL and VH domains of an MIC antibody. The two domains of the Fv fragment, i.e., the VL and VH, are encoded by separate coding regions, but can be further linked to each other using a synthetic linker, such as a poly-G4S amino acid sequence ("(G4S)n," disclosed as SEQ ID NO: 17, where n = 1 to 5), allowing them to be prepared as a single protein chain in which the VL and VH domains combine to form a monovalent molecule (known as a single-chain Fv (ScFv)). The term "antigen-binding portion" of an antibody is also intended to include such single-chain antibodies. Other forms of single-chain antibodies, such as "diabodies," are also included herein. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker connecting the VH and VL domains is too short to allow the two domains to bind on the same chain, thereby allowing the VH and VL domains to pair with complementary domains on separate chains (VL and VH, respectively) to form two antigen-binding sites (see, e.g., Holliger, R, et al. (1993) Proc. Natl. Acad. Sci. USA 90:64446448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0057] The immunoglobulin constant region refers to the constant region of a heavy or light chain. Human heavy chain constant region amino acid sequences and human light chain constant region amino acid sequences are well known in the art. The constant region can be of any suitable type selected from the immunoglobulin classes IgA, IgD, IgE, IgG, and IgM. Some immunoglobulin classes can be further divided into isotypes, for example, IgG1, IgG2, IgG3, IgG4, or IgA1 and IgA2. Heavy chain constant regions (Fc) corresponding to different immunoglobulin classes can be α, δ, ε, γ, and μ, respectively. Light chains can be either kappa or κ or lambda or λ.

[0058] In some embodiments, the constant region can have an IgG1 isotype. In some embodiments, the constant region can have an IgG2 isotype. In some embodiments, the constant region can have an IgG3 isotype. In some embodiments, the constant region can have an IgG4 isotype. In some embodiments, the Fc domain can have a hybrid isotype comprising constant regions from more than one isotype. In some embodiments, the immunoglobulin constant region can be an IgG1 or IgG4 constant region.

[0059] In some embodiments, the MIC antibody heavy chain is of the IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the MIC antibody light chain is of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO: 8.

[0060] Additionally, the MIC antibody or antigen-binding portion thereof may be part of a larger binder formed by covalent or noncovalent binding of the antibody or antibody portion to one or more other proteins or peptides. Such binders involve the use of streptavidin core regions to prepare tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidinyl peptides, such as a hexahistidinyl tag (the "hexahistidinyl tag" disclosed as SEQ ID NO: 18), to produce bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0061] With respect to the amino acid sequences of VH and VL, those skilled in the art will recognize that individual substitutions, deletions, or additions (insertions) to the nucleic acid encoding VH or VL, or amino acids in a polypeptide that alter a single amino acid or a small percentage of amino acids in the encoded sequence, are "conservatively modified variants," which result in the substitution of an amino acid with a chemically similar amino acid (conservative amino acid substitution), and the modified polypeptide retains the ability to specifically bind to MIC with greater binding affinity than antibody B10G5.

[0062] In some embodiments, conservatively modified variants of MIC antibodies or antigen-binding portions thereof can have alterations in the FRs (i.e., outside of the CDRs); for example, conservatively modified variants of MIC antibodies have the VH and VL CDR amino acid sequences (represented by SEQ ID NOS: 11-16) and at least one conservative amino acid substitution in the FRs. In some embodiments, the VH and VL amino acid sequences (represented by SEQ ID NOS: 1 and 2, respectively) collectively have 8, 6, 4, 2, or 1 or fewer conservative amino acid substitutions in the FRs compared to the VH and VL amino acid sequences (represented by SEQ ID NOS: 1 and 2, respectively). In some embodiments, the VH and VL amino acid sequences (represented by SEQ ID NOS: 1 and 2, respectively) have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conservative amino acid substitutions in the FRs compared to the VH and VL amino acid sequences (represented by SEQ ID NOS: 1 and 2, respectively). In a further aspect of any of these embodiments, conservatively modified variants of MIC antibodies, antigen-binding portions thereof, or other binding agents exhibit a binding affinity for MIC that is greater than the binding affinity of antibody B10G5.

[0063] Conservative amino acid substitutions involve the replacement of a given amino acid with a residue having similar physiochemical properties, such as the substitution of one aliphatic residue for another (e.g., Ile, Val, Leu, or Ala for each other), or the substitution of one polar residue for another (e.g., Lys for Arg, Glu for Asp, or Gln for Asn). Other such conservative amino acid substitutions are well known, such as full-length substitutions with similar hydrophobic properties. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, e.g., antigen-binding activity and specificity, of the native or reference polypeptide, i.e., for MIC (sMIC and / or membrane-bound MIC), is retained.

[0064] For conservative substitutions, amino acids can be grouped according to the similarity of their side chain properties (A.L. Lehninger, Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic Asp (D) and Glu (E); and (4) basic Lys (K), Arg (R), and His (H).

[0065] Alternatively, for conservative substitutions, naturally occurring residues can be divided into groups based on shared side chain properties: (1) hydrophobic norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic Cys, Ser, Thr, Asn, Gln; (3) acidic Asp, Glu; (4) basic His, Lys, Arg; (5) chain orientation-influencing residues Gly, Pro; and (6) aromatic Trp, Tyr, Phe. Non-conservative substitutions would involve exchanging members of one of these classes or another.

[0066] Particular conservative substitutions include, for example, Ala to Gly or Ser, Arg to Lys, Asn to Gln or His, Asp to Glu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Ala or Pro, His to Asn or Gln, Ile to Leu or Val, Leu to Ile or Val, Lys to Arg, Gln, or Glu, Met to Leu, Tyr, or Ile, Phe to Met, Leu, or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp, and / or Phe to Val, Ile, or Leu.

[0067] In some embodiments, conservatively modified variants of a MIC antibody or antigen-binding portion thereof are preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a reference VH or VL sequence, wherein the VH and VL CDRs (SEQ ID NOS: 11-16) are unaltered. The degree of homology (percent identity) between a reference sequence and a modified sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0068] In some embodiments, the VH and VL amino acid sequences (represented in SEQ ID NOs: 1 and 2, respectively) collectively have no more than 8, or 6, or 4, or 2, or 1 conservative amino acid substitutions in the framework regions compared to the VH and VL amino acid sequences (represented in SEQ ID NOs: 1 and 2, respectively). ... amino acid substitutions, deletions, or insertions in the framework regions compared to the VH and VL amino acid sequences (represented in SEQ ID NOs: 1 and 2, respectively). In some embodiments, the VH and VL amino acid sequences (represented by SEQ ID NOs: 1 and 2, respectively) have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conservative amino acid substitutions in the framework regions compared to the VH and VL amino acid sequences (represented by SEQ ID NOs: 1 and 2, respectively). In some embodiments, the VH and VL amino acid sequences (represented by SEQ ID NOs: 1 and 2, respectively) collectively have 8 or 6 or 4 or 2 or 1 or fewer amino acid substitutions, deletions, or insertions compared to the VH and VL amino acid sequences (represented by SEQ ID NOs: 1 and 2, respectively).

[0069] Modification of a native (or reference) amino acid sequence can be achieved by any of a number of techniques known to those skilled in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the desired mutant sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes a variant with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-targeted site-directed mutagenesis procedures can be used to provide altered nucleotide sequences with specific codons altered according to the desired substitution, deletion, or insertion. Techniques for making such modifications are well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986), Bauer et al. (Gene 37:73, 1985), Craik (BioTechniques, January 1985, 12-19), Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981), and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties.

[0070] In some embodiments, the MIC antibody or antigen-binding portion thereof has a fully human constant region. In some embodiments, the MIC antibody or antigen-binding portion thereof has a non-human constant region. In some embodiments, the MIC antibody heavy chain is an IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the MIC antibody light chain is a kappa isotype and has the amino acid sequence set forth in SEQ ID NO:8.

[0071] In some embodiments, the MIC antibody or antigen-binding portion thereof has an altered constant region (Fc region) or Fc domain. The Fc domain (e.g., CH1, CH2, CH3, and optionally CH4) is a portion of the Fc region. The Fc domain or a portion of the Fc region can bind to an Fc receptor (FcR) on a cell. FcRs are organized into classes (e.g., gamma (γ), alpha (α), and epsilon (ε)) based on the class of antibody they recognize. The FcaR class can bind IgA and includes several isoforms, such as FcaRI (CD89). The FcyR class can bind IgG and includes several isoforms, FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). FcyRIIIA (CD16a) has two major variants, F158 or V158.

[0072] Binding of an Fc domain or Fc region to an FcR can modify the immune response relative to a reference. Similarly, lack of binding of an Fc domain to an FcR can modify the immune response relative to a reference.

[0073] MIC antibodies can have an Fc domain with a sequence that has been modified, relative to a wild-type or reference sequence, to alter at least one constant domain or constant region-mediated biological effector function associated with the corresponding wild-type or reference sequence. For example, in some embodiments, the Fc domain can be modified to reduce or increase at least one constant domain or constant region-mediated biological effector function associated with an unmodified Fc domain, e.g., to reduce or increase binding to an Fc receptor (FcR). FcR binding can be reduced or increased, e.g., by modifying the immunoglobulin constant region segment of the antibody at a particular site(s) involved in (e.g., required for, or affecting) FcR interactions. In some embodiments, the Fc domain is altered to reduce binding to one or more Fc gamma receptors (e.g., one or more of FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, and / or FcRN).

[0074] In some embodiments, the antibody constant region or constant domain is modified to acquire or improve at least one constant region-mediated biological effector function relative to the unmodified Fc domain, e.g., to enhance FcyR interaction. For example, the antibody constant region or constant domain can be modified to bind FcyRIIA, FcyRIIB, and / or FcyRIIIA with higher affinity than the corresponding wild-type Fc domain or Fc region.

[0075] Modifications in the amino acid sequence of an Fc domain can alter FcR recognition of the Fc domain or Fc region, although such modifications still allow signaling through the FcR. The modification(s) can be a substitution of an amino acid at a residue with a different amino acid at that residue. The modification(s) can enable the FcR to bind to a site on the Fc domain or Fc region to which the FcR would not normally bind. The modification(s) can increase the binding affinity of the FcR to the Fc domain or Fc region compared to binding of a reference sequence. The modification(s) can decrease the binding affinity of the FcR to a site on the Fc domain compared to binding of a reference sequence.

[0076] In some embodiments, modification(s) in the amino acid sequence of the Fc domain may alter the recognition of one or more FcRs for the Fc domain or Fc region. Such modification(s) can alter the ability of an antibody or antigen-binding portion to interact with immune cells. Such a modification or series of modifications to the Fc domain or Fc region can enable selective binding of the Fc domain to an FcR on an immune cell, or can reduce or eliminate the interaction of an antibody or antigen-binding portion having the modified domain with an immune cell. For example, a modification to the Fc domain can reduce binding of the Fc domain to Fc gamma receptors, while retaining the ability of the Fc domain to bind to FcRn.

[0077] The modified Fc region or domain can have at least one amino acid change compared to the sequence of the wild-type Fc region or domain. The amino acid change in the Fc region can enable the antibody, or antigen-binding portion thereof, to bind to at least one Fc receptor with higher affinity compared to the wild-type or reference Fc region. The amino acid change in the Fc domain can enable the antibody to bind to at least one Fc receptor with higher affinity compared to the wild-type or reference Fc domain. The amino acid change in the Fc region can enable the antibody to bind to at least one Fc receptor with lower affinity compared to the wild-type or reference Fc region. The amino acid change in the Fc domain can enable the antibody to bind to at least one Fc receptor with lower affinity compared to the wild-type or reference Fc domain.

[0078] In some embodiments, the MIC antibody or antigen-binding portion thereof can have an Fc domain or Fc region comprising the sequence of an IgG1 isoform modified from the wild-type IgG1 sequence. The modification can include substitutions at one or more amino acid residues in the Fc domain, such as five different amino acid residues comprising L235V / F243L / R292P / Y300L / P396L (IgG1VLPLL) according to the Kabat EU index. The modification can include substitutions at one or more different amino acid residues in the Fc domain, such as two different amino acid residues comprising S239D / I332E (IgG1DE) according to the Kabat EU index. The modification can include substitutions at one or more different amino acid residues in the Fc domain, such as three different amino acid residues comprising S298A / E333A / K334A (IgG1 AAA) according to the Kabat EU index.

[0079] In some embodiments, the Fc domain or Fc region of an MIC antibody or antigen-binding portion thereof may exhibit reduced binding affinity for one or more Fc receptors. In some embodiments, the Fc domain or Fc region may exhibit reduced binding affinity for one or more Fc gamma receptors. In some embodiments, the Fc domain or Fc region may exhibit reduced binding affinity for the FcRn receptor. In some embodiments, the Fc domain or Fc region may exhibit reduced binding affinity for Fc gamma receptors and FcRn receptors. In some embodiments, the Fc domain is an Fc null domain or region. As used herein, "Fc null" refers to a domain that exhibits weak to no binding to any of the Fc gamma receptors. In some embodiments, the Fc null domain or region exhibits at least a 1000-fold decrease in binding affinity (eg, an increased Kd) for an Fc gamma receptor.

