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-associated molecule (sMIC) and a non-blocking sMIC neutralizing antibody. Also provided are methods of using such complexes to activate CD8 T cells and methods of treating MIC-negative cancers and viral infections.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 890,933, filed on August 23, 2019, the disclosure of which is incorporated by reference in its entirety.

[0003] Government Support Statement

[0004] This invention was made with government support under R01CA208246 and R41CA206688-01A1 awarded by the National Institute of Health (NIH). The government has certain rights in this invention.

[0005] Incorporation by Reference of Electronically Submitted Materials

[0006] Incorporated by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing filed concurrently with this article and identified as follows: File name: 2019-146_Seqlisting.txt; Size: 75,481 bytes; Creation date: August 21, 2020. Background Art

[0008] Effective T cell co-stimulation is essential for the primary induction and subsequent maintenance of antigen-specific T cell responses. In addition to increased co-inhibitory signals, an insufficient co-stimulatory tumor microenvironment is also an important reason for the poor activation and maintenance of tumor-killing CD8 T cells. Therefore, one of the main goals of cancer immunotherapy is to provide sustainable co-stimulatory signals to enhance the generation and persistence of effective tumor-killing CD8 T cells and ultimately achieve durable tumor control. However, with the exception of engineered CAR-T cells containing co-stimulatory motifs in the engineered TCR, means to enhance sustained in situ CD8 T cell co-stimulation remain far from expectations due to the unsustainable expression of the canonical and activation-induced families of co-stimulatory receptors on CD8 T cells in the tumor microenvironment. Background Art

[0009] Effective T cell co-stimulation is essential for the primary induction and subsequent maintenance of antigen-specific T cell responses 1-3 Inadequate co-stimulation is the main reason for poor activation and maintenance of tumor-killing antigen-specific CD8 T cells 2,3. Therefore, one of the main goals of cancer immunotherapy is to provide sustainable co-stimulatory signals to enhance the generation and persistence of effective tumor-killing CD8 T cells and ultimately achieve durable tumor control. However, with the exception of engineered CAR-T cells containing co-stimulatory motifs in the engineered TCR, means to enhance sustained in situ CD8 T cell co-stimulation remain far from ideal due to: 1) unsustainable expression of the canonical and activation-induced families of co-stimulatory receptors on CD8 T cells in the tumor microenvironment; 2) considerable autoimmune cytotoxicity potentially generated by undesired co-stimulation of lymphocytes compared to cytotoxic T cells 4 .

[0010] Natural Killer Group 2D (NKG2D) is an activating receptor expressed by all human NK cells, also defined as human NKT, CD8T, and T cells. 5-10 . Similar to the classical co-stimulatory molecule CD28 and the activation-induced TNF-R superfamily of co-stimulatory molecules, NKG2D co-stimulation amplifies the amplitude of CD3 / TCR signaling. Unlike these well-studied co-stimulatory molecules, NKG2D expression is not associated with T cell activation or functional status and is not found on CD4 T cells or B cells under normal conditions. 5-11 Compelling evidence shows that NKG2D costimulation not only enhances CD8 T cell effector function but is also important for memory CD8 T cell development and rescue 11-13 These insights have recognized NKG2D as an instrumental co-stimulatory molecule that can generate potent and durable tumor-killing antigen-specific CD8 T cells. Summary of the invention

[0011] In one aspect, described herein is a natural killer group 2D (NKG2D) complex comprising a soluble MHC I chain-associated molecule (sMIC) and a non-blocking sMIC neutralizing antibody. In some embodiments, the non-blocking antibody in the complex comprises the CDRs shown in SEQ ID NO: 4-9. In some embodiments, the non-blocking antibody in the complex comprises the light chain variable region shown in SEQ ID NO: 11. In some embodiments, the non-blocking antibody in the complex comprises the heavy chain variable region shown in SEQ ID NO: 10. In some embodiments, the non-blocking antibody in the complex comprises the CDRs shown in SEQ ID NO: 12-17. In some embodiments, the non-blocking antibody in the complex comprises the light chain variable region shown in SEQ ID NO: 19. In some embodiments, the non-blocking antibody in the complex comprises the heavy chain variable region shown in SEQ ID NO: 18.

[0012] In some embodiments, the soluble MIC in the complex is sMICA. In some embodiments, sMICA comprises the amino acid sequence shown in one of SEQ ID NO: 1 and 20-77. In some embodiments, the soluble MIC in the complex is sMICB. In some embodiments, sMICB comprises the amino acid sequence shown in one of SEQ ID NO: 2 and 78-100.

[0013] Also contemplated are compositions comprising a NKG2D complex described herein and a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0014] 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 I chain-related molecule (sMIC) and a non-blocking sMIC neutralizing antibody.

[0015] In some embodiments, the subject suffers from a viral infection. In some embodiments, the viral infection is caused by a DNA virus (e.g., herpes virus, such as herpes simplex virus, Epstein-Barr virus, cytomegalovirus; pox virus, such as variola (small pox) virus; hepatitis virus (e.g., hepatitis B virus); papillomavirus; adenovirus); RNA virus (e.g., HIV I, II; HTLV I, II; poliovirus; hepatitis A; coronavirus, such as acute respiratory syndrome (SARS); orthomyxovirus (e.g., influenza virus); paramyxovirus (e.g., measles virus); rabies virus (e.g., rabies virus); virus); hepatitis C virus); flavivirus, influenza virus; calicivirus; or rabies virus, rinderpest virus, and arena virus. In some embodiments, the viral infection is caused by lymphocytic choriomeningitis (LCMV).

[0016] In some embodiments, the subject has cancer. Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, cancer), gastrointestinal cancer, glioblastoma (GBM), hepatocarcinoma, hepatoma, intraepithelial neoplasm, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, lymphoma including Hodgkin's lymphoma and non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer Cancer of the uterus or endometrium, cancer of the urinary system, vulvar cancer, B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's Macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorder (PTLD).

[0017] In some embodiments, the subject has an MHC I chain-associated molecule (MIC) negative cancer. In some embodiments, the subject has a viral infection.

[0018] In some embodiments, the method described herein further includes 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

[0019] Figure 1 The sMIC / anti-MIC D4H3 mAb complex was shown to provide strong co-stimulation to amplify CD3-mediated CD8 T cell activation.

[0020] Figure 2 shows that co-stimulation of sMIC / NO4 mAb complexes amplifies antigen-specific TCR signaling. Human tyrosinase-specific HLA-A2-restricted TIL13831 was co-cultured with APC T2-A2 cells overnight under the indicated conditions prior to functional assays.

[0021] Figure 3 Co-stimulation with sMIC / D4H3 mAb complexes was shown to amplify antigen-specific TCR signaling. Human tyrosinase-specific HLA-A2-restricted TIL13831 was co-cultured with APC T2-A2 cells overnight (o / n) under the indicated conditions prior to functional assays.

[0022] Figure 4 is a graph showing that treatment with sMIC / D4H3 inhibits the growth of MIC colon tumors. All agents (control IgG, antibody D4H3, and sMIC / D4H3 complex) were administered 4 mg / Kg BW intraperitoneally (ip) twice weekly. *, P<0.01.

[0023] Figure 5 Figures A and 5B provide graphs showing the detection of sMIC(A / B) / D4H3 complex or sMIC(A / B) / NO4 complex binding to NKG2D. Recombinant soluble NKG2D-His tag (2 μg / ml) was immobilized on a 96-well plate overnight at 4°C. 50 μl of the indicated concentrations of recombinant sMICA or sMICB were mixed with different amounts of D4H3 ( Figure 5 A) or NO4( Figure 5B) Mixing. The X-axis indicates the concentration of D4H3 or NO4. After incubation and washing, the binding of SMIC / D4H3 or sMIC / NO4 complexes to NKG2D was detected by HRP-conjugated goat anti-mouse IgG.

[0024] FIG. 6 depicts the MICA alleles shown in SEQ ID NOs: 20-77.

[0025] Figure 7 The MICB alleles shown in SEQ ID NOs: 78-100 are depicted.

[0026] Figure 8 is a graph showing that administration of sMIC / D4H3 complex to mice inoculated with lymphocytic choriomeningitis virus (LCMV) significantly reduced the LCMV titer of the mice. DETAILED DESCRIPTION

[0027] This article describes a natural killer group 2D (NKG2D) complex that is soluble M HC I chain Related molecules (sMIC) and non-blocking sMIC neutralizing antibodies. A fusion protein is also described herein, which comprises an sMIC connected to the heavy chain (or light chain) of a non-blocking sMIC neutralizing antibody by a polylinker. As shown in the embodiments provided herein: 1) compared with soluble NKG2D ligands, sMIC / anti-sMIC complexes provide a lasting NKG2D costimulatory amplitude to amplify TCR / CD3 signaling; 2) sMIC / anti-sMIC complexes and CD28 agonists produce additive costimulatory effects; 3) in contrast to the negative effects of soluble NKG2D ligands that downregulate NKG2D expression, sMIC / anti-sMIC costimulation stabilizes NKG2D expression on CD8 T cells.

