MIC antibodies, binders, and methods for using them.

Abstract

The present invention provides MIC antibodies, antigen-binding portions thereof, and MIC binders thereof for the treatment of cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 049,012, filed on 7 July 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, granted by the U.S. Small Business Administration. The government has certain rights to this invention. [Background technology]

[0003] Major histocompatibility complex class I chain-related molecules A and B (MICA and MICB, respectively, commonly referred to as MICs) are a family of proteins that bind to NKG2D. NKG2D is an activated immune receptor expressed by natural killer (NK) cells, NKT cells, a subset of gamma delta T cells, and human CD8 T cells. When MICs bind to NKG2D on NK cells or T cells, they have other effects, but in vitro, NK cells are activated and CD8 T cells and gamma delta T cells are co-stimulated.

[0004] MIC family molecules are expressed on tumor cells and are thought to be involved in suppressing the immune response against tumor cells. MICA is expressed more frequently and in larger quantities on the surface of tumor cells than MICB. MIC proteins are found in both membrane-bound and soluble (sMIC) forms, the latter being detached from tumor cells. Elevated serum levels of sMIC are associated with many types of cancer, including solid tumors such as melanoma, prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, kidney cancer, gastrointestinal (GI) cancer, and head and neck cancer, as well as hematological malignancies such as lymphoma and multiple myeloma, and bone and soft tissue cancers such as sarcomas. Membrane-bound MIC(B) has been reported to maintain protective anti-tumor immunity via NKG2D in mice, while the soluble form correlated with tumor progression resulting from decreased NKG2D expression in NK cells and CD8 T cells and impaired peripheral maintenance of NK cells. Therefore, drugs that bind to sMIC may be a therapeutic approach for patients. On the other hand, there is still a need for drugs optimized for the treatment of MIC+ cancer in humans. [Overview of the project]

[0005] The inventions disclosed herein are, in part, based on MIC-binding antibodies, their antigen-binding moieties, and associated binders that specifically bind to soluble MICs (sMICs) and / or cell membrane-bound MICs (also known as membrane-bound MICs) and exhibit improved therapeutic properties. MICs, and sMICs in particular, are important and advantageous therapeutic targets for the treatment of certain cancers. These MIC-binding antibodies, their antigen-binding moieties, and binders provide compositions and methods based on the use of such antibodies, antigen-binding moieties, and associated binders in the treatment of MIC+ cancers. Accordingly, the present invention provides methods, compositions, kits, and products related to MIC binders.

[0006] In some embodiments, a binder is provided 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, wherein the framework regions of the heavy chain and light chain are optionally modified by substitution, deletion, or insertion of 1 to 8 amino acids in the framework region. The binder specifically binds to MIC. In some embodiments, the binder specifically binds to MIC with a higher binding affinity than antibody B10G5. In some embodiments, the binder comprises (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. The binder specifically binds to MIC. In some embodiments, the binder specifically binds to MIC with a higher binding affinity than antibody B10G5.

[0007] In some embodiments, a binder is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises a complementarity-determining region HCDR1 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, and the VL region comprises LCDR1 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 VL region each comprise a humanized framework region. In some embodiments, the humanized VH framework region is derived from a human germ cell gene having the amino acid sequence represented by 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 region is derived from a human germ cell gene having an amino acid sequence 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). The binder specifically binds to the MIC. In some embodiments, the binder specifically binds to the MIC with a higher binding affinity than the antibody B10G5.

[0008] In some embodiments, the binder is an antibody or its antigen-binding portion. In some embodiments, the binder 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 binder has a heavy chain variable region linked to the 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 the heavy chain constant region have the amino acid sequence represented by SEQ ID NO: 3. In some embodiments, the binder has a light chain variable region linked to the 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 the light chain constant region have the amino acid sequence represented by SEQ ID NO: 4. In some embodiments, the heavy chain constant region further includes an amino acid modification that increases binding affinity to at least human Fc gamma RIII. In some embodiments, the heavy chain constant region further includes an amino acid modification that increases antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, the heavy chain constant region further includes at least one amino acid modification that increases CDC activity.

[0010] In some embodiments, the binder is monospecific. In some embodiments, the binder is monospecific and divalent. In some embodiments, the binder is divalent. In some embodiments, the binder is divalent and bispecific or polyvalent and multiplespecific.

[0011] In some embodiments, pharmaceutical compositions are provided that comprise a binder of any of the embodiments described herein and a pharmaceutically acceptable carrier.

[0012] In some embodiments, there is provided a nucleic acid encoding the heavy chain variable region of a binder having the amino acid sequence represented by SEQ ID NO: 1, optionally having the nucleic acid sequence represented by SEQ ID NO: 21. In some embodiments, there is provided a nucleic acid encoding the light chain variable region of a binder having the amino acid sequence represented by SEQ ID NO: 2, optionally having the nucleic acid sequence represented by SEQ ID NO: 22. In some embodiments, there is provided a nucleic acid encoding any of the binders of the embodiments described herein, optionally having the nucleic acid sequences represented by SEQ ID NO: 21 and SEQ ID NO: 22. In some embodiments, there is provided a vector comprising any of the nucleic acids encoding the MIC binder polypeptides described herein. In some embodiments, there is provided a cell comprising a nucleic acid encoding any of the binder polypeptides described herein, or a vector comprising such nucleic acid(s).

[0013] In some embodiments, there is provided a method of treating MIC+ cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a binder 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, 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 binder specifically binds to sMIC and / or membrane-bound MIC. In some embodiments, the binder specifically binds to MIC with a binding affinity higher than that of antibody B10G5.

[0014] In some embodiments, there is provided a method of treating MIC+ cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a binder 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, wherein the binder specifically binds to MIC. In some embodiments, the binder specifically binds to MIC with a binding affinity higher than that of antibody B10G5.

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

[0016] In some embodiments, the cancer is a carcinoma, sarcoma, neuroendocrine tumor, or malignant blood disease. 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, kidney cancer, and head and neck cancer. In some embodiments, the malignant blood disease is lymphoma, leukemia, or multiple myeloma.

[0017] In some embodiments, the method further comprises administering an 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 antibodies that specifically bind 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 a step of not administering chemotherapy to the subject for at least 4 weeks, at least 6 weeks, or at least 8 weeks prior to the administration of the binder. In some embodiments, the binder is administered intravenously. In some embodiments, the binder is administered in doses 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, a method is provided for reducing the level of circulating sMIC in a subject having cancer, comprising administering a therapeutically effective amount of a binder of any embodiment described herein, or a pharmaceutical composition of any embodiment of the binder described herein, wherein the binder specifically binds to the circulating sMIC. In some embodiments, cancer is a carcinoma, sarcoma, neuroendocrine tumor, or malignant hematological disorder. In some embodiments, 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, malignant hematological disorder is lymphoma, leukemia, or multiple myeloma.

[0020] In some embodiments, a method is provided for improving the treatment outcomes of subjects receiving immunotherapy, comprising administering an effective dose of immunotherapy to a subject with cancer and administering to the subject a therapeutically effective dose of a conjugate of any embodiment described herein or a pharmaceutical composition of any embodiment of the conjugate described herein, wherein the conjugate specifically binds to circulating sMICs and / or cell membrane-bound MICs, and the treatment outcomes of the subject are improved compared to administration of immunotherapy alone. In some embodiments, the improved treatment outcome is an objective response selected from stable, partial response or complete response. In some embodiments, the improved treatment outcome is a reduction in tumor volume. In some embodiments, the improved treatment 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 includes an antibody that specifically binds to human PD-1, human PD-L1, or CTLA4. In some embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, semiprimab, or ipilimumab.

[0021] In some embodiments, no chemotherapy is administered to the subject for at least 4 weeks, at least 6 weeks, or at least 8 weeks prior to the administration of the binder. In some embodiments, the binder is administered intravenously. In some embodiments, the binder is administered in doses of approximately 0.01 mg / kg to approximately 100 mg / kg, or approximately 0.01 mg / kg to approximately 25 mg / kg, or approximately 0.01 mg / kg to approximately 20 mg / kg, or approximately 0.01 mg / kg to approximately 15 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 100 mg / kg, or approximately 0.1 mg / kg to approximately 25 mg / kg, or approximately 0.1 mg / kg to approximately 20 mg / kg, or approximately 0.1 mg / kg to approximately 15 mg / kg, or approximately 0.1 mg / kg to approximately 10 mg / kg.

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

[0023] [Figure 1] This shows the FACS binding affinity analysis of humanized variants of antibody B10G5 (linked to the Fc region of human IgG1). The two humanized variants, antibody G and antibody K, showed higher mean fluorescence intensity (MFI) than antibody J (Ab-J, a B10G5 chimeric antibody composed of the F(ab)2 of mouse B10G5 antibody and the Fc domain of human IgG1). [Figure 2A] Antibody B10G5 (Figure 2A) has a lower (weaker) binding affinity than antibody K (Ab-K, Figure 2B), which was determined by biolayer interferometry using Octet Red96 (ForteBio) to be Kd = 12.1 nM vs. 7.2 nM, respectively. [Figure 2B] Antibody B10G5 (Figure 2A) has a lower (weaker) binding affinity than antibody K (Ab-K, Figure 2B), which was determined by biolayer interferometry using Octet Red96 (ForteBio) to be Kd = 12.1 nM vs. 7.2 nM, respectively. [Figure 3A] Antibody K (Ab-K) shows higher activity than Ab-J (chimeric B10G5) in enhancing the killing of MIC+ thyroid oncocytoma UC1 tumor cells (Figure 3A) and pancreatic PL12 cells (Figure 3B) by IL-2 activated primary NK cells. [Figure 3B] Antibody K (Ab-K) shows higher activity than Ab-J (chimeric B10G5) in enhancing the killing of MIC+ thyroid oncocytoma UC1 tumor cells (Figure 3A) and pancreatic PL12 cells (Figure 3B) by IL-2 activated primary NK cells. [Figure 4A]Figure 4C shows the levels of antibody monomers and aggregates for mouse antibody B10G5 (Figure 4A), chimeric Ab-K antibody (ch-Ab-K, Ab-K variable domain and mouse IgG1-FC, Figure 4B), and antibody K (Ab-K, humanized, Figure 4C), as determined by dynamic light scattering (DLS) assay. [Figure 4B] Figure 4C shows the levels of antibody monomers and aggregates for mouse antibody B10G5 (Figure 4A), chimeric Ab-K antibody (ch-Ab-K, Ab-K variable domain and mouse IgG1-FC, Figure 4B), and antibody K (Ab-K, humanized, Figure 4C), as determined by dynamic light scattering (DLS) assay. [Figure 4C] Figure 4C shows the levels of antibody monomers and aggregates for mouse antibody B10G5 (Figure 4A), chimeric Ab-K antibody (ch-Ab-K, Ab-K variable domain and mouse IgG1-FC, Figure 4B), and antibody K (Ab-K, humanized, Figure 4C), as determined by dynamic light scattering (DLS) assay. [Modes for carrying out the invention]

[0024] definition For convenience, specific terms in the specification, examples, and claims are defined herein. Unless otherwise stated or implicitly indicated in the context, the following terms and phrases have the meanings provided below. The definitions are provided to aid in describing specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited solely by the claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains.

[0025] When used herein, unless otherwise specified, the terms "a" and "an" shall be interpreted as meaning "one," "at least one," or "one or more." Unless otherwise required by context, singular terms used herein shall include the plural form, and plural terms shall include the singular form.

[0026] Unless the context clearly requires a different interpretation, terms such as “comprise” and “comprising” throughout the specification and claims should be interpreted in a comprehensive sense, not exclusive or exhaustive; that is, “including, but not limited to.”

[0027] In this specification, the terms “reduce,” “mitigate,” “reduced,” “decrease,” “decrease,” and “suppress” are all used in general to mean a decrease of a statistically significant amount relative to a standard.

[0028] In this specification, the terms “increased,” “enhance,” “boost,” and “activate” are all used in general to mean an increase of a statically significant amount relative to a standard.

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

[0030] As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a set of amino acid residues linked 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 protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used in reference to relatively large polypeptides, the term “peptide” is often used in reference to small polypeptides, but the use of these terms in the art is overlapping. The terms “protein” and “polypeptide” are used interchangeably herein when referring to encoded gene products and their fragments. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologues, paralogs, fragments and other equivalents, variants, fragments, and the aforementioned analogs.