[0080] In some embodiments, the Fc domain or region of the MIC antibody is an Fc null domain or region comprising a flexible sequence such as GGGS. In some embodiments, the MIC antibody is an IgG1 isoform, and the flexible sequence is inserted between G237 and G238 of the human IgG1 heavy chain. In some embodiments, incorporation of a flexible sequence into the MIC antibody results in little to no effect on the binding affinity of the antibody. In some embodiments, inclusion of a flexible sequence in the MIC antibody reduces the ADCC activity of the antibody. In some embodiments, incorporation of a flexible sequence into the MIC antibody results in little to no loss of glycosylation and / or significant alteration(s) of the glycan profile of the antibody. The glycosylation and / or glycan profile of an antibody can be measured, for example, by subjecting the antibody to PNGaseF digestion and analyzing the enzymatic products using hydrophilic interaction chromatography (HILIC) with a fluorescence detector.

[0081] In certain embodiments, the Fc domain has reduced binding affinity for one or more of FcyRI (CD64), FcyRIIA (CD32), FcyRIIIA (CD16a), FcyRIIIB (CD16b), or any combination thereof. To decrease the binding affinity of the Fc domain or Fc region to an Fc receptor, the Fc domain or Fc region may comprise one or more amino acid substitutions that reduce the binding affinity of the Fc domain or Fc region to an Fc receptor.

[0082] In some embodiments, the one or more substitutions comprise any one or more of the IgG1 heavy chain substitutions corresponding to E233P, L234V, L234A, L235A, L235E, AG236, G237A, E318A, K320A, K322A, A327G, A330S, and / or P331S according to the EU index of Kabat numbering. In some embodiments, the modification can be a substitution of E233, L234, and L235, such as E233P / L234V / L235A or E233P / L234V / L235A / AG236 according to the EU index of Kabat. In some embodiments, the modification can be a substitution of P238, such as P238A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of D265, such as D265A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of N297, such as N297A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of A327, such as A327Q, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of P329, such as P239A, according to the EU index of Kabat.

[0083] In some embodiments, the IgG Fc domain or IgG Fc region comprises at least one amino acid substitution that reduces its binding affinity to FcyRI compared to a wild-type or reference IgG Fc domain. In some embodiments, the modification may comprise a substitution at F241, such as F241A, according to the Kabat EU index. In some embodiments, the modification may comprise a substitution at F243, such as F243A, according to the Kabat EU index. In some embodiments, the modification may comprise a substitution at V264, such as V264A, according to the Kabat EU index. In some embodiments, the modification may comprise a substitution at D265, such as D265A, according to the Kabat EU index.

[0084] In some embodiments, the IgG Fc domain or IgG Fc region comprises at least one amino acid substitution that increases its binding affinity to FcyRI compared to a wild-type or reference IgG Fc domain, hi some embodiments, the modification may comprise substitutions at A327 and P329, such as A327Q / P329A, according to the EU index of Kabat.

[0085] In some embodiments, the IgG Fc modification(s) comprise one or more amino acid substitutions that reduce the binding affinity of the IgG Fc domain or region for the FcyRII and FcyRIIIA receptors. In some embodiments, the modification can be a substitution of D270, such as D270A, according to the Kabat EU index. In some embodiments, the modification can be a substitution of Q295, such as Q295A, according to the Kabat EU index. In some embodiments, the modification can be a substitution of A327, such as A237S, according to the Kabat EU index.

[0086] In some embodiments, the modification comprises a substitution of one or more amino acids that increases the binding affinity of the IgG Fc domain or IgG Fc region for the FcyRII and FcyRIIIA receptors. In some embodiments, the modification can be a substitution of T256, such as T256A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of K290, such as K290A, according to the EU index of Kabat.

[0087] In some embodiments, the modification comprises a substitution of one or more amino acids that increases the binding affinity of the IgG Fc domain or IgG Fc region for the FcyRII receptor. In some embodiments, the modification can be a substitution of R255, such as R255A, according to the Kabat EU index. In some embodiments, the modification can be a substitution of E258, such as E258A, according to the Kabat EU index. In some embodiments, the modification can be a substitution of S267, such as S267A, according to the Kabat EU index. In some embodiments, the modification can be a substitution of E272, such as E272A, according to the Kabat EU index. In some embodiments, the modification can be a substitution of N276, such as N276A, according to the Kabat EU index. In some embodiments, the modification can be a substitution of D280, such as D280A, according to the Kabat EU index. In some embodiments, the modification can be a substitution of H285, such as H285A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of N286, such as N286A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of T307, such as T307A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of L309, such as L309A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of N315, such as N315A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of K326, such as K326A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of P331, such as P331A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of S337, such as S337A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of A378, such as A378A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of E430, such as E430, according to the Kabat EU index.

[0088] In some embodiments, the modification comprises a substitution of one or more amino acids that increases the binding affinity of the IgG Fc domain or IgG Fc region for the FcyRII receptor and decreases its binding affinity for the FcyRIIIA receptor. In some embodiments, the modification can be a substitution of H268, such as H268A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of R301, such as R301A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of K322, such as K322A, according to the EU index of Kabat.

[0089] In some embodiments, the modification comprises one or more amino acid substitutions that reduce the binding affinity of the IgG Fc domain or IgG Fc region for the FcyRII receptor but do not significantly affect the binding affinity for the FcyRIIIA receptor. In some embodiments, the modification can be a substitution of R292, such as R292A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of K414, such as K414A, according to the EU index of Kabat.

[0090] In some embodiments, the modification comprises a substitution of one or more amino acids that reduces the binding affinity of the IgG Fc domain or IgG Fc region for the FcyRII receptor and increases its binding affinity for the FcyRIIIA receptor. In some embodiments, the modification can be a substitution of S298, such as S298A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of S239, I332, and A330, such as S239D / I332E / A330L. In some embodiments, the modification can be a substitution of S239 and I332, such as S239D / I332E.

[0091] In some embodiments, the modification comprises a substitution of one or more amino acids that reduces the binding affinity of the IgG Fc domain or IgG Fc region for the FcyRIIIA receptor, hi some embodiments, the modification can be a substitution of F241 and F243, such as F241S / F243S or F241I / F243I, according to the EU index of Kabat.

[0092] In some embodiments, the modification comprises one or more amino acid substitutions that reduce the binding affinity of the IgG Fc domain or IgG Fc region for the FcyRIIIA receptor without significantly affecting the binding affinity for the FcyRII receptor. In some embodiments, the modification may be a substitution of S239, such as S239A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of E269, such as E269A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of E293, such as E293A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of Y296, such as Y296F, according to the Kabat EU index. In some embodiments, the modification may be a substitution of V303, such as V303A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of A327, such as A327G, according to the Kabat EU index. In some embodiments, the modification can be a substitution of K338, such as K338A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of D376, such as D376A, according to the EU index of Kabat.

[0093] In some embodiments, the modification comprises one or more amino acid substitutions that increase the binding affinity of the IgG Fc domain or IgG Fc region for the FcyRIIIA receptor without affecting the binding affinity for the FcyRII receptor. In some embodiments, the modification can be a substitution of E333, such as E333A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of K334, such as K334A, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of A339, such as A339T, according to the EU index of Kabat. In some embodiments, the modification can be a substitution of S239 and 1332, such as S239D / I332E, according to the EU index of Kabat.

[0094] In some embodiments, the modification comprises a substitution of one or more amino acids that increases the binding affinity of the IgG Fc domain or IgG Fc region for the FcyRIIIA receptor. In some embodiments, the modification can be a substitution of L235, F243, R292, Y300, and P396 according to the Kabat EU index, such as L235V / F243L / R292P / Y300L / P396L (IgG1 VLPLL). In some embodiments, the modification can be a substitution of S298, E333, and K334 according to the Kabat EU index, such as S298A / E333A / K334A. In some embodiments, the modification can be a substitution of K246 according to the Kabat EU index, such as K246F.

[0095] Other substitutions in the IgG Fc domain that affect its interaction with one or more Fc gamma receptors are disclosed in U.S. Pat. Nos. 7,317,091 and 8,969,526, the disclosures of which are incorporated herein by reference.

[0096] In some embodiments, the IgG Fc domain or IgG Fc region comprises at least one amino acid substitution that reduces binding affinity to FcRn compared to a wild-type or reference IgG Fc domain. In some embodiments, the modification may comprise a substitution at H435, such as H435A, according to the Kabat EU index. In some embodiments, the modification may comprise a substitution at I253, such as I253A, according to the Kabat EU index. In some embodiments, the modification may comprise a substitution at H310, such as H310A, according to the Kabat EU index. In some embodiments, the modification may comprise substitutions at I253, H310, and H435, such as I253A / H310A / H435A, according to the Kabat EU index.

[0097] In some embodiments, the modification may comprise a substitution of a single amino acid residue that increases the binding affinity of the IgG Fc domain for FcRn compared to a wild-type or reference IgG Fc domain. In some embodiments, the modification may comprise a substitution at V308, such as V308P, according to the Kabat EU index. In some embodiments, the modification may comprise a substitution at M428, such as M428L, according to the Kabat EU index. In some embodiments, the modification may comprise a substitution at N434, such as N434A, according to the Kabat EU index, or N434H, according to the Kabat EU index. In some embodiments, the modification may comprise a substitution at T250 and M428, such as T250Q and M428L, according to the Kabat EU index. In some embodiments, the modification may comprise a substitution at M428 and N434, such as M428L and N434S, N434A, or N434H, according to the Kabat EU index. In some embodiments, the modification may include substitutions at M252, S254, and T256, such as M252Y / S254T / T256E, according to the EU index of Kabat. In some embodiments, the modification may be a substitution of one or more amino acids selected from P257L, P257N, P257I, V279E, V279Q, V279Y, A281S, E283F, V284E, L306Y, T307V, V308F, Q31IV, D376V, and N434H. Other substitutions in the IgG Fc domain that affect its interaction with FcRn are disclosed in U.S. Patent No. 9,803,023, the disclosure of which is incorporated herein by reference.

[0098] In some embodiments, the MIC antibody or antigen-binding portion thereof has an altered constant region (Fc region) or Fc domain that alters complement-dependent cytotoxicity (CDC) activity. CDC is a cell killing method that can be directed by an antibody. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are highly effective at inducing CDC via the classical complement activation pathway.

[0099] In some embodiments, the Fc region has a modification that reduces CDC activity at one or more amino acid positions E318, K320, K322, P329, and / or P331 of IgG1 according to the EU index of Kabat, such as E318A, K320A, K322A, P329A, and / or P331A. In some embodiments, the Fc region has a modification that increases CDC activity at one or more amino acid positions E430, E345, and S440 of IgG1 according to the EU index of Kabat, such as one or more of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and / or S440W.

[0100] In various embodiments, MIC antibodies, antigen-binding portions thereof, and other binding agents can be produced in cell lines of human, murine, or other animal origin. Recombinant DNA expression can be used to produce MIC antibodies, antigen-binding portions thereof, and other binding agents. This allows for the production of a spectrum of MIC antibodies, as well as MIC antigen-binding portions and other binding agents (including fusion proteins), in a selected host species. Production of MIC antibodies, antigen-binding portions thereof, and other binding agents in bacteria, yeast, transgenic animals, and chicken eggs are also alternatives to cell-based production systems. A major advantage of transgenic animals is the potential high yield from renewable resources.

[0101] In some embodiments, the MIC VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 is encoded by a nucleic acid. In some embodiments, the MIC VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 is encoded by a nucleic acid. In some embodiments, the MIC VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 is encoded by a nucleic acid having a sequence set forth in SEQ ID NO: 21. In some embodiments, the MIC VL polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 is encoded by a nucleic acid having a sequence set forth in SEQ ID NO: 22.

[0102] As used herein, the terms "nucleic acid" or "nucleic acid sequence" or "polynucleotide sequence" or "nucleotide" refer to a polymer incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be a single strand of denatured double-stranded DNA. In some embodiments, a nucleic acid can be cDNA, e.g., a nucleic acid lacking introns.

[0103] Nucleic acid molecules encoding the amino acid sequences of MIC antibodies, antigen-binding portions thereof, and other binding agents can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding MIC antibodies, antigen-binding portions thereof, or other binding agents. Additionally, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding MIC antibodies or antigen-binding portions thereof and other binding agents. Nucleic acid sequences encoding at least the MIC antibodies, antigen-binding portions thereof, binding agents, or polypeptides thereof described herein can be recombined into vector DNA according to conventional techniques, such as, for example, restriction enzyme digestion to provide blunt or staggered ends for ligation, suitable termini, filling in appropriate sticky ends, alkaline phosphatase treatment to avoid undesired ligations, and ligation with an appropriate ligase. Techniques for such manipulations are disclosed, for example, by Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993, and can be used to generate nucleic acid sequences and vectors encoding MIC antibodies or antigen-binding portions thereof or VH or VL polypeptides thereof.

[0104] Nucleic acid molecules, such as DNA, contain nucleotide sequences containing transcriptional and translational regulatory information, and are said to be "capable of expressing" a polypeptide when such sequences are "operably linked" to a nucleotide sequence encoding the polypeptide. An operable linkage is one in which the regulatory DNA sequences and the DNA sequence to be expressed (e.g., a MIC antibody or antigen-binding portion thereof) are linked in a manner that allows for gene expression of recoverable quantities of the polypeptide(s) or antigen-binding portion. The precise nature of the regulatory regions required for gene expression can vary from organism to organism, as is well known in the art. See, e.g., Sambrook et al., 1989; Ausubel et al., 1987-1993.