[0028] Major histocompatibility complex class I chain-related (MIC) polypeptide

[0029] 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. The presence of MIC polypeptides on the cell surface can signal tumor immune destruction to the immune receptor NKG2D, usually through natural killer cells (NK cells) and cytotoxic T cells (CTL). However, in many tumors, MIC is shed from the tumor surface, resulting in reduced host immunity to tumor cells and promoting tumor escape and progression. MIC polypeptides include but are not limited to human MICA (e.g., NCBI Ref Seqs NP_000238 (SEQ ID NO: 1) and 001170990) and human MICB (e.g., NCBI Ref Seq: NP_005922 (SEQ ID NO: 2)). In some embodiments, the MIC polypeptide comprises MICA. In some embodiments, the MIC polypeptide may comprise MICB. In some embodiments, the MIC polypeptide comprises the following amino acid sequence:

[0030] EPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRYDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNVETEEWTVPQSSRAQTLA MNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESSVVLRRRVPPMVNVTRSEALEGNITVTCGASSFYPRNITLTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPS(SEQ ID NO:3).

[0031] In some embodiments, the NKG2D complex (or fusion protein) comprises a soluble MIC (sMIC) polypeptide. As used herein, "soluble MIC" or "sMIC" refers to a portion of a MIC polypeptide lacking a transmembrane domain, such as an extracellular portion of a MIC that has been cleaved from a transmembrane domain. In some embodiments, the soluble MIC may comprise about, for example, amino acids 24-260 of SEQ ID NO: 1 or 2. In some embodiments, the soluble MIC may comprise about, for example, 20 or more amino acids of residues 24-260 of SEQ ID NO: 1 or 2, such as 20, 50, 100, 150 or more amino acids of residues 24-260 of SEQ ID NO: 1 or 2.

[0032] In some embodiments, the NK2GD agonist complex (or fusion protein) comprises a MICA allele set forth in one of SEQ ID NOs: 20-77. In some embodiments, the NK2GD agonist complex (or fusion protein) comprises a MICB allele set forth in one of SEQ ID NOs: 78-100.

[0033] Anti-MIC antibody

[0034] In some embodiments, the NKG2D agonist complex (or fusion protein) described herein comprises a non-blocking antibody or antigen binding portion thereof that selectively binds to a MIC polypeptide. In some embodiments, the MIC polypeptide is a soluble MIC polypeptide (sMIC).

[0035] The term "non-blocking antibody" as used herein refers to a sMIC neutralizing antibody that does not block the interaction of NKG2D with membrane-bound MIC or soluble MIC, and thus does not interfere with the sensitivity of NKG2D-mediated NK cell cytolytic activity to MIC+ cells.

[0036] As used herein, the term "antibody" refers to an immunoglobulin molecule. The term also refers to antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, and the various forms, including full-length antibodies and antigen-binding portions thereof; including, for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, single domain antibodies (dAbs), diabodies, multispecific antibodies, bispecific antibodies, anti-idiotypic antibodies, bispecific antibodies, functionally active epitope-binding fragments thereof, bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol 17, 105 (1987)), and single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference). (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), which are incorporated herein by reference).

[0037] Each heavy chain consists of a variable region of the heavy chain (abbreviated herein 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 consists of a variable region of the light chain (abbreviated herein as LCVR or VL) and a constant region of the light chain. The light chain constant region consists of the CL domain. The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs) and interspersed with conserved regions called framework regions (FRs). Each VH and VL region is therefore composed of three CDRs and four FRs, which are arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

[0038] As used herein, the term "CDR" refers to the complementary determining region in the variable sequence of an antibody. There are three CDRs in each variable region of heavy and light chains, which are designated as CDR1, CDR2 and CDR3 for each variable region. The accurate boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest" (National Institutes of Health, Bethesda, Maryland, Md.), (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Other boundaries defining the CDRs overlapping with Kabat CDRs are defined by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J. Molecular Biology (J. Mol. Biol. The CDR boundary definitions described herein may not strictly follow one of the above systems, but will nevertheless overlap with the Kabat CDRs, but the boundaries may be shortened or lengthened in light of predictions or experimental findings that a particular residue or group of residues, or even an entire CDR, does not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, but preferred embodiments use CDRs defined by Kabat.

[0039] As used herein, "selective binding" or "specific binding" refers to the ability of an anti-MIC binding peptide (e.g., an antibody or a portion thereof) described herein to bind to a target, such as a MIC molecule present on the surface of a cell, with a KD of 10 -5 M (10000nM) or less, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 11 M, 10 -12 M or less (or any range that includes any of these values ​​as endpoints). Specific binding can be affected by, for example, the affinity and avidity of the polypeptide agent and the concentration of the polypeptide agent. One of ordinary skill in the art can use any suitable method, such as titrating the polypeptide agent in a suitable cell binding assay to determine the appropriate conditions for the polypeptide agent described herein to selectively bind to the target. A polypeptide that specifically binds to a target will not be displaced by a non-similar competitor. In certain embodiments, when an antibody or its antigen-binding portion preferentially recognizes a target antigen in a complex mixture of proteins and / or macromolecules, the antibody or its antigen-binding portion is referred to as specifically binding to the antigen. In some embodiments, the antibody or its antigen-binding portion is expressed as 10 -5 M (10000nM) or less, 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 The dissociation constant (KD) of M binds to the sMIC polypeptide.

[0040] The terms "antigen-binding fragment" or "antigen-binding portion" of an antibody are used interchangeably herein and refer to one or more fragments of an antibody as described herein that still exhibit binding affinity as defined above. Fragments of intact antibodies have been shown to be able to perform the antigen-binding function of an antibody. Examples of antigen-binding fragments include, but are not limited to, (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments interconnected by a disulfide bond in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the FL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of the VH domain or VH, CH1, CH2, DH3 or VH, CH2, CH3 (dAbs or single domain antibodies (comprising only the VL domain) have also been shown to specifically bind to a target epitope). Although the two domains of the Fv fragment (i.e., VL and VH) are encoded by separate genes, they can be further linked to each other using synthetic linkers (e.g., poly G4S amino acid sequences) and recombinant methods, making it possible to prepare them as a single protein chain in which the VL and VH regions combine to form a monovalent molecule (called single-chain Fv (ScFv); see, e.g., 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 (e.g., "diabodies") are also included herein. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short for the two domains to be able to combine on the same chain is used, thereby forcing the domains to pair with complementary domains of different chains and form two antigen binding sites (see, e.g., Holliger, R et al. (1993) Proc. Natl. Acad. Sci. USA 90:64446448; Poljak, RJ et al. (1994) Structure 2:1121-1123). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy and light chain constant domain amino acid sequences are known in the art.

[0041] In addition, antibodies or antigen-binding portions thereof as described herein can be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Related to such immunoadhesion molecules are the use of streptavidin core regions to prepare tetrameric scFv molecules (Kipriyanov, SM et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides and C-terminal polyhistidines, such as hexahistidine tags ('hexahistidine tags' disclosed as SEQ ID NO: 18) to produce divalent and biotinylated scFv molecules (Kipriyanov, SM et al. (1994) Mol. Immunol. 31: 10471058).

[0042] In some embodiments, the antibody is an IgG, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a multispecific antibody, a bispecific antibody, an anti-idiotypic antibody or a bispecific antibody. In some embodiments, the antigen-binding portion of the antibody is a Fab, Fab', F(ab')2, Fv, a disulfide-linked Fv, scFv, a single domain antibody, a diabody or a functionally active epitope-binding fragment thereof.

[0043] The term "human antibody" refers to an antibody whose variable region and constant region correspond to or are derived from the immunoglobulin sequence of human germline, as described by, for example, Kabat et al. (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, human antibodies may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR, and particularly in CDR3. Recombinant human antibodies as described herein have variable regions and may also include constant regions of immunoglobulin sequences derived from human germline (see Kabat, EA 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 specific embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo mutagenesis in recipient cells if an animal transgenic for human Ig sequences is used) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while related to or derived from VH and VL sequences of the human germline, do not naturally occur in the human antibody germline repertoire in vivo. According to specific embodiments, such recombinant antibodies are the result of selective mutagenesis or back mutation or both. Preferably, the mutagenesis results in an affinity for the target that is greater than the affinity of the parent antibody, and / or an affinity for non-target structures that is less than the affinity of the parent antibody.