[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 sequence 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, MIC polypeptide refers to MICA. In some embodiments, MIC polypeptide refers to MICB. In some embodiments, 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 an MIC polypeptide (MICA or MICB) that includes the alpha-1 and alpha-2 domains and the portion up to the alpha-3 domain or the proteolytic cleavage site of the alpha-3 domain, and lacks a transmembrane domain (e.g., the extracellular portion of the MIC). In some embodiments, the soluble MICA may include the amino acid sequence represented by Genbank accession number CAA77031.1 (SEQ ID NO: 27), or variants thereof, such as amino acid residues 24-297 of Genbank accession numbers 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 number CAE45581.1, or amino acids 1-273 of Genbank accession numbers AAD52069.1, AAD52070.1, or QDW65494.1 (these disclosures are incorporated herein by reference). In some embodiments, the soluble MICB may include amino acid sequences represented by Genbank accession numbers ARB08539.1 (SEQ ID NO: 28), AAB71646.1, AAB71647.1, or AAB71644.1, or variants thereof, such as amino acid residues 24-297 of Genbank accession numbers ABO16470.1, ABB51802.1, AAB42011.1, AAB71643.1, or Q29980.1, or reference sequence NP_005922.2, or amino acid sequences represented by amino acids 1-273 of Genbank accession numbers AAC39848.1, AEK67483.1, AFR7773.1, AXY93666.1, CAB72098.1, or AAC39849.1 (these disclosures are incorporated herein by reference). Unless otherwise specified, the term "MIC" is intended to refer to both the cell membrane-bound and soluble forms of MIC.

[0033] As used herein, antibody B10G5 refers to the MIC antibody of the same name as described in U.S. Patent No. 9,803,017 (the disclosure thereof is incorporated herein by reference for all purposes).

[0034] As used herein, “epitope” refers to an amino acid typically bound by an immunoglobulin VH / VL pair, such as antibodies and conjugates described herein. Epitopes can be formed on polypeptides from adjacent amino acids or non-adjacent amino acids paralleled by tertiary folding of the protein. Epitopes formed from adjacent amino acids are usually retained even when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, about nine, or about eight to ten amino acids in their unique spatial conformation. Epitopes define the minimum binding site for an antibody or other conjugate and thus represent the target of the specificity of the antibody, its antigen-binding portion, or other immunoglobulin conjugate. In the case of a single-domain antibody, an epitope represents a structural unit bound by an isolated variable domain.

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

[0036] In some embodiments, the MIC antibodies or antigen-binding portions thereof or other binders described herein have a -5 dissociation constant (KD) of 10 M (10000 nM) or less, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 and specifically bind to the MIC polypeptide. In some embodiments, the MIC antibodies or antigen-binding portions thereof or other binders described herein have a dissociation constant (KD) of about 10 -5 M to 10 -6 M and specifically bind to the MIC polypeptide. In some embodiments, the MIC antibodies or antigen-binding portions thereof or other binders described herein have a dissociation constant (KD) of about 10 [[ID=​​​​​​​​M~10 -9 It specifically binds to the MIC polypeptide with a dissociation constant (KD) of M. In some embodiments, the MIC antibody or its antigen-binding moiety or other binder described herein is about 10 -9 M~10 -10 It specifically binds to the MIC polypeptide with a dissociation constant (KD) of M. In some embodiments, the MIC antibody or its antigen-binding moiety or other binder described herein is about 10 -10 M~10 -11 It specifically binds to the MIC polypeptide with a dissociation constant (KD) of M. In some embodiments, the MIC antibody or its antigen-binding moiety or other binder described herein is about 10 -11 M~10 -12 It specifically binds to the MIC polypeptide with a dissociation constant (KD) of M. In some embodiments, the MIC antibody or its antigen-binding moiety or other binder described herein is 10 -12 It specifically binds to MIC polypeptides with a dissociation constant (KD) of less than M.

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

[0038] As used herein, the term “essentially consisting of” refers to an element necessary for a given embodiment. The term permits the presence of elements that do not substantially affect the basic, novel, or functional features of that embodiment.

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

[0040] Unless otherwise indicated in the examples or elsewhere, all figures representing ingredient amounts or reaction conditions used herein should be understood to be modified in all cases by the term “approximately.” The term “approximately” used in relation to percentages may mean + / - 1%.

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

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

[0043] This specification provides MIC-binding antibodies (also called MIC antibodies or MIC-binding antibodies) and their antigen-binding moieties that specifically bind to MIC. Surprisingly, the MIC antibodies exhibit improved properties compared to antibody B10G5. In some embodiments, the MIC antibodies reduce the level of free sMIC circulating in a subject. In some embodiments, the MIC-binding antibody or its antigen-binding moiety includes (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. In some embodiments, the MIC-binding antibody or its antigen-binding moiety includes (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, wherein the variable framework regions of the heavy-chain and light-chain are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions in the framework region, and the CDR of the heavy-chain or light-chain variable region is not modified. In some embodiments, the MIC-conjugated antibody or its antigen-binding moiety comprises (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, wherein the variable framework regions of the heavy and light chains 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 region, and the CDR of the heavy or light-chain variable region is not modified. In further embodiments of any of these embodiments, the MIC-conjugated antibody or its antigen-binding moiety binds specifically to the MIC with a higher binding affinity than antibody B10G5.

[0044] In some embodiments, a binder is provided herein 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, wherein the binder specifically binds to the MIC. In some embodiments, a binder is provided herein 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, wherein the variable framework regions of the heavy chain and light chain are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions in the framework region, and the CDR of the heavy chain or light chain variable region is not modified. In some embodiments, a binder is provided herein 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, wherein the variable framework regions of the heavy chain and light chain 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 region, and the CDR of the heavy chain or light chain variable region is not modified. As described herein, the binder may comprise an MIC antibody or its antigen-binding moiety(s) and may include other peptides or polypeptides covalently bound to the MIC antibody or its antigen-binding moiety. In any of these embodiments, the binder binds specifically to the MIC. In some embodiments, the binder binds specifically to the MIC with a higher binding affinity than antibody B10G5.

[0045] In some embodiments, a binder is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises a complementarity-determining region HCDR1 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, and the VL region comprises LCDR1 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 each VH and VL comprises a humanized framework region. In some embodiments, the VH framework region is derived from a human germ cell gene having the amino acid sequence represented by 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 region is derived from a human germ cell gene having an amino acid sequence 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).

[0046] In some embodiments, a binder is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises a complementarity-determining region HCDR1 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, and 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 binder is provided comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL region comprises LCDR1 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 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, a binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises a complementarity-determining region HCDR1 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, and the VH region 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 less than the amino acid sequence of SEQ ID NO: 1. A binder is provided which contains an amino acid sequence that is at least 99% identical, and the VL region comprises LCDR1 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 VL region contains 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, a binder comprising a heavy chain including a heavy chain variable (VH) region and a light chain including a light chain variable (VL) region, wherein the VH region comprises a complementarity determining region HCDR1 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, and the VH region comprises the amino acid sequence of SEQ ID NO: 1, and the VL region comprises LCDR1 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 VL region comprises the amino acid sequence of SEQ ID NO: 2 A binder is provided, comprising an amino acid sequence, wherein the heavy chain 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: 3, and the light chain 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: 4. In some embodiments, a binder 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 binder is an antibody.

[0050] In some embodiments, the binders 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 by Tm and Tag, which can be obtained, for example, by intrinsic protein fluorescence (IPF) (266 nm excitation, 280–450 nm radiation scan) and static light scattering (SLS) at 473 nm using the Uncle system (Unchained Labs). 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 may 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% as a percentage of the total amount of the MIC antibody, based on the peak area of ​​the antibody monomer and HMW aggregates. That is, 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 exists in monomeric form when in solution. 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 its antigen-binding moiety or other binder specifically binds to structural epitopes on MICA and MICB located within the range of amino acid positions approximately 66-77, 136-144, and 247-258 of the amino acid sequence represented by SEQ ID NO: 27 or 28. In certain embodiments, the MIC antibody or its antigen-binding moiety or other binder competes for specific binding with antibody B10G5, which has (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, and an optionally substituted framework region as described herein, and specifically binds to structural epitopes on MICA and MICB located within the range of amino acids approximately 66-77, 136-144, and 247-258 of the amino acid sequence represented by SEQ ID NO: 27 and 28.

[0052] In some embodiments, the compositions and methods described herein relate to the suppression of the immunosuppressive effect of sMICs in vivo by MIC antibodies, their antigen-binding moieties, or other binders (e.g., reducing the level and / or activity of sMICs available for interaction with the cell receptor NKG2D). In some embodiments, sMIC suppression may be a reduction in serum levels of unbound MICs, as well as a restoration of cell surface NKG2D expression on NK and CD8 T cells. In some embodiments, sMIC suppression may be a reduction in the level of MICs (e.g., the level of circulating sMICs).

[0053] As used herein, the term “antibody” refers to an immunoglobulin molecule and a molecule containing an immunoactive portion of the immunoglobulin molecule, i.e., an antigen-binding site that specifically binds to an antigen. Generally, this term refers to an antibody consisting of a full-length antibody (having heavy and light chain constant regions) and its antigen-binding site, comprising two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions. For example, intact monoclonal antibody, Fab, Fab′, F(ab′) 2This includes Fv, disulfide-bonded Fv, scFv, single-domain antibodies (dAb), diabodies, multispecific antibodies, dual-specific antibodies, bispecific antibodies, and single chains (see, for example, Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988) (these are incorporated herein by reference)).

[0054] Each heavy chain consists of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may contain three domains CH1, CH2, and CH3, and optionally a fourth domain CH4. Each light chain consists of a variable region (abbreviated as VL) and a constant region. The light chain constant region is the CL domain. The VH and VL regions may be further divided into hypervariable regions called complementarity-determining regions (CDRs), and conserved regions called framework regions (FRs) may be incorporated. 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 described in SEQ ID NO: 1, with 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 described in SEQ ID NO: 2, with 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 heavy chain or light chain variable region CDR is not modified" refers to these VH and VL CDRs (SEQ ID NOs: 11-16), which contain no amino acid substitutions, deletions, or insertions.

[0056] As used herein, the “antigen-binding moiety” of an MIC antibody refers to the portion of the MIC antibody described herein that has the VH and VL sequences (represented by SEQ ID NOs: 1 and 2, and optionally modified as described herein). According to the term “antigen-binding moiety” of an antibody, examples of antigen-binding moieties include Fab, Fab′, F(ab′). 2 This includes Fv, disulfide-linked Fv, scFv, single-domain antibodies (dAb), diabodies, and single chains. As used herein, the terms Fab, F(ab′)2, and Fv refer to: (i) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′)2 fragments, i.e., bivalent fragments containing two Fab fragments linked to each other at a hinge region via disulfide crosslinks; and (iii) Fv fragments consisting of the VL and VH domains of an MIC antibody. The two domains of the Fv fragment, i.e., VL and VH, are encoded by separate coding regions, but these can be further linked to each other using synthetic linkers, e.g., polyG4S amino acid sequences (disclosed as SEQ ID NO: 17, "(G4S)n", where n=1 to 5), allowing them to be prepared as single protein chains to which the VL and VH regions bind to form a monovalent molecule (known as single-chain Fv (ScFv)). The term “antigen-binding site” of an antibody is also intended to include such single-chain antibodies. Other forms of single-chain antibodies, such as “diabodies,” are similarly included here. 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, thus pairing the VH and VL domains with complementary domains on separate chains (VL and VH, respectively) to form two antigen-binding sites (see, for example, 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 the heavy or light chain. The amino acid sequences of the human heavy chain constant region and the human light chain constant region are well known in the art. The constant region can be any suitable type that can be selected from the classes of immunoglobulin, IgA, IgD, IgE, IgG, and IgM. Some immunoglobulin classes can be further divided into isotypes, e.g., IgG1, IgG2, IgG3, IgG4, or IgA1 and IgA2. The heavy chain constant regions (Fc) corresponding to different classes of immunoglobulin may be α, δ, ε, γ, and μ, respectively. The light chain may be either kappa (κ) or lambda (λ).