[0105] Thus, expression of the MIC antibodies or antigen-binding portions thereof described herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, avian, and mammalian cells, in vivo or in situ, or host cells derived from mammals, insects, birds, or yeast. Mammalian cells or tissues can be derived from humans, primates, hamsters, rabbits, rodents, cattle, pigs, sheep, horses, goats, dogs, or cats, although any other mammalian cells may also be used. Furthermore, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, by using the yeast ubiquitin hydrolase system. The fusion proteins so produced can be processed in vivo or purified and processed in vitro, allowing for the synthesis of the MIC antibodies or antigen-binding portions thereof described herein with specific amino-terminal sequences. Furthermore, problems associated with retaining the methionine residue from the start codon in direct yeast (or bacterial) expression can be avoided. (See, e.g., Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).) Recombinant MIC antibodies, or antigen-binding portions thereof, can be produced using any of a range of yeast gene expression systems that incorporate promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

[0106] Production of MIC antibodies or antigen-binding portions thereof in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express the polypeptide by methods known to those of skill in the art. See Ausubel et al., 1987-1993.

[0107] In some embodiments, the introduced nucleic acid sequence (encoding the MIC antibody or antigen-binding portion thereof or polypeptide) is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host cell. Any of a wide variety of vectors can be used for this purpose and are known and available to those of skill in the art. See, e.g., Ausubel et al., 1987-1993. Important factors in selecting a particular plasmid or viral vector include the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector, the copy number of the vector desired in a particular host, and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0108] Representative prokaryotic vectors known in the art include plasmids capable of replication in E. coli. Other gene expression elements useful for expressing DNA encoding an MIC antibody or antigen-binding portion thereof include, but are not limited to, (a) viral transcription promoters and enhancer elements such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus long terminal repeat (Gorman et al., 79 PNAS 6777 (1982)), and Moloney murine leukemia virus long terminal repeat (Grosschedl et al., 41 Cell 885 (1985)), (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayama et al., 1983), and (c) polyadenylation sites such as those from SV40 (Okayama et al., 1983). Immunoglobulin-encoding DNA genes can be expressed using the expression elements SV40 early promoter and enhancer, mouse immunoglobulin heavy chain promoter enhancer, SV40 late region mRNA splicing, rabbit S-globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation element, as described by Liu et al., infra, and Weidle et al., 51 Gene 21 (1987).

[0109] In the case of a nucleotide sequence encoding an immunoglobulin, the transcription promoter can be, for example, human cytomegalovirus and the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin.

[0110] In some embodiments, for expression of a DNA coding region in rodent cells, the transcription promoter can be a viral LTR sequence, and the transcription promoter enhancer can be either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, as well as a polyadenylation region and a transcription termination region. In other embodiments, DNA sequences encoding other proteins are combined with the above expression elements to achieve protein expression in mammalian cells.

[0111] Each coding region or gene fusion is assembled into or inserted into an expression vector. Recipient cells capable of expressing the MIC variable region(s) or antigen-binding portion thereof (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof, as described herein) are then transfected with nucleotides encoding the MIC antibody or antibody polypeptide or antigen-binding portion thereof, either alone, or co-transfected with polynucleotide(s) encoding the VH and VL chain coding regions. The transfected recipient cells are cultured under conditions that allow expression of the integrated coding regions, and the expressed antibody chains or intact antibody or antigen-binding portion thereof are recovered from the culture.

[0112] In some embodiments, nucleic acids containing coding regions encoding a MIC antibody or antigen-binding portion thereof (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 2 or variants thereof as described herein) are assembled into separate expression vectors and then used to co-transfect recipient host cells. Each vector can contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, one designed for selection in a bacterial system and a second designed for selection in a eukaryotic system, with each vector carrying a set of coding regions. This strategy results in vectors that first direct the production of nucleotide sequences in a bacterial system and allow for amplification. The DNA vectors thus produced and amplified in the bacterial host are then used to co-transfect eukaryotic cells, allowing for the selection of co-transfected cells carrying the desired transfected nucleic acid (e.g., including the heavy and light chains of a MIC antibody). Non-limiting examples of selectable genes for use in bacterial systems are genes that confer resistance to ampicillin and genes that confer resistance to chloramphenicol. Selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo). Alternatively, fused nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

[0113] For transfection of the expression vector and production of the MIC antibody or antigen-binding portion thereof, the recipient cell line can be a Chinese hamster ovary cell line (e.g., DG44) or a myeloma cell. The myeloma cell can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin genes and possess the machinery for immunoglobulin glycosylation. For example, in some embodiments, the recipient cell is recombinant Ig-producing myeloma cell SP2 / 0 (ATCC#CRL 8287). SP2 / 0 cells produce only the immunoglobulin encoded by the transfected gene. The myeloma cells can be grown in culture or in the peritoneal cavity of mice, and the secreted immunoglobulin can be obtained from the ascites fluid.

[0114] An expression vector encoding a MIC antibody or antigen-binding portion thereof (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof described herein) can be introduced into a suitable host cell by any of a variety of suitable means, including biochemical means such as transformation, transfection, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and microprojectile bombardment. See Johnston et al., 240 Science 1538 (1988), which are known to those skilled in the art.

[0115] Yeast offers advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeast carries out post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies utilizing strong promoter sequences and high copy number plasmids exist and can be used to produce desired proteins in yeast. Yeast recognizes leader sequences in cloned mammalian gene products and secretes polypeptides bearing leader sequences (i.e., prepolypeptides). See, e.g., Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0116] Yeast gene expression systems can be routinely evaluated for antibody production, secretion, and stability levels, as well as for assembled MIC antibodies and their antigen-binding portions. A variety of yeast gene expression systems are available that incorporate promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Another example is the translation elongation factor 1 alpha promoter. Several approaches can be taken to evaluate the optimal expression plasmid for immunoglobulin expression in yeast. See II DNA Cloning 45, (Glover, ed., IRL Press, 1985) and, e.g., U.S. Publication No. US2006 / 0270045A1.

[0117] Bacterial strains can also be used as hosts for the production of the antibody molecules or antigen-binding portions thereof described herein. Examples include E. coli K12 strains such as E. coli W3110 (ATCC 27325), Bacillus species, Enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species. In connection with these bacterial hosts, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used. The vectors contain replication sites as well as specific genes capable of providing phenotypic selection in transformed cells. Several approaches can be taken to evaluate expression plasmids for the production of MIC antibodies and antigen-binding portions thereof in bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996).

[0118] Host mammalian cells can be grown in vitro or in vivo and provide post-translational modifications to immunoglobulin molecules, including removal of leader peptides, folding and assembly of VH and VL chains, glycosylation of antibody molecules, and secretion of functional antibodies and / or antigen-binding portions thereof.

[0119] Mammalian cells that may be useful as hosts for the production of antibody proteins include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells, in addition to the cells of lymphoid origin described above. Exemplary eukaryotic cells that can be used to express immunoglobulin polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PERC6™ cells (Crucell); and NSO cells. In some embodiments, particular eukaryotic host cells are selected based on their ability to make desired post-translational modifications to the heavy and / or light chains. For example, in some embodiments, CHO cells produce polypeptides with higher sialylation levels than the same polypeptides produced in 293 cells.

[0120] In some embodiments, one or more MIC antibodies or antigen-binding portions thereof (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 2 or variants thereof described herein) can be produced in vivo in an animal engineered or transfected with one or more nucleic acid molecules encoding the polypeptides according to any suitable method.

[0121] In some embodiments, the antibody or antigen-binding portion thereof (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 2 or a variant thereof described herein) is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0122] Many vector systems are available for the expression of VH and VL chains (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 2, or a variant thereof, as described herein) in mammalian cells (see Glover, 1985). To obtain intact antibodies, various approaches can be followed. As described above, VH and VL chains, and optionally associated constant regions, can be co-expressed in the same cell to achieve intracellular association and linkage of the VH and VL chains into a complete tetrameric H2L2 antibody, or antigen-binding portion thereof. Co-expression can occur by using the same or different plasmids in the same host. Nucleic acids encoding VH and VL chains, or antigen-binding portions thereof (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 1 and a VL having the amino acid sequence set forth in SEQ ID NO: 2, or a variant thereof, as described herein) can be placed on the same plasmid, which can then be transfected into cells, thereby directly selecting cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., a VL chain, and the resulting cell line can then be transfected with a VH chain plasmid containing a second selectable marker. Cell lines producing antibodies or antigen-binding portions thereof can be transfected, via either route, with plasmids encoding additional copies of the peptide, VH, VL, or VH plus VL chain (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 2, or a variant thereof, as described herein) along with additional selectable markers to generate cell lines with enhanced properties, such as higher production of the assembled MIC antibody or antigen-binding portion thereof, or improved stability of the transfected cell line.

[0123] Furthermore, plants have emerged as a convenient, safe, and economical alternative expression system for recombinant antibody production based on large-scale microbial or animal cell culture. MIC-binding antibodies or antigen-binding portions can be expressed in plant cell culture or in conventionally grown plants. Expression in plants can be systemic, restricted to intracellular plastids, or restricted to seeds (endosperm). See, for example, U.S. Patent Publication No. 2003 / 0167531, U.S. Patent No. 6,080,560, U.S. Patent No. 6,512,162, and WO0129242. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, for example, Biolex, NC).

[0124] In the case of intact antibodies, the variable regions (VH and VL) of an MIC antibody (e.g., a VH having the amino acid sequence set forth in SEQ ID NO: 1 and / or a VL having the amino acid sequence set forth in SEQ ID NO: 2, or variants thereof, as described herein) are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. Human constant region DNA sequences can be isolated from various human cells, such as immortalized B cells, according to well-known procedures (WO 87 / 02671, incorporated herein by reference in its entirety). MIC-binding antibodies can include both light and heavy chain constant regions. The heavy chain constant region can include CH1, hinge, CH2, CH3, and optionally CH4 regions. In some embodiments, the CH2 domain can be deleted or omitted.

[0125] Alternatively, techniques described for the production of single-chain antibodies (see, e.g., U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989), which are incorporated herein by reference in their entireties) can be adapted to produce single-chain antibodies that specifically bind to MIC. Single-chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region (having the amino acid sequences set forth in SEQ ID NOS: 1 and 2 herein, or variants thereof (e.g., optionally modified by substitution, deletion, and / or insertion of 1 to 8 amino acids)) via an amino acid bridge to obtain a single-chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (see, eg, Skerra et al., Science 242:1038-1041 (1988) ; incorporated herein by reference in its entirety).

[0126] Intact (e.g., complete) antibodies, their dimers, individual light and heavy chains, or antigen-binding portions thereof can be recovered and purified by known techniques, such as immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof. See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982). Substantially pure MIC-binding antibodies or antigen-binding portions thereof having at least about 90% to 95% homogeneity, as well as those having 98% to 99% or greater homogeneity, are advantageous, particularly for pharmaceutical uses. Once purified to partial or the desired homogeneity, the intact MIC antibodies or antigen-binding portions thereof can then be used therapeutically, or for developing and implementing assay procedures, immunofluorescence staining, and the like. See generally, Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981).

[0127] Furthermore, as described herein, for human therapy, MIC antibodies or antigen-binding portions thereof can be further optimized to reduce potential immunogenicity while maintaining functional activity. In some embodiments, the optimized MIC-binding antibody or antigen-binding portion thereof is derived from an MIC antibody comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the optimized MIC-binding antibody or antigen-binding portion thereof is derived from a MIC-binding antibody comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the heavy and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In this context, functional activity refers to a MIC-binding antibody or antigen-binding portion thereof that can exhibit one or more known functional activities associated with a MIC-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2. In any of these embodiments, the functional activity of the MIC-binding antibody or antigen-binding portion thereof includes specifically binding to MIC with a binding affinity greater than that of antibody B10G5. Additional functional activities include inhibition of MIC and / or anti-cancer activity.Furthermore, a MIC antibody or antigen-binding portion thereof having functional activity means that the polypeptide exhibits an activity, with or without dose dependency, similar to or better than the activity of a reference antibody or antigen-binding portion thereof described herein (e.g., a MIC-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2, or a variant thereof, as described herein). If a dose dependency exists, it need not be identical to the dose dependency of the reference antibody or antigen-binding portion thereof, but rather will exhibit a dose dependency for a given activity that is substantially similar to or better than the dose dependency for the reference antibody or antigen-binding portion thereof described herein (i.e., the candidate polypeptide will exhibit greater activity relative to the reference antibody).

[0128] Another aspect of the MIC antibodies and antigen-binding portions thereof or other binding agents relates to compositions comprising the active ingredient (i.e., the MIC antibodies or antigen-binding portions thereof or other binding agents described herein, or nucleic acids encoding the antibodies or antigen-binding portions thereof or other binding agents described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier approved for use in the pharmaceutical industry. As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0129] The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited in any way based on a particular formulation. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, although solid forms suitable for rehydration or suspension in liquid prior to use can also be prepared. Preparations can also be emulsified or provided as liposomal compositions. The MIC antibody or antigen-binding portion thereof or other binding agent can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Additionally, if desired, pharmaceutical compositions can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance or maintain the effectiveness of the active ingredient (e.g., the MIC antibody or antigen-binding portion thereof). The pharmaceutical compositions described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and the like), and organic bases (e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like). Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing an active ingredient (e.g., an MIC antibody and / or antigen-binding portion thereof) and water, as well as sterile aqueous solutions which may contain a buffer such as sodium phosphate at a physiological pH, physiological saline, or both (e.g., phosphate-buffered saline). Furthermore, aqueous carriers may contain two or more buffer salts, further salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol, and other solutes. Liquid compositions may also contain liquid phases in addition to and in addition to water.Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0130] In some embodiments, a pharmaceutical composition comprising a MIC antibody or antigen-binding portion thereof described herein, or a nucleic acid encoding a MIC antibody or antigen-binding portion thereof described herein, can be a lyophilizate.

