Materials and methods for activating antigen-specific T cell responses

Abstract

Described herein are natural killer group 2D (NKG2D) agonist complexes comprising a soluble MHC I-chain-related molecule (sMIC) and a non-blocking sMIC-neutralizing antibody. Methods for activating CD8+ T cells and methods for treating MIC-negative cancers and viral infections using such complexes are also provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 890,933, filed on August 23, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Statement of Government Support This invention was made with government support under R01CA208246 and R41CA206688 - 01A1 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Materials A computer - readable nucleotide / amino acid sequence listing, submitted simultaneously with this specification and identified as File Name: 2019 - 146_Seqlisting.txt, Size: 75,481 bytes, Creation Date: August 21, 2020, is incorporated herein by reference in its entirety.

Background Art

[0004] Effective T - cell co - stimulation is crucial for primary induction and subsequent maintenance of antigen - specific T - cell responses. In addition to increased co - inhibitory signals, an insufficient co - stimulatory tumor microenvironment accounts for much of the sub - optimal activation and maintenance of tumor - killing CD8 T cells. Thus, one of the major goals in cancer immunotherapy is to provide a sustainable co - stimulatory signal to enhance the generation and persistence of effective tumor - killing CD8 T cells and ultimately achieve durable tumor control. Nevertheless, beyond engineered CAR - T cells that contain engineered TCR co - stimulatory motifs, means to enhance persistent in - situ CD8 T - cell co - stimulation are still far from expectations due to the non - sustainable expression of the co - stimulatory receptor family on CD8 T cells in the standard activation - induced tumor microenvironment.

[0005] Effective T cell co-stimulation is crucial for primary induction and subsequent maintenance of antigen-specific T cell responses. 1~3 Insufficient co-stimulation accounts for much of the suboptimal activation and maintenance of tumor-killing antigen-specific CD8 T cells. 2、3 Therefore, one of the major goals in cancer immunotherapy is to provide a sustainable co-stimulation signal to enhance the generation and persistence of effective tumor-killing CD8 T cells and ultimately achieve durable tumor control. However, beyond engineered CAR-T cells containing co-stimulatory motifs within the engineered TCR, means to enhance the co-stimulation of CD8 T cells in situ persistently are still far from expectations for the following reasons: 1) the unsustainable expression of co-stimulatory receptors on CD8 T cells in the tumor microenvironment and the family induced by standard and activation, 2) significant autoimmunity cytotoxicity may be due to unwanted co-stimulation of lymphocytes rather than cytotoxic T cells. 4 。

[0006] Natural killer group 2D (NKG2D), an activating receptor expressed by all human NK cells, is also defined as a co-stimulatory factor for human NKT, CD8 T, and γδ T cells. 5~10 Similar to the standard co-stimulatory molecule CD28 and the activation-induced TNF-R superfamily of co-stimulatory molecules, NKG2D co-stimulation amplifies the magnitude of CD3 / TCR signaling. Different from these well-studied co-stimulatory molecules, the expression of NKG2D is independent of T cell activation or functional state and is not seen in normal CD4 T cells or B cells under normal conditions. 5~11 Compelling evidence has demonstrated that NKG2D co-stimulation not only enhances CD8 T cell effector function but is also important for the development and rescue of memory CD8 T cells. 11~13 These understandings support NKG2D as a co-stimulatory molecule useful for generating effective and persistent tumor-killing antigen-specific CD8 T cells. SUMMARY OF THE INVENTION

[0007] In one aspect, a natural killer group 2D (NKG2D) complex comprising a soluble MHC class I chain-related molecule (sMIC) and a non-blocking sMIC neutralizing antibody is described herein. In some embodiments, the non-blocking antibody in the complex comprises CDRs shown in SEQ ID NOs: 4-9. In some embodiments, the non-blocking antibody in the complex comprises a light chain variable region shown in SEQ ID NO: 11. In some embodiments, the non-blocking antibody in the complex comprises a heavy chain variable region shown in SEQ ID NO: 10. In some embodiments, the non-blocking antibody in the complex comprises CDRs shown in SEQ ID NOs: 12-17. In some embodiments, the non-blocking antibody in the complex comprises a light chain variable region shown in SEQ ID NO: 19. In some embodiments, the non-blocking antibody in the complex comprises a heavy chain variable region shown in SEQ ID NO: 18.

[0008] In some embodiments, the soluble MIC in the complex is sMICA. In some embodiments, sMICA comprises an amino acid sequence set forth in one of SEQ ID NOs: 1 and 20-77. In some embodiments, the soluble MIC in the complex is sMICB. In some embodiments, sMICB comprises an amino acid sequence shown in one of SEQ ID NOs: 2 and 78-100.

[0009] Also contemplated herein are compositions comprising the NKG2D complex described herein and a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0010] In another aspect, described herein is a method of activating CD8 T cells in a subject in need thereof, comprising administering to the subject a complex comprising a soluble MHC class I chain-related molecule (sMIC) and a non-blocking sMIC neutralizing antibody.

[0011] In some embodiments, the subject is suffering from a viral infection. In some embodiments, the viral infection is caused by a DNA virus (e.g., a herpes virus such as herpes simplex virus, Epstein-Barr virus, cytomegalovirus; a poxvirus such as Variola (smallpox) virus; a hepadnavirus (e.g., hepatitis B virus); a papillomavirus; an adenovirus); an RNA virus (e.g., HIV I, II; HTLV I, II; poliovirus; hepatitis A; a coronavirus such as severe acute respiratory syndrome (SARS); an orthomyxovirus (e.g., influenza virus); a paramyxovirus (e.g., measles virus); rabies virus; hepatitis C virus), a flavivirus, an influenza virus; a calicivirus; or a rabies virus, rinderpest virus and an arenavirus. In some embodiments, the viral infection is caused by lymphocytic choriomeningitis (LCMV).

[0012] In some embodiments, the subject has cancer. Exemplary cancers include basal cell carcinoma, biliary cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma multiforme (GBM), hepatocellular carcinoma, hepatoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary tract cancer, vulvar cancer, B cell lymphomas (including low grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade non-cleaved cell NHL, large tumor lesion NHL, mantle cell lymphoma), AIDS-related lymphomas, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), but are not limited thereto.

[0013] In some embodiments, the subject has an MHC class I chain-related molecule (MIC)-negative cancer. In some embodiments, the subject has a viral infection.

[0014] In some embodiments, the methods described herein further comprise administering an immune checkpoint inhibitor to a subject. In some embodiments, the immune checkpoint inhibitor is MGA27, ipilimumab, pembrolizumab, nivolumab, atezolizumab, IMP321, IPH2101, tremelimumab, pidilizumab, MPDL3280A, MEDI4736, MSB0010718C, AUNP12, avelumab, durvalumab or TSR-022.

Brief Description of the Drawings

[0015]

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Mode for Carrying Out the Invention

[0016] The natural killer group 2D (NKG2D) complex, which is a soluble MHC class I chain-related molecule (sMIC), and non-blocking sMIC neutralizing antibodies are described herein. Also described herein are fusion proteins comprising sMIC bound to the heavy (or light) chain of a non-blocking sMIC neutralizing antibody having a peptide linker. As shown in the examples provided herein: 1) In contrast to soluble NKG2D ligands, the sMIC / anti-sMIC complex provides durable, large-sized NKG2D co-stimulation to amplify TCR / CD3 signaling, 2) The sMIC / anti-sMIC complex and CD28 agonist produce an additive co-stimulatory effect, 3) In contrast to the negative effect of soluble NKG2D ligands that down-modulate NKG2D expression, sMIC / anti-sMIC co-stimulation stabilizes NKG2D expression on CD8 T cells.

[0017] Major histocompatibility complex class I chain-related (MIC) polypeptide The NKG2D superagonist complex (or fusion protein) described herein comprises a major histocompatibility complex class I chain-related (MIC) polypeptide. MIC is a surface transmembrane protein. When the MIC polypeptide is present on the cell surface, it can signal to the immunoreceptor NKG2D, typically for tumor immune destruction by natural killer cells (NK cells) and cytotoxic T lymphocytes (CTLs). However, in many tumors, MIC detaches from the tumor surface, reducing host immunity against tumor cells and promoting tumor escape and progression. MIC polypeptides include, but are not limited to, human MICA (e.g., NCBI reference sequences NP_000238 (SEQ ID NO: 1) and 001170990) and human MICB (e.g., NCBI reference sequence NP_005922 (SEQ ID NO: 2)). In some embodiments, the MIC polypeptide comprises MICA. In some embodiments, the MIC polypeptide can comprise MICB. In some embodiments, the MIC polypeptide comprises the following amino acid sequence: EPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRYDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNVETEEWTVPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESSVVLRRRVPPMVNVTRSEALEGNITVTCGASSFYPRNITLTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPS (SEQ ID NO: 3).

[0018] In some embodiments, the NKG2D complex (or fusion protein) comprises an available MIC (sMIC) polypeptide. As used herein, "soluble MIC" or "sMIC" refers to a portion of an MIC polypeptide lacking a transmembrane domain, such as, for example, the extracellular portion of MIC cleaved from the transmembrane domain. In some embodiments, the soluble MIC comprises, for example, amino acids 24-260 of SEQ ID NO: 1 or 2. In some embodiments, the soluble MIC can comprise, for example, about 20 or more amino acids of residues 24-260 of SEQ ID NO: 1 or 2, such as, for example, 20, 50, 100, 150 or more amino acids of residues 24-260 of SEQ ID NO: 1 or 2.

[0019] In some embodiments, the NK2GD agonist complex (or fusion protein) comprises a MICA allele shown in one of SEQ ID NOs: 20-77. In some embodiments, the NK2GD agonist complex (or fusion protein) comprises a MICB allele shown in one of SEQ ID NOs: 78-100.

[0020] anti-MIC antibody In some embodiments, the NKG2D agonist complex (or fusion protein) described herein comprises a non-blocking antibody or an antibody-binding portion thereof that selectively binds to an MIC polypeptide. In some embodiments, the MIC polypeptide is a soluble MIC polypeptide (sMIC).

[0021] As used herein, the term "non-blocking antibody" refers to an sMIC neutralizing antibody that does not block the interaction of NKG2D with membrane-bound or soluble MIC and thus does not interfere with the susceptibility of MIC+ cells to NKG2D-mediated NK cell lysis activity.

[0022] As used herein, the term "antibody" refers to an immunoglobulin molecule. This term also refers to various forms including, for example, an immunoglobulin molecule, monoclonal antibody, chimeric antibody, CDR-grafted antibody, humanized antibody, single domain antibody (dAb), diabody, multispecific antibody, bispecific antibody, anti-idiotypic antibody, bispecific antibody, its functionally active epitope-binding fragment, bifunctional hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol 17, 105 (1987), incorporated herein by reference) and single chain (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85. 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988)), an antibody consisting of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as full-length antibodies and antigen-binding portions thereof. (See generally, Hood et al, Immunology, Benjamin, NY, 2ND ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature, 323, 15-16 (1986), incorporated herein by reference).