[0044] The term "chimeric antibody" refers to an antibody containing heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies with mouse heavy and light chain variable regions connected to human constant regions. Humanized antibodies have variable region framework residues substantially from human antibodies (referred to as acceptor antibodies) and complementary determining regions substantially from non-human antibodies (e.g., mouse antibodies) (referred to as donor immunoglobulins). See, Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90 / 07861, U.S. Pat. No. 5,693,762, No. 5,693,761, No. 5,585,089, No. 5,530,101 and Winter, U.S. Pat. No. 5,225,539, which are incorporated herein by reference in their entirety. If present, the constant region is also substantially or completely from human immunoglobulins. Human variable domains are usually selected from human antibodies, and their framework sequences exhibit a high degree of sequence identity with the (mouse) variable region domains of the CDR source. The heavy and light chain variable region framework residues may be substantially similar to the regions of the same or different human antibody sequences. The human antibody sequence may be the sequence of a naturally occurring human antibody or may be a consensus sequence of several human antibodies. See Carter et al., WO 92 / 22653, which is incorporated herein by reference in its entirety.

[0045] In some embodiments, the antibodies described herein are not naturally occurring biomolecules. For example, in the absence of human intervention and manipulation (e.g., a manufacturing step performed by a person), mouse antibodies produced against human antigens do not occur in nature. Chimeric antibodies are also not naturally occurring biomolecules, for example, because they contain sequences obtained from multiple species and assembled into recombinant molecules. In certain specific embodiments, human antibody reagents described herein are not naturally occurring biomolecules, for example, fully human antibodies against human antigens will be subjected to negative selection in nature and are not naturally present in the human body.

[0046] Those of ordinary skill in the art will recognize that individual substitutions, deletions or additions of the amino acid sequence that change a single amino acid or a small number of amino acids in the coding sequence are "conservatively modified variants," wherein the changes result in the substitution of an amino acid with a chemically similar amino acid and retain the ability to specifically bind to a target antigen of a MIC polypeptide (e.g., an epitope present on sMIC). Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs and alleles consistent with the present disclosure.

[0047] In some embodiments, the antibody, or antigen binding portion thereof, specifically binds to a sMIC polypeptide and comprises one or more heavy and light chain complementarity determining regions (CDRs) selected from the group consisting of: (a) a light chain CDR1 having an amino acid sequence of SEQ ID NO:4; (b) a light chain CDR2 having an amino acid sequence of SEQ ID NO:5; (c) a light chain CDR3 having an amino acid sequence of SEQ ID NO:6; (d) a heavy chain CDR1 having an amino acid sequence of SEQ ID NO:7; (e) a heavy chain CDR2 having an amino acid sequence of SEQ ID NO:8; (f) a heavy chain CDR3 having an amino acid sequence of SEQ ID NO:9. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise one or more CDRs, e.g., 1 CDR, 2 CDRs, 3 CDRs, 4 CDRs, 5 CDRs, or 6 CDRs, selected from the group consisting of: (a) a light chain CDR1 having an amino acid sequence of SEQ ID NO:4; (b) a light chain CDR2 having an amino acid sequence of SEQ ID NO:5; (c) a light chain CDR3 having an amino acid sequence of SEQ ID NO:6; (d) a heavy chain CDR1 having an amino acid sequence of SEQ ID NO:7; (e) a heavy chain CDR2 having an amino acid sequence of SEQ ID NO:8; (f) a heavy chain CDR3 having an amino acid sequence of SEQ ID NO:9. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain or a portion thereof comprising one or more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs, selected from the group consisting of: a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 7; a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 8; and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain or a portion thereof comprising one or more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs, selected from the group consisting of: a light chain CDR1 having an amino acid sequence of SEQ ID NO: 4; a light chain CDR2 having an amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 6.

[0048] In some embodiments, the antibody or antigen binding portion thereof comprises a light chain complementary determining region (CDR): (a) a light chain CDR1 having an amino acid sequence of SEQ ID NO: 12; (b) a light chain CDR2 having an amino acid sequence of SEQ ID NO: 13; (c) a light chain CDR3 having an amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or antigen binding portion thereof comprises a heavy chain complementary determining region (CDR): (d) a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 15; (e) a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 16; (f) a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise one or more CDRs, e.g., 1 CDR, 2 CDRs, 3 CDRs, 4 CDRs, 5 CDRs, or 6 CDRs, selected from the group consisting of: (a) a light chain CDR1 having an amino acid sequence of SEQ ID NO: 12; (b) a light chain CDR2 having an amino acid sequence of SEQ ID NO: 13; (c) a light chain CDR3 having an amino acid sequence of SEQ ID NO: 14; (d) a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 15; (e) a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 16; (f) a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain or a portion thereof comprising one or more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs, selected from the group consisting of: a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 15; a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 16; and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain or a portion thereof comprising one or more CDRs, e.g., 1 CDR, 2 CDRs, or 3 CDRs, selected from the group consisting of: a light chain CDR1 having an amino acid sequence of SEQ ID NO: 12; a light chain CDR2 having an amino acid sequence of SEQ ID NO: 13; and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 14.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10.

[0050] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18.

[0051] In embodiments where the antibodies as described herein comprise at least one CDR that is different from the sequence of SEQ ID NO: 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, "Antibody affinity maturation by random mutagenesis", Methods in Molecular Biology: Antibody Engineering 248: 345-349 (which is incorporated herein by reference in its entirety), particularly in Figure 2 and Section 3.3, describes a method for generating a library for any CDR of interest. This allows one of ordinary skill in the art to identify alternative CDRs, including conservative substitution variants of specific CDR sequences described herein, which, when present in an antibody or antigen-binding fragment thereof described herein, will produce an antigen or antigen-binding fragment thereof that will bind to a MIC polypeptide, but will not block the binding of the MIC polypeptide to other antibodies. The methods described in Fujii et al. also allow one of ordinary skill in the art to screen light chain sequences that will result in the desired binding behavior when combined with a known heavy chain fragment, or vice versa.

[0052] In some embodiments, the antibodies and / or antigen-binding portions thereof described herein may be variants of the sequences described herein, such as conservative substitution variants of antibody polypeptides. In some embodiments, the variant is a conservatively modified variant. For example, a conservatively substituted variant can be obtained by mutation of a natural nucleotide sequence. As referred to herein, a "variant" is a polypeptide substantially homologous to a natural or reference polypeptide, but has an amino acid sequence different from the amino acid sequence of a natural or reference polypeptide due to one or more deletions, insertions or substitutions. The DNA sequence encoding the variant polypeptide encompasses a sequence comprising one or more nucleotide additions, deletions or substitutions compared to a natural or reference DNA sequence, encoding a variant protein or fragment thereof that retains activity, such as an antigen-specific binding activity of a related target polypeptide (e.g., sMIC polypeptide). A variety of PCR-based site-specific mutagenesis methods are also known in the art and can be applied by ordinary technicians.

[0053] Examples of substitution variants include conservative substitutions of amino acids that do not change the sequence of the CDRs, such as H or V L Conservative substitutions in sequences not encompassed by the CDRs may be substitutions relative to wild-type or naturally occurring sequences, such as human or murine framework and / or constant region antibody sequences.

[0054] In some embodiments, a conservatively modified variant of an antibody may contain changes other than the CDRs, for example, a conservatively modified variant of an antibody agent may contain CDRs having the sequence of one or more of SEQ ID NOs: 4-9 and 12-17.

[0055] A given amino acid may be replaced by a residue having similar physiochemical properties, for example, replacing one aliphatic residue for another (e.g., Ile, Val, Leu or Ala), or replacing one polar residue for another (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative substitutions (e.g., substitutions of entire regions having similar hydrophobic properties) are well known. Polypeptides comprising conservative amino acid substitutions may be tested in any of the assays described herein to confirm that the desired activity, such as antigen binding activity and specificity of a native or reference polypeptide, is retained.

[0056] Amino acids can be grouped according to the similarity of their side chain properties (A Lehninger, Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York, (1975)): (1) Non-polar: 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).

[0057] Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would entail exchanging a member of one of these classes for another.

[0058] Specific conservative substitutions include, for example: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0059] In some embodiments, the CRH peptide 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 mutant sequences can be determined, for example, by comparing the two sequences using a freely available computer program commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0060] The change of the native amino acid sequence can be achieved by any of a variety of techniques known to those skilled in the art. For example, mutations can be introduced by synthesizing oligonucleotides containing mutant sequences at a specific locus, and the mutant sequences are flanked by restriction sites that can be connected to fragments of the native sequence. After connection, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide a nucleotide sequence with a change in a specific codon that changes according to a desired substitution, deletion or insertion. The techniques for making such changes are well established and include, for example, those disclosed in Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37: 73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety.

[0061] Any cysteine ​​residue not involved in maintaining the correct conformation of the polypeptide may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, one or more cysteine ​​bonds may be added to a polypeptide to improve its stability or promote oligomerization.