[0131] In some embodiments, a syringe containing a therapeutically effective amount of a MIC antibody or antigen-binding portion thereof, or a pharmaceutical composition described herein is provided.

[0132] Cancer treatment In some aspects, the MIC antibody or antigen-binding portion thereof or other binding agent described herein can be used in a method or methods comprising administering the MIC antibody or antigen-binding portion thereof or other binding agent described herein to a subject in need thereof. In some embodiments, the MIC-binding antibody or antigen-binding portion thereof comprises (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the MIC-binding antibody or antigen-binding portion thereof comprises (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the MIC-binding antibody, or antigen-binding portion thereof, comprises (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the heavy and light chain variable framework regions are optionally modified by substitution, deletion, or insertion of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In any of these embodiments, the MIC-binding antibody specifically binds to MIC with a higher binding affinity than antibody B10G5.

[0133] In some embodiments, the subject is in need of treatment for cancer and / or malignancy. In some embodiments, the subject is in need of treatment for an MIC+ cancer or MIC+ malignancy, such as, for example, an epithelial cell cancer, an MIC+ solid tumor, or an MIC+ hematopoietic malignancy. In some embodiments, the method is for treating a subject with an MIC+ cancer or malignancy. In some embodiments, the method is for treating an epithelial cell cancer or hematopoietic malignancy in a subject. In some embodiments, the method is for treating an MIC+ epithelial cell cancer or an MIC+ hematopoietic malignancy in a subject. As used herein, "epithelial cell cancer" refers to a cancer that arises from epithelial cells.

[0134] The methods described herein include administering a therapeutically effective amount of a MIC-binding antibody or antigen-binding portion thereof or other binding agent. As used herein, the phrase "therapeutically effective amount," "effective amount," or "effective amount" refers to an amount of a MIC antibody or antigen-binding portion thereof or other binding agent described herein that provides a therapeutic benefit in the treatment, management, or prevention of recurrence of a tumor or malignancy, e.g., an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of a tumor or malignancy. Determining a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art. Generally, a therapeutically effective amount can vary depending on the subject's medical history, age, condition, and sex, as well as the severity and type of the subject's medical condition and the administration of other pharmaceutically active agents.

[0135] The terms "cancer" and "malignant tumor" refer to the uncontrolled growth of cells that interferes with the normal function of bodily organs and systems. Cancer or malignant tumors can be primary or metastatic, meaning they become invasive and seed tumor growth in tissues distant from the original tumor site. A "tumor" refers to the uncontrolled growth of cells that interferes with the normal function of bodily organs and systems. A subject with cancer is one who has objectively measurable cancer cells present in the subject's body. This definition includes benign tumors and malignant cancers, as well as potentially dormant tumors and micrometastases. Cancers that migrate from their original location and seed other vital organs can ultimately lead to the subject's death due to impaired function of the affected organ. Hematologic malignancies (blood cancers), such as leukemia and lymphoma, can, for example, overwhelm a subject's normal hematopoietic compartment, thereby causing hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia) and ultimately death.

[0136] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer; choriocarcinoma, colorectal cancer (colon cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (gastrointestinal cancer and stomach cancer). cancer), glioblastoma (GBM), hepatocellular carcinoma, liver cancer, intraepithelial neoplasia, renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoma including Hodgkin's lymphoma and non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, oral cavity, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer; and other carcinomas and sarcomas, and B-cell lymphoma (low-grade / follicular non-Hodgkin's) These include, but are not limited to, lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (cerebral tumor edema), and Meigs' syndrome.

[0137] In some embodiments, the carcinoma is selected from solid tumors, including but not limited to melanoma, prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, kidney cancer, and head and neck cancer.

[0138] In some embodiments, the cancer or malignant tumor is MIC-positive (MIC+). As used herein, the terms "MIC-positive" or "sMIC+" are used to describe cancer cells, cancer cell populations, tumor masses, or metastatic cells that express MIC on their cell surface (membrane-bound MIC) and / or produce sMIC protein, which is released from the cancer cell(s). These terms are intended to encompass all cancer cells and / or tumor masses that release all or part of the extracellular domain of the MIC protein into the intratumoral space or the circulatory or lymphatic system. Thus, these cells may only display MIC protein on their surface for a short period of time. That is, the term encompasses cancer cells and tumor cells that shed sMIC protein or secrete sMIC via exosomes or other mechanisms, regardless of whether detectable MIC protein is present on their cell surface. However, any cancer cell or tumor that can evade innate immune rejection by shedding MIC is considered a "MIC-positive cancer" as that term is used herein. Some non-limiting examples of MIC-positive cancers include epithelial cell cancers and hematopoietic malignancies. In some embodiments, the MIC-positive cancer or malignancy can be MIC-positive prostate cancer and / or metastases thereof.

[0139] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cats, canine species, e.g., dogs, foxes, wolves, birds, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "patient," "individual," and "subject" are used interchangeably herein.

[0140] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects, for example, to represent animal models of various cancers. Furthermore, the methods described herein can be used to treat livestock and / or pets. The subject may be male or female. In certain embodiments, the subject is human.

[0141] A subject may have previously been diagnosed with or identified as having an sMIC+ or MIC+ cancer and is in need of treatment, but need not have already received treatment for the sMIC+ or MIC+ cancer. Alternatively, a subject may also be a subject who has not previously been diagnosed with an sMIC+ or MIC+ cancer in need of treatment. A subject may exhibit one or more risk factors for a condition or one or more complications associated with an sMIC+ or MIC+ cancer, or a subject who does not exhibit risk factors. A "subject in need" of treatment for a particular sMIC+ cancer may be a subject who has the condition or has been diagnosed with the condition. In other embodiments, a subject "at risk of developing" a condition refers to a subject who has been diagnosed as being at risk for developing a condition (e.g., an sMIC+ or MIC+ cancer).

[0142] As used herein, the terms "treat," "treatment," "treating," or "amelioration," when used in reference to a disease, disorder, or medical condition, refer to therapeutic treatment for a condition whose purpose is to reverse, alleviate, improve, inhibit, slow, or halt the progression or severity of the symptom or condition. The term "treat" includes reducing or alleviating at least one adverse event or symptom of the condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the condition is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the halting or at least slowing or worsening of the progression of symptoms that would be expected in the absence of treatment. Beneficial or desirable clinical results include, but are not limited to, a reduction in a subject's free sMIC levels, alleviation of one or more symptoms, a reduction in the extent of the defect, stabilization (i.e., not worsening) of the cancer or malignant tumor condition, a delay or slowing of tumor growth and / or metastasis, and an increase in lifespan compared to the condition expected in the absence of treatment. As used herein, the term "administering" means providing a MIC-binding antibody or antigen-binding portion thereof or other binding agent described herein, or a nucleic acid encoding the MIC antibody or antigen-binding portion thereof or other binding agent described herein, to a subject by a method or route that results in binding of the MIC-binding antibody or antigen-binding portion thereof or other binding agent to the MIC. Similarly, a pharmaceutical composition comprising a MIC-binding antibody or antigen-binding portion thereof or other binding agent described herein, or a nucleic acid encoding a MIC antibody or antigen-binding portion thereof or other binding agent disclosed herein, can be administered by any suitable route that results in effective treatment in the subject.

[0143] The dosage range of the MIC-binding antibody or antigen-binding portion thereof depends on potency and includes an amount sufficient to produce the desired effect, e.g., lowering the MIC level, slowing tumor growth, or reducing tumor size. The dosage should not be so high as to cause unacceptable adverse side effects. Generally, the dosage will vary depending on the age, condition, and sex of the subject and can be determined by one of ordinary skill in the art. The dosage can also be adjusted by an individual physician if complications arise. In some embodiments, the dosage ranges from 0.01 mg / kg to 10 mg / kg body weight. In some embodiments, the dosage ranges from 0.05 mg / kg to 5 mg / kg body weight. In some embodiments, the dosage ranges from 0.01 mg / kg to 10 mg / kg body weight. In some embodiments, the dosage ranges from 0.05 mg / kg to 5 mg / kg body weight. Alternatively, the dosage can be scaled to maintain serum levels between 1 pg / mL and 1000 μg / mL. For systemic administration, a subject can receive a therapeutic amount such as, for example, 0.01 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg or more.

[0144] The administration of the above doses can be repeated. In practice, to the extent that removing free or unbound MICs can promote immune attack against tumors, cancer cells or malignant cells, for example, long-term administration is intended to first treat tumors, cancers or malignant tumors themselves, and then provide continuous monitoring for the development of tumor cells that acquire the ability to release MICs. In a preferred embodiment, the above doses are administered weekly, every two weeks, every three weeks, or monthly for several weeks or months. The duration of treatment depends on the clinical course of the subject and their responsiveness to treatment.

[0145] In some embodiments, the dose can be from about 0.01 mg / kg to about 100 mg / kg. In some embodiments, the dose can be from about 0.01 mg / kg to about 25 mg / kg. In some embodiments, the dose can be from about 0.01 mg / kg to about 20 mg / kg. In some embodiments, the dose can be from about 0.01 mg / kg to about 15 mg / kg. In some embodiments, the dose can be from about 0.01 mg / kg to about 100 mg / kg. In some embodiments, the dose can be from about 0.01 mg / kg to about 25 mg / kg. In some embodiments, the dose can be from about 0.01 mg / kg to about 20 mg / kg. In some embodiments, the dose can be from about 0.01 mg / kg to about 15 mg / kg. In some embodiments, the dose can be from about 0.1 mg / kg to about 10 mg / kg. In some embodiments, the dose can be from about 1 mg / kg to about 100 mg / kg. In some embodiments, the dose can be about 1 mg / kg to about 25 mg / kg. In some embodiments, the dose can be about 1 mg / kg to about 20 mg / kg. In some embodiments, the dose can be about 1 mg / kg to about 15 mg / kg. In some embodiments, the dose can be about 2 mg / kg. In some embodiments, the dose can be about 4 mg / kg. In some embodiments, the dose can be about 5 mg / kg. In some embodiments, the dose can be about 6 mg / kg. In some embodiments, the dose can be about 8 mg / kg. In some embodiments, the dose can be about 10 mg / kg. In some embodiments, the dose can be about 15 mg / kg. In some embodiments, the dose can be about 100 mg / kg. 2 ~about 700mg / m 2 In some embodiments, the dose may be about 250 mg / m 2 In some embodiments, the dose may be about 375 mg / m 2 In some embodiments, the dose may be about 400 mg / m 2 In some embodiments, the dose may be about 500 mg / m 2 It could be.

[0146] In some embodiments, the dose may be administered intravenously. In some embodiments, the intravenous administration may be an infusion administered over a period of about 10 minutes to about 4 hours. In some embodiments, the intravenous administration may be an infusion administered over a period of about 30 minutes to about 90 minutes.

[0147] In some embodiments, the dose may be administered weekly. In some embodiments, the dose may be administered every other week. In some embodiments, the dose may be administered about every two weeks. In some embodiments, the dose may be administered about every three weeks. In some embodiments, the dose may be administered every three weeks. In some embodiments, the dose may be administered every four weeks.

[0148] In some embodiments, a total of about 2 to about 10 doses are administered to the subject. In some embodiments, a total of 4 doses are administered. In some embodiments, a total of 5 doses are administered. In some embodiments, a total of 6 doses are administered. In some embodiments, a total of 7 doses are administered. In some embodiments, a total of 8 doses are administered. In some embodiments, a total of 9 doses are administered. In some embodiments, a total of 10 doses are administered. In some embodiments, more than a total of 10 doses are administered.

[0149] Pharmaceutical compositions containing MIC-binding antibodies or antigen-binding portions thereof or other MIC-binding agents can be administered in unit doses. When used with reference to pharmaceutical compositions, the term "unit dose" refers to a physically discrete unit suitable for unitary administration to a subject, each unit containing a predetermined amount of active agent (e.g., MIC-binding antibody or antigen-binding portion thereof) calculated to produce a desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0150] In some embodiments, the MIC-binding antibody or antigen-binding portion thereof, or a pharmaceutical composition of either or both thereof, is administered with immunotherapy. As used herein, "immunotherapy" refers to a therapeutic strategy designed to induce or enhance a subject's own immune system to fight cancer or malignant tumors. Examples of immunotherapies include, but are not limited to, adoptive cell therapy (e.g., autologous NK cells, allogeneic NK cells, autologous T cells, CAR-modified T cells, and CAR-modified NK cells), antibodies such as checkpoint inhibitors, antibodies that disrupt metabolic signals, immunocytokines such as the gamma chain family cytokines IL-2, IL15, and their variants, chemotoxins, radiation therapy, and epigenetic modifiers. See, e.g., Rohaan et al., Virchows Archiv 474:449-461 (2019); Magalhaes et al., Expert Opinion on Biological Therapy 19:8, 811-827 (2019); and Ott et al., 2019 ASCO Educational Book, Developmental Immunotherapy and Tumor Immunobiology, e70-78 (2020), the disclosures of which are incorporated herein by reference.