[0023] Each heavy chain is composed of a variable region of the heavy chain (here abbreviated as HCVR or VH) and a constant region of the heavy chain. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain includes a variable region of the light chain (here abbreviated as LCVR or VL) and a constant region of the light chain. The light chain constant region is composed of the CL domain. The VH and VL regions are further divided into hypervariable regions called complementarity-determining regions (CDRs), and conserved regions called framework regions (FRs) are interspersed. Thus, each VH and VL region is composed of three CDRs and four FRs, and is arranged from the N-terminus to the C-terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well-known to those skilled in the art.

[0024] As used herein, "CDR" refers to the complementarity determining regions within the antibody variable sequences. Each variable region of the heavy and light chains has three CDRs, which are referred to as CDR1, CDR2, and CDR3 for each variable region. The exact boundaries of the CDRs vary by system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) provides not only a clear residue numbering system applicable to any variable region of an antibody, but also the exact residue boundaries that define the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Other boundaries that define CDRs overlapping with Kabat CDRs are described by Padlan (FASEBJ. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)) and Chothia (J. Mol. Biol. 196:901-917 (1987) and Nature 342:877-883 (1989)). Still other definitions of CDR boundaries may not strictly follow one of the above systems, but nevertheless overlap with Kabat's CDRs and can be shortened or extended from the perspective of predictions or experimental results that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, but in a preferred embodiment, the CDRs defined by Kabat are used.

[0025] As used herein, "selectively binds" or "specifically binds" means that an anti-MIC binding peptide (e.g., an antibody or a portion thereof) described herein has a KD of 10 -5 M (10000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10M, 10 11 M, 10 -12 At M or below (or any range including these values as endpoints), it shows the ability to bind to a target such as an MIC molecule present on the cell surface. Specific binding can be affected, for example, by the affinity and binding force of the polypeptide agent, as well as the concentration of the polypeptide agent. Those skilled in the art can determine the appropriate conditions for the polypeptide agent described herein to selectively bind to the target using any appropriate method such as dosing the polypeptide agent in an appropriate cell binding assay. The polypeptide specifically bound to the target cannot be replaced by a non-similar competitor. In certain embodiments, an antibody or its antigen-binding portion is said to specifically bind to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, the antibody or its antigen-binding portion has a dissociation constant (KD) of 10 -5 M (10000 nm) or below, for example, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 11 M, 10 -12 M or below and binds to the sMIC polypeptide.

[0026] The terms "antigen-binding fragment" or "antigen-binding portion" of an antibody used interchangeably herein refer to one or more fragments of the antibodies described herein that still demonstrate the binding affinity defined above herein. Fragments of a full antibody have been shown to be able to perform the antigen-binding function of the antibody. Examples of antigen-binding fragments include (i) Fab fragment, i.e., a monovalent fragment composed of VL, VH, CL, and CH1 domains, (ii) F(ab’)2 fragment, i.e., a divalent fragment containing two Fab fragments linked to each other in the hinge region via a disulfide bridge, (iii) Fd fragment composed of VH domain and CH1 domain, (iv) Fv fragment composed of the FL and VH domains of a single arm of the antibody, (v) dAb fragment consisting of VH domain or VH, CH1, CH2, DH3, or VH, CH2, CH3 (dAbs, or single-domain antibodies: those containing only the VL domain have also been shown to specifically bind to the target epitope) (Ward et al., (1989) Nature 341:544-546), but are not limited thereto. The two domains of the Fv fragment, namely VL and VH, are encoded by separate genes, but synthetic linkers, such as poly G4S amino acid sequences, and recombinant methods are used to enable them to be prepared as a single protein chain in which the VL region and VH region bind to form a monovalent molecule (known as single-chain Fv (ScFv)), and they can be further linked to each other. See, for example, Bird et al, (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). The term "antigen-binding portion" of an antibody is also intended to include such single-chain antibodies. Other forms of single-chain antibodies, such as "diabodies", are also included herein.The diabody is a bivalent bispecific antibody in which the VH and VL domains are expressed as a single polypeptide chain, but since a linker that is too short is used for the two domains to bind to the same chain, the domains are paired with complementary domains of different chains to form two antigen-binding sites. (See, for example, Holliger, R, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, Poljak, R.J, et al. (1994) Structure 2:1121-1123). Immunoglobulin constant domains denote heavy or light chain constant domains. The amino acid sequences of human IgG heavy and light chain constant domains are known in the art.

[0027] Furthermore, the antibodies or antigen-binding portions thereof described herein can be part of larger immunoadhesion molecules formed by covalent or non-covalent attachment of the antibody portion to the antibody or one or more additional proteins or peptides. Associated with such immunoadhesion molecules are the use of streptavidin core regions for preparing tetrameric scFv molecules (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine residues, marker peptides, and C-terminal polyhistidyls, such as a hexahistidinyl tag (the "hexahistidinyl tag" disclosed as SEQ ID NO: 18), for generating bivalent biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol. Immunol. 31:1047-1058).

[0028] In some embodiments, the antibody is an IgG, monoclonal antibody, chimeric antibody, CDR-grafted antibody, humanized antibody, multispecific antibody, bispecific antibody, anti-idiotype antibody or bispecific antibody. In some embodiments, the antigen-binding portion of the antibody is a Fab, Fab’, F(ab’) 2 , Fv, disulfide-bonded Fv, scFv, single-domain antibody, diabody or a functionally active epitope-binding fragment thereof.

[0029] The term "human antibody" refers to an antibody whose variable and constant regions correspond to or are derived from human germline immunoglobulin sequences, as described, for example, by Kabat (see Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, a human antibody can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or somatic mutation in vivo), for example, in the CDRs, particularly in CDR3. The recombinant human antibodies described herein can have variable regions and can include constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, according to certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if transgenic animals with human Ig sequences are used, somatic in vivo mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibody are related to or derived from human germline VH and VL sequences but are sequences that do not naturally occur in vivo within the human antibody germline repertoire. According to certain embodiments, this type of recombinant antibody is the result of either or both of selective mutagenesis or back-mutation. Preferably, the mutagenesis results in greater affinity for the target and / or less affinity for non-target structures than the parental antibody.

[0030] The term "chimeric antibody" refers to an antibody that includes the variable region sequences of the heavy and light chains from one species and the constant region sequences from another species, for example, an antibody having mouse heavy and light chain variable regions linked to human constant regions. A humanized antibody has variable region framework residues substantially derived from a human antibody (referred to as the acceptor antibody) and complementarity determining regions substantially derived from a non-human antibody such as a mouse antibody (referred to as the donor immunoglobulin). See Queen et al., Proc Natl Acad Sci USA 86:10029-10033 (1989) and WO90 / 07861, U.S. Patent Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101, and Winter, U.S. Patent No. 5,225,539, which are incorporated herein by reference in their entirety. The constant region, when present, is also substantially or completely derived from human immunoglobulins. The human variable domain is usually selected from a human antibody whose framework sequence exhibits a high degree of sequence identity with the (mouse) variable region domain from which the CDRs are derived. The heavy and light chain variable region framework residues can be substantially similar to regions of the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be a consensus sequence of several human antibodies. See Carter et al., WO92 / 22653, which is incorporated herein by reference in its entirety.

[0031] In some embodiments, the antibodies described herein are not naturally occurring biomolecules. For example, a mouse antibody produced against a human-derived antigen would not occur in nature without human intervention and manipulation such as manufacturing processes performed by humans. Chimeric antibodies are also not naturally occurring biomolecules in that they, for example, are obtained from multiple species and contain sequences assembled into recombinant molecules. In certain embodiments, the human antibody reagents described herein are not naturally occurring biomolecules, and, for example, fully human antibodies against human antigens are essentially the subject of negative selection and are not found naturally in the human body.

[0032] One of ordinary skill in the art will recognize that individual substitutions, deletions or additions to an amino acid sequence which alter a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" where such alteration results in substitution of an amino acid with a chemically similar amino acid and retains the ability to specifically bind to the target antigen of the MIC polypeptide (e.g., an epitope present in sMIC). Such conservatively modified variants are added to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0033] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an sMIC polypeptide comprises one or more heavy and light chain complementarity determining regions (CDRs) selected from the group consisting of: (a) a light chain CDR1 having the amino acids of SEQ ID NO: 4, (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, (d) a heavy chain CDR1 having the amino acids of SEQ ID NO: 7, (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment thereof described herein is one or more CDRs selected from the group consisting of (a) a light chain CDR1 having the amino acids of SEQ ID NO: 4, (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, (d) a heavy chain CDR1 having the amino acids of SEQ ID NO: 7, (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 9, e.g., 1 CDR, 2 CDRs, 3 CDRs, 4 CDRs, 5 CDRs, or 6 CDRs. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain or a portion thereof and one or more CDRs selected from the group consisting of a heavy chain CDR1 having the amino acids of SEQ ID NO: 7, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 9, e.g., 1 CDR, 2 CDRs, or 3 CDRs. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain or a portion thereof and one or more CDRs selected from the group consisting of a chain CDR1 having the amino acids of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, e.g., 1 CDR, 2 CDRs, or 3 CDRs.

[0034] In some embodiments, the antibody or antigen-binding portion thereof comprises light chain complementarity determining regions (CDRs): (a) a light chain CDR1 having the amino acids of SEQ ID NO: 12, (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 13, and (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding portion thereof comprises heavy chain complementarity determining regions (CDRs): a heavy chain CDR1 having the amino acids of SEQ ID NO: 15, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 16, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody or antigen-binding fragment thereof described herein comprises one or more CDRs selected from the group consisting of: (a) a light chain CDR1 having the amino acids of SEQ ID NO: 12, (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 13, (c) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 14, (d) a heavy chain CDR1 having the amino acids of SEQ ID NO: 15, (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 16, and (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 17, e.g., 1 CDR, 2 CDRs, 3 CDRs, 4 CDRs, 5 CDRs, or 6 CDRs. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain or a portion thereof and comprises one or more CDRs selected from the group consisting of: a heavy chain CDR1 having the amino acids of SEQ ID NO: 15, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 16, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 17, e.g., 1 CDR, 2 CDRs, or 3 CDRs. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain or a portion thereof and comprises one or more CDRs selected from the group consisting of: a light chain CDR1 having the amino acids of SEQ ID NO: 13, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 14, e.g., 1 CDR, 2 CDRs, or 3 CDRs.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 11 Light chainIt includes a variable region. In some embodiments, the antibody or its antigen-binding fragment includes the amino acid sequence of SEQ ID NO: 10 Heavy chain It includes a variable region. In some embodiments, the antibody or its antigen-binding fragment includes the amino acid sequence of SEQ ID NO: 11 Light chain It includes. In some embodiments, the antibody or its antigen-binding fragment includes the amino acid sequence of SEQ ID NO: 10 Heavy chain It includes.