[0058] In some embodiments, the steady region may have an IgG1 isotype. In some embodiments, the steady region may have an IgG2 isotype. In some embodiments, the steady region may have an IgG3 isotype. In some embodiments, the steady region may have an IgG4 isotype. In some embodiments, the Fc domain may have a hybrid isotype comprising steady regions from two or more isotypes. In some embodiments, the immunoglobulin steady region may be an IgG1 or IgG4 steady region.

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

[0060] Furthermore, the MIC antibody or its antigen-binding moiety may be part of a larger binding agent formed by covalent or non-covalent bonding between the antibody or antibody moiety and one or more other proteins or peptides. Such binding agents are related to the use of streptavidin core regions for preparing 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 the hexahistidinyl tag (disclosed as SEQ ID NO: 18, "hexahistidinyl tag") for producing divalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol.Immunol.31:10471058).

[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 alterations in the amino acids in a polypeptide that change the proportion of a single amino acid or a small number of amino acids in the encoded sequence, are "conservative modified variants." This modification results 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, a conservative variant of the MIC antibody or its antigen-binding moiety may have modifications in the FR (i.e., outside the CDR), for example, a conservative variant of the MIC antibody has the amino acid sequences of the VH and VL CDRs (represented by SEQ ID NOs: 11-16) and has at least one conservative amino acid substitution in the FR. 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 FR compared to the amino acid sequences of VH and VL (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 8-1, 6-1, 4-1, or 2-1 conservative amino acid substitutions in the FR compared to the amino acid sequences of VH and VL (represented by SEQ ID NOs: 1 and 2, respectively). In further embodiments of any of these embodiments, a conservative variant of the MIC antibody, its antigen-binding moiety, or other binder exhibits a binding affinity to MIC that is greater than the binding affinity of antibody B10G5.

[0063] In conservative amino acid substitutions, a given amino acid is replaced by a residue with similar physiological and chemical properties, for example, by substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala), or by substituting one polar residue for another (such as Lys for Arg, Glu for Asp, or Gln for Asn). Other such conservative amino acid substitutions, such as substitutions of entire regions with similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested with any of the assays described herein to confirm that the desired activities of the native polypeptide or reference polypeptide, such as antigen-binding activity and specificity, are retained, i.e., retained against MICs (sMICs and / or membrane-bound MICs).

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

[0065] Alternatively, in the case of conservative substitutions, naturally occurring residues can be grouped based on common 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) residues that affect chain orientation, Gly, Pro; and (6) aromatic Trp, Tyr, Phe. Non-conservative substitutions involve replacing members of one or more of these classes.

[0066] Certain conservative substitutions include, for example, substitutions of 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, Hisn 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, the conservative modified variant on the MIC antibody or its antigen-binding moiety is preferably identical to the reference VH or VL sequence by 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, and the VH and VL CDRs (SEQ ID NOs. 11-16) are unmodified. The degree of homology (identity percentage) between the reference sequence and the modified sequence can be determined, for example, by comparing the two sequences using a freely available computer program 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 by SEQ ID NOs. 1 and 2, respectively) collectively have 8, 6, 4, 2, or 1 or fewer conserved amino acid substitutions in the framework region 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 8-1, 6-1, 4-1, or 2-1 conserved amino acid substitutions in the framework region 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, 6, 4, 2, or 1 or fewer amino acid substitutions, deletions, or insertions in the framework region 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 8-1, 6-1, 4-1, or 2-1 conserved amino acid substitutions in the framework region 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 collectively 8, 6, 4, 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 the many techniques known to those skilled in the art. Mutations can be introduced to a specific locus, for example, by synthesizing an oligonucleotide containing the desired mutant sequence, flanked by a restriction site that allows ligation to a fragment of the native sequence. After ligation, the resulting reconstructed sequence encodes a variant having the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-targeted site-specific mutagenesis procedure can be used to provide a modified nucleotide sequence having a specific codon altered according to the desired substitution, deletion, or insertion. Methods 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. Patents 4,518,584 and 4,737,462, all of which are incorporated herein by reference.

[0070] In some embodiments, the MIC antibody or its antigen-binding moiety has a fully human constant region. In some embodiments, the MIC antibody or its antigen-binding moiety has a non-human constant region. In some embodiments, the MIC antibody heavy chain is the IgG1 isotype and has the amino acid sequence represented by SEQ ID NO: 7. In some embodiments, the MIC antibody light chain is the kappa isotype and has the amino acid sequence represented by SEQ ID NO: 8.

[0071] In some embodiments, the MIC antibody or its antigen-binding moiety has a modified constant region (Fc region) or Fc domain. The Fc domain (e.g., CH1, CH2, CH3, and optionally CH4) is part of the Fc region. The Fc domain or part of the Fc region can bind to Fc receptors (FcRs) on cells. 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 to IgA and includes several isoforms such as FcaRI(CD89). The FcyR class can bind to IgG and includes several isoforms, FcyRI(CD64), FcyRIIA(CD32a), FcyRIIB(CD32b), FcyRIIIA(CD16a), and FcyRIIIB(CD16b). FcyRIIIA(CD16a) has two main variants: F158 and V158.

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

[0073] MIC antibodies may have an Fc domain that has been modified to alter the biological effector function mediated by at least one constant domain or constant region associated with the corresponding wild-type or reference sequence, compared to the wild-type or reference sequence. For example, in some embodiments, the Fc domain can be modified to reduce or increase the biological effector function mediated by at least one constant domain or constant region associated with the unmodified Fc domain, for example, to reduce or increase binding to an Fc receptor (FcR). FcR binding can be reduced or increased, for example, by modifying the immunoglobulin constant region segment of the antibody at specific sites (maybe multiple sites) involved in (e.g., required for or influencing) FcR interaction. In some embodiments, the Fc domain is modified 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 constant region or domain of an antibody is modified to acquire or improve biological effector function via at least one constant region associated with an unmodified Fc domain, for example, to enhance FcyR interactions. For example, the constant region or domain of an antibody can be modified to bind to FcyRIIA, FcyRIIB, and / or FcyRIIIA with higher affinity than the corresponding wild-type Fc domain or Fc region.

[0075] Modifications to the amino acid sequence in the Fc domain can alter the recognition of FcR to the Fc domain or Fc region. However, FcR-mediated signaling is still possible even after such modifications. Modifications may involve substituting an amino acid at a particular residue with a different amino acid at that residue. Modifications may enable FcR to bind to sites on the Fc domain or Fc region that FcR cannot normally bind to. Modifications may increase the binding affinity of FcR to the Fc domain or Fc region compared to binding to the reference sequence. Modifications may decrease the binding affinity of FcR to sites on the Fc domain compared to binding to the reference sequence.

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

[0077] A modified Fc region or domain may have at least one amino acid change compared to the sequence of the wild-type Fc region or domain. Amino acid changes in the Fc region may enable the antibody or its antigen-binding portion to bind to at least one Fc receptor with higher affinity compared to the wild-type or reference Fc region. Amino acid changes in the Fc domain may enable the antibody to bind to at least one Fc receptor with higher affinity compared to the wild-type or reference Fc domain. Amino acid changes in the Fc region may enable the antibody to bind to at least one Fc receptor with lower affinity compared to the wild-type or reference Fc region. Amino acid changes in the Fc domain may 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 its antigen-binding moiety may have an Fc domain or Fc region containing a sequence of an IgG1 isoform modified from a wild-type IgG1 sequence. The modification may include substitutions at one or more amino acid residues in the Fc domain, such as five different amino acid residues including L235V / F243L / R292P / Y300L / P396L (IgG1VLPLL) according to Kabat's EU index. The modification may include substitutions at one or more amino acid residues, such as two different amino acid residues in the Fc domain including S239D / I332E (IgG1DE) according to Kabat's EU index. The modification may include substitutions at one or more amino acid residues, such as three different amino acid residues in the Fc domain including S298A / E333A / K334A (IgG1 AAA) according to Kabat's EU index.

[0079] In some embodiments, the Fc domain or Fc region of an MIC antibody or its antigen-binding moiety may exhibit reduced binding affinity to one or more Fc receptors. In some embodiments, the Fc domain or Fc region may exhibit reduced binding affinity to one or more Fc gamma receptors. In some embodiments, the Fc domain or Fc region may exhibit reduced binding affinity to FcRn receptors. In some embodiments, the Fc domain or Fc region may exhibit reduced binding affinity to both Fc gamma receptors and FcRn receptors. In some embodiments, the Fc domain is an Fc null domain or Fc null region. As used herein, "Fc null" refers to a domain that exhibits weak binding to non-binding to any of the Fc gamma receptors. In some embodiments, the Fc null domain or Fc null region exhibits at least a 1000-fold decrease in binding affinity to the Fc gamma receptor (e.g., an increase in Kd).

[0080] In some embodiments, the Fc domain or Fc region of the MIC antibody is an Fc null domain or Fc null region containing 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, the incorporation of the flexible sequence into the MIC antibody results in little to no effect on the antibody's binding affinity. In some embodiments, the inclusion of the flexible sequence in the MIC antibody reduces the antibody's ADCC activity. In some embodiments, the incorporation of the flexible sequence into the MIC antibody results in little to no loss of glycosylation and / or significant alteration(s) of the antibody's glycan profile. The antibody's glycosylation and / or glycan profile can be measured, for example, by subjecting the antibody to PNGaseF digestion and analyzing the enzyme product using hydrophilic interaction chromatography (HILIC) with a fluorescence detector.

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

[0082] In some embodiments, one or more substitutions are made by the EU index of Kabat numbering, E233P, L234V, L234A, L235A, L235E 、 G236 A , comprising any one or more of the IgG1 heavy chain substitutions corresponding to G237A, E318A, K320A, K322A, A327G, A330S, and / or P331S. In some embodiments, the modification is E233P / L234V / L235A or E233P / L234V / L235A according to Kabat's EU index. / G236 A These may be substitutions for E233, L234, and L235. In some embodiments, the modification may be a substitution for P238, such as P238A, according to the Kabat EU index. In some embodiments, the modification may be a substitution for D265, such as D265A, according to the Kabat EU index. In some embodiments, the modification may be a substitution for N297, such as N297A, according to the Kabat EU index. In some embodiments, the modification may be a substitution for A327, such as A327Q, according to the Kabat EU index. In some embodiments, the modification may be a substitution for P329, such as P239A, according to the Kabat EU index.

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

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

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

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

[0087] In some embodiments, the modification involves the substitution of one or more amino acids that increase the binding affinity of the IgG Fc domain or IgG Fc region to the FcyRII receptor. In some embodiments, the modification may be a substitution of R255, such as R255A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of E258, such as E258A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of S267, such as S267A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of E272, such as E272A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of N276, such as N276A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of D280, such as D280A, according to the Kabat EU index. In some embodiments, the modification may 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 involves the substitution of one or more amino acids that increase the binding affinity of the IgG Fc domain or IgG Fc region to the FcyRII receptor and decrease the binding affinity to the FcyRIIIA receptor. In some embodiments, the modification may be a substitution of H268, such as H268A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of R301, such as R301A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of K322, such as K322A, according to the Kabat EU index.

[0089] In some embodiments, the modification involves the substitution of one or more amino acids that reduces the binding affinity of the IgG Fc domain or IgG Fc region to the FcyRII receptor but does not significantly affect the binding affinity to the FcyRIIIA receptor. In some embodiments, the modification may be a substitution of R292, such as R292A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of K414, such as K414A, according to the Kabat EU index.

[0090] In some embodiments, the modification involves the substitution of one or more amino acids that decrease the binding affinity of the IgG Fc domain or IgG Fc region to the FcyRII receptor and increase the binding affinity to the FcyRIIIA receptor. In some embodiments, the modification may be a substitution of S298, such as S298A, according to Kabat's EU index. In some embodiments, the modification may be a substitution of S239, I332, and A330, such as S239D / I332E / A330L. In some embodiments, the modification may be a substitution of S239 and I332, such as S239D / I332E.

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

[0092] In some embodiments, the modification involves the substitution of one or more amino acids that reduces the binding affinity of the IgG Fc domain or IgG Fc region to the FcyRIIIA receptor, but does not significantly affect the binding affinity to 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 may be a substitution of K338, such as K338A, according to the EU index of Kabat. In some embodiments, the modification may be a substitution of D376, such as D376A, according to the EU index of Kabat.