[0062] Methods for preparing antibodies

[0063] Traditionally, monoclonal antibodies have been produced as natural molecules in mouse hybridoma systems. In addition to the technology, the methods and compositions described herein provide recombinant DNA expression of monoclonal antibodies. This allows the production of humanized antibodies and a series of antibody derivatives and complexes in the selected host species. Producing antibodies in bacteria, yeast, transgenic animals and eggs is also an alternative to hybridoma-based production systems. The main advantage of transgenic animals is the potential high yield from renewable resources.

[0064] 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 cassette mutagenesis of variants or non-variant versions prepared earlier by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and antibodies. Can be based on conventional techniques, including blunt or staggered end connection ends, restriction enzyme digestion to provide appropriate ends, appropriate filling of sticky ends, alkaline phosphatase treatment to avoid undesirable engagement and appropriate ligase connection, can be encoded at least one antibody as described herein, part or polypeptide nucleotide sequence and carrier DNA recombinant. The technology used for this type of operation is disclosed in, for example, Maniatis et al., "Molecular Cloning Laboratory Manual (Molecular Cloning, Lab.Manual)" (New York Cold Spring Harbor Laboratory Press (Cold Spring Harbor Lab.Press, NY), 1982 and 1989) and Ausubel, 1987, 1993, and can be used to construct a nucleic acid sequence encoding an antibody molecule or its antigen binding region.

[0065] In some embodiments, the introduced nucleotide sequence is incorporated into a plasmid or viral vector that can replicate autonomously in a recipient host. Any of a variety of vectors can be used for this purpose and are known and available to those skilled in the art or ordinary skill. See, for example, Ausubel et al., 1987, 1993. Important factors in selecting a particular plasmid or viral vector include: the ease with which recipient cells containing the vector can be identified and selected from those that do not contain the vector; the number of copies of the vector required in a particular host; and whether it is necessary to be able to "shuttle" the vector between host cells of different species.

[0066] Exemplary prokaryotic vectors known in the art include plasmids, such as those capable of replication in E. coli. Other gene expression elements for expressing cDNA encoding antibodies or antigen-binding portions thereof include, but are not limited to, (a) viral transcriptional promoters and enhancer elements thereof, such as SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)) and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splicing regions and polyadenylation sites, such as those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites, such as those in SV40 (Okayama et al., 1983) immunoglobulin cDNA genes can be expressed as described by Liu et al., see below, and Weidle et al., 51 Gene 21 (1987), using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S-globin insertion sequence, the immunoglobulin and rabbit S-globin polyadenylation sites, and the SV40 polyadenylation element as expression elements.

[0067] Each fusion gene is assembled or inserted into an expression vector. Recipient cells capable of expressing the chimeric immunoglobulin chain gene product are then transfected with the antibody, its antigen binding portion, or chimeric H or chimeric L chain encoding gene alone, or co-transfected with chimeric H and chimeric L chain genes. The transfected recipient cells are cultured under conditions that allow expression of the incorporated genes, and the expressed immunoglobulin chains or complete antibodies or fragments are recovered from the culture.

[0068] Expression vectors carrying antibodies or antigen-binding portions thereof as described herein can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, conjugation, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988), as known to those of ordinary skill in the art.

[0069] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin molecules, including leader peptide removal, H chain and L chain folding and assembly, antibody glycosylation, and secretion of functional antibody proteins.

[0070] Mammalian cells that can be used as hosts for producing antibody proteins include, in addition to the lymphoid cells described above, cells of fibroblastic 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 COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6 TM In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modification on the heavy chain and / or light chain. For example, in some embodiments, the sialylation level of a polypeptide produced by CHO cells is higher than that of the same polypeptide produced by 293 cells.

[0071] In some embodiments, one or more antibodies, or antigen-binding portions thereof, as described herein can be produced according to any suitable method in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding a polypeptide.

[0072] In some embodiments, an antibody or antigen-binding portion thereof as described herein is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, for example, 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 entirety.

[0073] In some aspects, provided herein are methods and systems for producing humanized antibodies, the humanized antibodies being prepared by a process comprising: maintaining a host transformed with a first expression vector encoding a humanized antibody light chain and a second expression vector encoding a humanized antibody heavy chain under conditions such that each chain is expressed, and isolating the humanized antibodies formed by assembling the chains so expressed. The first and second expression vectors may be the same vector. Also provided herein are DNA sequences encoding light or heavy chains of humanized antibodies; expression vectors incorporating the DNA sequences; and hosts transformed with the expression vectors.

[0074] Those of ordinary skill in the art can generate humanized antibodies based on the sequences and information provided herein without undue experimentation. In one approach, four general steps are used to humanize monoclonal antibodies, see, for example, U.S. Patent Nos. 5,585,089; 6,835,823; 6,824,989. The steps are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light chain and heavy chain variable domains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanization process; (3) actual humanization methods / techniques; (4) transfection and expression of the humanized antibody.

[0075] Typically, the CDR regions in humanized antibodies and human antibody variants are substantially identical, and more typically, identical to the corresponding CDR regions in the mouse or human antibody from which they are derived. Although generally undesirable, one or more conservative amino acid substitutions may sometimes be made to CDR residues without significantly affecting the binding affinity of the resulting humanized immunoglobulin or human antibody variant. Sometimes, substitutions in CDR regions may enhance binding affinity.

[0076] In addition, techniques developed for producing "chimeric antibodies" by splicing genes from mice or other species, antibody molecules with appropriate antigenic specificity, and genes from human antibody molecules with appropriate biological activity can be used (see Morrison et al., Proc. Natl. Acad. Sci. USA 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); which are incorporated herein by reference in their entirety). Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable regions derived from murine monoclonal antibodies and human immunoglobulin constant regions, such as humanized antibodies.

[0077] The variable segment of a chimeric antibody is generally connected to at least a portion of an immunoglobulin constant region (Fc), generally a constant region of a human immunoglobulin. Human constant region DNA sequences can be isolated from a variety of human cells, such as immortalized B cells (WO 87 / 02671; which is incorporated herein by reference in its entirety) according to well-known methods. The antibody may contain a light chain and a heavy chain constant region. The heavy chain constant region may include a CH1 region, a hinge region, a CH2 region, a CH3 region, and sometimes a CH4 region. For therapeutic purposes, the CH2 domain may be deleted or omitted.

[0078] Alternatively, techniques described for producing single-chain antibodies (see, e.g., U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989); which are incorporated herein by reference in their entirety) can be used to produce single-chain antibodies. Single-chain antibodies are formed by connecting the heavy and light chain fragments of the Fv region by an amino acid bridge, thereby producing a single-chain polypeptide. Techniques for assembling functional Fv fragments in E. coli (see, e.g., Skerra et al., Science 242:1038-1041 (1988); which are incorporated herein by reference in their entirety) can also be used.

[0079] Treatment

[0080] In one aspect, described herein is a method of activating CD8 T cells in a subject in need thereof, comprising administering to the subject a complex (or fusion protein) comprising a soluble MHC I chain-associated molecule (sMIC) and a non-blocking sMIC neutralizing antibody.

[0081] In some embodiments, the subject suffers from a viral infection. CD8+ T cells of many viruses have been shown to play a role in protection. See Huber et al., Front《Immunol.》5:171,2014. In some embodiments, the subject suffers from a viral infection caused by: DNA virus (e.g., herpes virus, such as herpes simplex virus, Epstein-Barr virus, cytomegalovirus; pox virus, such as smallpox virus; hepatitis virus (e.g., hepatitis B virus); papillomavirus; adenovirus); RNA virus (e.g., HIV I, II; HTLV I, II; poliovirus; hepatitis A; coronavirus, such as sudden acute respiratory syndrome (SARS); orthomyxovirus (e.g., influenza virus); paramyxovirus (e.g., measles virus); rabies virus; hepatitis C virus); flavivirus, influenza virus; calicivirus; rabies virus, rinderpest virus, arenavirus, etc. In some embodiments, the viral infection is caused by lymphocytic choriomeningitis (LCMV). In some embodiments, the subject suffers from a virus-related disease. Exemplary virus-related diseases include, but are not limited to, acquired immunodeficiency; hepatitis; gastroenteritis; hemorrhagic disease; enteritis; carditis; encephalitis; paralysis; bronchiolitis; upper and lower respiratory tract disease; respiratory papillomatosis; arthritis; disseminated disease; meningitis; and mononucleosis.

[0082] In some embodiments, the subject has cancer or a malignant tumor. In some embodiments, the activation of CD8 T cells is determined by enzyme-linked immunospot (ELISPOT), flow cytometry activation sorting (FACS), or target killing cytotoxicity assay. Other methods for determining T cell activation, such as those described in Plebanski et al., Expert. Rev. Vaccines, 9: 595-600, 2010.