[0036] In some embodiments, the antibody or its antigen-binding fragment includes the amino acid sequence of SEQ ID NO: 19 Light chain It includes a variable region. In some embodiments, the antibody or its antigen-binding fragment includes SEQ ID NO: 18 Heavy chain It includes a variable region. In some embodiments, the antibody or its antigen-binding fragment includes the amino acid sequence of SEQ ID NO: 19 Light chain It includes. In some embodiments, the antibody or its antigen-binding fragment includes the amino acid sequence of SEQ ID NO: 18 Heavy chain It includes.

[0037] In embodiments where the antibody described herein comprises at least one CDR that is not identical to the sequences of SEQ ID NOs: 4-9 or 12-17, the amino acid sequence of the at least one CDR can be selected by methods well known to those skilled in the art. For example, Fujii, 2004, Methods in Molecular Biology: Antibody Engineering 248:345-349, “Antibody affinity maturation by random mutagenesis” (which is hereby incorporated by reference in its entirety), particularly Figures 2 and Section 3.3, describe methods for generating libraries of any desired CDR. Thereby, one skilled in the art can identify alternative CDRs that, when present in the antibodies or antigen-binding fragments thereof described herein, result in binding to the MIC polypeptide, but will not block binding of the MIC polypeptide to other antibodies, and that include conservative substitution variants of the specific CDR sequences described herein. The methods described in Fujii et al. also enable one skilled in the art to screen for light chain sequences that, when combined with known heavy chain fragments, will give the desired binding behavior, and vice versa.

[0038] In some embodiments, the antibodies and / or antigen-binding portions thereof described herein can be variants of the sequences described herein, e.g., conservative substitution variants of the antibody polypeptides. In some embodiments, in some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained, for example, by mutation of the native nucleotide sequence. As used herein, a “variant” is a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide includes sequences that contain one or more additions, deletions, or substitutions of nucleotides as compared to the native or reference DNA sequence, but includes, for example, sequences that encode a variant protein or fragment thereof that retains activity (e.g., antigen-specific binding activity to a relevant target polypeptide such as an sMIC polypeptide). A variety of PCR-based site-directed mutagenesis approaches are also known in the art and can be applied by those skilled in the art.

[0039] Examples of substitution variants include conservative substitutions of amino acids within a V H or V L domain that do not change the sequence of the CDRs. Conservative substitutions in sequences not included in the CDRs can be substitutions with respect to wild-type or naturally occurring sequences, e.g., the framework and / or constant regions of human or mouse antibody sequences.

[0040] In some embodiments, conservatively modified variants of the antibody can include changes other than in the CDRs, e.g., a conservatively modified variant of an antibody reagent can include CDRs having one or more of the sequences of SEQ ID NOs: 4-9 and 12-17.

[0041] For example, a given amino acid can be replaced with a residue having similar physicochemical properties, such as replacing one aliphatic residue with another (e.g., substituting Ile, Val, Leu, or Ala for each other), or replacing one polar residue with another (e.g., between Lys and Arg, Glu and Asp, or Gln and Asn). Other such conservative substitutions, such as substitution of an entire region having similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm the desired activity, e.g., the antigen-binding activity and specificity of the native or reference polypeptide are retained.

[0042] Amino acids can be grouped according to the similarity of the properties of their side chains (A.L. Lehninger, Biochemistry, second ed., pp. 73 - 75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).

[0043] Alternatively, naturally occurring residues can be grouped into classes based on general 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 affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions require the exchange of a member of one of these classes for another.

[0044] For certain conservative substitutions, for example, substituting Ala with Gly or Ser, Arg with Lys, Asn with Gln or His, Asp with Glu, Cys with Ser, Gln with Asn, Glu with Asp, Gly with Ala or Pro, His with Asn or Gln, Ile with Leu or Val, Leu with Ile or Val, Lys with Arg, Gln or Glu, Met with Leu, Tyr or Ile, Phe with Met, Leu or Tyr, Ser with Thr, Thr with Ser, Trp with Tyr, Tyr with Trp, and / or Phe with Val, Ile or Leu, is included.

[0045] In some embodiments, the antibody comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to any of the CDR sequences shown in SEQ ID NOs: 4-9 and 12-17, or any of the variable region amino acid sequences shown in SEQ ID NOs: 10, 11, 18 and 19. The degree of homology (percent identity) between the native and variant sequences can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0046] Modifications of the native amino acid sequence can be achieved by any of several techniques known to those skilled in the art. Mutations can be introduced, for example, at specific loci by synthesizing oligonucleotides containing the mutant sequence, flanked by restriction sites that enable ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes analogs having the desired amino acid insertions, substitutions, or deletions. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide a modified nucleotide sequence having specific codons altered according to the required substitutions, deletions, or insertions. Techniques for making such modifications are well established and are incorporated herein by reference in their entirety, including, for example, those disclosed by Walder et al. (Gene 42:133, 1986), Bauer et al. (Gene 37:73, 1985), Craik (BioTechniques, Jan. 1985, 12-19), Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981), and U.S. Patent Nos. 4,518,584, and 4,737,462.

[0047] Any cysteine residues not involved in maintaining the proper three-dimensional structure of the polypeptide can also generally be substituted with serine in order to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds can be added to the polypeptide to improve its stability or promote oligomerization.

[0048] Method for producing an antibody Traditionally, monoclonal antibodies have been produced as natural molecules in murine hybridoma lines. In addition to that technology, the methods and compositions described herein provide for the recombinant DNA expression of monoclonal antibodies. This enables the production of humanized antibodies and a series of antibody derivatives and conjugates in selected host species. Antibody production in bacteria, yeast, transgenic animals, and chicken eggs are also alternative means to hybridoma-based production systems. The main advantage of transgenic animals is the potential to obtain high yields from renewable resources.

[0049] Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared variants or non-variant versions of the antibody. The nucleic acid sequences encoding at least one antibody, portion, or polypeptide described herein can be recombined with vector DNA according to conventional techniques, including blunt-ending or sticky-ending for ligation, restriction enzyme digestion to provide appropriate ends, filling in of sticky ends if necessary, alkaline phosphatase treatment to avoid unwanted ligation, and ligation with an appropriate ligase. Techniques for such manipulations are disclosed, for example, in Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel, 1987, 1993, and can be used to construct nucleic acid sequences encoding monoclonal antibodies or antigen-binding regions thereof.

[0050] In some embodiments, the introduced nucleotide sequence is incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host. Any of a variety of vectors can be used for this purpose, and they are known to and available to those of skill in the art. See, for example, Ausubel et al., 1987, 1993. Important factors in selecting a particular plasmid or viral vector include that recipient cells containing the vector are readily recognizable and selectable from recipient cells that do not contain the vector, the copy number of the vector desired in a particular host, and whether it is desirable to be able to "shuttle" the vector between different species of host cells.

[0051] Examples of prokaryotic vectors known in the art include plasmids that can replicate in, for example, E. coli. Other gene expression elements useful for the expression of cDNAs encoding antibodies or antigen-binding portions thereof include (a) viral transcriptional promoters such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), the Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)), and the Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)) and their enhancer elements, and (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites such as SV40 (Okayama et al., 1983), but are not limited thereto. As described by Liu et al., and Weidle et al., 51 Gene 21 (1987) below, the SV40 early promoter and its enhancer, the murine immunoglobulin H chain promoter enhancer, SV40 late region mRNA splicing, the rabbit S-globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and the SV40 polyadenylation element can be used as expression elements to express immunoglobulin cDNA genes.

[0052] Each fusion gene is incorporated or inserted into an expression vector. Next, recipient cells capable of expressing the chimeric immunoglobulin chain gene product are transfected with the gene encoding the antibody, its antigen-binding portion, or the chimeric H or chimeric L chain alone, or the chimeric H and chimeric L chain genes are co-transfected. The transfected recipient cells are cultured under conditions that allow expression of the incorporated gene, and the expressed immunoglobulin chain or intact antibody or fragment is recovered from the culture.

[0053] The antibody described in this specification, or the expression vector carrying the antibody-binding portion thereof, can be introduced into a suitable host cell by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and particle gun. Johnston et al., 240 Science 1538 (1988), which is known to those skilled in the art.

[0054] The host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including removal of the leader peptide, folding and assembly of the H and L chains, glycosylation of the antibody molecule, and secretion of the functional antibody protein.

[0055] Mammalian cells that can be useful as hosts for the production of antibody proteins include, in addition to the above-mentioned lymphoid-derived cells, cells of fibroblast origin such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Exemplary eukaryotic cells that can be used to express 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, PER.C6™ cells (Crucell), and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modifications on the heavy and / or light chains. For example, in some embodiments, CHO cells produce a polypeptide with a higher level of sialylation than the same polypeptide produced in 293 cells.

[0056] In some embodiments, one or more antibodies or antigen-binding portions thereof disclosed herein can be produced in vivo in an animal engineered or transfected with one or more nucleic acid molecules encoding a polypeptide according to any suitable method.

[0057] In some embodiments, the antibodies or antigen-binding portions thereof described herein are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009), Spirin, Trends Biotechnol. 22:538-45 (2004), Endo et al., Biotechnol. Adv. 21:695-713 (2003), the disclosures of which are incorporated herein by reference in their entireties.

[0058] In some aspects, provided herein are methods and systems for the production of a humanized antibody, prepared by a process comprising maintaining a host transformed with a first expression vector encoding a light chain of a humanized antibody and a second expression vector encoding a heavy chain of the humanized antibody under conditions such that each chain is expressed, and isolating the humanized antibody formed by the collection of chains so expressed. The first and second expression vectors can be the same vector. Also provided herein are DNA sequences encoding a light or heavy chain of a humanized antibody, expression vectors incorporating the DNA sequences, and hosts transformed with the expression vectors.

[0059] Generating humanized antibodies from the sequences and information provided herein can be carried out by those skilled in the art without performing more experiments than necessary. In one approach, four general steps are used to humanize monoclonal antibodies. See, for example, U.S. Pat. Nos. 5,585,089, 6,835,823, 6,824,989. These are: (1) determination of the nucleotide and predicted amino acid sequences of the light and heavy chain variable domains of the starting antibody; (2) design of the humanized antibody, i.e., determination of the antibody framework regions to be used during the humanization process; (3) actual humanization methodology / techniques; and (4) transfection and expression of the humanized antibody.

[0060] Typically, the CDR regions of humanized antibodies and human antibody variants are substantially identical, and more generally, identical to the corresponding CDR regions of the mouse or human antibodies from which they are derived. Although not usually desirable, it may be possible to make one or more conservative amino acid substitutions of CDR residues without significantly affecting the binding affinity of the resulting humanized immunoglobulin or human antibody variant. In some cases, substitution of the CDR region can enhance the binding affinity.

