Anti-NKG2D antibodies and uses thereof

Abstract

The present disclosure describes several anti-NKG2D (Nature Killer Group 2, member D) antibodies. The complementarity determining regions (CDRs) of the anti-NKG2D antibodies are further disclosed. The anti-NKG2D antibodies disclosed herein can be used to treat various diseases, such as cancer, autoimmune diseases, viral infections, bacterial infections, or diseases associated with overexpression of NKG2D.

Description

[Technical field]

[0001] Description of sequence listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on May 2, 2022, is named P-604135-PC_SL.txt and is 21 kilobytes in size.

[0002] The present disclosure relates generally to antibodies. In one embodiment, the present disclosure describes the generation and use of antibodies against NKG2D. [Background technology]

[0003] NKG2D (Nature Killer Group 2, member D) is an activation cell surface protein that is expressed primarily on cytotoxic immune cells. NKG2D is abundant on all NK cells, NKT cells, and a subset of γδT cells. Naive human CD8 + T cells express NKG2D, but in mice, they upregulate its expression only after activation. + T cells generally do not express NKG2D even after activation, but in humans, its expression can be induced under certain pathological conditions, such as Crohn's disease, juvenile-onset lupus, and cytomegalovirus infection. The molecular structure of NKG2D allows it to bind several structurally distinct MHC-I-like ligands. NKG2D ligands have in common that their expression is generally low under homeostatic conditions. In contrast, their expression can be highly induced upon cellular stress, such as infection or oncogenic transformation. In humans, NKG2D ligands are six members of the MICA, MICB, and ULBP families.

[0004] NKG2D is a homodimer of two disulfide-linked transmembrane proteins with a very short intracellular domain that has no signaling properties. In mice, NKG2D uses the adaptor molecules DAP10 and DAP12 to relay its signaling, whereas in humans, NKG2D associates exclusively with DAP10. DAP10 and DAP12 initiate distinct signaling cascades. DAP10 has a YIM motif that allows binding of phosphatidylinositol-3 kinase (PI3K) to p85. Furthermore, DAP10 binds to Grb2, which associates with Vav1. DAP12 contains an immunoreceptor tyrosine-based activation motif, which is phosphorylated by Src kinase upon NKG2D triggering. This event allows binding and activation of the tyrosine kinases, Syk and Zap70.

[0005] NKG2D plays a key role in the recognition and elimination of potentially dangerous cells. It has been shown to mediate immune responses against tumors, virus-infected cells, and organ transplants. For this reason, NKG2D was originally thought to primarily mediate direct cytotoxicity in response to ligand encounters on stressed target cells. However, in most cases, NKG2D can only mediate immune cell activation if it occurs within an inflammatory context. Both NK cells and T cells generally require a secondary signal before NKG2D can mediate a measurable effect. Thus, the primary function of NKG2D appears to be the regulation of signaling through other receptors. Its unique feature is that it can both inhibit and enhance the signaling of multiple receptors in multiple ontologically distinct immune cell subsets and during different stages of the immune cell life cycle, such as hematopoietic development, priming, and effector responses.

[0006] NKG2D has great potential as a therapeutic target because of its potency in enhancing cytolytic immune responses against important diseases such as cancer. Considering the important role of NKG2D, it is necessary to develop anti-NKG2D antibodies to explore the therapeutic applications of NKG2D. Summary of the Invention

[0007] In one embodiment, the disclosure provides several anti-NKG2D antibodies, each of which comprises three complementarity determining regions (CDRs) on the heavy chain (HCDR1, HCDR2, and HCDR3) and three CDRs on the light chain (LCDR1, LCDR2, and LCDR3), HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 12 to 14, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 16 to 18, or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 20 to 22, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 24 to 26, or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 28 to 30, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 32 to 34, or HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 36 to 38, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 40 to 42.

[0008] In one embodiment, each of the anti-NKG2D antibodies comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region may be one of the following pairs: SEQ ID NOs: 11 and 15, SEQ ID NOs: 19 and 23, SEQ ID NOs: 27 and 31, or SEQ ID NOs: 35 and 39.

[0009] In one embodiment, the disclosure provides a composition comprising a pharma- ceutically acceptable carrier and an anti-NKG2D antibody disclosed herein.