[0083] As used herein, "tumor" refers to the uncontrolled growth of cells that interfere with the normal function of body organs and systems. A subject suffering from cancer or tumor is a subject in which objectively measurable cancer cells are present in the subject. This definition includes benign and malignant tumors, as well as potential dormant tumors or micrometastases. Cancers that migrate from their original location and seed other vital organs will eventually lead to the death of the subject through functional degradation of the affected organs. Hematopoietic cancers (e.g., leukemias) can overcome the normal hematopoietic compartment of the subject, resulting in hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia), ultimately leading to death.

[0084] Exemplary cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include, but are not limited to, basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatoma; hepatoma; intraepithelial neoplasm; kidney / renal cancer cancer); laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphomas including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colorectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; vulvar cancer; and other carcinomas and sarcomas; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD).

[0085] In some embodiments, the tumor or malignancy is MIC negative. As used herein, the term "MIC negative tumor" is used to describe tumor cells, tumor cell clusters or tumor masses that do not produce MIC protein. The term is intended to cover all tumor cells and / or tumor masses that do not display MIC protein on the surface of tumor cells, so these cells do not shed MIC protein. In other words, subjects with MIC negative cancer should have no detectable sMIC except background noise. MIC negative tumors can be identified by measuring serum level MIC (e.g., sMICA or sMICb) using standard MICA or MICB detection ELISA, as described in, for example, Ghadially et al., "British Journal of Cancer (Br.J.Cancer)", 116: 1208-1217, 2017, the disclosure of which is incorporated herein by reference. For tumors available for biopsy, tumors with negative MIC expression can also be selected or confirmed, and immunohistochemistry shows no cross-reaction with anti-MIC antibodies.

[0086] As used herein, "subject" refers to a person or an animal. In some embodiments, the animal is a vertebrate, such as a primate, a rodent, a domestic animal, or a game animal. Primates include chimpanzees, crab-eating monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo; feline species, such as house cats; and canine species, such as dogs, foxes, wolves; bird species, such as chickens, emus, ostriches; and fish, such as trout, catfish, and salmon. Patients or subjects include any subset of the foregoing, such as the above-mentioned owners, but do not include 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.

[0087] In some embodiments, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. For example, mammals other than humans can be advantageously used as subjects representing animal models of, for example, various cancers. In addition, the present description and methods can be used to treat domesticated animals and / or pets. The subject can be female or male.

[0088] In some embodiments, the subject has been previously diagnosed with or identified as suffering from or suffering from a condition (e.g., cancer) that requires treatment or one or more complications associated with such a condition, and optionally, but not necessarily, has undergone treatment for the condition or one or more complications associated with the condition. Alternatively, the subject has not been previously diagnosed as having a condition that requires treatment or one or more complications associated with such a condition. For example, the subject may be a subject who exhibits one or more risk factors for the condition or one or more complications associated with the condition or a subject who does not exhibit risk factors. A "subject" who "needs" to be treated for a particular condition may be a subject who suffers from the condition, is diagnosed with the condition, or is at risk of developing the condition.

[0089] As used herein, when used with respect to a disease, disorder or medical condition, the term "treat / treatment / treating" or "improvement" refers to the therapeutic treatment of the condition, wherein the purpose is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a symptom or condition. The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of the condition. If one or more symptoms or clinical markers are reduced, then the treatment is usually "effective". Alternatively, if the progression of the condition is reduced or stopped, then the treatment is "effective". That is, "treatment" includes not only the improvement of symptoms or markers, but also stops or at least slows down the symptom progression or deterioration expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, the alleviation of one or more symptoms, the alleviation of the degree of defects, the stable (i.e., non-deterioration) state of a tumor or malignancy, the delay or slowing of tumor growth and / or metastasis, and the extension of life expectancy compared to the life expectancy without treatment.

[0090] As used herein, the term "administering" refers to placing an agonist complex (or fusion protein) described herein in a subject's body by a method or approach that allows the agent to be at least partially localized to a desired site. A pharmaceutical composition comprising an agonist complex (or fusion protein) described herein can be administered by any suitable route to produce effective treatment in a subject.

[0091] Pharmaceutical compositions and routes of administration

[0092] Compositions comprising a natural killer group 2D (NKG2D) agonist complex (or fusion protein) are also contemplated, the complex comprising a soluble MHC I chain-associated molecule (sMIC) and a non-blocking sMIC neutralizing antibody as described herein. In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and a carrier acceptable for use in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, and are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment.

[0093] The preparation of pharmaceutical compositions comprising active ingredients dissolved or dispersed therein is well known in the art and does not need to be limited based on the formulation. Typically, such compositions are prepared as injectable liquid solutions or suspensions, however, solid forms suitable for solutions or suspensions in liquids before use can also be prepared. The preparation can also be emulsified or present in the form of liposome compositions. The active ingredient can be mixed with an excipient, which is pharmaceutically acceptable and compatible with the active ingredient, and is in an amount suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, glucose, glycerol, ethanol, etc. and combinations thereof. In addition, if necessary, the composition may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., which enhance or maintain the effectiveness of the active ingredient. The therapeutic composition as described herein may include pharmaceutically acceptable salts of the components thereof. Pharmaceutically acceptable salts include acid addition salts (formed by the free amino groups of the polypeptide), which are formed by inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed by 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-ethylamine ethanol, histidine, procaine, etc. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no material other than active ingredients and water or contain buffers such as sodium phosphate at physiological pH, physiological saline or both, such as phosphate buffered saline. Still further, aqueous carriers can contain more than one buffer salt and salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain a liquid phase other than water and excluding water. Examples of such additional liquid phases are glycerol, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of the active agent used in the present invention that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0094] For example, therapeutic compositions containing at least one pharmaceutical agent can be routinely administered in unit doses. When used in relation to therapeutic compositions, the term "unit dose" refers to physically discrete units suitable as unit dosages for subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect together with the required physiologically acceptable diluent (i.e., carrier or excipient).

[0095] The exact amount of active ingredient to be administered depends on the judgment of the practitioner and is specific to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable administration regimens are also variable, but are represented by an initial administration followed by repeated administration at intervals of one or more hours by subsequent injections or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain the concentration in the blood within the range specified for in vivo therapy is contemplated.

[0096] 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, such as an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of a tumor or malignancy. The determination of a therapeutically effective amount is well within the capabilities of those skilled in the art. In general, a therapeutically effective amount may vary with the subject's medical history, age, condition, sex, and the severity and type of the subject's medical condition, as well as the administration of other pharmaceutically active agents.

[0097] The dosage range of medicament depends on efficacy, and encompasses an amount sufficient to produce the desired effect, such as slowing tumor growth or reducing tumor size. The dosage should not be so large as to cause unacceptable adverse side effects. Typically, the dosage will vary with the patient's age, condition, and gender and can be determined by those skilled in the art. In the event of any complications, the dosage can also be adjusted by an individual doctor. In some embodiments, the dosage range is between 0.001 mg / kg body weight and 0.5 mg / kg body weight. Alternatively, the dosage range can be adjusted to maintain serum levels between 1 mg / mL and 1000 mg / mL. For systemic administration, a therapeutic amount, such as 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 a subject.

[0098] The administration of the above dosage can be repeated. In some embodiments, the dosage is administered once a day or multiple times a day. In some embodiments, the dosage is administered daily for several weeks or months. The duration of treatment depends on the clinical progress of the subject and the response to treatment.

[0099] In some embodiments, the dosage is 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.

[0100] In some embodiments, the dose can be administered intravenously. In some embodiments, intravenous administration can be an infusion performed over a period of about 10 minutes to about 3 hours. In some embodiments, intravenous administration can be an infusion performed over a period of about 30 minutes to about 90 minutes.

[0101] In some embodiments, the dosage may be administered approximately once a week. In some embodiments, the dosage may be administered weekly. In some embodiments, the dosage may be administered weekly for approximately 12 to approximately 18 weeks. In some embodiments, the dosage may be administered approximately once every 2 weeks. In some embodiments, the dosage may be administered approximately once every 3 weeks. In some embodiments, the dosage may be approximately 2 mg / kg to approximately 15 mg / kg administered approximately once every 2 weeks. In some embodiments, the dosage may be approximately 2 mg / kg to approximately 15 mg / kg administered approximately once every 3 weeks. In some embodiments, the dosage may be approximately 2 mg / kg to approximately 15 mg / kg administered intravenously approximately once every 2 weeks. In some embodiments, the dosage may be approximately 2 mg / kg to approximately 15 mg / kg administered intravenously approximately once every 3 weeks.

[0102] In some embodiments, the dosage may be about 1 mg to about 2000 mg. In some embodiments, the dosage may be about 3 mg. In some embodiments, the dosage may be about 10 mg. In some embodiments, the dosage may be about 30 mg. In some embodiments, the dosage may be about 1000 mg. In some embodiments, the dosage may be about 2000 mg. In some embodiments, the dosage may be about 3 mg given daily by intravenous infusion. In some embodiments, the dosage may be about 10 mg given daily by intravenous infusion. In some embodiments, the dosage may be about 30 mg given three times a week by intravenous infusion.