[0093] In some embodiments, the modification involves the substitution of one or more amino acids that increases the binding affinity of the IgG Fc domain or IgG Fc region to the FcyRIIIA receptor, without affecting the binding affinity to the FcyRII receptor. In some embodiments, the modification may be a substitution of E333, such as E333A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of K334, such as K334A, according to the Kabat EU index. In some embodiments, the modification may be a substitution of A339, such as A339T, according to the Kabat EU index. In some embodiments, the modification may be a substitution of S239 and S332, such as S239D / I332E, according to the Kabat EU index.

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

[0095] Other substitutions affecting the interaction of the IgG Fc domain with one or more Fc gamma receptors are disclosed in U.S. Patents 7,317,091 and 8,969,526 (these disclosures are incorporated herein by reference).

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

[0097] In some embodiments, the modification may include the substitution of a single amino acid residue that increases the binding affinity of the IgG Fc domain to FcRn compared to the wild-type or reference IgG Fc domain. In some embodiments, the modification may include the substitution at V308, such as V308P, according to Kabat's EU index. In some embodiments, the modification may include the substitution at M428, such as M428L, according to Kabat's EU index. In some embodiments, the modification may include the substitution at N434, such as N434A, or N434H, according to Kabat's EU index. In some embodiments, the modification may include the substitution at T250 and M428, such as T250Q and M428L, according to Kabat's EU index. In some embodiments, the modification may include the substitution at M428 and N434, such as M428L and N434S, N434A, or N434H, according to Kabat's EU index. In some embodiments, modifications may include substitutions at M252, S254, and T256, such as M252Y / S254T / T256E, according to Kabat's EU index. In some embodiments, modifications may be substitutions 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 (which is incorporated herein by reference).

[0098] In some embodiments, the MIC antibody or its antigen-binding moiety has a modified constant region (Fc region) or Fc domain that modifies complement-dependent cell-mediated 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 in inducing CDC via the classical complement activation pathway.

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

[0100] In various embodiments, MIC antibodies, their antigen-binding moieties, and other binders can be produced in human, mouse, or other animal cell lines. Recombinant DNA expression can be used to produce MIC antibodies, their antigen-binding moieties, and other binders. This allows for the production of a spectrum of MIC antibodies, as well as MIC antigen-binding moieties and other binders (including fusion proteins) in a selected host species. Production of MIC antibodies, their antigen-binding moieties, and other binders in bacteria, yeast, transgenic animals, and chicken eggs is also an alternative to cell-based production systems. The main 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 represented by SEQ ID NO: 1 is encoded by a nucleic acid. In some embodiments, the MIC VL polypeptide having the amino acid sequence represented by SEQ ID NO: 2 is encoded by a nucleic acid. In some embodiments, the MIC VH polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is encoded by a nucleic acid having the sequence represented by SEQ ID NO: 21. In some embodiments, the MIC VL polypeptide having the amino acid sequence represented by SEQ ID NO: 2 is encoded by a nucleic acid having the sequence represented by SEQ ID NO: 22.

[0102] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide sequence,” or “nucleotide” refer to a macromolecule incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogues. Nucleic acids can be single-stranded or double-stranded. Single-stranded nucleic acids may be single-stranded nucleic acids of denatured double-stranded DNA. In some embodiments, nucleic acids may be cDNA, for example, nucleic acids lacking introns.

[0103] Nucleic acid molecules encoding amino acid sequences of MIC antibodies, their antigen-binding moieties, and other binders can be prepared by various methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding MIC antibodies, antigen-binding moieties, or other binders. Furthermore, nucleotide sequences encoding MIC antibodies or antigen-binding moieties and other binders can be prepared using oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis. Nucleic acid sequences encoding at least an MIC antibody, its antigen-binding moiety, a binder, or its polypeptide described herein can be recombined into vector DNA according to conventional techniques such as blunt-end or staggered-end ligation, restriction enzyme digestion to provide suitable ends, packing of appropriate attached ends, alkaline phosphatase treatment to avoid undesirable conjugation, and ligation with a suitable ligase. Techniques for such operations 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 create nucleic acid sequences and vectors encoding MIC antibodies or their antigen-binding moieties or their VH or VL polypeptides.

[0104] Nucleic acid molecules such as DNA contain nucleotide sequences that carry regulatory information for transcription and translation. If such sequences are "operably linked" to nucleotide sequences encoding polypeptides, the polypeptide is said to be "expressible." An operable linkage is one in which a regulatory DNA sequence and the DNA sequence to be expressed (e.g., an MIC antibody or its antigen-binding portion) are linked in such a way that a recoverable amount of polypeptide(s) or antigen-binding portion can be expressed. The precise properties of the regulatory regions required for gene expression can vary from organism to organism, as is well known in similar techniques. See, for example, Sambrook et al., 1989; Ausubel et al., 1987–1993.

[0105] Therefore, the expression of the MIC antibodies or their antigen-binding moieties described herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, bird, and mammalian cells in vivo or in situ, or host cells derived from mammals, insects, birds, or yeast. Mammalian cells or tissues may be derived from humans, primates, hamsters, rabbits, rodents, cattle, pigs, sheep, horses, goats, dogs, or cats, but any other mammalian cells may 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 thus produced can be processed in vivo or purified and processed in vitro to enable the synthesis of the MIC antibodies or their antigen-binding moieties described herein having specific amino-terminal sequences. In addition, problems associated with the retention of methionine residues derived from the start codon in direct yeast (or bacterial) expression can be avoided. (See, for example, Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).) Recombinant MIC antibodies or their antigen-binding moieties can be obtained using one of a series of yeast gene expression systems that incorporate promoter and termination elements from actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, promoter and terminator signals of phosphoglycerate kinase genes can be used.

[0106] The production of MIC antibodies or their antigen-binding moieties in insects can be achieved, for example, by infecting an insect host with a baculovirus engineered to express a polypeptide by a method known to those skilled in the art. See Ausubel et al., 1987–1993.

[0107] In some embodiments, the nucleic acid sequence to be introduced (encoding an MIC antibody or its antigen-binding portion or its polypeptide) is incorporated into a plasmid or viral vector capable of autonomous replication in recipient host cells. A wide variety of vectors can be used for this purpose and are known and available to those skilled in the art. See, for example, 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 without the vector, the desired copy number of the vector in a particular host, and whether it is desirable that the vector can be "shuttle" between host cell species.

[0108] Representative prokaryotic vectors known in this field include plasmids that can replicate in E. coli. Other gene expression elements useful for expressing MIC antibodies or DNA encoding their antigen-binding portions include, but are not limited to, (a) viral transcription promoters and their enhancer elements such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)), and Moloney mouse leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)), (b) splice regions and polyadenylation sites derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites in SV40 (Okayama et al., 1983), etc. Immunoglobulin-encoding DNA genes can be expressed using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S-globin intercalated sequence, the immunoglobulin and rabbit S-globin polyadenylation sites, and the SV40 polyadenylation element, as described by Liu et al., infra, and Weidle et al., 51 Gene 21 (1987).

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

[0110] In some embodiments, the transcription promoter can be a viral LTR sequence for the expression of a DNA coding region in rodent cells, and the transcription promoter enhancer can be either or both a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, as well as a polyadenylated region and a transcription termination region. In other embodiments, a DNA sequence encoding another protein is 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 its antigen-binding moiety (e.g., VH having the amino acid sequence represented by SEQ ID NO: 1 and / or VL having the amino acid sequence represented by SEQ ID NO: 2, or a variant thereof) are then transfected alone with a nucleotide encoding the MIC antibody or antibody polypeptide or its antigen-binding moiety, or co-transfected with a polynucleotide(s) encoding the VH and VL chain coding regions. The transfected recipient cells are cultured under conditions that allow expression of the incorporated coding region, and the expressed antibody chain or intact antibody or antigen-binding moiety is recovered from the culture.

[0112] In some embodiments, nucleic acids containing coding regions encoding an MIC antibody or its antigen-binding portion (e.g., VH having the amino acid sequence represented by SEQ ID NO: 1 and / or VL having the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof) are assembled into separate expression vectors and subsequently used to cotransfect recipient host cells. Each vector may contain one or more selectable genes. For example, in some embodiments, two selectable genes are used: a first selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic cell system, with each vector having a set of coding regions. This strategy results in a vector that first directs the production of a nucleotide sequence in a bacterial system and enables amplification. The DNA vector thus produced and amplified in the bacterial host is then used to cotransfect eukaryotic cells, enabling the selection of cells to be cotransfected that carry the desired transfected nucleic acid (e.g., the heavy and light chains of an MIC antibody). Non-limiting examples of selectable genes for use in a bacterial system 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 (named gpt) and the Tn5-derived phosphotransferase gene (named neo). Alternatively, fusion nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

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

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

[0115] Yeast offers superior advantages over bacteria in the production of heavy and light chains of immunoglobulins. Yeast performs post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies exist that utilize strong promoter sequences and high-copy-number plasmids and can be used to produce desired proteins in yeast. Yeast recognizes the leader sequence of a cloned mammalian gene product and secretes a polypeptide (i.e., pre-polypeptide) containing the leader sequence. See, for example, Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0116] Yeast gene expression systems can be used to evaluate the levels of antibody production, secretion, and stability, as well as the assembled MIC antibodies and their antigen-binding moieties, according to specifications. Various yeast gene expression systems can be utilized, incorporating promoters and termination elements from actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast is grown in glucose-rich media. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be used. 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, for example, U.S. Publication US2006 / 0270045A1.

[0117] Bacterial strains can also be used as hosts for the production of antibody molecules or their antigen-binding moieties as described herein, including E. coli K12 strains such as E. coli W3110 (ATCC 27325), Bacillus species, Enterobacteria such as Salmonella tiphimurium or Serratia marcesens, and various Pseudomonas species. In relation to these bacterial hosts, plasmid vectors containing replicons and regulatory sequences derived from species compatible with the host cells are used. These vectors have specific genes that can provide phenotypic selection in transformed cells as well as replication sites. Several approaches can be taken to evaluate expression plasmids for the production of MIC antibodies and their antigen-binding moieties 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. Mammalian cells 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 their antigen-binding moieties.

[0119] Mammalian cells that may be useful as hosts for antibody protein production include fibroblast-derived cells such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells, in addition to the lymphoid-derived cells mentioned 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, specific eukaryotic host cells are selected based on their ability to perform 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 by 293 cells.

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

[0121] In some embodiments, the antibody or its antigen-binding moiety (e.g., VH having the amino acid sequence represented by SEQ ID NO: 1 described herein and / or VL having the amino acid sequence represented by SEQ ID NO: 2 or its variants) 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); and 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., VH having the amino acid sequence represented by SEQ ID NO: 1 described herein and / or VL having the amino acid sequence represented by SEQ ID NO: 2 or its variants) in mammalian cells (see Glover, 1985). Various approaches can be followed to obtain intact antibodies. As described above, it is possible to achieve intracellular association and linkage of the VH and VL chains by co-expressing the VH and VL chains, and optionally related constant regions, in the same cell to form a complete tetrameric H2L2 antibody or its antigen-binding moiety. Co-expression can occur by using the same or different plasmids in the same host. The nucleic acids encoding the VH and VL chains or their antigen-binding moieties (e.g., VH having the amino acid sequence represented by SEQ ID NO: 1 described herein and VL having the amino acid sequence represented by SEQ ID NO: 2 or its variants) can be placed in the same plasmid, and then these can be transfected into cells to directly select cells expressing both chains. Alternatively, cells may be initially transfected with a plasmid encoding one chain, such as a VL chain, and then the resulting cell line may be transfected with a VH chain plasmid containing a second selectable marker. Cell lines producing antibodies or their antigen-binding moieties may be transfected via either pathway with plasmids encoding additional copies of the peptide, VH, VL, or VH-plus-VL chain (e.g., VH having the amino acid sequence represented by SEQ ID NO: 1 described herein and / or VL or a variant thereof having the amino acid sequence represented by SEQ ID NO: 2), along with additional selectable markers, to generate cell lines with enhanced properties, such as higher production of assembled MIC antibodies or their antigen-binding moieties, or improved stability of the transfected cell line.