[0151] In some embodiments, adoptive cell therapy is a T cell therapy, such as CAR T cell therapy, in which T cells are removed from a subject's blood, genetically modified to express a chimeric antigen receptor for an appropriate target on cancer cells, and then readministered to the subject. See, e.g., WO2019 / 018603; WO2019 / 090003; WO2020 / 033927; WO2019089969; WO2015 / 164675; WO2016064929; WO2019 / 032929; WO2016 / 115559; WO2016 / 033570; WO2014 / 130657; WO2016028896; WO2015 / 090230; and WO2014 / 153270.

[0152] In some embodiments, adoptive cell therapy is a cell therapy in which PBMCs are removed from a subject's blood, primed to respond to a specific antigen, and then readministered to the subject (e.g., sipuleucel-T). (See, e.g., WO2001 / 039594; WO2001 / 074855; and WO1999 / 063050.)

[0153] In some embodiments, the adoptive cell therapy is NK cell therapy. NK cells can be engineered to express a chimeric antigen receptor for an appropriate target on cancer cells, and the engineered NK cells are then administered to a subject. (See, e.g., WO2006 / 103569; WO2018 / 165291; WO2016 / 201304; WO2017 / 100709; WO2019 / 028337; WO2016 / 176651 and WO2018 / 183385.)

[0154] In some embodiments, the immunotherapy comprises administration of a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BMA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, LILRB1, LILRB2, CD47, CD137, CD70, 2B4, CD160, TIGIT, CGEN-15049, CHK1, CHK2, SIGLEC-15, NKG2A, CD39, CD73, A2AR, and A2BR. In some embodiments, immune checkpoint inhibitors include agents that inhibit CTLA-4, PD-1, PD-L1, etc. Suitable anti-CTLA-4 therapeutic agents include, for example, anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, murine anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, ipilimumab, tremelimumab, anti-CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA-4 mAbs, heavy chain anti-CTLA-4 mAbs, light chain anti-CTLA-4 mAbs, inhibitors of CTLA-4 that stimulate the costimulatory pathway, antibodies disclosed in PCT Publication No. WO2001 / 014424, antibodies disclosed in PCT Publication No. WO2004 / 035607, antibodies disclosed in U.S. Publication No. 2005 / 0201994, and antibodies disclosed in European Patent No. EP1212422B1. Additional anti-CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720, PCT Publication Nos. WO01 / 14424 and WO00 / 37504, and U.S. Publication Nos. 2002 / 0039581 and 2002 / 086014.Other anti-CTLA-4 antibodies that can be used in the methods of the present invention include those disclosed in, for example, WO 98 / 42752, U.S. Patent Nos. 6,682,736 and 6,207,156, Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998), Camacho et al., J. Clin. Oncology, 22(145):Abstract No. 2505 (2004) (antibody CP-675206), Mokyr et al., Cancer Res, 58:5301-5304 (1998), U.S. Patent Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281.

[0155] Suitable anti-PD-1 and anti-PD-L1 therapeutics include, for example, anti-PD-1 and anti-PD-L1 antibodies, human anti-PD-1 and anti-PD-L1 antibodies, murine anti-PD-1 and anti-PD-L1 antibodies, mammalian anti-PD-1 and anti-PD-L1 antibodies, humanized anti-PD-1 and anti-PD-L1 antibodies, monoclonal anti-PD-1 and anti-PD-L1 antibodies, polyclonal anti-PD-1 and anti-PD-L1 antibodies, chimeric anti-PD-1 and anti-PD-L1 antibodies, anti-PD-1 Adnectins and anti-PD-L1 Adnectins, anti-PD-1 domain antibodies and anti-PD-L1 domain antibodies, single-chain anti-PD-1 and single-chain anti-PD-L1 mAbs, heavy-chain anti-PD-1 and heavy-chain anti-PD-L1 mAbs, and light-chain anti-PD-1 and light-chain anti-PD-L1 mAbs. In certain embodiments, anti-PD-1 therapies include nivolumab, pembrolizumab, pidilizumab, MEDI0680, and combinations thereof. In other certain embodiments, anti-PD-L1 therapies include atezolizumab, BMS-936559, MEDI4736, MSB0010718C, and combinations thereof.

[0156] Suitable anti-PD-1 and anti-PD-L1 antibodies are also described in Topalian, et al., Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy, Cancer Cell 27:450-61 (April 13, 2015), which is incorporated by reference in its entirety.

[0157] In some embodiments, the checkpoint inhibitor is ipilimumab (Yervoy), nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi).

[0158] In some embodiments, methods are provided for improving therapeutic outcomes in subjects receiving immunotherapy. The methods generally include administering an effective amount of immunotherapy to a subject with cancer and administering to the subject a therapeutically effective amount of a binding agent or pharmaceutical composition thereof, wherein the binding agent specifically binds to circulating MIC, thereby improving the therapeutic outcome in the subject compared to administering immunotherapy alone. In some embodiments, the binding agent comprises (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2, wherein the framework regions of the heavy and light chains are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids in the framework regions, and the binding agent specifically binds to MIC with a higher binding affinity than antibody B10G5. In some embodiments, the binding agent comprises (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2, wherein the binding agent specifically binds to MIC. In some embodiments, the binding agent is an antibody or an antigen-binding portion thereof. In some embodiments, the binding agent is a monoclonal antibody, a Fab, a Fab', a F(ab'), an Fv, a disulfide-linked Fc, an scFv, a single domain antibody, a diabody, a bispecific antibody, or a multispecific antibody.

[0159] In some embodiments, the improved outcome is an objective response selected from stable disease, partial response, or complete response as determined by standard medical criteria for the cancer being treated. In some embodiments, the improved outcome is a reduction in tumor burden. In some embodiments, the improved outcome is progression-free survival or disease-free survival.

[0160] In some embodiments, the method comprises administering a chemotherapy or other treatment that impairs the immune system by depleting endogenous NK cells or T cells or their precursors, followed by the administration of a MIC antibody or antigen-binding portion thereof at least 4 weeks, at least 6 weeks, or at least 8 weeks later. In some such embodiments, the chemotherapy or treatment is selected from the group consisting of radiation therapy or chemotherapy. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as thiotepa and CYTOXAN™ cyclophosphamide, alkyl sulfonates, such as busulfan, improsulfan, and piposulfan, aziridines, such as benzodopa, carboquone, meturedopa, and uredopa, ethyleneimines and methylameramines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine, acetogenins (e.g., bullatacin and bullatacinone), camptothecins (such as the synthetic analog topotecan), bryostatin, kallistatin, CC-1065 (such as its adozelesin, carzelesin, and bezelesin synthetic analogs), cryptophycins (e.g., cryptophycin 1 and cryptophycin 8), dolastatin, and the like. cin, duocarmycin (such as synthetic analogs KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, and uracil mustard, nitrosoureas such as camrustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine, antibiotics such as enediyne antibiotics (e.g., calicheamicin, specifically calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Agnew, Chem. Intl. Ed. Engl.,33:183-186(1994)); dynemicins (including dynemicin A); bisphosphonates (such as clodronate); esperamicin, as well as neocarzinostatin chromophores and related chromoprotein (enediyne antibiotic chromophores), aclacinomycins, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM.Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, querramycin, rodorubicin, streptonigrin, streptozocin antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (such as denopterin, methotrexate, pteropterin, trimetrexate); purine analogs (such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine); pyrimidine analogs (such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxycycline, Fluridine, enocitabine, floxuridine, etc.); androgens (calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, etc.); antiadrenal agents (aminoglutethimide, mitotane, trilostane, etc.); folic acid supplements (floric acid, etc.); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; Demecolcine; diaziquone; elfomitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids (such as maytansine and ansamitocin); mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharide complex (JHS Natural Products, Eugene, Oreg.).); razoxane, rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids (e.g., TAXOL™ paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ Cremophor albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ docetaxel (Rhone-Poulenc Rorer, Antony, France), chlorambucil, GEMZAR™ gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin, oxaliplatin, and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, NAVELBINE™ vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, Xeloda, ibandronate, irinotecan (Camptosar, CPT-11) (5-FU and leucovorin treatment regimens), the topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, capecitabine, combretastatin, leucovorin (LV), oxaliplatin such as oxaliplatin treatment regimens (FOLFOX), lapatinib (Tykerb™), PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva™)), and VEGF-A inhibitors that reduce cell proliferation, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0161] "Radiation therapy" refers to the use of directed gamma or beta rays to induce sufficient damage to cells to limit their ability to function normally or to destroy the cells entirely.

[0162] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments of, and examples for, the present disclosure are described herein for illustrative purposes; however, those skilled in the relevant art will recognize that various equivalent modifications are possible within the scope of the present disclosure. The teachings of the disclosure provided herein can be applied to other procedures or methods, as appropriate. Various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to adopt the structure, function, and concepts of the above references and applications to provide further embodiments of the present disclosure. These and other changes can be made to the present disclosure in light of the detailed description.

[0163] Certain elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure are described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages, to fall within the scope of the present disclosure.

[0164] All patents and other identified publications are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the procedures described in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or depiction of the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the date or accuracy of the contents of these documents.

[0165] Non-limiting exemplary embodiments This invention is further illustrated by the following embodiments, which should not be construed as limiting. 1. A binding agent comprising (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 1 and (ii) a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the framework regions of the heavy and light chains are optionally modified by substitution, deletion or insertion of 1 to 8 amino acids in the framework regions.

[0166] 2. A binding agent comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1, and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2.

[0167] 3. A binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions HCDR1 having the amino acid sequence set forth in SEQ ID NO: 11, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 13, and the VL region comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 16, and the VH region and the VL region each comprise a humanized framework region.

[0168] 4. The binding agent of embodiment 3, wherein the humanized VH framework regions are derived from human germline genes having amino acid sequences set forth in IMGT IGHV4-59*11 (SEQ ID NO: 29) and IGHJ4*01 (SEQ ID NO: 30) or IGHV4-30-4*01 (SEQ ID NO: 31) and IGHJ4*01 (SEQ ID NO: 30).

[0169] 5. The binding agent of embodiment 3 or embodiment 4, wherein the humanized VL framework regions are derived from human germline genes having the amino acid sequences represented by IMGT IGKV1-NL1*01 (SEQ ID NO: 32) and IMGT IGKJ1*01 (SEQ ID NO: 33), IMGT IGKV1-33*01 (SEQ ID NO: 34) and IMGT IGKJ1*01 (SEQ ID NO: 33), or IMGT IGKV1-5*01 (SEQ ID NO: 35) and IMGT IGKJ1*01 (SEQ ID NO: 33).

[0170] 6. The binding agent of any one of embodiments 1 to 5, wherein the binding agent specifically binds to an MIC.

[0171] 7. The binding agent of any one of embodiments 1 to 6, wherein the binding agent is an antibody or an antigen-binding portion thereof.

[0172] 8. The binding agent of embodiment 7, wherein said binding agent is a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody.

[0173] 9. The binding agent of any one of the preceding embodiments, wherein the heavy chain variable region further comprises a heavy chain constant region.

[0174] 10. The binding agent of embodiment 9, wherein said heavy chain constant region is of the IgG isotype.

[0175] 11. The binding agent of embodiment 10, wherein said heavy chain constant region is an IgG1 constant region.

[0176] 12. The binding agent of embodiment 10, wherein said heavy chain constant region is an IgG4 constant region.

[0177] 13. The binding agent of embodiment 11, wherein the heavy chain variable region and the heavy chain constant region have the amino acid sequence set forth in SEQ ID NO:3.

[0178] 14. The binding agent of any one of the preceding embodiments, wherein the light chain variable region further comprises a light chain constant region.

[0179] 15. The binding agent of embodiment 14, wherein said light chain constant region is of the kappa isotype.

[0180] 16. The binding agent of embodiment 15, wherein the light chain variable region and the light chain constant region have the amino acid sequence set forth in SEQ ID NO:4.

[0181] 17. The binding agent of any one of embodiments 9 to 16, wherein said heavy chain constant region further comprises amino acid modifications that increase binding affinity to at least human Fc gamma RIII.

[0182] 18. The binding agent of any one of embodiments 9 to 17, wherein the heavy chain constant region further comprises at least one amino acid modification that reduces binding to one or more Fc gamma receptors.

[0183] 19. The binding agent of any one of embodiments 9 to 17, wherein the heavy chain constant region further comprises at least one amino acid modification that increases CDC activity.

[0184] 20. The binding agent of any one of embodiments, wherein the binding agent is monospecific.

[0185] 21. The binding agent of any one of embodiments 1 to 20, wherein the binding agent is bivalent.

[0186] 22. The binding agent of embodiment 21, wherein the binding agent comprises a second binding domain and the binding agent is bispecific.

[0187] 23. The binding agent of any one of embodiments 1 to 22, wherein the binding agent specifically binds to soluble MIC (sMIC).

[0188] 24. The binding agent of any one of embodiments 1 to 23, wherein the binding agent specifically binds to MIC with a higher binding affinity than antibody B10G5.

[0189] 25. A pharmaceutical composition comprising the binding agent of any of the preceding embodiments and a pharmaceutically acceptable carrier.

[0190] 26. A nucleic acid encoding a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, optionally having the nucleic acid sequence set forth in SEQ ID NO: 21.

[0191] 27. A nucleic acid encoding a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2, optionally having the nucleic acid sequence set forth in SEQ ID NO:22.

[0192] 28. A nucleic acid encoding a binding agent according to any one of embodiments 1 to 24, optionally having the nucleic acid sequences set out in SEQ ID NO: 21 and SEQ ID NO: 22.

[0193] 29. A vector comprising a nucleic acid according to any one of embodiments 26 to 28.