[0061] Furthermore, techniques developed for the production of "chimeric antibodies" (see Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984), Neuberger et al., Nature 312:604-608 (1984), Takeda et al., Nature 314:452-454 (1985), which are hereby incorporated by reference in their entirety) can be used by splicing genes from mice or other species together with genes from human antibody molecules of appropriate biological activity to obtain antibody molecules of appropriate antigen specificity. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable regions derived from mouse monoclonal antibodies and human immunoglobulin constant regions such as humanized antibodies.

[0062] The variable segments of chimeric antibodies are typically linked to at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. Human constant region DNA sequences can be isolated from various human cells such as immortalized B cells (WO87 / 02671, which is hereby incorporated by reference in its entirety). Antibodies can include constant regions of both the light and heavy chains. The heavy chain constant region can include the CH1, hinge, CH2, CH3, and optionally the CH4 regions. For therapeutic purposes, the CH2 domain can be deleted or omitted.

[0063] Alternatively, the techniques described for the production of single-chain antibodies (see, e.g., 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), which are hereby incorporated by reference in their entirety) can be adapted to produce single-chain antibodies. Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide. Techniques for assembling functional Fv fragments in E. coli can also be used (see, e.g., Skerra et al., Science 242:1038-1041 (1988), which is hereby incorporated by reference in its entirety).

[0064] Methods of treatment In one aspect, a method of activating CD8 T cells in a subject in need thereof, the method comprising administering to the subject a complex (or fusion protein) comprising a soluble MHC I chain-related molecule (sMIC) and a non-blocking sMIC neutralizing antibody, is described herein.

[0065] In some embodiments, the subject has a viral infection. There are many viruses for which CD8+ T cells have been shown to play a role in defense. See, e.g., Huber et al., Immunol., 5:171, 2014. In some embodiments, the subject has a DNA virus (e.g., herpes viruses such as herpes simplex virus, Epstein-Barr virus, cytomegalovirus; pox viruses such as Variola (smallpox) virus; hepadnaviruses (e.g., hepatitis B virus); papillomaviruses; adenoviruses); RNA viruses (e.g., HIV I, II; HTLV I, II; poliovirus; hepatitis A; coronaviruses such as severe acute respiratory syndrome (SARS); orthomyxoviruses (e.g., influenza virus); paramyxoviruses (e.g., measles virus); rabies virus; hepatitis C virus), flaviviruses, influenza virus; caliciviruses; or a viral infection caused by rabies virus, rinderpest virus, and arenaviruses. In some embodiments, the viral infection is caused by lymphocytic choriomeningitis (LCMV). In some embodiments, the subject has a virus-related disease. Exemplary virus-related diseases include, but are not limited to, acquired immunodeficiency, hepatitis, gastroenteritis, hemorrhagic diseases, enteritis, carditis, encephalitis, paralysis, bronchiolitis, upper and lower respiratory diseases, respiratory papillomatosis, arthritis, disseminated diseases, meningitis, and mononucleosis.

[0066] In some embodiments, the subject has cancer or a malignancy. In some embodiments, activation of CD8 T cells is determined by enzyme-linked immunosorbent spot (ELISPOT), fluorescence-activated cell sorting (FACS), or a target-killing cytotoxicity assay. Other methods for determining T cell activation as described in Plebanski et al., Expert.Rev.Vaccines, 9:595-600, 2010.

[0067] As used herein, "tumor" refers to the uncontrolled growth of cells that interfere with the normal function of the body's organs and systems. A subject having cancer or a tumor is a subject in whom cancer cells can be objectively measured within the subject's body. This definition includes benign tumors and malignant cancers, as well as potentially dormant tumors or micrometastases. Cancers that migrate from their original site and seed other vital organs can ultimately lead to the death of the subject through impairment of the function of the affected organs. Hematopoietic cancers such as leukemia can destroy the subject's normal hematopoietic compartment, thereby causing hematopoietic disorders (in the form of anemia, thrombocytopenia, and neutropenia), and ultimately leading to death.

[0068] Exemplary cancers 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 and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma multiforme (GBM), hepatoma, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma), lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary tract cancer, vulvar cancer, and other glandular cancers and sarcomas, mantle cell lymphoma, AIDS-related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), but are not limited to these.

[0069] In some embodiments, the tumor or malignancy is MIC negative. As used herein, the term "MIC-negative tumor" is used to describe tumor cells, clusters of tumor cells, or tumor masses that do not produce the MIC protein. This term is intended to encompass all tumor cells and / or tumor masses that do not present the MIC protein on the surface of the tumor cells, and thus these cells do not secrete the MIC protein. In other words, a subject suffering from MIC-negative cancer should not have detectable sMIC above background noise. MIC-negative tumors can be identified, for example, by assaying serum levels of MIC (e.g., sMICA or sMICb) using a standard MICA or MICB detection ELISA as described in Ghadially et al., Br. J. Cancer, 116:1208-1217, 2017, the disclosure of which is incorporated herein by reference. In the case of tumors where a biopsy is available, tumors with negative MIC expression can also be selected or confirmed by immunohistochemistry that does not show cross-reactivity with anti-MIC antibodies.

[0070] As used herein, the term "subject" means a human or an animal. In some embodiments, the animal is a vertebrate such as a primate, rodent, domestic animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic animals and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, cat species, such as house cats, and dog species, such as dogs, foxes, wolves, bird species, such as chickens, emus, ostriches, and fish, such as salmon, catfish, and trout. A patient or subject includes, but is not limited to, the aforementioned subsets, such as all of the above, excluding one or more groups or species such as humans, primates, or rodents. 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.

[0071] In some embodiments, the subject has cancer. The mammal can be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. Non-human mammals can be advantageously used, for example, as subjects representing animal models of various cancers. Further, the methods described herein can be used to treat domesticated animals and / or pets. The subject can be male or female.

[0072] In some embodiments, the subject has or has previously been diagnosed or identified as having a condition (e.g., cancer) that requires treatment or one or more complications associated with such a condition, and optionally, need not have already received treatment for a condition or one or more complications associated with such a condition. Alternatively, the subject has not previously been diagnosed as having a condition that requires treatment or one or more complications associated with such a condition. For example, the subject can be one that exhibits one or more risk factors for a condition, or one or more complications associated with a condition, or a subject that exhibits no risk factors. A "subject in need of treatment" for a particular condition can be a subject having the condition, a subject diagnosed as having the condition, or a subject at risk of developing the condition.

[0073] As used herein, the terms "treating," "treatment," "to treat," or "ameliorating" when used with respect to a disease, disorder, or condition, refer to a therapeutic intervention with respect to the condition, with the goal of reversing, alleviating, improving, inhibiting, slowing down, or halting the progression or severity of the disease or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of the condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the condition is reduced or halted. That is, "treatment" includes not only improvement of symptoms or markers, but also halting or at least slowing the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, diminution of the extent of a deficit, stable (i.e., not worsening) state of a tumor or malignancy, delay or retardation of tumor growth and / or metastasis, and extension of lifespan as compared to that expected without treatment.

[0074] As used herein, the term "administering" refers to the placement of an agonist complex (or fusion protein) described herein into a subject by a method or route that results in at least partial localization of the agent to a desired site. A pharmaceutical composition containing an agonist complex (or fusion protein) described herein can be administered by any suitable route that results in an effective treatment in a subject.

[0075] Pharmaceutical Compositions and Routes of Administration Also contemplated are natural killer group 2D (NKG2D) agonist complexes (or fusion proteins) comprising soluble MHC class I chain-related molecules (sMIC) and non-blocking sMIC neutralizing antibodies as described herein. According to some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to an active agent combined with a carrier approved for use in the pharmaceutical industry. As used herein, the expression "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0076] The preparation of pharmaceutical compositions containing an active ingredient dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectables as either liquid solutions or suspensions, although solid forms suitable for solution or suspension in a liquid prior to use can also be prepared. The preparations can also be emulsified or presented as liposomal compositions. The active ingredient can be mixed with pharmaceutically acceptable excipients that are compatible with the active ingredient in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Further, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, which enhance or maintain the effectiveness of the active ingredient. The therapeutic compositions described herein can contain pharmaceutically acceptable salts of the ingredients therein. Pharmaceutically acceptable salts include, for example, acid addition salts formed with inorganic acids such as hydrochloric or phosphoric acid, or with organic acids such as acetic, tartaric, or mandelic acid (formed with the free amino groups of polypeptides). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials other than the active ingredient and water, or contain both, such as a buffer solution like sodium phosphate at physiological pH values, saline, or phosphate-buffered saline. Still further, the aqueous carrier can contain two or more buffering salts, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain, in addition to water, and without water, a liquid phase. Examples of such additional liquid phases are glycerol, vegetable oils such as cottonseed oil, and water-oil emulsions.The amount of the active compound used in the present invention that is effective for the treatment of a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0077] A therapeutic composition containing at least one agent can, for example, conventionally be administered in unit doses. The term "unit dose" as used in connection with a therapeutic composition refers to a physically discrete unit suitable as a unit dose for a subject, each unit containing a predetermined quantity of the active substance calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle.

[0078] The exact amount of the active ingredient that needs to be administered depends on the judgment of the practitioner and is specific to each individual. However, dosage ranges suitable for systemic application are disclosed herein and depend on the route of administration. The regimen suitable for administration can also vary, but typically it is an initial administration followed by repeated administrations at intervals of more than one hour by injection or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain the blood concentration within the range specified for in vivo therapy is contemplated.

[0079] As used herein, the phrases "therapeutically effective amount", "effective amount" or "effective dose" refer to an amount that provides a therapeutic or aesthetic benefit in the treatment, prevention, or management of a tumor or malignancy, e.g., an amount that results in a statistically significant decrease in at least one symptom, sign, or marker of the tumor or malignancy. The determination of a therapeutically effective amount is well within the ability of one of ordinary skill in the art. Generally, the therapeutically effective amount can vary depending on the subject's medical history, age, condition, gender, as well as the severity and type of the subject's medical condition, and the administration of other pharmaceutically active agents.

[0080] The dosage range of the agent depends on effectiveness and includes an amount sufficient to produce a desired effect, such as delaying tumor growth or reducing tumor size. The dosage should not be so high as to cause unacceptable adverse side effects. Generally, the dosage varies depending on the patient's age, condition, and gender and can be determined by those skilled in the art. In the event of complications, the individual physician can adjust the dosage. In some embodiments, the dosage is in the range of 0.001 mg / kg body weight to 0.5 mg / kg body weight. Alternatively, the dosage range can be set such that the serum level is maintained at 1 mg / mL to 1000 mg / mL. In the case of systemic administration, a therapeutically effective amount such as, for example, 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, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more can be administered to the subject.

[0081] The administration of the above dosage can be repeated. In some embodiments, the dosage is given once a day or multiple times a day. In some embodiments, the dosage is administered daily for several weeks or months. The treatment period depends on the clinical progression of the subject and the response to the therapy.