[0103] A therapeutically effective amount is an amount of an agent sufficient to produce a statistically significant, measurable change in tumor size, tumor growth, etc. (measurements of efficacy are described below). Such effective amounts can be measured in clinical trials as well as in animal studies.

[0104] The medicament can be administered intravenously by injection or gradual infusion over time. Given an appropriate formulation for a given route, the medicaments useful in the methods and compositions described herein, for example, can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavitary, and, if desired, delivered by peristaltic means or by other means known to those skilled in the art. The compounds used herein are preferably administered orally, intravenously or intramuscularly to patients suffering from cancer. Local administration directly to a tumor mass is also specifically contemplated.

[0105] Combination therapy

[0106] The combination of the described NKG2D agonist complex (or fusion protein) and additional therapeutic agent or therapy is particularly envisioned. In some embodiments, additional therapeutic agent is effective in cancer treatment. Exemplary additional therapeutic agent or therapy include but are not limited to surgical therapy, chemotherapy (e.g., administering protein kinase inhibitors or EGFR targeted therapy), radiotherapy, cryotherapy, hyperthermia, phototherapy, radioablation therapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenesis therapy, cytokine therapy or biotherapy, such as monoclonal antibodies, siRNA, miRNA, antisense oligonucleotides, ribozymes or gene therapy. Biotherapy can be but is not limited to gene therapy, such as tumor suppressor gene therapy, cell death protein gene therapy, cell cycle regulation 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-angiogenesis factor gene therapy.

[0107] Combination therapy can be before or after other agents are treated, at intervals of several minutes to several weeks. In the embodiment of another agent and combination therapy applied to cells separately, it is usually ensured that there is no expiration for a considerable period of time between each delivery time, so that the agent and expression construct will still be able to produce a favorable combination effect to the cell. In this case, it is envisioned that cells can be contacted in two ways within about 12-24 hours, more preferably within about 6-12 hours. In some cases, it may be necessary to significantly extend the treatment period; however, wherein between each administration, through several days (for example, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days) to several weeks (for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks).

[0108] In some embodiments, the additional therapeutic agent or therapy comprises chemotherapy. Cisplatin (CDDP); carboplatin; procarbazine; mechlorethamine; cyclophosphamide; camptothecin; ifosfamide; melphalan; chlorambucil; busulfan; nitrosurea; dactinomycin; daunorubicin ); doxorubicin; bleomycin; puromycin; mitomycin; etoposide (VP16); tamoxifen; raloxifene; estrogen receptor binding agents; paclitaxel; gemcitabine; vinorelbine; farnesyl protein transferase inhibitors; stransplatinum; 5-fluorouracil; vincristine; vinblastine astine and methotrexate; Temozolomide (the aqueous form of DTIC); alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, and melamine. thylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin;CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine oxide, mechlorethamine oxide hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma II and calicheamicin omega II; dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis , actinomycin D, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid (such as mycophenolic acid), acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folate, opterin, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;Androgens (e.g., calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone); antiadreners (e.g., mitotane, trilostane); folic acid supplements (e.g., frolinic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid (triaziquone); 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel gemcitabine; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, puromycin, gemcitabine, vinorelbine, farnesyl protein transferase inhibitors, transplatin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. ;

[0109] 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 Gleevec (e.g., Gleevec can be administered to a patient at about 400 mg / day to about 800 mg / day). In some embodiments, one or more chemotherapeutic agents can be used in combination with the agonist complex described herein.

[0110] In some embodiments, additional therapeutic agents or therapies include radiotherapy. Other factors that cause DNA damage and have been widely used include the directed delivery of commonly referred to as gamma rays, X-rays and / or radioisotopes to tumor cells. Other forms of DNA damaging factors are also known, such as microwaves and UV irradiation. It is likely that all of these factors will produce extensive damage to DNA, DNA precursors, replication and repair of DNA, and assembly and maintenance of chromosomes. The dosage range of X-rays varies from a daily dose of 50 roentgens to 200 roentgens for a long time (3 weeks to 4 weeks) to a single dose of 2000 roentgens to 6000 roentgens. The dosage range of radioisotopes varies greatly and depends on the half-life of the isotope, the intensity and type of radiation, and the absorption of tumor cells.

[0111] In some embodiments, the additional therapeutic agent of therapy includes immunotherapy.Immunotherapy usually relies on the use of immune effector cells and molecules to target and destroy cancer cells.Immuno effectors can be antibodies that are, for example, specific to certain markers on the surface of tumor cells.Single antibody can be used as an effector for treatment or it can recruit other cells to actually affect cell killing.Antibodies can also be combined with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and used only as targeting agents.Or, effectors can be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets.Various effector cells include cytotoxic T cells and NK cells and genetically engineered variants of these cell types, which are modified to express chimeric antigen receptors.

[0112] 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 therapies (e.g., interferon α, β, and γ; interleukins (IL-1, IL-2), GM-CSF, and TNF) (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998). 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. It has anti-tumor activity and has been approved for the treatment of malignant tumors (Dillman, 1999). Combination therapy of cancer with Herceptin and chemotherapy has been shown to be more effective than individual therapies. Therefore, it is contemplated that one or more anti-cancer therapies may be used with the combination therapy described herein.

[0113] It is contemplated that other immunotherapies used in the methods of the present disclosure include those described by Tchekmedyian et al., 2015, which are incorporated herein by reference.Immunotherapy may include suppressing T regulatory cells (Treg), myeloid-derived suppressor cells (MDSC) and cancer-associated fibroblasts (CAF). In some embodiments, 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 to specific tumor antigens with chimeric antigen receptors (CAR) or T cell receptors (TCR). As used herein, chimeric antigen receptors (or CAR) may refer to any engineered receptors specific for the antigen of interest, and when expressed in T cells, it gives the specificity of CAR to T cells. Once produced using standard molecular techniques, T cells expressing chimeric antigen receptors can be introduced into patients, as, for example, the technology of adoptive cell transfer. In some aspects, the T cells are activated CD4 and / or CD8 T cells in an individual, characterized by CD4 and / or CD8 T cells producing IFNγ and / or enhanced cytolytic activity compared to before administration of the combination. The CD4 and / or CD8 T cells may exhibit increased cytokine release selected from IFN-γ, TNF-α and interleukins. The CD4 and / or CD8 T cells may be effector memory T cells. In certain embodiments, the CD4 and / or CD8 effector memory T cells are characterized by having CD44 高 CD62 低 Express.

[0114] Examples of monoclonal antibodies that can be used in combination with the compositions provided herein include, but are not limited to, trastuzumab (anti-HER2 / neu antibody); pertuzumab (anti-HER2 mAb); cetuximab (chimeric monoclonal antibody to 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); obinutuzumab (anti-CD20 mAb); Ofatumumab (anti-CD20 mAb); Tositumumab-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); AMG386 (angiopoietin-1 and angiopoietin-2 binding peptide fused to IgG1 Fc); Dalotuzumab (anti-IGF-1R mAb); Gemtuzumab ozogamicin (anti-CD33 mAb); alemtuzumab (anti-Campath-1 / CD52 mAb); brentuximab vedotin (anti-CD30 mAb); catumaxomab (a bispecific mAb targeting epithelial cell adhesion molecule and CD3); naptumomab (anti-5T4 mAb); girentuximab (anti-carbonic anhydrase ix); or fatuzumab (anti-folate receptor).Other examples include antibodies such as Panorex.TM. (17-1A) (mouse monoclonal antibody); Panorex (@ (17-1A) (chimeric mouse monoclonal antibody); BEC2 (anti-idiotypic mAb, mimics GD epitope) (with BCG); Oncolym (Lym-1 monoclonal antibody); SMART M195 Ab, humanized 13'1LYM-1 (Oncolym), Ovarex (B43.13, anti-idiotypic mouse mAb); 3622W94 mAb that binds to the pan-cancer antigen EGP40 (17-1A) on adenocarcinoma; Zenapax (SMART anti-Tac (IL-2 receptor); SMART M195 Ab, humanized Ab, humanized); NovoMAb-G2 (pan-cancer specific); TNT (chimeric mAb for histone antigens); TNT (chimeric mAb for histone antigens); Gliomab-H (monoclonal-humanized Abs); GNI-250Mab; EMD-72000 (chimeric-EGF antagonist); LymphoCide (humanized IL.L.2 antibody); and MDX-260 bispecific, target GD-2, ANA Ab, SMART IDIO Ab, SMART ABL364Ab or ImmuRAIT-CEA. Examples of antibodies include those disclosed in U.S. Pat. Nos. 5,736,167, 7,060,808 and 5,821,337.