[0123] Furthermore, plants are emerging as a convenient, safe, and economical alternative expression system for recombinant antibody production based on large-scale cultures of microbial or animal cells. MIC-conjugated antibodies or antigen-binding moieties can be expressed in plant cell cultures or in normally grown plants. Expression in plants may be systemic, limited to intracellular plastids, or limited to seeds (endosperm). See, for example, U.S. Patent Publication 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 an intact antibody, the variable regions (VH and VL) of an MIC antibody (e.g., VH having the amino acid sequence represented by SEQ ID NO: 1 described herein and / or VL having the amino acid sequence represented by SEQ ID NO: 2 or a variant thereof) are typically ligated to at least a portion of the immunoglobulin constant region (Fc), typically to at least a portion of human immunoglobulin. Human constant region DNA sequences can be isolated from various human cells, such as immortalized B cells, by well-known procedures (WO87 / 02671; the whole of which is incorporated herein by reference). MIC-conjugated antibodies may contain both light-chain and heavy-chain constant regions. The heavy-chain constant region may include the CH1, hinge, CH2, CH3, and possibly CH4 regions. In some embodiments, the CH2 domain may be deleted or omitted.

[0125] Alternatively, the methods described for the production of single-chain antibodies (see, for example, U.S. Patent 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); these are incorporated herein by reference in their entirety) can be adapted to produce single-chain antibodies that specifically bind to MICs. Single-chain antibodies are formed by linking the heavy chain and light chain variable regions of the Fv region (having the amino acid sequences represented by Sequence IDs 1 and 2 described herein, or variants thereof (e.g., modified by optional substitutions, deletions, and / or insertions of 1 to 8 amino acids)) via amino acid crosslinks to obtain a single-chain polypeptide. The technique for assembling functional Fv fragments in E. coli can also be used (see, for example, Skerra et al., Science 242:1038-1041 (1988); this entire work is incorporated herein by reference).

[0126] Intact (e.g., complete) antibodies, their dimers, individual light and heavy chains, or their antigen-binding moieties can be recovered and purified by known methods, such as immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high-performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof. See Scopes, Protein Purification (Springer-Verlag, NY, 1982) for general information. Substantially pure MIC-conjugated antibodies or their antigen-binding moieties with at least approximately 90%–95% homogeneity are particularly advantageous for pharmaceutical applications, as are those with 98%–99% or higher homogeneity. Once partially or to the desired level of homogeneity, the intact MIC antibodies or their antigen-binding moieties can then be used therapeutically, in the development and implementation of assay procedures, immunofluorescence staining, etc. See Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981) for general information.

[0127] Furthermore, as described herein, for therapeutic use in humans, the MIC antibody or its antigen-binding moiety can be further optimized to reduce potential immunogenicity while maintaining functional activity. In some embodiments, the optimized MIC-binding antibody or its antigen-binding moiety is derived from an MIC antibody 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, wherein the variable framework regions of the heavy chain and light chain are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions in the framework region, and the CDR of the heavy chain or light chain variable region is not modified. In some embodiments, the optimized MIC-binding antibody or its antigen-binding moiety is derived from an MIC-binding antibody 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, wherein the variable framework regions of the heavy and light chains are optionally modified by substitutions, deletions, or insertions of 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids in the framework regions, and the CDR of the heavy-chain or light-chain variable region is not modified. In this context, functional activity means an MIC antibody or its antigen-binding moiety that can exhibit one or more known functional activities related to the MIC-binding antibody or its antigen-binding moiety 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. In any of these embodiments, the functional activity of the MIC-binding antibody or its antigen-binding moiety includes specific binding to the MIC with a binding affinity greater than that of antibody B10G5. Additional functional activities include inhibition of the MIC and / or anticancer activity.Furthermore, a functionally active MIC antibody or its antigen-binding moiety means that, when measured in a specific assay, such as a biological assay, the polypeptide exhibits activity similar to or better than that of a reference antibody or its antigen-binding moiety described herein (for example, an MIC-conjugated antibody or its antigen-binding moiety 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 or a variant thereof, as described herein, with or without dose dependence). If dose dependence exists, it does not need to be identical to the dose dependence of the reference antibody or its antigen-binding moiety, but rather substantially similar to or better than the dose dependence of a given activity compared to the reference antibody or its antigen-binding moiety described herein (i.e., the candidate polypeptide will exhibit greater activity than the reference antibody).

[0128] Other embodiments of MIC antibodies and their antigen-binding moieties or other binders relate to compositions comprising an active ingredient (i.e., a nucleic acid encoding the MIC antibody or its antigen-binding moiety or other binder described herein, or the antibody or its antigen-binding moiety or other binder described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term “pharmaceutical composition” refers to an activator in combination with a pharmaceutically acceptable carrier approved for use in the pharmaceutical industry. As used herein, “pharmaceutically acceptable” means a compound, material, composition and / or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of safe medical judgment, and that is commensurate with a reasonable benefit / risk ratio.

[0129] The preparation of pharmacological compositions containing an active ingredient dissolved or dispersed therein is well understood in the art and does not need to be limited in any way based on a particular formulation. Typically, such compositions are prepared as injectable liquid solutions or suspensions, or as solid forms suitable for rehydration or suspension in liquid before use. Preparations may also be emulsified or provided as liposome compositions. MIC antibodies or their antigen-binding moieties or other binders 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 include, for example, water, saline, dextrose, glycerol, ethanol or similar, and combinations thereof. Furthermore, if necessary, the pharmaceutical composition may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers and similar, which enhance or maintain the effect of the active ingredient (e.g., MIC antibodies or their antigen-binding moieties). The pharmaceutical compositions described herein may contain pharmaceutically acceptable salts of their components. Examples of pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, or mandelic acid (formed with free amino groups of polypeptides). Salts formed with free carboxyl groups may also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine). Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers include sterile aqueous solutions containing an active ingredient (e.g., an MIC antibody and / or its antigen-binding moiety) and water, as well as sterile aqueous solutions that may contain a buffer such as sodium phosphate, physiological saline, or both (e.g., phosphate-buffered saline) at a physiological pH. Furthermore, aqueous carriers may contain two or more buffer salts, as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol, and other solutes. In addition to water, the liquid composition may also contain a liquid phase.Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active ingredient effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0130] In some embodiments, a pharmaceutical composition comprising the MIC antibody or its antigen-binding moiety described herein, or a nucleic acid encoding the MIC antibody or its antigen-binding moiety described herein, may be a lyophilized product.

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

[0132] Cancer treatment In some embodiments, the MIC antibody or its antigen-binding moiety or other binder described herein can be used in a manner comprising administering the MIC antibody or its antigen-binding moiety or other binder described herein to a person requiring it. In some embodiments, the MIC-binding antibody or its antigen-binding moiety comprises (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. In some embodiments, the MIC-binding antibody or its antigen-binding moiety comprises (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, wherein the variable framework regions of the heavy and light chains 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 CDR of the heavy or light-chain variable region is not modified. In some embodiments, the MIC-conjugated antibody or its antigen-binding moiety includes (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, wherein the variable framework regions of the heavy and light chains 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 region, and the CDR of the heavy or light-chain variable region is not modified. In any of these embodiments, the MIC-conjugated antibody binds specifically to the MIC with a higher binding affinity than antibody B10G5.

[0133] In some embodiments, the subject requires treatment for cancer and / or malignant tumors. In some embodiments, the subject requires treatment for MIC+ cancer or MIC+ malignant tumors, such as epithelial cell carcinoma, MIC+ solid tumors, or MIC+ hematopoietic malignancies. In some embodiments, the method is for treating a subject with MIC+ cancer or malignant tumors. In some embodiments, the method is for treating epithelial cell carcinoma or hematopoietic malignancies in a subject. In some embodiments, the method is for treating MIC+ epithelial cell carcinoma or MIC+ hematopoietic malignancies in a subject. As used herein, “epithelial cell carcinoma” means cancer that originates from epithelial cells.

[0134] The methods described herein involve administering a therapeutically effective dose of an MIC-binding antibody or its antigen-binding moiety or other binder. As used herein, the terms “therapeutic dose,” “effective dose,” or “effective dose” refer to the amount of the MIC antibody or its antigen-binding moiety or other binder described herein that provides therapeutic utility in the treatment, management, or prevention of recurrence of a tumor or malignant tumor, for example, an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of the tumor or malignant tumor. Determining the therapeutically effective dose is well within the capabilities of those skilled in the art. Generally, the therapeutically effective dose 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 proliferation of cells that interfere with the normal functioning of organs and systems of the body. Cancer or malignant tumors can be primary or metastatic, meaning they can become invasive and spread tumor growth to tissues far from the original site of tumor growth. “Tumor” refers to the uncontrolled proliferation of cells that interfere with the normal functioning of organs and systems of the body. A person with cancer is a person who has objectively measurable cancer cells present in their 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 spread to other vital organs can ultimately lead to the death of the person due to the deterioration of the function of the affected organ. Hematological malignancies (blood cancers), such as leukemia and lymphoma, can, for example, overwhelm the person’s normal hematopoietic compartment, thereby causing hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia) and ultimately leading to death.

[0136] Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer 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). This includes cancers such as glioblastoma (GBM), hepatocellular carcinoma, liver cancer, neoplasia in situ, kidney 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), lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, oral cavity, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, respiratory cancers, salivary gland cancers, sarcomas, skin cancers, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary tract cancers, vulvar cancer; and other carcinomas and sarcomas, as well as B-cell lymphomas (low-grade / follicular non-Hodgkin This includes, but is not limited to, lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, giant lesion NHL, mantle cell lymphoma, AIDS-associated lymphoma, and Waldenstorm macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), pilaris cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis, edema (brain tumor edema) associated with nevus syndromes, 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, cancer or malignant tumors are 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 proteins released from cancer cells(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 tumor space or the circulatory or lymphatic system. Thus, these cells may exhibit the MIC protein on their surface only for a short period. That is, the term encompasses cancer cells and tumor cells that excrete or secrete sMIC proteins via exosomes or other mechanisms, regardless of whether a 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 to be an “MIC-positive cancer” as the term is used herein. Some non-limiting examples of MIC-positive cancers include epithelial cell carcinomas and hematopoietic malignancies. In some embodiments, MIC-positive cancer or malignant tumor may be MIC-positive prostate cancer and / or its metastases.

[0139] As used herein, “Subject” refers to a human or an animal. Typically, animals are vertebrates such as primates, rodents, domesticated animals, or game animals. Primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domesticated animals and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canid species such as dogs, foxes, wolves, birds such as chickens, emus, ostriches, and fish such as trout, catfish, and salmon. In certain embodiments, the subject is a mammal, such as a primate, such as 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, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals can be advantageously used, for example, as subjects representing 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] Participants are those who have been previously diagnosed with or identified as having sMIC+ cancer or MIC+ cancer and require treatment, but do not need to have already received treatment for sMIC+ cancer or MIC+ cancer. Alternatively, participants may also be those who have not been previously diagnosed with sMIC+ cancer or MIC+ cancer and require treatment. Participants may be those who exhibit one or more risk factors for a condition or one or more complications associated with sMIC+ cancer or MIC+ cancer, or those who do not exhibit any risk factors. Participants who “require treatment” for a particular sMIC+ cancer may be those who have that condition or have been diagnosed with that condition. In other embodiments, participants who are “at risk of developing” a condition refer to those who have been diagnosed as being at risk of developing the condition (e.g., sMIC+ cancer or MIC+ cancer).

[0142] When used herein, the terms “to treat,” “to treat,” “to treat,” or “to improve” mean, when used in relation to a disease, disorder, or medical condition, a therapeutic action for a condition aimed at reversing, alleviating, improving, inhibiting, slowing or stopping the progression or severity of symptoms or conditions. The term “to treat” includes reducing or alleviating at least one adverse event or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Or, treatment is “effective” if the progression of a condition is reduced or stopped. In other words, “treatment” includes not only improvement of symptoms or markers but also stopping or at least slowing or worsening of the progression of symptoms that would be expected without treatment. Beneficial or desirable clinical outcomes include, but are not limited to, a decrease in the subject’s free sMIC levels, relief of one or more symptoms, reduction in the extent of a defect, stabilization of a cancer or malignant condition (i.e., no worsening), delay or slowing of tumor growth and / or metastasis, and an extension of life compared to the condition that would be expected without treatment. As used herein, the term “administer” means providing to a subject by a method or route that results in the binding of the MIC-conjugating antibody or its antigen-binding moiety or other binder described herein, or the nucleic acid encoding the MIC-conjugating antibody or its antigen-binding moiety or other binder described herein, to a MIC. Similarly, a pharmaceutical composition comprising the MIC-conjugating antibody or its antigen-binding moiety or other binder described herein, or the nucleic acid encoding the MIC-conjugating antibody or its antigen-binding moiety or other binder disclosed herein, may be administered by any suitable route that results in an effective treatment in a subject.