[0082] In some embodiments, the dosage is from about 2 mg / kg to about 15 mg / kg. In some embodiments, the dosage is about 2 mg / kg. In some embodiments, the dosage is about 4 mg / kg. In some embodiments, the dosage is about 5 mg / kg. In some embodiments, the dosage is about 6 mg / kg. In some embodiments, the dosage is about 8 mg / kg. In some embodiments, the dosage is about 10 mg / kg. In some embodiments, the dosage is about 15 mg / kg.

[0083] In some embodiments, the dosage can be administered intravenously. In some embodiments, the intravenous administration can be an infusion carried out over a period of about 10 minutes to about 3 hours. In some embodiments, the intravenous administration can be an infusion carried out over a period of about 30 minutes to about 90 minutes.

[0084] In some embodiments, the dosage can be administered approximately weekly. In some embodiments, in some embodiments, the dosage can be administered intravenously weekly. In some embodiments, the dosage can be administered weekly for a period of about 12 weeks to about 18 weeks. In some embodiments, the dosage can be administered every about 2 weeks. In some embodiments, the dosage can be administered every about 3 weeks. In some embodiments, the dosage can be about 2 mg / kg to about 15 mg / kg, administered every about 2 weeks. In some embodiments, the dosage can be about 2 mg / kg to about 15 mg / kg, administered every about 3 weeks. In some embodiments, the dosage can be from about 2 mg / kg to about 15 mg / kg, administered intravenously every about 2 weeks. In some embodiments, the dosage can be from about 2 mg / kg to about 15 mg / kg, administered intravenously every about 3 weeks.

[0085] In some embodiments, the dosage can be about 1 mg to about 2000 mg. In some embodiments, the dosage can be about 3 mg. In some embodiments, the dosage can be about 10 mg. In some embodiments, the dosage can be about 30 mg. In some embodiments, the dosage can be about 1000 mg. In some embodiments, the dosage can be about 2000 mg. In some embodiments, the dosage can be about 3 mg administered by daily intravenous infusion. In some embodiments, the dosage can be about 10 mg administered by daily intravenous infusion. In some embodiments, the dosage can be about 30 mg administered by intravenous infusion three times a week.

[0086] A therapeutically effective amount is an amount of a drug sufficient to produce, for example, a statistically significant and measurable change in tumor size, tumor growth, etc. (Measurement of effectiveness is described below in this specification). Such effective amounts can be measured not only in animal experiments but also in clinical trials.

[0087] The drug can be administered intravenously by injection or by gradual infusion over time. Given a formulation appropriate for a given route, for example, drugs useful in the methods and compositions described herein can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and can be delivered, if desired, by peristaltic means or by other means known to those of skill in the art. The compounds used herein are preferably administered orally, intravenously or intramuscularly to a patient having cancer. Direct local administration to the tumor mass is also specifically contemplated.

[0088] Combination Therapy Specifically contemplated is a combination of the described NKG2D agonist complex (or fusion protein) with an additional therapeutic agent or therapy. In some embodiments, the additional therapeutic agent is effective in the treatment of cancer. Exemplary additional therapeutic agents or therapies include, but are not limited to, surgical therapy, chemotherapy (e.g., administration of a protein kinase inhibitor or an EGFR-targeted therapeutic agent), radiation therapy, cryotherapy, hyperthermia, phototherapy, radiosurgery, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenesis therapy, cytokine therapy, or biological therapies such as monoclonal antibodies, siRNA, miRNA, antisense oligonucleotides, ribozymes or gene therapy. Biological therapy can be gene therapy such as tumor suppressor gene therapy, cell death protein gene therapy, cell cycle regulatory gene therapy, cytokine gene therapy, toxin gene therapy, immune gene therapy, suicide gene therapy, prodrug gene therapy, anti-cell proliferation gene therapy, enzyme gene therapy, or anti-angiogenic factor gene therapy, but is not limited thereto.

[0089] Combination therapy can be administered before or after other pharmaceutical treatments at intervals ranging from a few minutes to a few weeks. In embodiments where the other agent and the combination therapy are applied separately to the cell, generally, a significant period of time does not elapse between each delivery to ensure that the agent and the expression construct can still exert an advantageous combined effect on the cell. In such cases, it is contemplated that both modalities can contact the cell within about 12 to 24 hours of each other, more preferably within about 6 to 12 hours of each other. However, depending on the situation, it may be desirable to significantly extend the treatment period such that several days (e.g., 2, 3, 4, 5, 6, or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapse between each administration.

[0090] In some embodiments, the additional therapeutic agent or therapy includes chemotherapy. Exemplary chemotherapies include cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, etoposide receptor binding agent, taxol, gemcitabine, navelbine, famesyl protein transferase inhibitor, transplatin, 5-fluorouracil, vincristine, vinblastine, and methotrexate, temozolomide (aqueous form of DTIC); alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, uredopa; ethylene imines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bratasin and bratasinone), camptothecin (including synthetic analog topotecan), bryostatin, calistatin, CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs), cryptophycins (especially cryptophycin 1 and cryptophycin 8), dolastatin, duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1), erythrobin, pancratistatin, sarcodictyin, spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novobiocin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chloroozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma I and calicheamicin omega I; dynemicin including dynemicin A;Bisphosphonates such as clodronate; esperamicin, as well as neocarzinostatin chromophore, and related chromoprotein enediyne antibiotics chromophore, aclacinomycin, actinomycin, actinomycin, azaserine, bleomycin, cactinomycin, calvisine, calminomycin, cardifilin, chromomycin, daunorubicin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolidino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin; mitomycins such as mitomycin C; mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, promycin, keramycin, rhodomycin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epitestanol, mepitiostane, testolactone; antiadrenal such as mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demeclocycline, diaziquone, elfomithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine; maytansinoids such as maytansine and ansamitocin;Mitoguazone, mitoxantrone, mopidamol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxane, rizoxin, schizophyllan, spirogermanium, tenuazonic acid, triaziquone, 2,2’,2”-trichlorotriethylamine, trichothecene (especially T-2 toxin, verracurin A, loline A, anguidine), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gasitocin, arabinoside (“Ara-C”), cyclophosphamide; taxoid, for example, paclitaxel and docetaxel gemcitabine; 6-thioguanine, mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, carboplatin; vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatraxate, daunomycin, aminopterin, zeloda, ibandronic acid, irinotecan (e.g., CPT-11), topoisomerase inhibitor RFS2000, difluoromethylhironutin (DMFO); retinoids such as retinoic acid; capecitabine, carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl protein transferase inhibitor, transplatin, and pharmaceutically acceptable salts, acids or derivatives of any of the above, but not limited thereto.;

[0091] In some embodiments, the agonist complex described herein is used in combination with a histone deacetylase inhibitor. In some embodiments, the agonist complex described herein is used in combination with gefitinib. In some embodiments, the agonist complex described herein is used in combination with Iressa (e.g., about 400 to about 800 mg / day of Iressa can be administered to a patient). In some embodiments, one or more chemotherapeutic agents can be used in combination with the agonist complex described herein.

[0092] In some embodiments, the additional therapeutic agent or treatment includes radiation therapy. Other factors that cause DNA damage and have been widely used generally include those known as gamma rays, X-rays, and / or direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also known, such as microwave and ultraviolet irradiation. All of these factors are most likely to cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range of X-rays is from a dose of 50 to 200 roentgens per day for a long period (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dose range of radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by neoplastic cells.

[0093] In some embodiments, the additional therapeutic agent of the therapy includes immunotherapy. Immunotherapeutic agents generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector can be, for example, an antibody specific for a certain marker on the surface of tumor cells. The antibody may function as the effector of the treatment in some cases, or mobilize other cells to actually kill the cells in other cases. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function simply as a targeted agent. Alternatively, the effector can be a lymphocyte carrying surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells, as well as genetically engineered variants of these cell types modified to express chimeric antigen receptors.

[0094] Exemplary immunotherapies that can be combined with the agonist complexes described herein include immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds) (U.S. Pat. Nos. 5,801,005, 5,739,169, Hui and Hashimoto, 1998, Christodoulides et al., 1998), cytokine therapy (e.g., interferon alpha, beta, and gamma, interleukin (IL-1, IL-2), GM-CSF and TNF) (Bukowski et al., 1998, Davidson et al., 1998, Hellstrand et al., 1998) gene therapy (e.g., TNF, IL-1, IL-2, p53) (Qin et al., 1998, Austin-Ward and Villaseca, 1998, U.S. Pat. Nos. 5,830,880 and 5,846,945) and monoclonal antibodies (e.g., anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998, Hanibuchi et al., 1998, U.S. Pat. No. 5,824,311). Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. Herceptin has antitumor activity and is approved for use in the treatment of malignancies (Dillman, 1999). The combination therapy of Herceptin and chemotherapy for cancer has been shown to be more effective than the individual therapies. Thus, it is contemplated that one or more anti-cancer therapies can be used in conjunction with the combination therapies described herein.

[0095] Other immunotherapies contemplated for use in the methods of the present disclosure include those described by Tchekmedyian et al., 2015, which are incorporated herein by reference. Immunotherapy may include suppression of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts (CAFs). In some embodiments, the immunotherapy is a tumor vaccine (e.g., whole tumor cell vaccine, peptide, and recombinant tumor-associated antigen vaccine), or adoptive cell therapy (ACT) (e.g., T cells, natural killer cells, TIL, and LAK cells). T cells can be engineered with a chimeric antigen receptor (CAR) or a T cell receptor (TCR) against a specific tumor antigen. As used herein, a chimeric antigen receptor (or CAR) can refer to any engineered receptor specific for an antigen of interest that, when expressed in a T cell, confers the specificity of the CAR to the T cell. When created using standard molecular techniques, T cells expressing a chimeric antigen receptor can be introduced into a patient, similar to techniques such as adoptive cell transfer. In some aspects, the T cells are activated CD4 and / or CD8 T cells of an individual that are characterized by enhanced cytolytic activity compared to before administration of IFNγ-producing CD4 and / or CD8 T cells and / or combinations. The CD4 and / or CD8 T cells may show an increase in the release of cytokines selected from the group consisting of IFN-γ, TNF-α, and interleukin. The CD4 and / or CD8 T cells can be effector memory T cells. In certain embodiments, the CD4 and / or CD8 effector memory T cells are CD44 高 CD62 低 characterized by having expression.