[0194] 30. A cell line comprising a nucleic acid according to any one of embodiments 26 to 28, or a vector according to embodiment 29.

[0195] 31. A method for treating MIC+ cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a binding agent according to any one of embodiments 1 to 24, or a pharmaceutical composition according to embodiment 25.

[0196] 32. The method of embodiment 31, wherein the cancer is a carcinoma, a sarcoma, a neuroendocrine tumor, or a malignant hematological disease.

[0197] 33. The method of embodiment 33, wherein the cancer is a carcinoma.

[0198] 34. The method of embodiment 33, wherein the carcinoma is selected from prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, and head and neck cancer.

[0199] 35. The method of embodiment 32, wherein the cancer is a malignant hematological disease.

[0200] 36. The method of embodiment 33, wherein the malignant hematological disease is lymphoma or multiple myeloma.

[0201] 37. The method of any one of embodiments 31-36, further comprising administering immunotherapy to the subject.

[0202] 38. The method of embodiment 37, wherein the immunotherapy comprises adoptive cellular therapy or a checkpoint inhibitor.

[0203] 39. The method of embodiment 38, wherein said immunotherapy comprises adoptive cell therapy.

[0204] 40. The method of embodiment 39, wherein said adoptive cell therapy is selected from autologous NK cells, allogeneic NK cells, autologous T cells, CAR-modified T cells, and CAR-modified NK cells.

[0205] 41. The method of embodiment 38, wherein the immunotherapy comprises a checkpoint inhibitor.

[0206] 42. The method of embodiment 41, wherein the checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4.

[0207] 43. The method of embodiment 42, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

[0208] 44. The method of any one of embodiments 31-43, wherein said subject has not been administered chemotherapy for at least 4 weeks prior to said administration of said binding agent.

[0209] 45. The method of any one of embodiments 31-44, wherein the binding agent is administered intravenously.

[0210] 46. ​​The method of any one of embodiments 31-45, wherein the binding agent is administered at a dose of about 0.1 mg / kg to about 100 mg / kg.

[0211] 47. A method for reducing the level of circulating sMIC in a subject with cancer, comprising administering a therapeutically effective amount of a binding agent described in any one of embodiments 1 to 24, or a pharmaceutical composition described in embodiment 25.

[0212] 48. The method of embodiment 47, wherein the cancer is a carcinoma, a sarcoma, a neuroendocrine tumor, or a malignant hematological disease.

[0213] 49. The method of embodiment 48, wherein the cancer is a carcinoma.

[0214] 50. The method of embodiment 49, wherein the carcinoma is selected from prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, and head and neck cancer.

[0215] 51. The method of embodiment 48, wherein the cancer is a malignant hematological disease.

[0216] 52. The method of embodiment 51, wherein the malignant hematological disease is lymphoma or multiple myeloma.

[0217] 53. A method for improving the therapeutic outcome of a subject undergoing immunotherapy, comprising: a. administering to said subject having cancer an effective amount of immunotherapy; b. administering to the subject a therapeutically effective amount of the binding agent of any one of embodiments 1-24 or the pharmaceutical composition of embodiment 25, wherein the binding agent specifically binds to an MIC; The method, wherein the subject's outcome is improved compared to administration of the immunotherapy alone.

[0218] 54. The method of embodiment 53, wherein the improved outcome is an objective response selected from stable disease, partial response, or complete response.

[0219] 55. The method of embodiment 53, wherein the improved outcome is a reduction in tumor burden.

[0220] 56. The method of embodiment 53, wherein the improved outcome is progression-free survival or disease-free survival.

[0221] 57. The method of embodiment 53, wherein the immunotherapy is an adoptive cell therapy or a checkpoint inhibitor.

[0222] 58. The method of embodiment 57, wherein said immunotherapy is adoptive cell therapy.

[0223] 59. The method of embodiment 58, wherein the adoptive cell therapy comprises autologous NK cells, allogeneic NK cells, autologous T cells, CAR-modified T cells, and CAR-modified NK cells.

[0224] 60. The method of embodiment 57, wherein the immunotherapy is a checkpoint inhibitor.

[0225] 61. The method of embodiment 60, wherein the checkpoint inhibitor comprises an antibody that specifically binds to human PD-1, human PD-L1, or CTLA4.

[0226] 62. The method of embodiment 61, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

[0227] 63. The method of any one of embodiments 53-62, wherein said subject has not been administered chemotherapy for at least 4 weeks prior to said administration of said binding agent.

[0228] 64. The method of any one of embodiments 53-63, wherein the binding agent is administered intravenously.

[0229] 65. The method of any one of embodiments 53-64, wherein the binding agent is administered at a dose of about 0.1 mg / kg to about 10 mg / kg.

[0230] 66. Use of the binding agent according to any one of embodiments 1 to 24 or the pharmaceutical composition according to embodiment 25 for the treatment of MIC+ cancer in a subject.

[0231] 67. Use of a binding agent according to any one of embodiments 1 to 24 or a pharmaceutical composition according to embodiment 25 for the treatment of MIC+ cancer in a subject undergoing immunotherapy.

[0232] 68. The binding agent of any one of embodiments 1 and 3 to 24, wherein the VH is selected from heavy chain variable regions having amino acid sequences represented by SEQ ID NOs: 1, 23, and 24, and the VL is selected from light chain variable regions having amino acid sequences represented by SEQ ID NOs: 2, 25, and 26.

[0233] 69. The binding agent of embodiment 68, wherein the VH has the amino acid sequence represented by SEQ ID NO: 24, and the VL has the amino acid sequence represented by SEQ ID NO: 2. [Example]

[0234] Example 1: Generation of humanized antibodies The following example describes the preparation of a humanized version of antibody B10G5. Three humanized light chains and three humanized heavy chains were designed based on two different heavy and light chain human acceptor frameworks. The first humanized chain of each parent chain utilizes the first framework and contains most human sequences, including minimal parent antibody framework sequences (designated humanized HC1, LC1). The second humanized chain of each parent chain uses the same framework as the previous one but contains additional parent sequences (designated humanized HC2, LC2). The third humanized chain of each utilizes the respective second framework and, like HC2 / LC2, also contains additional parent sequences fused to the human framework (designated humanized HC3, LC3).

[0235] Next, we were ready to combine the humanized light and heavy chains to create variant fully humanized antibodies. As described in the Examples below, all possible combinations of humanized light and heavy chains were tested for their expression levels and binding affinities.

[0236] Full-length antibody genes were constructed by first synthesizing the variable region sequences. The sequences were optimized for expression in mammalian cells. These variable region sequences were then cloned into an expression vector that already contained a human Fc domain. For the heavy chain, an IgG1 constant region was utilized as required. Additionally, for comparison, the heavy and light chains of the parent antibody were constructed as full-length chimeric chains using the same main chain Fc sequence. The antibodies were identified as shown in Table 1 below. [Table 1]

[0237] Example 2: Evaluation of humanized antibodies in small-scale production All nine humanized antibodies underwent small-scale production at 0.01 liters. The B10G5 antibody was also scaled up for direct comparison. HEK293 cells, cultured in suspension in a chemically defined medium in the absence of serum, were transfected with the indicated heavy and light chain plasmids. Five days after transfection, conditioned medium from each production run was collected and clarified. Antibodies in the conditioned medium were purified using MabSelectSuRe™ Protein A medium (GE Healthcare). The final yields are shown below (Table 2). [Table 2]

[0238] The antibodies were subjected to CE-SDS analysis under reducing conditions using a LabChip GXII (Perkin Elmer), and the resulting electropherograms were analyzed. Separate peaks for the heavy and light chains were observed for the purified humanized antibodies. The peak heights (or areas under the curve for the heavy and light chains) were similar. Antibodies C (HC2 / LC2) and K (HC2 / LC3) produced the highest antibody yields.

[0239] Example 3: Binding affinity testing of humanized antibodies The purified antibodies from Example 2 were analyzed for binding affinity by FACS against the human prostate tumor cell line M12. Three different antibody concentrations were tested (0.1 μg / mL, 0.05 μg / mL, and 0.01 μg / mL). To compare binding affinity, the MFI of each antibody at each concentration was determined, and the results were plotted as shown in Figure 1. At the 0.1 μg / mL concentration, from top to bottom, the antibodies are Ab-G, Ab-K, Ab-E, Ab-J, Ab-I, and Ab-H (overlap), Ab-D, Ab-B, Ab-A, and Ab-C. Surprisingly, two antibodies, Ab-G and Ab-K, had higher MFI than antibody J (chimeric B10G5 with a human IgG1 Fc region).

[0240] Example 4: BLI analysis of antibody K by BLI Biolayer interferometry (BLI) was performed using Octet Red 96 (ForteBio) as generally described in Kamat and Rafique, Analytical Biochemistry 536:16-31 (2017). Antibody samples, Ab-K and Ab-J (chimeric B10G5), were captured on a kinetic-grade biosensor. The loaded biosensor was then immersed in a dilution series of the sample containing the antigen (MICB) serially diluted in PBS buffer, 0.1% BSA, 0.02% Tween-20, pH 7.2. Association was observed for 150 seconds, followed by dissociation for 200 seconds. Repeat injections of the same antigen concentration showed good overlap. Kinetic analysis was performed using Scrubber software, using a 1:1 binding model and global fitting with mass transport constraints. Referring to Figures 2A and 2B, Ab-K (Figure 2B) had a higher affinity (Kd = 7.2 nM) than the chimeric antibody B10G5 (Ab-J, Kd = 12.1 nM) (Figure 2A).

[0241] Example 5: NK cell cytotoxicity assay The ability of antibody K to stimulate NK cell cytotoxicity was measured. NK cells were negatively selected from healthy donor PBMCs using an isolation kit from Stem Cell Technologies (Vancouver, BC, Canada). UC1 tumor cells or PL-12 cells were preincubated with 10 μg / ml of control human IgG, antibody Ab-J (chimeric B10G5 with a human IgG1 Fc region), or antibody K (Ab-K) for 30 minutes at 37°C. Purified NK cells were activated with IL-2 (1000 U / mL) for 18 hours before use in cytotoxicity assays against UC1 tumor cells or PL-12 cells. NK cell-mediated cytotoxicity was measured using a standard 4-hour incubation period. 51 The Cr release assay (Jewett A. et al., Hum. Immunol., 2003; 64: 505-520) was used to determine the NK cell activity. 51The cells were co-cultured with Cr-labeled UC1 or PL12 cells at a ratio of 10:1 in a cell culture incubator for 4 hours. After 4 hours of incubation, supernatants were collected from each sample and counted in a gamma counter. The percentage of specific cytotoxicity was calculated using the formula: % cytotoxicity = (experimental cpm - spontaneous cpm) / (total cpm - spontaneous cpm). NK cell killing activity was calculated as 30 / 10 lytic units, calculated by multiplying the reciprocal of the number of NK cells required to lyse 30% of the target cells by 100. 6 Expressed as cells. Five replicates of each condition were included in the experiment.

[0242] Referring to Figures 3A and 3B, antibody K (Ab-K) had an MIC + It exhibits greater activity than the chimeric antibody B10G5 in enhancing IL-2-activated primary NK cell killing of thyroid oncocytoma UC1 tumor cells (Figure 3A) and pancreatic PL12 cells (also called Panc 10.05, Figure 3B).

[0243] Example 6: Aggregation Assay Samples of murine antibody B10G5, chimeric Ab-K antibody (ch-Ab-K, humanized variable region and mouse Fc), and antibody K (Ab-K, humanized) were assayed using dynamic light scattering (DLS) with a quasi-elastic light scattering instrument. B10G5, ch-Ab-K, or Ab-K were each diluted to a concentration of 1.0 mg / mL in PBS buffer in a volume of 50 μL. All samples were filtered through a spin filter (SpinX® Cat. #8160) to remove unwanted large particles and degassed prior to assay. 5 μL of sample was used to load each disposable cuvette for DLS assay using an Unchained Lab nanoDLS pUNK machine. Details of the assay are described, for example, in Berne et al., Dynamic Light Scattering with Applications to Chemistry, Biology and Physics, Courier Dover Publications, ISBN 0-486-41155-9 (2000). In this assay, DLS measures the fluctuations in scattered light intensity by diffusing particles. Particles aggregate over time, which is seen as an increase in hydrodynamic diameter (x-axis). Peak areas represent the levels of each different antibody species (e.g., antibody monomer or aggregates) as a percentage of the total amount of antibody.

[0244] Figures 4A-4C show the antibody monomer and aggregate levels for murine antibody B10G5 (murine IgG1, Figure 4A), chimeric Ab-K antibody (ch-Ab-K, composed of the Ab-K humanized variable region with murine IgG1-Fc) (Figure 4B), and Ab-K (humanized) (Figure 4C), respectively. Both chimeric antibody ch-Ab-K and fully humanized antibody Ab-K are more stable in solution than murine antibody B10G5, as indicated by the high monomer levels of antibody chimeric Ab-K (Figure 4B, 99.5%) and fully humanized antibody Ab-K (Figure 4C, 99.95%).

[0245] Example 7: Clinical Trial of Immunotherapy Phase I clinical trials will be conducted in cancer patients with MIC+ tumor samples or serum sMIC+. Safety, maximum tolerated dose (MTD), and primary efficacy of the MIC antibody will be determined using an adaptive dose-escalation 3+3 design or a time-to-event Bayesian optimal interval design. The patient population includes chemotherapy-naive patients who have failed standard treatment. The dose range of the MIC antibody tested will be 0.01 mg / kg to 100 mg / kg, administered intravenously every 2–4 weeks for up to 90 days. Patients may be followed for up to one year to determine the MTD or recommended phase 2 dose (RP2D). Patients who do not experience significant adverse events will undergo extended infusions with up to two years of clinical follow-up to determine primary efficacy.