[0143] The dosage range of the MIC-binding antibody or its antigen-binding moiety depends on the potency and includes an amount sufficient to produce the desired effect, such as a decrease in MIC levels, slowing of tumor growth, or reduction in tumor size. The dosage should not be so high as to cause unacceptable adverse side effects. Generally, the dosage varies depending on the age, condition, and sex of the subject and can be determined by those skilled in the art. The dosage may also be adjusted by the individual physician if complications occur. In some embodiments, the dosage ranges from 0.01 mg / kg body weight to 10 mg / kg body weight. In some embodiments, the dose range is from 0.05 mg / kg body weight to 5 mg / kg body weight. In some embodiments, the dosage ranges from 0.01 mg / kg body weight to 10 mg / kg body weight. In some embodiments, the dose range is from 0.05 mg / kg body weight to 5 mg / kg body weight. Alternatively, the dose range can be determined to maintain serum levels between 1 pg / mL and 1000 ug / mL. In systemic administration, subjects can be administered therapeutic doses such as 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 higher.

[0144] The above doses can be administered repeatedly. In practice, long-term administration is intended to promote an immune attack against tumors, cancer cells, or malignant cells by removing free or unbound MICs, for example, to first treat the tumor, cancer, or malignant tumor itself, and then provide continuous monitoring for the development of tumor cells that acquire the ability to release MICs. In preferred embodiments, the above doses are administered weekly, bi-weekly, every three weeks, or monthly, over several weeks or months. The duration of treatment depends on the patient's clinical course and response to treatment.

[0145] In some embodiments, the dose may be about 0.01 mg / kg to about 100 mg / kg. In some embodiments, the dose may be about 0.01 mg / kg to about 25 mg / kg. In some embodiments, the dose may be about 0.01 mg / kg to about 20 mg / kg. In some embodiments, the dose may be about 0.01 mg / kg to about 15 mg / kg. In some embodiments, the dose may be about 0.01 mg / kg to about 100 mg / kg. In some embodiments, the dose may be about 0.01 mg / kg to about 25 mg / kg. In some embodiments, the dose may be about 0.01 mg / kg to about 20 mg / kg. In some embodiments, the dose may be about 0.01 mg / kg to about 15 mg / kg. In some embodiments, the dose may be about 0.1 mg / kg to about 10 mg / kg. In some embodiments, the dose may be about 1 mg / kg to about 100 mg / kg. In some embodiments, the dose may be approximately 1 mg / kg to approximately 25 mg / kg. In some embodiments, the dose may be approximately 1 mg / kg to approximately 20 mg / kg. In some embodiments, the dose may be approximately 1 mg / kg to approximately 15 mg / kg. In some embodiments, the dose may be approximately 2 mg / kg. In some embodiments, the dose may be approximately 4 mg / kg. In some embodiments, the dose may be approximately 5 mg / kg. In some embodiments, the dose may be approximately 6 mg / kg. In some embodiments, the dose may be approximately 8 mg / kg. In some embodiments, the dose may be approximately 10 mg / kg. In some embodiments, the dose may be approximately 15 mg / kg. In some embodiments, the dose may be approximately 100 mg / kg. 2 ~about 700mg / m 2 This is possible. In some embodiments, the dose is approximately 250 mg / m². 2 It is possible. In some embodiments, the dose is approximately 375 mg / m². 2 This is possible. In some embodiments, the dose is approximately 400 mg / m². 2 This is possible. In some embodiments, the dose is approximately 500 mg / m². 2 It is possible.

[0146] In some embodiments, the dose may be administered intravenously. In some embodiments, the intravenous administration may be an infusion carried out over a period of about 10 minutes to about 4 hours. In some embodiments, the intravenous administration may be an infusion carried out 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 approximately every two weeks. In some embodiments, the dose may be administered approximately 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 approximately 2 to 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, a total of more than 10 doses are administered.

[0149] A pharmaceutical composition containing an MIC-conjugated antibody or its antigen-binding moiety or other MIC-binding agent may be administered in unit doses. When used in reference to a pharmaceutical composition, the term "unit dose" refers to a physically discrete unit suitable as a unit dose to a subject, each unit containing a predetermined amount of the active substance (e.g., an MIC-conjugated antibody or its antigen-binding moiety) calculated to produce the desired therapeutic effect in relation to the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0150] In some embodiments, a pharmaceutical composition comprising an MIC-conjugated antibody or its antigen-binding moiety, or either or both thereof, is administered in conjunction with immunotherapy. As used herein, “immunotherapy” refers to a therapeutic strategy designed to induce or enhance the subject’s own immune system to fight cancer or malignant tumors. Examples of immunotherapies include, but are not limited to, adoptive cell therapies (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, as well as antibodies that disrupt metabolic signaling, immune cytokines such as gamma-chain family cytokines IL-2, IL-15, and their variants, chemotoxins, radiotherapy, and epigenetic modifiers. For example, see 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). These disclosures 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 extracted from the subject's blood, genetically modified to express chimeric antigen receptors against appropriate targets on cancer cells, and then re-administered to the subject. See, for example, 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 privately produced cell cells (PBMCs) are extracted from the subject's blood, primed to respond to a specific antigen, and then re-administered to the subject (e.g., cyproisel T). (See, for example, WO2001 / 039594; WO2001 / 074855; and WO1999 / 063050.)

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

[0154] In some embodiments, immunotherapy includes the administration of checkpoint inhibitors. In some embodiments, the checkpoint inhibitors are selected from inhibitors or from 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 drugs 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, mouse 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 adnectin, anti-CTLA-4 domain antibodies, single-chain anti-CTLA-4 mAbs, heavy-chain anti-CTLA-4 mAbs, light-chain anti-CTLA-4 mAbs, CTLA-4 inhibitors that stimulate the costimulatory pathway, antibodies disclosed in PCT Publication WO2001 / 014424, antibodies disclosed in PCT Publication WO2004 / 035607, antibodies disclosed in U.S. Publication 2005 / 0201994, and antibodies disclosed in European Patent EP1212422B 1. 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 Publications WO01 / 14424 and WO00 / 37504, and U.S. Publications 2002 / 0039581 and 2002 / 086014.Other anti-CTLA-4 antibodies that can be used in the method of the present invention include, for example, those disclosed in WO98 / 42752, U.S. Patents 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. Patents 5,977,318, 6,682,736, 7,109,003 and 7,132,281.

[0155] Suitable anti-PD-1 and anti-PD-L1 therapeutic agents include, for example, anti-PD-1 and anti-PD-L1 antibodies, human anti-PD-1 and anti-PD-L1 antibodies, mouse 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 adnectin and anti-PD-L1 adnectin, anti-PD-1 domain antibodies and anti-PD-L1 domain antibodies, single-chain anti-PD-1 mAbs and single-chain anti-PD-L1 mAbs, heavy-chain anti-PD-1 mAbs and heavy-chain anti-PD-L1 mAbs, and light-chain anti-PD-1 mAbs and light-chain anti-PD-L1 mAbs. In certain embodiments, anti-PD-1 therapeutic agents include nivolumab, pembrolizumab, pizilizumab, MEDI0680, and combinations thereof. In other specific embodiments, anti-PD-L1 therapeutic agents 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 herein 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 (Infinzi).

[0158] In some embodiments, a method is provided for improving treatment outcomes in subjects receiving immunotherapy. This method generally includes administering an effective dose of immunotherapy to a subject with cancer and administering a therapeutically effective dose of a binder or a pharmaceutical composition thereof to the subject, wherein the binder specifically binds to the circulating MIC, and the treatment outcomes of the subject are improved compared to administration of immunotherapy alone. In some embodiments, the binder comprises (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, 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 region, and the binder specifically binds to the MIC with a higher binding affinity than the antibody B10G5. In some embodiments, the binder comprises (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, and the binder specifically binds to the MIC. In some embodiments, the binder is an antibody or its antigen-binding portion. In some embodiments, the binder is a monoclonal antibody, Fab, Fab′, F(ab′), Fv, disulfide-linked Fc, scFv, single-domain antibody, diabody, bispecific antibody, or multispecific antibody.

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

[0160] In some embodiments, the method involves administering chemotherapy or other treatment that impairs the immune system by depleting endogenous NK cells or T cells or their precursors, followed by further administration of an MIC antibody or its antigen-binding moiety 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 radiotherapy or chemotherapy. Non-exclusive examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and pigosulfan; aziridines, e.g., benzodopa, carbocon, metsuredopa, and uredopa; ethyleneimines and methylamelamines, e.g., altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (specifically, bratacin and bratacinone); camptothecin (synthetic analogs such as topotecan); bryostatin; calistatin; CC-1065 (its adzelesin, karzelesin, and beizelesin synthetic analogs, etc.); cryptophycin (specifically, cryptophycin 1 and cryptophycin 8); and drasta Tin, duocalmycin (synthetic analogs KW-2189 and CB1-TM1, etc.), eryuterobin, pancratistatin, sarcodictiin, spongistatin, nitrogen mustard, e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, and uracil mustard, nitrosourea, e.g., camulstine, chlorozotosin, fotemustine, lomustine, nimustine, and ranimustine, antibiotics, e.g., engine antibiotics (e.g., calicheamicin, specifically calicheamicin gamma II and calicheamicin omega II (e.g., Agnew, Chem. Intl. Ed. Engl.),33:183-186(1994)); Dynemicin (including Dynemicin A); Bisphosphonates (such as clodronate); Esperamicin, and neocardinostatin chromophores and related pigment proteins (endiin antibiotic chromophores), acrasinomycins, actinomycin, ausramycin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, 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, marcelomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozosin Tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites (methotrexate and 5-fluorouracil (5-FU), etc.); folic acid analogs (denopterin, methotrexate, pteropterin, trimethrexate, etc.); purine analogs (fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.); pyrimidine analogs (ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyl Fluridine, enocitabine, phloxuridine, etc.; androgens (carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.); anti-adrenal agents (aminoglutethimide, mitotane, trilostane, etc.); folic acid supplements (floric acid, etc.); acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren; edatraxate; defofamine; Demecoltin; Diadiquan; Elfomitin; Erliptinium acetate; Epotilon; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Ronidynin; Maytansinoids (such as Maytansine and Anthamitosine); Mitoguazone; Mitoxantrone; Mopidamnol; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (Trademark) Polysaccharide Complex (JHS Natural Products, Eugene, Oreg.)); Lazoxane, Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2”-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Beraclin A, Loridine A, and Anguidin); Urethanes; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinosides ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids (e.g., TAXOL® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE.RTM. Cremoforalbumin-Modified Nanoparticle Formulation of Paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® Docetaxel (Rhone-Poulenc) Rorer, Antony, France), chlorambucil, GEMZAR (trademark) gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs, e.g., cisplatin, oxaliplatin and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, NAVELBINE (trademark) vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, Xeloda, ibandronate, irinotecan (Camptosar, CPT-11) (5-FU and irinotecan) Examples include treatment regimens with leucovorin, 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 any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0161] "Radiation therapy" refers to the use of directed gamma or beta rays to induce sufficient damage to cells so that they either continue to function normally or their ability to be completely destroyed is limited.

[0162] The description of embodiments of this disclosure is not intended to be exhaustive or to limit the disclosure to the exact form disclosed. Specific embodiments of the disclosure and examples thereof are described herein for illustrative purposes, but various equivalent modifications are possible within the scope of the disclosure, as will be recognized by those skilled in the art. The teachings of the disclosure provided herein may be applied to other procedures or methods as needed. Further embodiments may be provided by combining the various embodiments described herein. Aspects of the disclosure may be modified as needed to provide further embodiments of the disclosure by adopting the structure, function, and concepts of the above-mentioned references and applications. These and other modifications may be made to the disclosure in light of the detailed description.

[0163] Certain elements of any of the embodiments described above can be combined or substituted with elements of other embodiments. Furthermore, while advantages related to specific embodiments of this disclosure are described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments are required to exhibit such advantages so that they fall within the scope of this disclosure.