[0096] Examples of monoclonal antibodies that can be used in combination with the compositions provided herein include trastuzumab (anti-HER2 / neu antibody), pertuzumab (anti-HER2 mAb), cetuximab (chimeric monoclonal antibody against epidermal growth factor receptor EGFR), panitumumab (anti-EGFR antibody), nimotuzumab (anti-EGFR antibody), zalutumumab (anti-EGFR mAb), necitumumab (anti-EGFR mAb), MDX-210 (humanized anti-HER-2 bispecific antibody), MDX-210 (humanized anti-HER-2 bispecific antibody), MDX-447 (humanized anti-EGF receptor bispecific antibody), rituximab (chimeric mouse / human anti-CD20 mAb), ofatumumab (anti-CD20 mAb), obinutuzumab (anti-CD20 mAb), tositumomab-I131 (anti-CD20 mAb), ibritumomab tiuxetan (anti-CD20 mAb), bevacizumab (anti-VEGF mAb), ramucirumab (anti-VEGFR2 mAb), ranibizumab (anti-VEGF mAb), aflibercept (extracellular domains of VEGFR1 and VEGFR2 fused to IgG1 Fc), AMG 386 (angiopoietin-1 and -2 binding peptide fused to IgG1 Fc), daratumumab (anti-IGF-1R mAb), gemtuzumab ozogamicin (anti-CD33 mAb), alemtuzumab (anti-campath-1 / CD52 mAb), brentuximab vedotin (anti-CD30 mAb), catumaxomab (bispecific mAb targeting epithelial cell adhesion molecule and CD3), napumomab (anti-5T4 mAb), dirlotuximab (anti-carbonic anhydrase ix), or farletuzumab (anti-folate receptor), but are not limited thereto.Other examples include Panorex.TM. (17-1A) (mouse monoclonal antibody), Panorex(@ (17-1A) (chimeric mouse monoclonal antibody), BEC2 (anti-idiotype mAb, mimicking GD epitope) (including BCG), Oncolym (Lym-1 monoclonal antibody), SMART M195 Ab, humanized 13’1 LYM-1 (Oncolym), Ovarex (B43.13, anti-idiotype mouse mAb), EGP40 (17-1A) 3622W94 mAb that binds to adenocarcinoma-like spreading tumor antigen, Zenapax (SMART Anti-Tac (IL-2 receptor), SMART M195 Ab, humanized Ab, humanized), NovoMAb-G2 (tumor-specific Ab), TNT (chimeric mAb against histone antigen), TNT (chimeric mAb against histone antigen), Gliomab-H (monoclonal-humanized Ab), GNI-250 Mab, EMD-72000 (chimeric-EGF antagonist), LymphoCide (humanized IL.L.2 antibody), and bispecific GD-2 targeting MDX-260, ANA Ab, SMART IDIO Ab, SMART ABL 364 Ab or ImmuRAIT-CEA and the like. Examples of antibodies include those disclosed in U.S. Patent Nos. 5,736,167, 7,060,808, and 5,821,337.

[0097] Further examples of antibodies include anti-human OX40 agonist antibody (Genentech), zanolimumab (anti-CD4 mAb), keliximab (anti-CD4 mAb), ipilimumab (MDX-101, anti-CTLA-4 mAb), tremelimumab (anti-CTLA-4 mAb), daclizumab (anti-CD25 / IL-2R mAb), basiliximab (anti-CD25 / IL-2R mAb), MDX-1106 (anti-PD1 mAb); antibodies against GITR GC1008 (anti-TGF-β antibody), metelimumab / CAT-192 (anti-TGF-β antibody), lerdelimumab / CAT-152 (anti-TGF-β antibody), ID11 (anti-TGF-β antibody), denosumab (anti-RANKL mAb), BMS-663513 (humanized anti-4-1BB mAb), SGN-40 (humanized anti-CD40 mAb), CP870,893 (human anti-CD40 mAb), infliximab (chimeric anti-TNF mAb, adalimumab (human anti-TNF mAb), certolizumab (humanized Fab anti-TNF), golimumab (anti-TNF), etanercept (extracellular domain of TNFR fused to IgG1 Fc), belatacept (extracellular domain of CTLA-4 fused to Fc), abatacept (extracellular domain of CTLA-4 fused to Fc), belimumab (anti-B lymphocyte stimulator), muromonab-CD3 (anti-CD3 mAb), otelixizumab (anti-CD3 mAb), tepelizumab (anti-CD3 mAb), tocilizumab (anti-IL6R mAb), REGN88 (anti-IL6R mAb), ustekinumab (anti-IL-12 / 23 mAb), briakinumab (anti-IL-12 / 23 mAb), natalizumab (anti-α4 integrin), vedolizumab (anti-α4β7 integrin mAb), T1h (anti-CD6 mAb); epratuzumab (anti-CD22 mAb), efalizumab (anti-CD11a mAb), and Atacicept (extracellular domain of transmembrane activator and calcium-modulating ligand interactor fused to Fc).

[0098] It is contemplated that other agents can be used in combination with the compositions provided herein to improve the therapeutic efficacy of the treatment. These additional agents include immunomodulators, agents that affect cell surface receptor and GAP junction upregulation, cytostatic and differentiating agents, inhibitors of cell adhesion, or agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing substances. Immunomodulators include tumor necrosis factor, interferon alpha, beta, and gamma, and IL-2 and other cytokines, F42K and other cytokine analogs, or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines. Further, upregulation of cell surface receptors or their ligands (such as Fas / Fas ligand, DR4 or DR5 / TRAIL, etc.) is contemplated to enhance the apoptosis-inducing ability of the compositions provided herein by establishing an autocrine or paracrine effect on hyperproliferative cells. Increasing intercellular signaling by increasing the number of GAP junctions increases the antiproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic and differentiating agents can be used in combination with the compositions provided herein to enhance the antiproliferative effectiveness of the treatment. Inhibitors of cell adhesion are contemplated to improve the effectiveness of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Further, other agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, are contemplated to be used in combination with the compositions provided herein to improve the therapeutic effect.

[0099] In further embodiments, the other agent can be one or more oncolytic viruses. Examples of oncolytic viruses include adenovirus, adeno-associated virus, retrovirus, lentivirus, herpes virus, poxvirus, vaccinia virus, vesicular stomatitis virus, poliovirus, Newcastle disease virus, Epstein-Barr virus, influenza virus, and reovirus. In certain embodiments, the other agent is talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus genetically engineered to express GM-CSF. Talimogene laherparepvec, HSV-1 [JS1 strain] ICP34.5− / ICP47− / hGM-CSF (previously known as OncoVEX GM CSF), is an intratumoral delivery oncolytic immunotherapy that includes an immune-enhancing HSV-1 that selectively replicates in solid tumors. (Lui et al., 2003; U.S. Pat. Nos. 7,223,593 and 7,537,924, which are incorporated herein by reference).

[0100] In certain embodiments, hormone therapy can also be used in combination with this embodiment or in combination with any of the other previously described cancer therapies. The use of hormones may be used in the treatment of certain cancers, such as breast cancer, prostate cancer, ovarian cancer, or cervical cancer, to lower the levels of certain hormones, such as testosterone or estrogen, or to block their effects. This treatment is often used in combination with at least one other cancer therapy as an option for treatment or to reduce the risk of metastasis.

[0101] In some embodiments, the additional anti-cancer agent is a protein kinase inhibitor or monoclonal antibody that inhibits a protein kinase or a receptor involved in a growth factor signaling pathway such as an EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitor. Non-limiting examples of protein kinases or growth factor signaling pathway inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, sorafenib, sunitinib, trastuzumab, vandetanib, AP23451, vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, Alvocidib, Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016 or mixtures thereof

[0102] In some embodiments, the PI3K inhibitor is buparlisib, idelalisib, BYL-719, duvelisib, PF-05212384, pictilisib, copanlisib, copanlisib dihydrochloride, ZSTK-474, GSK-2636771, duvelisib, GS-9820, PF-04691502, SAR-245408, SAR-245409, sonolisib, alisertib, GDC-0032, GDC-0980, apitolisib, piraralisib, DLBS 1425, PX-866, buparlisib, AZD-8186, BGT-226, DS-7423, GDC-0084, GSK-2126458, INK-1117, SAR-260301, SF-1126, AMG-319, BAY-1082439, CH-5132799, GSK-2269557, P-7170, PWT-33597, CAL-263, RG-7603, LY-3023414, RP-5264, RV-1729, teselisib, TGR-1202, GSK-418, INCB-040093, panulisib, GSK-1059615, CNX-1351, AMG-511, PQR-309, 17 beta-hydroxywortmannin, AEZS-129, AEZS-136, HM-5016699, IPI-443, ONC-201, PF-4989216, RP-6503, SF-2626, X-339, XL-499, PQR-401, AEZS-132, CZC-24832, KAR-4141, PQR-311, PQR-316, RP-5090, VS-5584, X-480, AEZS-126, AS-604850, BAG-956, CAL-130, CZC-24758, ETP-46321, ETP-47187, GNE-317, GS-548202, HM-032, KAR-1139, LY-294002, PF-04979064, PI-620, PKI-402, PWT-143, RP-6530, 3-HOI-BA-01, AEZS-134, AS-041164, AS-252424, AS-605240, AS-605858, AS-606839, BCCA-621C, CAY-10505, CH-5033855, CH-5108134, CUDC-908, CZC-1It is selected from the group of PI3K inhibitors consisting of 9945, D-106669, D-87503, DPT-NX7, ETP-46444, ETP-46992, GE-21, GNE-123, GNE-151, GNE-293, GNE-380, GNE-390, GNE-477, GNE-490, GNE-493, GNE-614, HMPL-518, HS-104, HS-106, HS-116, HS-173, HS-196, IC-486068, INK-055, KAR1141, KY-12420, Wortmannin, Lin-05, NPT-520-34, PF-04691503, PF-06465603, PGNX-01, PGNX-02, PI620, PI-103, PI-509, PI-516, PI-540, PIK-75, PWT-458, RO-2492, RP-5152, RP-5237, SB-2015, SB-2312, SB-2343, SHBM-1009, SN 32976, SR-13179, SRX-2523, SRX-2558, SRX-2626, SRX-3636, SRX-5000, TGR-5237, TGX-221, UCB-5857, WAY-266175, WAY-266176, EI-201, AEZS-131, AQX-MN100, KCC-TGX, OXY-111A, PI-708, PX-2000, and WJD-008.