[0115] Other examples of antibodies include anti-human antibody OX40 agonist antibody (Genentech); Zanulimumab (anti-CD4 mAb); Keliximab (anti-CD4 mAb); Ipilimumab (MDX-101; anti-CTLA-4 mAb); Tremilimumab (anti-CTLA-4 mAb); Daclizumab (anti-CD25 / IL-2R mAb); Basiliximab (anti-CD25 / IL-2R mAb); MDX-1106 (anti-PD1 mAb); GITR antibody; 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 (humanized 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); Teplizumab (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); T1 h (anti-CD6 mAb);Epratuzumab (anti-CD22 mAb); Efalizumab (anti-CD11a mAb); and Atacicept (extracellular domain of transmembrane activating factor and calcium-regulated ligand-interacting factor fused to Fc).

[0116] It is contemplated that other agents may be used in combination with the compositions provided herein to improve the efficacy of treatment. These additional agents include immunomodulators, agents that affect the upregulation of cell surface receptors and GAP connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of hyperproliferative cells to apoptosis inducing agents. Immunomodulators include tumor necrosis factor; interferon α, β and γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands (e.g., Fas / Fas ligand, DR4 or DR5 / TRAIL) will 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 connections will increase the anti-hyperproliferative effect on neighboring hyperproliferative cell groups. In other embodiments, cell growth inhibitors or differentiation agents may be used in combination with the compositions provided herein to improve the anti-hyperproliferative efficacy of treatment. Consider cell adhesion inhibitors to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is also contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis (eg, antibody c225) can be used in combination with the compositions provided herein to improve therapeutic efficacy.

[0117] In a further embodiment, other agents may be one or more oncolytic viruses. Examples of oncolytic viruses include adenovirus, adeno-associated virus, retrovirus, slow virus, herpes virus, pox virus, vaccinia virus, vesicular stomatitis virus, polio virus, Newcastle disease virus (Newcastle's Disease viruses), Epstein-Barr virus (Epstein-Barr virus), influenza virus and reovirus. In a specific embodiment, another agent is talimogene laherparepvec (T-VEC), which is an oncolytic herpes simplex virus engineered to express GM-CSF. Latamogene (HSV-1 [strain JS1] ICP34.5- / ICP47- / hGM-CSF) (formerly known as OncoVEX.sup.GM CSF) is an oncolytic immunotherapy delivered intratumorally, including immune-enhancing HSV-1 selectively replicated in solid tumors. (Lui et al., 2003; US Pat. Nos. 7,223,593 and 7,537,924; incorporated herein by reference).

[0118] In certain embodiments, hormone therapy may also be used in combination with the present embodiment or in combination with any other cancer therapy previously described. The use of hormones can be used to treat certain cancers, such as breast cancer, prostate cancer, ovarian cancer, or cervical cancer, to reduce the level of certain hormones (such as testosterone or estrogen) or block their effects. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastasis.

[0119] In some aspects, the additional anti-cancer agent is a protein kinase inhibitor or a monoclonal antibody that inhibits a receptor involved in a protein kinase or 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 kinase or growth factor signaling pathway inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, nib), Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin, Ciro Temsirolimus, everolimus, ridaforolimus, Alvocidib, Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016 or a mixture thereof.

[0120] In some aspects, the PI3K inhibitor is selected from the group of PI3K inhibitors consisting of buparlisib, idelalisib, BYL-719, dactolisib, PF-05212384, pictilisib, copanlisib, copanlisib dihydrochloride, ZSTK-474, GSK-2636771, duvelisi b), GS-9820, PF-04691502, SAR-245408, SAR-245409, sonolisib, Archexin, GDC-0032, GDC-0980, apitolisib, pilaralisib, DLBS1425, PX-866, voxtalisib, AZD-8186, BGT-226, DS-7423, GDC-0084, GSK-21 26458, INK-1 1 17, SAR-260301, SF-1 1 26, AMG-319, BAY-1082439, CH-51 32799, GSK-2269557, P-71 70, PWT-33597, CAL-263, RG-7603, LY-3023414, RP-5264, RV-1729, taselisib, TGR-1 202, GSK-418, INCB-040093, Panulisib, GSK-105961 5, CNX-1351, AMG-51 1. PQR-309, 17beta-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-31 1. PQR-316, RP-5090, VS-5584, X-480, AEZS-126, AS-604850, BAG-956, CAL-130, CZC-24758, ETP-46321, ETP-471 87. 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-19945, 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.

[0121] It is contemplated that additional cancer therapies may include antibodies, peptides, polypeptides, small molecule inhibitors, siRNA, miRNA or gene therapy targeting, 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 alpha 2 chain (1L13Rα2), interleukin-6 (IL- 6), 1L-6 receptor, interleukin-4, IL-4 receptor, cytokine receptor, class I (hematopoietic family) and class II (interferon / 1L-10 family) receptor, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily (TNTRSF), death receptor family, TRAIL-receptor; testicular cancer (CT) antigen, lineage-specific antigen, differentiation antigen α-actinin-4, ARTC1, breakpoint clustering region-Abelson (Bcr-abl) fusion product B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNB1), cell division cycle 27 (C DC27), 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)n fusion protein, fibronectin (FN), GPNMB, low-density lipid receptor / GDP-L fucose; β-D-galactose 2-α-L fucosyltransferase (LDLR / FUT) fusion protein, HLA-A2, exchange of arginine with isoleucine at residue 170 of the α helix of the α2 domain in the HLA-A2 gene (HLA-A*201-R1700, MLA-A11,Heat shock protein 70-2 mutant (HSP70-2M), KIAA0205, MART2, melanoma universal mutant 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, myosin class 1, NFYC, OGT, OS-9, pml-RARα 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 recognition antigen on melanoma (CAMEL), MAGE-A1 (MAGE-1), MAGE-A2, MAGE- A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B 2. 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), kallikrein 4, mammaglobin-A, OA1, prostate-specific antigen (PSA), prostate-specific membrane antigen, TRP-1 / gp75, TRP-2, adipophilin, nielanorna 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), α-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, 1-2 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), carcinoembryonic antigen peptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B), human signetring tumor-2 (HST-2), human papillomavirus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsid antigen, others), Epstein-Barr virus (EBV) proteins (EBV latent membrane protein-LMP1, LMP2; others), hepatitis B or C virus proteins, and HIV proteins.

[0122] In some embodiments, the methods described herein include administering a 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 its antigen binding domain, or an antibody reagent. In some embodiments, the checkpoint inhibitor is an antibody or its antigen binding domain, or an antibody reagent binds to an immune checkpoint polypeptide and inhibits its activity. Common checkpoints for treatment 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 its antigen binding domain, or an antibody reagent binds to a PD-1, PD-L1, or PD-L2 polypeptide and inhibits its activity.

[0123] Inhibitors of 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 (checkpoint targets and manufacturers are noted in parentheses) may include: MGA271 (B7-H3: MacroGenics); Ipilimumab (CTLA-4; Bristol Meyers Squibb); Pembrolizumab (PD-1; Merck); Nivolumab (PD-1; Bristol Myers Squibb); Atezolizumab (PD-L1; Genentech); IMP321 (LAG3: Immuntep); BMS-986016 (LAG3; Bristol Myers Squibb); IPH2101 (KIR; Innate Pharmaceuticals); Pharma); tesimumab (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).

[0124] In some embodiments, the checkpoint inhibitor inhibits PD-1. PD-1 inhibitors include but are not limited to pembrolizumab (Keytruda TM ), 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.

[0125] Monitoring the effectiveness of treatment

[0126] The efficacy of a given cancer treatment can be determined by a skilled clinician. However, if, for example, any or all signs or symptoms of a tumor are altered in a beneficial manner or other clinically acceptable symptoms are improved or even modified, for example, by at least 10% after treatment with an agent described herein, then the treatment is considered to be "effective treatment" as used herein. Efficacy can also be measured by the individual not deteriorating or requiring medical intervention (e.g., cessation of progression of the disease) as assessed by hospitalization. Methods for measuring these indicators are known to those skilled in the art and / or described herein.

[0127] An effective amount for treating a disease refers to an amount sufficient to effectively treat the disease as defined herein when administered to a mammal in need thereof. The efficacy of an agent can be determined by assessing, for example, physical indicators of cancer, such as tumor size, tumor mass, tumor density, angiogenesis, tumor growth rate, etc. In addition, the efficacy of an agent can be measured by the reduction of circulating MIC peptides or fragments thereof in a subject treated with an agent comprising an antibody or antigen-binding portion thereof as described herein or a nucleic acid encoding an antibody or antigen-binding portion thereof as described herein.

[0128] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the disclosed precise form. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure as will be appreciated by those skilled in the relevant art. The teachings of the present disclosure provided herein may be appropriately applied to other programs or methods. Various embodiments described herein may be combined to provide other embodiments. If desired, aspects of the present disclosure may be modified to provide other other embodiments of the present disclosure using the composition, function and concept of the above references and applications. According to the detailed description, these and other changes may be made to the present disclosure.