[0164] All patents and other recognized publications are expressly incorporated herein by reference, for example, for the purpose of describing and disclosing procedures described in such publications that may be used in connection with the present invention. These publications are provided only for disclosures prior to the filing date of this application. Nothing in this regard should be construed as an acknowledgment by the inventors that they have no prior rights to such disclosures, either due to prior invention or for any other reason. Any date statements or descriptions of the contents of these documents, based on the information available to the applicant, do not constitute any acknowledgment of the accuracy of the dates or the contents of these documents.

[0165] Non-exclusive exemplary embodiments The present invention is further described by the following embodiments, which should not be construed as limiting. 1. A binder comprising (i) a heavy chain variable (VH) region having an amino acid sequence represented by SEQ ID NO: 1, and (ii) a light chain variable (VL) region having an amino acid sequence represented by SEQ ID NO: 2, 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.

[0166] 2. A binder 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.

[0167] 3. A binder comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises a complementarity-determining region HCDR1 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, and the VL region comprises LCDR1 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 humanization framework region.

[0168] 4. The humanized VH framework region is derived from a human germ cell gene having an amino acid sequence represented by 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), as described in Embodiment 3.

[0169] 5. The humanized VL framework region is derived from a human germ cell gene having an amino acid sequence 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), as described in Embodiment 3 or Embodiment 4.

[0170] 6. The binder is a binder according to any one of Embodiments 1 to 5, which specifically binds to MIC.

[0171] 7. The binder is an antibody or its antigen-binding portion, as described in any one of Embodiments 1 to 6.

[0172] 8. The binder according to Embodiment 7, wherein the binder is a monoclonal antibody, Fab, Fab′, F(ab′), Fv, disulfide-bonded Fc, scFv, single-domain antibody, diabody, bispecific antibody, or multispecific antibody.

[0173] 9. The binder according to any one of the prior embodiments, wherein the heavy chain variable region further comprises a heavy chain steady region.

[0174] 10. The binder according to Embodiment 9, wherein the heavy chain constant region is an IgG isotype.

[0175] 11. The binder according to Embodiment 10, wherein the heavy chain constant region is the IgG1 constant region.

[0176] 12. The binder according to Embodiment 10, wherein the heavy chain constant region is the IgG4 constant region.

[0177] 13. The binder according to Embodiment 11, wherein the heavy chain variable region and the heavy chain constant region have the amino acid sequence represented by Sequence ID No. 3.

[0178] 14. The binder according to any one of the prior embodiments, wherein the light chain variable region further comprises a light chain steady region.

[0179] 15. The binder according to Embodiment 14, wherein the light chain constant region is a kappa isotype.

[0180] 16. The binder according to Embodiment 15, wherein the light chain variable region and the light chain constant region have the amino acid sequence represented by Sequence ID No. 4.

[0181] 17. The binder according to any one of embodiments 9 to 16, wherein the heavy chain constant region further comprises an amino acid modification that increases binding affinity to at least human Fc gamma RIII.

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

[0183] 19. The binder according to 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 binder according to any one embodiment, wherein the binder has single specificity.

[0185] 21. The binder according to any one of Embodiments 1 to 20, wherein the binder is divalent.

[0186] 22. The binder according to Embodiment 21, wherein the binder comprises a second binding domain and the binder is bispecific.

[0187] 23. The binder is the binder according to any one of Embodiments 1 to 22, which specifically binds to soluble MIC (sMIC).

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

[0189] 25. A pharmaceutical composition comprising a binder described in any of the prior embodiments and a pharmaceutically acceptable carrier.

[0190] 26. A nucleic acid encoding a heavy chain variable region having the amino acid sequence represented by Sequence ID No. 1, wherein the nucleic acid optionally has the nucleic acid sequence represented by Sequence ID No. 21.

[0191] 27. A nucleic acid encoding a light chain variable region having the amino acid sequence represented by Sequence ID No. 2, wherein the nucleic acid optionally has the nucleic acid sequence represented by Sequence ID No. 22.

[0192] 28. A nucleic acid encoding a binder according to any one of Embodiments 1 to 24, wherein the nucleic acid optionally has nucleic acid sequences represented by Sequence ID No. 21 and Sequence ID No. 22.

[0193] 29. A vector comprising the nucleic acid described in 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 of treatment a therapeutically effective amount of a binder according to any one of Embodiments 1 to 24, or a pharmaceutical composition according to Embodiment 25.

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

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

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

[0199] 35. The method according to Embodiment 32, wherein the cancer is a malignant hematological disease.

[0200] 36. The method according to Embodiment 33, wherein the malignant hematological disease is lymphoma or multiple myeloma.

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

[0202] 38. The method according to Embodiment 37, wherein the immunotherapy comprises adoptive cell therapy or a checkpoint inhibitor.

[0203] 39. The method according to Embodiment 38, wherein the immunotherapy includes adoptive cell therapy.

[0204] 40. The adoptive cell therapy according to Embodiment 39, 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.

[0205] 41. The method according to Embodiment 38, wherein the immunotherapy comprises a checkpoint inhibitor.

[0206] 42. The method according to Embodiment 41, wherein the checkpoint inhibitor is selected from antibodies that specifically bind to human PD-1, human PD-L1, or human CTLA4.

[0207] 43. The method according to Embodiment 42, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, semiprimab, or ipilimumab.

[0208] 44. The method according to any one of embodiments 31 to 43, wherein no chemotherapy is administered to the subject for at least four weeks prior to the administration of the binder.

[0209] 45. The method according to any one of embodiments 31 to 44, wherein the binder is administered intravenously.

[0210] 46. ​​The method according to any one of Embodiments 31 to 45, wherein the binder is administered in a dose of approximately 0.1 mg / kg to approximately 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 binder according to any one of Embodiments 1 to 24 or a pharmaceutical composition according to Embodiment 25.

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

[0213] 49. The method according to Embodiment 48, wherein the cancer is a carcinoma.

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

[0215] 51. The method according to Embodiment 48, wherein the cancer is a malignant hematological disease.

[0216] 52. The method according to Embodiment 51, wherein the malignant hematological disease is lymphoma or multiple myeloma.

[0217] 53. A method for improving the treatment outcomes of patients receiving immunotherapy, a. Administering an effective dose of immunotherapy to the aforementioned subjects who have cancer, b. Administering to the subject a therapeutically effective amount of the binder described in any one of Embodiments 1 to 24, or the pharmaceutical composition described in Embodiment 25, wherein the binder or pharmaceutical composition is specifically bound to the MIC. The method wherein the treatment outcomes of the subject are improved compared to the administration of immunotherapy alone.

[0218] 54. The method according to Embodiment 53, wherein the improved treatment outcome is an objective response selected from stable, partial response, or complete response.

[0219] 55. The method according to embodiment 53, wherein the improved treatment outcome is a reduction in tumor volume.

[0220] 56. The method according to Embodiment 53, wherein the improved treatment outcome is progression-free survival or disease-free survival.

[0221] 57. The method according to Embodiment 53, wherein the immunotherapy is adoptive cell therapy or a checkpoint inhibitor.

[0222] 58. The method according to Embodiment 57, wherein the immunotherapy is adoptive cell therapy.

[0223] 59. The adoptive cell therapy according to Embodiment 58, comprising autologous NK cells, allogeneic NK cells, autologous T cells, CAR-modified T cells, and CAR-modified NK cells.

[0224] 60. The method according to Embodiment 57, wherein the immunotherapy is a checkpoint inhibitor.

[0225] 61. The method according to 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 according to Embodiment 61, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, semiprimab, or ipilimumab.

[0227] 63. The method according to any one of embodiments 53 to 62, wherein no chemotherapy is administered to the subject for at least four weeks prior to the administration of the binder.

[0228] 64. The method according to any one of embodiments 53 to 63, wherein the binder is administered intravenously.

[0229] 65. The method according to any one of Embodiments 53 to 64, wherein the binder is administered in a dose of approximately 0.1 mg / kg to approximately 10 mg / kg.

[0230] 66. Use of any one of Embodiments 1 to 24 or the pharmaceutical composition described in Embodiment 25 for the treatment of MIC+ cancer in subjects.

[0231] 67. Use of any one of Embodiments 1 to 24 or the pharmaceutical composition according to Embodiment 25 for the treatment of MIC+ cancer in subjects receiving immunotherapy.

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

[0233] 69. The binder according to Embodiment 68, wherein VH has the amino acid sequence represented by SEQ ID NO: 24, and VL has the amino acid sequence represented by SEQ ID NO: 2. [Examples]

[0234] Example 1: Preparation of humanized antibodies The following examples describe 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 parental chain utilizes the first framework and contains most of the human sequences, including the minimum parental antibody framework sequence (named humanized HC1, LC1). The second humanized chain of each parental chain uses the same framework as before but includes additional parent sequences (named humanized HC2, LC2). Each third humanized chain utilizes the respective second framework and, like HC2 / LC2, also includes additional parent sequences fused with the human framework (named humanized HC3, LC3).

[0235] Next, preparations were made to create fully humanized antibodies of the variant by combining the humanized light and heavy chains. As described in the following examples, all possible combinations of humanized light and heavy chains were tested for their expression levels and binding affinity.

[0236] The full-length antibody gene was first constructed by synthesizing the variable region sequence. This sequence was then optimized for expression in mammalian cells. Next, these variable region sequences were cloned into an expression vector that already contained a human Fc domain. For the heavy chain, the IgG1 constant region was used as required. Furthermore, for comparison, the heavy and light chains of the parental antibody were constructed as full-length chimeric chains using the same backbone 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 were produced in small batches of 0.01 liters. The B10G5 antibody was also scaled up for direct comparison. HEK293 cells, cultured in suspension in chemically defined media in the absence of serum, were transfected with the specified heavy and light chain plasmids. Five days after transfection, acclimatization media were collected from each production run and clarified. Antibodies in the acclimatization media were purified using MabSelectSuRe® Protein A medium (GE Healthcare). Final yields are shown below (Table 2). [Table 2]

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

[0239] Example 3: Humanized antibody binding affinity test The purified antibodies from Example 2 were analyzed for binding affinity to the human prostate tumor cell line M12 by FACS. Three different antibody concentrations were tested (0.1 ug / mL, 0.05 ug / mL, and 0.01 ug / 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 a concentration of 0.1 ug / mL, from top to bottom, the antibodies are Ab-G, Ab-K, Ab-E, Ab-J, Ab-I and Ab-H (double), 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 human IgG1 Fc region).

[0240] Example 4: BLI analysis of antibody K using BLI Biolayer interferometry (BLI) was performed using Octet Red96 (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 with a kinetic-grade biosensor. The loaded biosensor was then immersed in a series of sample dilutions containing antigen (MICB) serially diluted in PBS buffer at 0.1% BSA, 0.02% Tween-20, pH 7.2. Association was observed for 150 seconds, followed by dissociation for 200 seconds. Repeated injections of the same antigen concentration showed good overlap. Kinetic analysis was performed using Scrubber software with 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=7.2nM) than the chimeric antibody B10G5 (Ab-J, Kd=12.1nM) (Figure 2A).

[0241] Example 5: NK cell cytotoxicity assay The ability of antibody K to stimulate cytotoxicity in NK cells 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 pre-incubated at 37°C for 30 minutes with 10 ug / ml of control human IgG, antibody Ab-J (chimeric B10G5 with human IgG1 Fc region), or antibody K (Ab-K). 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 over a standard 4-hour period. 51 The results were determined using a Cr release assay (Jewett A. et al., Hum. Immunol, 2003; 64: 505-520). Specifically, NK cells were analyzed. 51Cr-labeled UC1 or PL12 cells were co-cultured in a 10:1 ratio in a cell culture incubator for 4 hours. After 4 hours of incubation, supernatant was collected from each sample and counted with 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 determined using 30 / 10 lysis units, calculated by multiplying the reciprocal of the number of NK cells required to lyse 30% of the target cells by 100. 6 The cells were represented. Five copies under each condition were included in the experiment.