[0103] Additional cancer therapies include, for example, epidermal growth factor receptor (EGFR, EGFR1, ErbB-1, HER1), ErbB-2 (HER2 / neu), ErbB-3 / HER3, ErbB-4 / HER4, EGFR ligand family, insulin-like growth factor receptor (IGFR) family, IGF binding protein (IGFBP), IGFR ligand family (IGF-1R), platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family, fibroblast growth factor receptor (FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGFR) family, VEGF family, HGF receptor family, TRK receptor family, ephrin (EPH) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, TIE receptor family, angiopoietin 1, 2, receptor tyrosine kinase-like orphan receptor (ROR) receptor family, discoidin domain receptor (DDR) family, RET receptor family, KLG receptor family, RYK receptor family, MuSK receptor family, transforming growth factor alpha (TGF-α), TGF-α receptor, transforming growth factor-beta (TGF-β), TGF-β receptor, interleukin 13 receptor alpha2 chain (1L13Ralpha2), interleukin-6 (IL-6), 1L-6 receptor, interleukin-4, IL-4 receptor, cytokine receptor, class I (hematopoietin family) and class II (interferon / 1L-10 family) receptors, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily (TNTRSF), cell death receptor family, TRAIL receptor; cancer-testis (CT) antigen, lineage-specific antigen, differentiation antigen, alpha-actinin-4, ARTC1, breakpoint cluster region-Abelson (Bcr-abl) fusion product, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), beta-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, elongation factor 2 (ELF2),Ets variant gene 6 / Acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low density lipoprotein receptor / GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase (LDLR / FUT) fusion protein, HLA-A2, exchange of arginine to isoleucine at residue 170 of the alpha helix of the alpha2 domain of the HLA-A2 gene (HLA-A*201-R170I), HLA-A11, heat shock protein 70-2 mutation (HSP70-2M), KIAA0205, MART2, melanoma ubiquitous mutations 1, 2, 3 (MUM-1, 2, 3), prostate acid phosphatase (PAP), neo-PAP, myosin class 1, NFYC, OGT, OS-9, pml-RARalpha fusion protein, PRDXS, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, triosephosphate isomerase, BAGE, BAGE-1, BAGE-2,3,4,5, GAGE-1,2,3,4,5,6,7,8, GnT-V (abnormal N-acetylglucosaminyltransferase V, MGATS), HERV-K-MEL, KK-LC, LAGE, LAGE-1, CTL recognized antigen on melanoma (CAMEL), MAGE-A1 (MAGE-1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1), MART-1 / Melan-A (MLANA), gp100, gp100 / Pme117 (S1LV), tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1 / LAGE-2, SAGE, Sp17, SSX-1,2,3,4, TRP2-1NT2, carcinoembryonic antigen (CEA), Kallikfein 4, mammaglobin-A, OA1, prostate specific antigen (PSA), prostate specific membrane antigen, TRP-1 / gp75, TRP-2, adipophilin,Absent in Nieranolna 2: Interferon-inducible protein (AIM-2), BING-4, CPSF, Cyclin D1, Epithelial cell adhesion molecule (Ep-CAM), EpbA3, Fibroblast growth factor-5 (FGF-5), Glycoprotein 250 (gp250 intestinal carboxylesterase (iCE), Alpha-fetoprotein (AFP), M-CSF, mdm-2, MUCI, p53 (TP53), PBF, FRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, Survivin (BIRCS), Human telomerase reverse transcriptase (hTERT), Telomerase, Wilms tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE, TPTE, Immunoglobulin idiotype, Bence Jones protein, Estrogen receptor (ER), Androgen receptor (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3, Cancer antigen 72-4 (CA 72-4), Cancer antigen 15-3 (CA 15-3), Cancer antigen 27-29 (CA 27-29), Cancer antigen 125 (CA 125), Cancer antigen 19-9 (CA 19-9), Beta-human chorionic gonadotropin, Beta-2 microglobulin, Squamous cell carcinoma antigen, Neuron-specific enolase, Heat shock protein gp96, GM2, Sargramostim, CTLA-4, 707 Alanine Proline (707-AP), Adenocarcinoma antigen recognized by T cells 4 (ART-4), Cancer embryogenic antigen peptide-1 (CAP-1), Calcium-activated chloride channel-2 (CLCA2), Cyclophilin B (Cyp-B), Human signal ring tumor-2 (HST-2), Human papillomavirus (HPV) proteins (HPV-E6, HPV-E7, Major or minor papilloma antigens, others), Epstein-Barr virus (EBV) proteins (EBV latent membrane protein-LMP1, LMP2; others), Hepatitis B or C virus proteins,It is contemplated that it may include antibodies, peptides, polypeptides, small molecule inhibitors, siRNA, miRNA or gene therapy targeting HIV proteins.

[0104] In some embodiments, the methods described herein further comprise administering an immune checkpoint inhibitor to a subject. In some embodiments, the checkpoint inhibitor is a small molecule, an inhibitory nucleic acid, an inhibitory polypeptide, an antibody or antigen-binding domain thereof, or an antibody reagent. In some embodiments, the checkpoint inhibitor is an antibody or antigen-binding domain thereof, or the antibody reagent binds to an immune checkpoint polypeptide and inhibits its activity. Common checkpoints targeted by therapeutic agents include, but are not limited to, PD-L1, PD-L2, PD-1, CTLA-4, TIM-3, LAG-3, VISTA, and TIGIT. In some embodiments, the checkpoint inhibitor is an antibody or antigen-binding domain thereof, or the antibody reagent binds to a PD-1, PD-L1, or PD-L2 polypeptide and inhibits its activity.

[0105] Known checkpoint regulators (e.g., PD-L1, PD-L2, PD-1, CTLA-4, TIM-3, LAG-3, VISTA, or TIGIT) are known in the art. Non-limiting examples of checkpoint inhibitors (with checkpoint target and manufacturer noted in parentheses) include the following: MGA271 (B7-H3: MacroGenics), ipilimumab (CTLA-4, Meyers Squibb), pembrolizumab (PD-1, Merck), nivolumab (PD-1, Bristol Meyers Squibb), atezolizumab (PD-L1, Genentech), IMP321 (LAG3: Immuntep), BMS-986016 (LAG3, Bristol Meyers Squibb), IPH2101 (KIR, Innate Pharma), tremelimumab (CTLA-4, Medimmune), pidilizumab (PD-1, Medivation), MPDL3280A (PD-L1, Roche), MEDI4736 (PD-L1, AstraZeneca), MSB0010718C (PD-L1, EMD Serono), AUNP12 (PD-1, Aurigene), avelumab (PD-L1, Merck), durvalumab (PD-L1, Medimmune), and TSR-022 (TIM3, Tesaro).

[0106] In some embodiments, the checkpoint inhibitor inhibits PD-1. PD-1 inhibitors include, but are not limited to, pembrolizumab (Keytruda™), nivolumab, AUNP-12, and pidilizumab. In another embodiment, the checkpoint inhibitor inhibits PD-L1. PD-L1 inhibitors include, but are not limited to, atezolizumab, MPDL3280A, avelumab, and durvalumab.

[0107] Monitoring the effectiveness of treatment The effectiveness of a given treatment for cancer can be determined by a skilled clinician. However, for example, after treatment with the agents described herein, if at least 10%, for example, any or all of the signs or symptoms of the tumor change in a beneficial way, or other clinically acceptable symptoms are improved, or if improved, when the term is used herein, the treatment is considered an "effective treatment". Effectiveness can also be measured by the individual not deteriorating, as evaluated by the elimination of the need for hospitalization or medical intervention (e.g., cessation of disease progression). Methods for measuring these metrics are known to those of skill in the art and / or are described herein.

[0108] An effective amount for the treatment of a disease means an amount sufficient to provide an effective treatment for that disease when administered to a mammal in need thereof, as defined herein. The effectiveness of an agent can be determined, for example, by evaluating physical indicators of cancer, such as tumor size, tumor mass, tumor density, angiogenesis, tumor growth rate, etc. Further, the effectiveness of an agent can be measured by a decrease in circulating MIC peptide or a fragment thereof in a subject treated with an agent comprising an antibody or an antigen-binding portion thereof described herein, or a nucleic acid encoding an antibody or antigen-binding portion described herein.

[0109] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Specific embodiments of the present disclosure and examples thereof are described herein for illustrative purposes, but various equivalent modifications are possible within the scope of the present disclosure, as will be recognized by those of ordinary skill in the relevant art. The teachings of the disclosure provided herein can be applied, as appropriate, to other procedures or methods. Combinations of the various embodiments described herein can provide further embodiments. Aspects of the present disclosure can be modified, as needed, using the compositions, functions, and concepts of the above references and applications to provide still further embodiments of the present disclosure. These and other changes can be added to the disclosure in light of the detailed description.

[0110] Specific elements of any of the foregoing embodiments can be combined with or replaced by elements of other embodiments. Further, the advantages associated with specific embodiments of the present disclosure have been described in the context of those embodiments, but other embodiments may exhibit such advantages as well, and not all embodiments necessarily exhibit such advantages within the scope of the present disclosure.

[0111] All identified patents and other publications are hereby expressly incorporated by reference herein for the purpose of, for example, explaining and disclosing the methodologies described in such publications that may be used in connection with the present invention. These publications are provided only for their disclosure prior to the filing date of the present application. It should not be construed that the inventors admit any right to antedate invention or other reason for such disclosure. All statements as to the date or representation of the content of these documents are based on the information available to the applicant and do not constitute an admission as to the accuracy of the date or content of these documents.

Examples

[0112] Materials and Methods Identification of sMIC / MIC-negative cancers: sMIC-negative tumors can be identified by assaying serum levels of sMICA or sMICB using standard MICA or MICB detection ELISAs. MIC-negative subjects should not contain detectable sMIC above background noise. In the case of tumors where biopsies are available, MIC expression can also be selected or confirmed as negative by immunohistochemistry, showing no cross-reactivity with anti-MIC antibodies.

[0113] Peptide-binding regions of the non-blocking anti-sMIC / MIC antibody D4H3: Chemical cross-linking, high-quality MALDI mass spectrometry, and nLC-Orbitrap mass spectrometry were used to characterize the interaction interface between the antigen and the antibody Ab-D4H3. The results showed that Ab-D4H3 binds to two regions of the antigen at the following amino acids: 68, 72, 75, 77 and 206, 207 and 209. These regions do not compete with the NKG2D-binding regions of the alpha-1 and alpha-2 domains of MIC (Li et cl., Nat Immunol. 2001 May;2(5):443-51, the entire disclosure of which is incorporated herein by reference).

[0114] Generation of sMIC / anti-sMIC complexes: Complexes were formed by mixing sMIC with anti-sMIC antibodies at room temperature or 37°C (molar ratio 1:1 or 2:1). Complexes can also be formed by binding sMIC to the heavy or light chains of anti-sMIC antibodies with a linker.

[0115] Example 1 - sMIC / anti-sMIC complexes enhance CD8 T cell activation via CD3 / TCR in vitro PBMCs from normal donors were stimulated for 3 days in the presence or absence of plate-bound CD3 in the presence of sMIC, anti-sMIC antibody (e.g., D4H3), or a complex of sMIC / anti-sMIC mAb, and CD8 T cell IFNγ production was assayed by intracellular staining. Soluble anti-CD28 stimulation was used as a positive control. As shown in Figure 1, similar to anti-CD28 stimulation, the sMIC / anti-MIC complex activated CD8 T cells upon CD3 ligation, suggesting a co-stimulatory effect of the sMIC / anti-MIC complex. Neither sMIC nor anti-MIC mAb alone had a similar effect. Furthermore, sMIC / anti-MIC and anti-CD28 produced an additive effect that amplified CD3-mediated CD8 T cell activation (Figure 1). Additionally, the carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay demonstrated that sMIC / anti-MIC co-stimulation also enhanced CD8 T cell proliferation (Figure 1).