[0246] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0247] Various publications, including patents, patent application publications, and scientific literature, are cited herein, the disclosures of which are incorporated by reference in their entireties for all purposes. Sequence Listing SEQ ID NO: 1 - VH amino acid sequence of HC2 EVQLQESGPG LVKPSQTLSL TCTVSGYSIT SDYAWNWIRQ PPGKGLEWIG YISYSSGSTNY NPSLKSRVTI SRDTSKNQFS LKLSSVTAAD TAVYYCARGG TYFDYWGQGT LVTVSS SEQ ID NO:2 - VL amino acid sequence of LC3 DVVMTQSPST LSASVGDRVT ITCRASAHIN NWLAWYQQKP GKAPKLLISD ATSLESGVPS RFSGSGSGKE YTLTISSLQP DDFATYYCQH YWSTPWTFGQ GTKVEIK SEQ ID NO: 3 - VH-IgG1 amino acid sequence EVQLQESGPG LVKPSQTLSL TCTVSGYSIT SDYAWNWIRQ PPGKGLEWIG YISYSSGSTNY NPSLKSRVTI SRDTSKNQFS LKLSSVTAAD TAVYYCARGG TYFDYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG SEQ ID NO: 4 - VL-Ig kappa amino acid sequence DVVMTQSPST LSASVGDRVT ITCRASAHIN NWLAWYQQKP GKAPKLLISD ATSLESGVPS RFSGSGSGKE YTLTISSLQP DDFATYYCQH YWSTPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO: 5 signal sequence MDPKGSLSWR ILLFLSLAFE LSYG SEQ ID NO: 6 signal sequence METDTLLLWV LLLWVPGSTG SEQ ID NO: 7 VH-IgG1 amino acid sequence with signal sequence MDPKGSLSWR ILLFLSLAFE LSYGEVQLQE SGPGLVKPSQ TLSLTCTVSG YSITSDYAWN WIRQPPGKGL EWIGYISYSG STNYNPSLKS RVTISRDTSK NQFSLKLSSV TAADTAVYYC ARGGTYFDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG SEQ ID NO: 8 VL-Ig kappa amino acid sequence with signal sequence METDTLLLWV LLLWVPGSTG DVVMTQSPST LSASVGDRVT ITCRASAHIN NWLAWYQQKP GKAPKLLISD ATSLESGVPS RFSGSGSGKE YTLTISSLQP DDFATYYCQH YWSTPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO: 9 Human MICA, isoform 1 MGLGPVFLLL AGIFPFAPPG AAAEPHSLRY NLTVLSWDGS VQSGFLTEVH LDGQPFLRCD RQKCRAKPQG QWAEDVLGNK TWDRETRDLT GNGKDLRMTL AHIKDQKEGL HSLQEIRVCE IHEDNSTRSS QHFYYDGELF LSQNLETKEW TMPQSSRAQT LAMNVRNFLK EDAMKTKTHY HAMHADCLQE LRRYLKSGVV LRRTVPPMVN VTRSEASEGN ITVTCRASGF YPWNITLSWR QDGVSLSHDT QQWGDVLPDG NGTYQTWVAT RICQGEEQRF TCYMEHSGNH STHPVPSGKV LVLQSHWQTF HVSAVAAAI FVIIIFYVRC CKKKTSAAEG PELVSLQVLD QHPVGTSDHR DATQLGFQPL MSDLGSTGST EGA SEQ ID NO: 10 Human MICB, isoform 1 MGLGRVLLFL AVAFPFAPPA AAAEPHSLRY NLMVLSQDGS VQSGFLAEGH LDGQPFLRYD RQKRRAKPQG QWAENVLGAK TWDTETEDLT ENGQDLRRTL THIKDQKGGL HSLQEIRVCE IHEDSSTRGS RHFYYDGELF LSQNLETQES TVPQSSRAQT LAMNVTNFWK EDAMKTKTHY RAMQADCLQK LQRYLKSGVA IRRTVPPMVN VTCSEVSEGN ITVTCRASSF YPRNITLTWR QDGVSLSHNT QQWGDVLPDG NGTYQTWVAT RIRQGEEQRF TCYMEHSGNH GTHPVPSGKA LVLQSQRTDF PYVSAAMPCF VIIIILCVPC CKKKTSAAEG PELVSLQVLD QHPVGTGDHR DAAQLGFQPL MSATGSTGST EGT SEQ ID NO: 11 VH CDR1 GYSITSDYA SEQ ID NO: 12 VH CDR2 GYISYSGST SEQ ID NO: 13 VH CDR3 ARGGTYFDY SEQ ID NO: 14 VL CDR1 RASAHINNW SEQ ID NO: 15 VL CDR2 DATSLES SEQ ID NO: 16 VL CDR3 QHYWSTPWT SEQ ID NO: 17 (Gly Gly Gly Gly Ser)n, where n = 1 to 5 SEQ ID NO: 18 His His His His His SEQ ID NO: 19 Coding sequence of HC2 ATG GAC CCC AAG GGC AGC CTG AGC TGG AGA ATC CTG CTG TTC CTG AGC CTG GCC TTC GAG CTG AGC TAC GGC GAA GTG CAG CTG CAG GAA TCT GGC CCT GGC CTC GTG AAG CCT TCC CAG ACC CTG TCT CTG ACC TGC ACC GTG TCC GGC TAC TCC ATC ACC TCC GAC TAC GCC TGG AAC TGG ATC CGG CAG CCT CCT GGC AAG GGA CTG GAA TGG ATC GGC TAC ATC TCC TAC TCC GGC TCC ACC AAC TAC AAC CCC AGC CTG AAG TCC AGA GTG ACC ATC TCC CGG GAC ACC TCC AAG AAC CAG TTC TCC CTG AAG CTG TCC TCC GTG ACC GCC GCT GAT ACC GCC GTG TAC TAC TGT GCT AGA GGC GGC ACC TAC TTC GAC TAC TGG GGC CAG GGC ACC CTC GTG ACC GTG TCA TCT GCT AGC ACC AAG GGC CCC AGC GTG TTC CCT CTG GCC CCC AGC AGC AAG AGC ACC AGC GGC GGA ACC GCC GCC CTG GGC TGC CTG GTG AAG GAC TAC TTC CCC GAG CCC GTG ACC GTG TCC TGG AAC AGC GGC GCT CTG ACC AGC GGA GTG CAC ACC TTC CCT GCC GTG CTG CAG AGC AGC GGC CTG TAC TCC CTG AGC AGC GTG GTG ACC GTG CCC AGC AGC AGC CTG GGC ACC CAG ACC TAC ATC TGC AAC GTG AAC CAC AAG CCC TCC AAC ACC AAG GTG GAC AAG AAG GTG GAG CCT AAG AGC TGC GAC AAG ACC CAC ACC TGC CCTCCC TGC CCC GCC CCC GAG CTG CTG GGC GGA CCC AGC GTG TTC CTG TTC CCT CCC AAG CCC AAG GAC ACC CTG ATG ATC AGC CGC ACC CCC GAG GTG ACC TGC GTG GTG GTG GAC GTG AGC CAC GAG GAC CCC GAG GTG AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAC AAC GCC AAG ACC AAG CCT CGG GAG GAG CAG TAC AAC TCC ACC TAC CGC GTG GTG AGC GTG CTG ACC GTG CTG CAC CAG GAC TGG CTG AAC GGC AAG GAG TAC AAG TGC AAG GTG AGC AAC AAG GCC CTG CCC GCT CCC ATC GAG AAG ACC ATC AGC AAG GCC AAG GGC CAG CCC CGG GAG CCT CAG GTG TAC ACC CTG CCC CCC AGC CGC GAC GAG CTG ACC AAG AAC CAG GTG AGC CTG ACC TGC CTG GTG AAG GGC TTC TAC CCC TCC GAC ATC GCC GTG GAG TGG GAG AGC AAC GGC CAG CCT GAG AAC AAC TAC AAG ACC ACC CCT CCC GTG CTG GAC AGC GAC GGC AGC TTC TTC CTG TAC AGC AAG CTG ACC GTG GAC AAG TCC CGG TGG CAG CAG GGC AAC GTG TTC AGC TGC AGC GTG ATG CAC GAG GCC CTG CAC AAC CAC TAC ACC CAG AAG AGC CTG AGC CTG AGC CCC GGA TAG TAA sequence no. 20 LC3 code sequence ATG GAG ACC GAC ACC CTG CTG CTC TGG GTG CTG CTC TGG GTG CCC GGC TCC ACC GGA GAC GTC GTG ATG ACC CAG TCC CCC TCC ACA CTG TCT GCC TCT GTG GGC GAC AGA GTG ACC ATC ACC TGT CGG GCC TCC GCC CAC ATC AAC AAC TGG CTG GCC TGG TAT CAG CAG AAG CCC GGC AAG GCC CCT AAG CTG CTG ATC TCT GAT GCC ACC TCC CTG GAA TCC GGC GTG CCC TCC AGA TTC TCC GGC TCT GGC TCT GGC AAG GAG TAT ACC CTG ACC ATC AGC TCC CTG CAG CCC GAT GAC TTC GCC ACC TAC TAC TGC CAG CAC TAC TGG TCC ACC CCC TGG ACC TTT GGC CAA GGC ACC AAG GTG GAA ATC AAG CGG ACC GTG GCC GCC CCC AGC GTG TTC ATC TTC CCT CCC AGC GAC GAG CAG CTG AAG TCT GGC ACC GCC AGC GTG GTG TGC CTG CTG AAC AAC TTC TAC CCC CGC GAG GCC AAG GTG CAG TGG AAG GTG GAC AAC GCC CTG CAG AGC GGC AAC AGC CAG GAG AGC GTG ACC GAG CAG GAC TCC AAG GAC AGC ACC TAC AGC CTG AGC AGC ACC CTG ACC CTG AGC AAG GCC GAC TAC GAG AAG CAC AAG GTG TAC GCC TGC GAG GTG ACC CAC CAG GGA CTG TCT AGC CCC GTG ACC AAG AGC TTC AAC CGG GGC GAG TGC TAA அக்க்கு நுர்க்கு21 VHக்கு மாட்ட்டி GAA GTG CAG CTG CAG GAA TCT GGC CCT GGC CTC GTG AAG CCT TCC CAG ACC CTG TCT CTG ACC TGC ACC GTG TCC GGC TAC TCC ATC ACC TCC GAC TAC GCC TGG AAC TGG ATC CGG CAG CCT CCT GGC AAG GGA CTG GAA TGG ATC GGC TAC ATC TCC TAC TCC GGC TCC ACC AAC TAC AAC CCC AGC CTG AAG TCC AGA GTG ACC ATC TCC CGG GAC ACC TCC AAG AAC CAG TTC TCC CTG AAG CTG TCC TCC GTG ACC GCC GCT GAT ACC GCC GTG TAC TAC TGT GCT AGA GGC GGC ACC TAC TTC GAC TAC TGG GGC CAG GGC ACC CTC GTG ACC GTG TCA TCT அக்க்கு நுர்க்கு22 VLCODE region GAC GTC GTG ATG ACC CAG TCC CCC TCC ACA CTG TCT GCC TCT GTG GGC GAC AGA GTG ACC ATC ACC TGT CGG GCC TCC GCC CAC ATC AAC AAC TGG CTG GCC TGG TAT CAG CAG AAG CCC GGC AAG GCC CCT AAG CTG CTG ATC TCT GAT GCC ACC TCC CTG GAA TCC GGC GTG CCC TCC AGA TTC TCC GGC TCT GGC TCT GGC AAG GAG TAT ACC CTG ACC ATC AGC TCC CTG CAG CCC GAT GAC TTC GCC ACC TAC TAC TGC CAG CAC TAC TGG TCC ACC CCC TGG ACC TTT GGC CAA GGC ACC AAG GTG GAA ATC AAG sequence no. 23 HC1 of VH amino acid sequence QVQLQESGPG LVKPSQTLSL TCTVSGYSIT SDYAWNWIRQ PPGKGLEWIG YISYSSGSTNY NPSLKSRVTI SVDTSKNQFS LKLSSVTAAD TAVYYCARGG TYFDYWGQGT LVTVSS SEQ ID NO: 24 VH amino acid sequence of HC3 EVQLVESGPG LVKPSETLSL TCTVSGYSIT SDYAWNWIRQ PPGKGLEWIG YISYSSGSTNY NPSLKSRVTI SRDTSKNQFS LKLSSVTAAD TAVYYCARGG TYFDYWGQGT TVTVSS SEQ ID NO: 25 VL amino acid sequence of LC1 DIQMTQSPSS LSASVGDRVT ITCRASAHIN NWLAWYQQKP GKAPKLLLSD ATSLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQH YWSTPWTFGG GTKVEIK SEQ ID NO: 26 VL amino acid sequence of LC2 DIVMTQSPSS LSASVGDRVT ITCRASAHIN NWLAWYQQKP GKAPKLLLSD ATSLESGVPS RFSGSGSGKD YTLTISSLQP EDFATYYCQH YWSTPWTFGG GTKVEIK SEQ ID NO: 27 Soluble MICA EPHSLRYNLT VLSWDGSVQS GFLAEVHLDG QPFLRCDRQK CRAKPQGQWA EDVLGNKTWD RETRDLTGNG KDLRMTLAHI KDQKEGLHSL QEIRVCEIHE DNSTRSSQHF YYDGELFLSQ NLETEEWTMP QSSRAQTLAM NIRNFLKEDA MKTKTHYHAM HADCLQELRR YLKSGVVLRR TVPPMVNVTR SEASEGNITV TCRASGFYPW NITLSWRQDG VSLSHDTQQW GDVLPDGNGT YQTWVATRIC QGEEQRFTCY MEHSGNHSTH PVPS SEQ ID NO: 28 Soluble MICB EPHSLRYNLM VLSQDGSVQS GFLAEGHLDG QPFLRYDRQK RRAKPQGQWA EDVLGAKTWD TETEDLTENG QDLRRTLTHI KDQKGGLHSL QEIRVCEIHE DSSTRGSRHF YYDGELFLSQ NLETQESTVP QSSRAQTLAM NVTNFWKEDA MKTKTHYRAM QADCLQKLQR YLKSGVAIRR TVPPMVNVTC SEVSEGNITV TCRASSFYPR NITLTWRQDG VSLSHNTQQW GDVLPDGNGT YQTWVATRIR QGEEQRFTCY MEHSGNHGTH PVPS SEQ ID NO: 29 IGHV4-59*11 (MK471385) QVQLQESGPG LVKPSETLSL TCTVSGGSIS SHYWSWIRQP PGKGLEWIGY IYYSGSTNYN PSLKSRVTIS VDTSKNQFSL KLSSVTAADT AVYYCAR SEQ ID NO: 30 IGHJ4*01 YFDYWGQGTL VTVSS SEQ ID NO: 31 IGHV4-30-4*01 (Z14238) QVQLQESGPG LVKPSQTLSL TCTVSGGSIS SGDYYWSWIR QPPGKGLEWI GYIYYSGSTYY NPSLKSRVTI VDTSKNQFSL KLSSVTAADT AVYYCAR SEQ ID NO: 32 IGKV1-NL1-4*01 (Y14865) DIQMTQSPSS LSASVGDRVT ITCRASQGIS NSLAWYQQKP GKAPKLLLYA ASRLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ YYSTP SEQ ID NO: 33 IGKJ1*01 (J00242) WTFGQGTKVE IK SEQ ID NO: 34 IGKV1-33*01 (M64856) DIQMTQSPSS LSASVGDRVT ITCQASQDIS NYLNWYQQKP GKAPKLLIYD ASNLETGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ YDNLP SEQ ID NO: 35 IGKV1-5*01 (Z00001) DIQMTQSPST LSASVGDRVT ITCRASQSIS SWLAWYQQKP GKAPKLLIYD ASSLESGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YNSYS SEQ ID NO: 36 B10G5 VH EVQLEESGPG LVKPSQSLSL TCTVTGYSIT SDYAWNWIRQ FPGNKLEWMG YISYSSGSTNY NPSLKSRISI TRDTSKNQFF LQLNSVITED TATYYCARGG TYFDYWGQGT TLTVSS SEQ ID NO: 37 B10G5 VL DIVLTQTTSY LSVSLGGRVT IACKASAHIN NWLAWYQQKP GNAPRLLISD ATSLETGVPS RFSGSGSGKD YTLSITSLQT EDVATYYCQH YWSTPWTFGG GTKLEIK