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

[0243] Example 6: Agglutination assay Samples of mouse 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 and prepared in 50 μL volumes. All samples were filtered through a spin filter (SpinX® Cat.#8160) to remove unwanted large particles and degassed before assay. 5 μL of sample was used to load each disposable cuvette for DLS assays using the Unchained Lab nanoDLS pUNK machine. The 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 variation in scattered light intensity due to diffusing particles. The particles aggregate over time, which is observed as an increase in the hydrodynamic diameter (x-axis). The peak area represents the respective levels of different antibody species (e.g., antibody monomer or aggregate) as a percentage of the total amount of antibody.

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

[0245] Example 7: Clinical trial of immunotherapy Phase I clinical trials will be conducted in cancer patients with MIC+ tumor samples or serum sMIC+. The 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 will include patients who have failed standard treatment but have not received chemotherapy. The study dose range for the MIC antibody will be intravenous infusion every 2-4 weeks for up to 90 days, from 0.01 mg / kg to 100 mg / kg, with follow-up of up to 1 year to determine the MTD or recommended Phase 2 dose (RP2D). Patients who do not experience serious adverse events will receive longer infusions with up to 2 years of clinical follow-up to determine primary efficacy.

[0246] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, not only those described herein, will be apparent to those skilled in the art from the above 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, and their disclosures are incorporated in their entirety by reference for all purposes. Sequence List Sequence ID 1 - VH amino acid sequence of HC2 EVQLQESGPG LVKPSQTLSL TCTVSGYSIT SDYAWNWIRQ PPGKGLEWIG YISYSSGSTNY NPSLKSRVTI SRDTSKNQFS LKLSSVTAAD TAVYYCARGG TYFDYWGQGT LVTVSS Sequence ID 2 - VL amino acid sequence of LC3 DVVMTQSPST LSASVGDRVT ITCRASAHIN NWLAWYQQKP GKAPKLLISD ATSLESGVPS RFSGSGSGKE YTLTISSLQP DDFATYYCQH YWSTPWTFGQ GTKVEIK Sequence ID 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 Sequence ID 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 Sequence ID 5 Signal Sequence MDPKGSLSWR ILLFLSLAFE LSYG Sequence ID 6: Signal Sequence METDTLLLWV LLLWVPGSTG Sequence ID 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 Sequence ID 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 Sequence ID 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 Sequence 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 Sequence ID 11 VH CDR1 GYSITSDYA Sequence ID 12 VH CDR2 GYISYSGST Sequence ID 13 VH CDR3 ARGGTYFDY Sequence ID 14 VL CDR1 RASAHINNW Sequence ID 15 VL CDR2 DATSLES Sequence ID 16 VL CDR3 QHYWSTPWT Sequence ID 17 (Gly Gly Gly Gly Ser)n, where n=1~5 Sequence ID 18 His His His His His His Code sequence of HC2, sequence number 19 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 மாட்ட்டை 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 VH amino acid sequence of SEQ ID NO: 24 HC3 EVQLVESGPG LVKPSETLSL TCTVSGYSIT SDYAWNWIRQ PPGKGLEWIG YISYSSGSTNY NPSLKSRVTI SRDTSKNQFS LKLSSVTAAD TAVYYCARGG TYFDYWGQGT TVTVSS VL amino acid sequence of SEQ ID NO: 25 LC1 DIQMTQSPSS LSASVGDRVT ITCRASAHIN NWLAWYQQKP GKAPKLLLSD ATSLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQH YWSTPWTFGG GTKVEIK Sequence ID 26: VL amino acid sequence of LC2 DIVMTQSPSS LSASVGDRVT ITCRASAHIN NWLAWYQQKP GKAPKLLLSD ATSLESGVPS RFSGSGSGKD YTLTISSLQP EDFATYYCQH YWSTPWTFGG GTKVEIK Sequence ID 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 Sequence 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 Sequence number 29 IGHV4-59*11 (MK471385) QVQLQESGPG LVKPSETLSL TCTVSGGSIS SHYWSWIRQP PGKGLEWIGY IYYSGSTNYN PSLKSRVTIS VDTSKNQFSL KLSSVTAADT AVYYCAR Sequence number 30 IGHJ4*01 YFDYWGQGTL VTVSS Sequence number 31 IGHV4-30-4*01 (Z14238) QVQLQESGPG LVKPSQTLSL TCTVSGGSIS SGDYYWSWIR QPPGKGLEWI GYIYYSGSTYY NPSLKSRVTI VDTSKNQFSL KLSSVTAADT AVYYCAR Sequence number 32 IGKV1-NL1-4*01 (Y14865) DIQMTQSPSS LSASVGDRVT ITCRASQGIS NSLAWYQQKP GKAPKLLLYA ASRLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ YYSTP Sequence number 33 IGKJ1*01 (J00242) WTFGQGTKVE IK Sequence number 34 IGKV1-33*01 (M64856) DIQMTQSPSS LSASVGDRVT ITCQASQDIS NYLNWYQQKP GKAPKLLIYD ASNLETGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ YDNLP Sequence ID 35 IGKV1-5*01 (Z00001) DIQMTQSPST LSASVGDRVT ITCRASQSIS SWLAWYQQKP GKAPKLLIYD ASSLESGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YNSYS Sequence ID 36 B10G5 VH EVQLEESGPG LVKPSQSLSL TCTVTGYSIT SDYAWNWIRQ FPGNKLEWMG YISYSSGSTNY NPSLKSRISI TRDTSKNQFF LQLNSVITED TATYYCARGG TYFDYWGQGT TLTVSS Sequence ID 37 B10G5 VL DIVLTQTTSY LSVSLGGRVT IACKASAHIN NWLAWYQQKP GNAPRLLISD ATSLETGVPS RFSGSGSGKD YTLSITSLQT EDVATYYCQH YWSTPWTFGG GTKLEIK

Claims

1. An isolated antibody or its antigen-binding moiety that specifically binds to MIC, wherein the antibody or its antigen-binding moiety comprises a heavy-chain variable (VH) region and a light-chain variable (VL) region, wherein the VH region comprises a complementarity-determining region 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, and 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, wherein the VH region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and the VL region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

2.

2. The isolated antibody or its antigen-binding portion according to claim 1, wherein the VH region includes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

1.

3. The isolated antibody or its antigen-binding portion according to claim 1 or 2, wherein the VH region comprises the amino acid sequence of SEQ ID NO:

1.

4. The isolated antibody or its antigen-binding portion according to any one of claims 1 to 3, wherein the VL region comprises the amino acid sequence of SEQ ID NO:

2.

5. An isolated antibody or its antigen-binding moiety according to any one of claims 1 to 4, which is an antibody selected from a monoclonal antibody, a bispecific antibody, and a multispecific antibody.

6. Fab, Fab', F(ab') ２ An isolated antibody or its antigen-binding portion according to any one of claims 1 to 4, wherein the antigen-binding portion is selected from Fv, disulfide-bonded Fv, scFv, and diabody.

7. An isolated antibody or its antigen-binding moiety according to any one of claims 1 to 5, wherein the antibody comprises a heavy chain including the VH region and a heavy chain constant region, and a light chain including the VL region and a light chain constant region.

8. The isolated antibody or its antigen-binding portion according to claim 7, wherein the heavy chain constant region is an IgG isotype.

9. The isolated antibody or its antigen-binding portion according to claim 7 or 8, wherein the heavy chain constant region is an IgG1 constant region or an IgG4 constant region.

10. The isolated antibody or its antigen-binding portion according to any one of claims 7 to 9, wherein the heavy chain constant region is Fc null.

11. The isolated antibody or its antigen-binding moiety according to any one of claims 7 to 10, wherein the heavy chain constant region is an IgG1 constant region and comprises at least one amino acid modification that reduces binding to one or more Fc gamma receptors.

12. The isolated antibody or its antigen-binding moiety according to any one of claims 7 to 11, wherein the heavy chain constant region is an IgG1 constant region and comprises (i) one or more substitutions that reduce the binding affinity of the Fc domain to the Fc receptor, at least one of which is selected from E233P, L234V, L234A, L235A, L235E, G236A, G237A, E318A, K320A, K322A, A327G, A330S, and P331S (according to the EU index of Kabat numbering); and / or (ii) GGGS between G237 and G238 (according to the EU index of Kabat numbering).

13. The isolated antibody or its antigen-binding moiety according to any one of claims 7 to 12, wherein the heavy chain constant region is an IgG1 constant region and includes substitutions of L234A and L235A (according to the EU index of Kabat numbering).

14. The isolated antibody or its antigen-binding moiety according to claim 13, wherein the heavy chain constant region includes a substitution of P329 (according to the EU index of Kabat numbering).

15. The heavy chain comprises the amino acid sequence represented by SEQ ID NO: 3, wherein the isolated antibody or its antigen-binding moiety is according to any one of claims 7 to 9.

16. The isolated antibody or its antigen-binding portion according to any one of claims 7 to 15, wherein the light chain constant region is a kappa isotype.

17. The light chain comprises the amino acid sequence represented by SEQ ID NO: 4, wherein the isolated antibody or its antigen-binding moiety is according to any one of claims 7 to 16.

18. A pharmaceutical composition comprising an isolated antibody or its antigen-binding moiety according to any one of claims 1 to 17, and a pharmaceutically acceptable carrier.

19. A nucleic acid encoding an isolated antibody or its antigen-binding portion according to any one of claims 1 to 17.

20. The nucleic acid according to claim 19, comprising the nucleic acid sequence represented by SEQ ID NO: 21 and / or the nucleic acid sequence represented by SEQ ID NO:

22.

21. A vector comprising the nucleic acid described in claim 19 or 20.

22. A cell line comprising the nucleic acid described in claim 19 or 20.

23. A cell line expressing an isolated antibody or its antigen-binding moiety according to any one of claims 1 to 17.

24. A method for producing an isolated antibody or its antigen-binding moiety, comprising culturing the cell line according to claim 22 or 23 under conditions suitable for expressing the isolated antibody or its antigen-binding moiety, and recovering the isolated antibody or its antigen-binding moiety from the culture.

25. A composition for treating MIC+ cancer in a subject, comprising an isolated antibody or its antigen-binding moiety according to any one of claims 1 to 17.

26. The composition according to claim 25, wherein the cancer is melanoma, prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, kidney cancer, sarcoma, pancreatic cancer, bladder cancer, endometrial cancer, brain tumor, esophageal cancer, gastric cancer, head and neck cancer, lymphoma, or multiple myeloma.

27. A composition for treating MIC+ cancer in a subject, comprising an isolated antibody or its antigen-binding moiety according to any one of claims 1 to 17, wherein the treatment comprises: administration of the isolated antibody or its antigen-binding moiety; and adoptive cell therapy.

28. The composition according to claim 27, wherein the cancer is a carcinoma, sarcoma, neuroendocrine tumor, or malignant hematological disease.

29. The composition according to claim 28, wherein the cancer is a carcinoma.

30. The composition according to claim 29, wherein the carcinoma is a solid tumor.

31. The composition according to claim 28, wherein the cancer is selected from melanoma, prostate cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, colon cancer, kidney cancer, sarcoma, pancreatic cancer, bladder cancer, endometrial cancer, brain tumor, esophageal cancer, gastric cancer, and head and neck cancer.

32. The composition according to claim 28, wherein the cancer is a malignant blood disorder.

33. The composition according to claim 32, wherein the malignant blood disorder is lymphoma or multiple myeloma.

34. A composition for improving the treatment outcomes of subjects receiving adoptive cell therapy for MIC+ cancer, wherein the composition comprises an isolated antibody or its antigen-binding moiety according to any one of claims 1 to 17, and the treatment outcomes of at least one of the subjects are improved compared to when the subject receives adoptive cell therapy alone.

35. The composition according to claim 34, wherein the at least one improved treatment outcome is an objective response selected from stable, partial response and complete response.

36. The composition according to claim 34, wherein the at least one improved therapeutic outcome is a reduction in tumor volume.

37. The composition according to claim 34, wherein the at least one improved treatment outcome is progression-free survival or disease-free survival.

38. The composition according to any one of claims 27 to 37, wherein the cells used in the adoptive cell therapy are selected from autologous NK cells, allogeneic NK cells, autologous T cells, CAR-modified T cells, and CAR-modified NK cells.

39. The composition according to any one of claims 27 to 38, wherein no chemotherapy is administered to the subject for at least four weeks prior to the administration of the isolated antibody or its antigen-binding portion.

40. The composition according to any one of claims 27 to 39, wherein the isolated antibody or its antigen-binding portion is administered intravenously to a subject.

Images