[0129] The specific elements of any of the foregoing embodiments may be combined or replace the elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit these advantages, and not all embodiments need to exhibit these advantages to fall within the scope of the present disclosure.

[0130] For the purpose of describing and disclosing the methods that may be used in conjunction with the present invention, for example, described in these publications, all patents and other publications are expressly incorporated herein by reference. These publications are only provided for disclosure prior to the filing date of the present application. Any content in this regard should not be construed as admitting that the inventor has no right to precede such disclosure by virtue of previous inventions or for any other reason. All statements about the dates or contents of these documents are based on information available to the applicant and do not constitute any recognition of the correctness of the dates or contents of these documents.

[0131] Example

[0132] Materials and methods

[0133] Identification of sMIC / MIC-negative cancers: sMIC-negative tumors can be identified by measuring serum levels of sMICA or sMICB using standard MICA or MICB detection ELISAs. MIC-negative subjects should not contain detectable sMICs above background noise. For tumors with biopsy availability, it is also possible to select tumors that are negative for MIC expression or have been confirmed by immunohistochemistry to have no cross-reactivity with anti-MIC antibodies.

[0134] Peptide binding regions of non-blocking anti-sMIC / MIC antibody D4H3: Chemical cross-linking, high-mass MALDI mass spectrometry, and nLC-Orbitrap mass spectrometry were used to characterize the interaction interface between the antigen and antibody Ab-D4H3. The results showed that Ab-D4H3 binds to two regions of the antigen with the following amino acids on the antigen: 68, 72, 75, 77 and 206, 207, and 209. These regions do not compete with the NKG2D binding regions in the α-1 and α-2 domains of MIC (Li et al., Nat Immunol. 2001 May; 2(5):443-51., the disclosure of which is incorporated herein by reference in its entirety).

[0135] 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 linking sMIC to the heavy or light chain of the anti-sMIC antibody using a polylinker.

[0136] Example 1 - sMIC / anti-sMIC complexes enhance CD3 / TCR-mediated CD8 T cell activation in vitro

[0137] PBMCs from normal donors were stimulated in the presence of sMIC, anti-sMIC antibodies (e.g., D4H3), or sMIC / anti-sMIC mAb complexes for 3 days in the presence or absence of plate-bound CD3, and CD8 T cell IFNγ production was determined by intracellular staining. Soluble anti-CD28 stimulation was used as a positive control. Figure 1 As shown, similar to anti-CD28 stimulation, sMIC / anti-MIC complex activated CD8 T cells after CD3 ligation, indicating the co-stimulatory effect of sMIC / anti-MIC complex. Neither sMIC nor anti-MIC mAb alone had a similar effect. In addition, sMIC / anti-MIC and anti-CD28 produced an additive effect to amplify CD3-mediated CD8 T cell activation ( Figure 1 ). In addition, carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay showed that sMIC / anti-MIC co-stimulation also enhanced CD8 T cell proliferation ( Figure 1 ).

[0138] In another embodiment, in the presence of sMIC, anti-sMIC antibodies (e.g., NO4) or sMIC / NO4 complexes, PBMCs are stimulated for 48 hours in the presence or absence of plate-bound CD3, and then CD8 T cell surface NKG2D expression and IFNγ production are determined by intracellular staining. Similarly, anti-CD28 agonist antibody stimulation is used as a positive control for CD8 T cell co-stimulation. Fig. 2 shows that complex stimulation amplifies CD3 / TCR activation, as measured by IFNγ production and proliferation performed by 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 short, the data provided in this embodiment show that sMIC / anti-sMIC complex co-stimulation is non-redundant with anti-Cd28 co-stimulation.

[0139] Example 2 - sMIC / anti-sMIC complexes amplify antigen-specific CD8 T cell responses.

[0140] In the presence or absence of sMIC(A) / D4H3 complex or tyrosinase peptide 369-377 In this case, TIL1383I cells (engineered to express CD34 as a reporter gene) were cross-linked with HLA-A2 + Instead of T2-A2 antigen presenting cells (APC), TIL13831 activation was assessed by intracellular staining for IFN, TNF, and CD107a (degranulation) after overnight culture. Figure 3As shown, sMIC / D4H3 complex significantly enhanced TIL13831 stimulation to HLA-A2 restricted tyrosinase peptide. sMIC / D4H3 complex together with scrambled OVA peptide did not stimulate TIL13831. Blocking NKG2D with mAb M585 abolished the co-stimulatory effect of sMIC / D4H3.

[0141] Example 3 - Therapy with sMIC / D4H3 complex inhibits the growth of MIC-negative tumors

[0142] MIC 阴性 MC38 colon tumor cells were implanted into syngeneic B6 / MICB male mice. When the tumor volume reached approximately 100 mm 3 At 400 g / kg, animals were treated with control IgG, recombinant rsMIC, D4H3, or sMIC / D4H3 complexes, either alone or in combination, by intraperitoneal administration twice a week at a dose of 4 mg / kg. Figure 4 As shown, sMIC / D4H3 complex significantly inhibited tumor growth. The combination therapy induced antigen-specific immune response.

[0143] Example 4 - Detection of MIC / anti-sMIC antibodies binding to receptor NKG2D by ELISA

[0144] The ELISA method was used to confirm that the complex composed of sMIC and non-blocking anti-sMIC antibody (eg, NO4 or D4H3) binds to the receptor NKG2D to activate NKG2D-mediated costimulatory signaling.

[0145] Figure 5 The complexes were shown to bind to NKG2D by ELISA. In this assay, recombinant soluble human NKG2D (rs-hNKG2D) was immobilized to a 96-well plate overnight, and after washing to remove unbound s-hNKG2D, the plates were incubated with various concentrations of the anti-sMIC monoclonal antibody D4H3 ( Figure 5 A) or NO4( Figure 5 B) (mouse IgG) complexed with indicated concentrations of recombinant sMICA (source: R&D systems) or recombinant MICB-His (source: Fisher). Binding of the complex to rs-hNKG2D was detected with HRP-conjugated goat anti-mouse antibody.

[0146] Example 5 - sMIC / D4H3 combination therapy rapidly clears LCMV virus infection.

[0147] Groups of C57BL / 6 mice were inoculated intravenously (iv) with 2x10 6PFU LCMV (lymphocytic choriomeningitis virus) Armstrong strain. Mice were administered (ip) sMIC / D4H3 complex (6 mg / Kg) or control PBS: Day 1 and Day 3 after virus inoculation. Blood samples were collected from mice on Day 1 and Day 5 after virus inoculation. Serum viral load was determined by plaque assay (as previously described in Welsh and Seedhom, Current Protocols in Microbiology (Curr Protoc Microbiol) February 2008; Section: Unit-15A.1). Figure 8 As shown, the LCMV titer of mice treated with sMIC / D4H3 complex was significantly reduced.

[0148] Any journal article or patent document cited herein is hereby incorporated by reference in its entirety.

Claims

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

9.

2. The complex of claim 1, wherein the non-blocking soluble MHC I chain-associated molecule neutralizing antibody comprises the light chain variable region shown in SEQ ID NO:

11.

3. The complex of claim 1, wherein the non-blocking soluble MHC I chain-associated molecule neutralizing antibody comprises the heavy chain variable region shown in SEQ ID NO:

10. The complex of claim 1 , wherein the soluble MHC I chain-associated molecule is sMICA. The complex of claim 1 , wherein the soluble MHC I chain-associated molecule is sMICB.

6. The complex of claim 1, wherein the soluble MHC I chain-related molecule comprises the amino acid sequence shown in one of SEQ ID NOs: 1 and 20-77.

7. The complex of claim 5, wherein the sMICB comprises the amino acid sequence shown in one of SEQ ID NOs: 2 and 78-100.

8. A pharmaceutical composition comprising the complex according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier, diluent or adjuvant.

9. Use of the complex of any one of claims 1 to 7 for the preparation of a medicament for activating CD8 T cells in a subject in need thereof, wherein the subject suffers from cancer, and wherein the cancer is colon and rectal cancer or melanoma.

10. Use of the complex of any one of claims 1 to 7 for the preparation of a medicament for activating CD8 T cells in a subject in need thereof, wherein the subject suffers from a viral infection, and wherein the viral infection is caused by lymphocytic choriomeningitis.

11. The use according to claim 9 or 10, wherein the drug further comprises an immune checkpoint inhibitor.

12. The use of claim 11, wherein the immune checkpoint inhibitor is MGA27, ipilimumab, pembrolizumab, nivolumab, atezolizumab, IMP321, IPH2101, tesimumab, pidilizumab, MPDL3280A, MEDI4736, MSB0010718C, AUNP12, avelumab, durvalumab or TSR-022.

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