Claims

1. 1. A binding agent comprising: (i) a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 1; and (ii) a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 2, wherein the framework regions of the heavy and light chains are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids in the framework regions.

2. A binding agent comprising (i) a heavy chain variable region having the amino acid sequence represented by SEQ ID NO: 1, and (ii) a light chain variable region having the amino acid sequence represented by SEQ ID NO:

2.

3. A binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence represented by SEQ ID NO: 11, HCDR2 having the amino acid sequence represented by SEQ ID NO: 12, and HCDR3 having the amino acid sequence represented by SEQ ID NO: 13; the VL region comprises an LCDR1 sequence having the amino acid sequence represented by SEQ ID NO: 14, LCDR2 having the amino acid sequence represented by SEQ ID NO: 15, and LCDR3 having the amino acid sequence represented by SEQ ID NO: 16; and the VH region and the VL region each comprise a humanized framework region sequence.

4. 4. The binding agent of claim 3, wherein the humanized VH framework regions are derived from a human germline gene having an amino acid sequence represented by IMGT IGHV4-59*11, IGHV4-30-4*01, or IGHV4-30-4*08.

5. 5. The binding agent of claim 3 or claim 4, wherein the humanized VL framework region is derived from a human germline gene having an amino acid sequence represented by IMGT IGKV1-5*01, IGKV1-5*02 or IGKV1-5*03.

6. 1. A binding agent comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises complementarity determining regions (HCDR1 sequence) having the amino acid sequence set forth in SEQ ID NO: 11, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 13; the VL region comprises an LCDR1 sequence having the amino acid sequence set forth in SEQ ID NO: 14, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 16; the VH region comprises an amino acid sequence at least 85%, at least 87%, at least 90%, at least 92%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 1; and the VL region comprises an amino acid sequence at least 85%, at least 87%, at least 90%, at least 92%, or at least 95% identical to the amino acid sequence of SEQ ID NO:

2.

7. The binding agent of any one of claims 1 to 6, wherein the binding agent specifically binds to MIC.

8. The binding agent of any one of claims 1 to 7, wherein the binding agent is an antibody or an antigen-binding portion thereof.

9. The binding agent of any one of claims 1 to 8, wherein the binding agent is an antibody or antigen-binding portion thereof that binds to MIC.

10. 10. The binding agent of claim 8 or claim 9, wherein the binding agent is a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody.

11. 10. The binding agent of any one of the preceding claims, wherein the binding agent comprises a heavy chain comprising the VH region and a heavy chain constant region.

12. The binding agent of claim 11 , wherein the heavy chain constant region is an IgG isotype.

13. The binding agent of claim 12, wherein the heavy chain constant region is an IgG1 constant region.

14. The binding agent of claim 12, wherein the heavy chain constant region is an IgG4 constant region.

15. The binding agent of any one of claims 11 to 14, wherein the heavy chain constant region comprises amino acid modifications that increase binding affinity to at least human Fc gamma RIII.

16. The binding agent of any one of claims 11 to 15, wherein the heavy chain constant region comprises at least one amino acid modification that increases CDC activity.

17. The binding agent of any one of claims 11 to 16, wherein the heavy chain constant region comprises at least one amino acid modification that reduces binding to one or more Fc gamma receptors.

18. The binding agent of claim 11 , wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:

3.

19. 10. The binding agent of any one of the preceding claims, wherein the binding agent comprises a light chain comprising the VL region and a light chain constant region.

20. 20. The binding agent of claim 19, wherein the light chain constant region is a kappa isotype.

21. 20. The binding agent of claim 19, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:

4.

22. 10. The binding agent of any one of the preceding claims, wherein the binding agent is monospecific.

23. The binder of any one of claims 1 to 22, wherein the binder is bivalent.

24. 24. The binding agent of claim 23, wherein the binding agent comprises a second binding domain and the binding agent is bispecific.

25. A binding agent that binds to MIC, the binding agent being an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:3 and a light chain comprising the amino acid sequence of SEQ ID NO:

4.

26. The binding agent according to any one of claims 1 to 25, which specifically binds to soluble MIC.

27. 27. The binding agent of any one of claims 1 to 26, wherein the binding agent specifically binds to MIC with a higher binding affinity than antibody B10G5, and comprises a VH region comprising the amino acid sequence of SEQ ID NO: 36 and a VL region comprising the amino acid sequence of SEQ ID NO:

37.

28. 10. A pharmaceutical composition comprising a binding agent according to any one of the preceding claims and a pharmaceutically acceptable carrier.

29. A nucleic acid encoding a heavy chain variable region having the amino acid sequence represented by SEQ ID NO:

1.

30. 30. The nucleic acid of claim 29, comprising the nucleic acid sequence represented by SEQ ID NO:

21.

31. A nucleic acid encoding a light chain variable region having the amino acid sequence represented by SEQ ID NO:

2.

32. 32. The nucleic acid of claim 31, comprising the nucleic acid sequence represented by SEQ ID NO:

22.

33. A nucleic acid encoding the binding agent of any one of claims 1 to 24.

34. 34. The nucleic acid of claim 33, comprising the nucleic acid sequences represented by SEQ ID NO: 21 and SEQ ID NO:

22.

35. A vector comprising the nucleic acid according to any one of claims 29 to 34.

36. A cell line comprising the nucleic acid of any one of claims 29 to 34 or the vector of claim 35.

37. A cell line expressing the binding agent of any one of claims 1 to 22.

38. 38. A method for producing a binding agent, comprising culturing a cell line according to claim 36 or claim 37 under conditions suitable for expressing said binding agent.

39. 39. The method of claim 38, further comprising recovering the binder from the culture.

40. 29. A method of treating MIC+ cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the binding agent of any of claims 1-27, or the pharmaceutical composition of claim 28.

41. 41. The method of claim 40, wherein the cancer is a carcinoma, a sarcoma, a neuroendocrine tumor, or a hematological malignancy.

42. 42. The method of claim 41, wherein the cancer is a carcinoma.

43. 43. The method of claim 42, wherein the carcinoma is optionally a solid tumor selected from melanoma, prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, renal cancer, and head and neck cancer.

44. 42. The method of claim 41, wherein the cancer is a hematological malignancy.

45. 45. The method of claim 44, wherein the malignant hematological disease is lymphoma or multiple myeloma.

46. 46. ​​The method of any of claims 40-45, further comprising administering immunotherapy to the subject.

47. 47. The method of claim 46, wherein the immunotherapy comprises adoptive cellular therapy or a checkpoint inhibitor.

48. 48. The method of claim 47, wherein the immunotherapy comprises adoptive cell therapy.

49. 49. The method of claim 48, wherein the adoptive cell therapy is selected from autologous NK cells, allogeneic NK cells, autologous T cells, CAR-modified T cells, and CAR-modified NK cells.

50. 47. The method of claim 46, wherein the immunotherapy comprises a checkpoint inhibitor.

51. 51. The method of claim 50, wherein the checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4.

52. 52. The method of claim 51, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

53. 53. The method of any one of claims 40-52, wherein the subject has not been administered chemotherapy for at least 4 weeks prior to the administration of the binding agent.

54. 54. The method of any one of claims 40 to 53, wherein the binding agent is administered intravenously.

55. 55. The method of any one of claims 40-54, wherein the binding agent is administered at a dose of about 0.1 mg / kg to about 10 mg / kg.

56. 29. A method of reducing the level of circulating sMIC in a subject with cancer, comprising administering a therapeutically effective amount of the binding agent of any one of claims 1 to 27, or the pharmaceutical composition of claim 28.

57. 57. The method of claim 56, wherein the cancer is a carcinoma, a sarcoma, a neuroendocrine tumor, or a hematological malignancy.

58. 58. The method of claim 57, wherein the cancer is a carcinoma.

59. 59. The method of claim 58, wherein the carcinoma is optionally a solid tumor selected from melanoma, prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, renal cancer, and head and neck cancer.

60. 58. The method of claim 57, wherein the cancer is a hematological malignancy.

61. 61. The method of claim 60, wherein the malignant hematological disease is lymphoma or multiple myeloma.

62. 1. A method for improving a treatment outcome in a subject undergoing immunotherapy for MIC+ cancer, comprising: a) administering to said subject having cancer an effective amount of an immunotherapy; b) administering to the subject a therapeutically effective amount of the binding agent of any one of claims 1 to 27 or the pharmaceutical composition of claim 28; The method, wherein at least one therapeutic outcome in the subject is improved compared to administration of the immunotherapy alone.

63. 63. The method of claim 62, wherein the at least one improved outcome is an objective response selected from stable disease, partial response, or complete response.

64. 64. The method of claim 62 or claim 63, wherein the at least one improved outcome is a reduction in tumor burden.

65. 65. The method of any one of claims 62 to 64, wherein the at least one improved outcome is progression-free survival or disease-free survival.

66. 66. The method of any one of claims 62 to 65, wherein the immunotherapy is adoptive cellular therapy or a checkpoint inhibitor.

67. 67. The method of claim 66, wherein the immunotherapy is adoptive cell therapy.

68. 68. The method of claim 67, wherein the adoptive cell therapy comprises autologous NK cells, allogeneic NK cells, autologous T cells, CAR-modified T cells, and CAR-modified NK cells.

69. 67. The method of claim 66, wherein the immunotherapy is a checkpoint inhibitor.

70. 70. The method of claim 69, wherein the checkpoint inhibitor comprises an antibody that specifically binds to human PD-1, human PD-L1, or CTLA4.

71. 71. The method of claim 70, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

72. 72. The method of any one of claims 62-71, wherein the subject has not been administered chemotherapy for at least 4 weeks prior to the administration of the binding agent.

73. 73. The method of any one of claims 62 to 72, wherein the binding agent is administered intravenously.

74. 74. The method of any one of claims 62-73, wherein the binding agent is administered at a dose of about 0.1 mg / kg to about 10 mg / kg.

75. Use of the binding agent of any one of claims 1 to 27 or the pharmaceutical composition of claim 28 for the treatment of MIC+ cancer in a subject.

76. Use of a binding agent according to any one of claims 1 to 27 or a pharmaceutical composition according to claim 28 for the treatment of sMIC+ cancer in a subject undergoing immunotherapy.

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