[0116] In another example, PBMCs were stimulated for 48 hours in the presence or absence of plate-bound CD3 in the presence of sMIC, anti-sMIC antibody (e.g., NO4) or a complex of sMIC / NO4, and then CD8 T cell surface NKG2D expression and IFNγ production were assayed by intracellular staining. Similarly, anti-CD28 agonist antibody stimulation was used as a positive control for CD8 T cell co-stimulation. Figure 2 demonstrates that complex stimulation amplified CD3 / TCR activation as measured by IFNγ production and proliferation by the CFSE dilution assay. Interestingly, CD3 and sMIC / NO4 complex stimulation increased CD8 T cell surface NKG2D expression compared to CD3 or CD3 / anti-CD28 stimulation. In summary, the data provided in this example demonstrated that sMIC / anti-sMIC complex co-stimulation is non-redundant with anti-Cd28 co-stimulation.

[0117] Example 2 - The sMIC / anti-sMIC complex amplifies antigen-specific CD8 T cell responses. TIL1383I cells (engineered to express CD34 as a reporter) were either the sMIC(A) / D4H3 complex or the tyrosine kinase peptide 369-377In the presence or absence of, HLA-A2 + TIL13831 was cultured with alternative T2-A2 antigen-presenting cells (APCs). After overnight culture, the activation of TIL13831 by intracellular staining of IFNγ, TNFα, and CD107a (degranulation) was evaluated. As shown in Figure 3, the sMIC / D4H3 complex significantly enhanced TIL13831 against HLA-A2-restricted tyrosine kinase peptide stimulation. The sMIC / D4H3 complex together with the scrambled OVA peptide did not stimulate TIL13831. Blocking NKG2D with mAb M585 abolished the co-stimulatory effect of sMIC / D4H3.

[0118] Example 3 - Therapy with the sMIC / D4H3 complex inhibits the growth of MIC-negative tumors MIC 陰性 MC38 colon tumor cells were transplanted into a cohort of syngeneic B6 / MICB male mice. When the tumors reached a volume of approximately 100 mm 3 in size, the animals were treated with control IgG, recombinant rsMIC, D4H3, or the sMIC / D4H3 complex by intraperitoneal administration at a dose of 4 mg / kg twice a week, either individually or in combination. As shown in Figure 4, the sMIC / D4H3 complex significantly inhibited tumor growth. The combination therapy elicited an antigen-specific immune response.

[0119] Example 4 - Detection of MIC / anti-sMIC antibodies that bind to the receptor NKG2D by ELISA Using the ELISA method, it was demonstrated that complexes composed of sMIC and non-blocking anti-sMIC antibodies (e.g., NO4 or D4H3) bind to the receptor NKG2D and activate NKG2D-mediated co-stimulatory signaling.

[0120] Figure 5 demonstrates that the complex binds to NKG2D by ELISA assay. In this assay, recombinant soluble human NKG2D (rs-hNKG2D) was immobilized overnight on a 96-well plate and washed with water to remove unbound rs-hNKG2D. Then, a given concentration of recombinant sMICA (source: R&D systems) or recombinant MICB-His (source: Fisher) complexed with various concentrations of anti-sMIC monoclonal antibody D4H3 (Figure 5A) or NO4 (Figure 5B) was added. Binding of the complex to rs-hNKG2D was detected with an HRP-conjugated goat anti-mouse antibody.

[0121] Example 5 - sMIC / D4H3 complex therapy rapidly resolves LCMV viral infection. A cohort of C57BL / 6 mice was inoculated intravenously (iv) with 2x10 6 PFU of LCMV (lymphocytic choriomeningitis virus) Armstrong strain. The sMIC / D4H3 complex (6 mg / Kg) or control PBS was administered to the mice (intraperitoneal injection) on days 1 and 3 post-viral inoculation. Blood samples were collected from the mice on days 1 and 5 post-viral inoculation. Serum viral load was assayed by plaque assay (as previously described in Curr Protoc Microbiol. 2008 Feb, CHAPTER: Unit-15A.1). As shown in Figure 8, the LCMV titer decreased significantly in mice receiving sMIC / D4H3 complex therapy.

[0122] All magazine articles or patent documents referenced herein are hereby incorporated by reference in their entirety.

Claims

**Claim 1** A natural killer group 2D (NKG2D) agonist complex comprising a soluble MHC class I chain-related molecule (sMIC) and a non-blocking sMIC neutralizing antibody, wherein in the non-blocking sMIC neutralizing antibody, a) the amino acid sequence of the light chain CDR1 is shown by SEQ ID NO: 4; b) the amino acid sequence of the light chain CDR2 is shown by SEQ ID NO: 5; c) the amino acid sequence of the light chain CDR3 is shown by SEQ ID NO: 6; d) the amino acid sequence of the heavy chain CDR1 is shown by SEQ ID NO: 7; e) the amino acid sequence of the heavy chain CDR2 is shown by SEQ ID NO: 8; and f) the amino acid sequence of the heavy chain CDR3 is shown by SEQ ID NO:

9. **Claim 2** The complex according to claim 1, wherein the non-blocking antibody comprises a light chain variable region shown by SEQ ID NO:

11. **Claim 3** The complex according to claim 1 or 2, wherein the non-blocking antibody comprises a heavy chain variable region shown by SEQ ID NO:

10. **Claim 4** A natural killer group 2D (NKG2D) agonist complex comprising a soluble MHC class I chain-related molecule (sMIC) and a non-blocking sMIC neutralizing antibody, wherein in the non-blocking sMIC neutralizing antibody, a) the amino acid sequence of the light chain CDR1 is shown by SEQ ID NO: 12; b) the amino acid sequence of the light chain CDR2 is shown by SEQ ID NO: 13; c) the amino acid sequence of the light chain CDR3 is shown by SEQ ID NO: 14; d) the amino acid sequence of the heavy chain CDR1 is shown by SEQ ID NO: 15; e) the amino acid sequence of the heavy chain CDR2 is shown by SEQ ID NO: 16; and f) the amino acid sequence of the heavy chain CDR3 is shown by SEQ ID NO:

17. **Claim 5** The complex according to claim 1 or 4, wherein the non-blocking antibody comprises a light chain variable region shown by SEQ ID NO:

19. **Claim 6** The complex according to claim 1, 4 or 5, wherein the non-blocking antibody comprises a heavy chain variable region shown by SEQ ID NO:

18. **Claim 7** The complex according to any one of claims 1 to 6, wherein the sMIC is sMICA. **Claim 8** The complex according to any one of claims 1 to 6, wherein the sMIC is sMICB. **Claim 9** The complex according to claim 7, wherein the sMICA comprises an amino acid sequence shown by one of SEQ ID NOs: 1 and 20-77. **Claim 10** The complex according to claim 8, wherein the sMICB comprises an amino acid sequence shown by one of SEQ ID NOs: 2 and 78-100. **Claim 11** A soluble MIC polypeptide (sMIC) neutralizing antibody, wherein the sMIC neutralizing antibody comprises a CDR comprising the amino acid sequences of SEQ ID NOs: 4-9, the sMIC neutralizing antibody.

12. The sMIC neutralizing antibody according to claim 11, wherein the sMIC neutralizing antibody comprises a light chain variable region comprising SEQ ID NO:

11.

13. The sMIC neutralizing antibody according to claim 11 or claim 12, wherein the sMIC neutralizing antibody comprises a heavy chain variable region comprising SEQ ID NO:

10.

14. A composition comprising the complex according to any one of claims 1 to 10, or the sMIC neutralizing antibody according to any one of claims 11 to 13, and a pharmaceutically acceptable carrier, diluent, or adjuvant.

15. A pharmaceutical composition for activating CD8 T cells in a subject in need thereof, comprising the complex according to any one of claims 1 to 10 or the sMIC neutralizing antibody according to any one of claims 11 to 13.

16. The pharmaceutical composition according to claim 15, wherein the subject is suffering from cancer.

17. The cancer is basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma (GBM), liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, lymphoma including Hodgkin lymphoma and non-Hodgkin lymphoma, melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary tract cancer, vulvar cancer, B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade non-cleaved cell NHL, large tumor lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD)), and is an abnormal angiogenesis associated with nevus, edema or Meigs syndrome, the pharmaceutical composition according to claim 16.

18. The pharmaceutical composition according to any one of claims 15 to 17, wherein the subject is suffering from an MHC class I chain-related molecule (MIC)-negative cancer.

19. The pharmaceutical composition according to claim 15, wherein the subject is suffering from a viral infection.

20. The pharmaceutical composition according to claim 19, wherein the viral infection is caused by a DNA virus; an RNA virus, a flavivirus, an influenza virus; a calicivirus; or a rabies virus, rinderpest virus and an arenavirus.

21. The pharmaceutical composition according to claim 20, wherein the DNA virus is a herpes virus, Epstein-Barr virus, cytomegalovirus; poxvirus, hepadnavirus, papillomavirus, or adenovirus.

22. The pharmaceutical composition according to claim 21, wherein the herpes virus is a herpes simplex virus.

23. The pharmaceutical composition according to claim 21, wherein the hepadnavirus is a hepatitis B virus.

24. The pharmaceutical composition according to claim 20, wherein the RNA virus is HIV I, HIV II, HTLV I, HTLV II; poliovirus, hepatitis A, coronavirus, orthomyxovirus, paramyxovirus, rabies virus or hepatitis C virus.

25. The pharmaceutical composition according to claim 24, wherein the coronavirus is severe acute respiratory syndrome (SARS).

26. The pharmaceutical composition according to claim 24, wherein the paramyxovirus is a measles virus.

27. The pharmaceutical composition according to claim 19, wherein the viral infectious disease is caused by lymphocytic choriomeningitis (LCMV).

28. A pharmaceutical composition for a viral infectious disease in a subject in need of treatment for a viral infectious disease, comprising the complex according to any one of claims 1 to 10 or the sMIC neutralizing antibody according to any one of claims 11 to 13.

29. A pharmaceutical composition for treating cancer in a subject in need of treatment for cancer, comprising the complex according to any one of claims 1 to 10 or the sMIC neutralizing antibody according to any one of claims 11 to 13.

30. The cancer is basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma (GBM), liver carcinoma, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, lymphoma including Hodgkin lymphoma and non-Hodgkin lymphoma, melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary tract cancer, vulvar cancer, B cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade non-cleaved cell NHL, large tumor lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD)), the pharmaceutical composition according to claim 29.

31. The pharmaceutical composition according to claim 29 or 30, wherein the subject has a MHC class I chain-related molecule (MIC)-negative cancer.

32. The pharmaceutical composition according to any one of claims 15-18 and 29-31, further comprising administering an immune checkpoint inhibitor to the subject.

33. The immune checkpoint inhibitor is MGA27, ipilimumab, pembrolizumab, nivolumab, atezolizumab, IMP321, IPH2101, tremelimumab, pidilizumab, MPDL3280A, MEDI4736, MSB0010718C, AUNP12, avelumab, durvalumab, and TSR-022, the pharmaceutical composition according to claim 32.

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