[0010] The present disclosure also provides polynucleotide sequences encoding the anti-NKG2D antibodies disclosed herein, as well as vectors and host cells containing such polynucleotide sequences.

[0011] In one embodiment, the anti-NKG2D antibodies disclosed herein can be used to treat diseases such as cancer, autoimmune diseases, GvHD, viral infections, or bacterial infections. In another embodiment, the anti-NKG2D antibodies disclosed herein can be used to treat diseases associated with NKG2D. In another embodiment, the anti-NKG2D antibodies disclosed herein can be used to treat diseases associated with overexpression of NKG2D.

[0012] These and other aspects of the anti-NKG2D antibodies will become apparent from the following description of the figures and detailed description of the anti-NKG2D antibodies. [Brief description of the drawings]

[0013] Some embodiments of anti-NKG2D antibodies and uses thereof are described herein, by way of example only, with reference to the accompanying drawings. With particular reference now to the drawings in detail, it is emphasized that the details shown are by way of example and are for the purpose of an illustrative discussion of the embodiments of anti-NKG2D antibodies and their uses. In this regard, the description taken in conjunction with the drawings will make apparent to those skilled in the art how the embodiments of anti-NKG2D antibodies and their uses may be put into practice. [Figure 1A] Binding of mouse sera test bleeds (TB) after immunization against recombinant human NKG2D-ECD-Fc (extracellular domain of NKG2D fused to human Fc) or pre-bleeds (PB) before immunization are shown. Figure 1A shows the results of ELISA binding of sera from Balb / c mice (mouse #8741, 8741, 8743, 8744, 8745) or SJL mice (mouse #8746, 8747, 8748, 8749, 8750). [Figure 1B]Figure 1B shows binding of mouse sera test bleed (TB) after immunization against recombinant human NKG2D-ECD-Fc (extracellular domain of NKG2D fused to human Fc) or pre-bleed (PB) before immunization. Figure 1B shows FACS binding of sera from Balb / c mice (mouse #8741, 8741, 8743, 8744, 8745) and SJL mice (mouse #8746, 8747, 8748, 8749, 8750) against human NKG2D / Dap10 overexpressed in CHO cells or against cyno-NKG2D / Dap10 overexpressed in CHO cells, whereas no reactivity was observed with CHO parental cells. hIgG1: negative human IgG control. mIgG1: negative mouse IgG control. NKG2D Ab: positive control hIgG mAb. [Diagram 2] The identities of selected mouse hybridoma clones are listed. [Diagram 3] Binding of selected purified mAbs (mAb001-mAb020) to the NKG2D-ECD-Fc antigen by ELISA is shown. [Figure 4A-1] Figure 4 shows FACS binding of selected purified mAbs (mAb001-mAb020) to CHO cells overexpressing human or cynoNKG2D / Dap10. Figure 4A shows binding to CHO cells overexpressing human NKG2D / Dap10. [Figure 4A-2] Figure 4 shows FACS binding of selected purified mAbs (mAb001-mAb020) to CHO cells overexpressing human or cynoNKG2D / Dap10. Figure 4A shows binding to CHO cells overexpressing human NKG2D / Dap10. [Figure 4B] Figure 4B shows FACS binding of selected purified mAbs (mAb001-mAb020) to CHO cells overexpressing human or cyno NKG2D / Dap10. Figure 4B shows binding to CHO cells overexpressing cyno NKG2D / Dap10. [Figure 4C]FACS binding of selected purified mAbs (mAb001-mAb020) to CHO cells overexpressing human or cynoNKG2D / Dap10 is shown. Figure 4C presents the binding to primary NK cells, showing dose-dependent binding for all mAbs (100 nM, left bar for each mAb; or 10 nM, right bar for each mAb) with various mean fluorescence intensities (MFI). Tab KYK2.0 is a positive anti-NKG2D-human IgG1 antibody with the LALA P329G mutation in the Fc. Human IgG1LALA P328G is a human IgG negative control Ab with the LALA P329G mutation in the Fc. [Diagram 5] The epitope binning analysis performed by ELISA is summarized and shows four different groups as follows: Group 1 (rectangle continuous line) collects mAbs 001, 002, 003, 004, 005, 006, 008, 011, 013, 015, 017, 018, 020; Group 2 (circle continuous line) collects mAbs 007, 010, 014; Group 3 (rectangle dashed line) collects mAbs 012, 016, 019; and Group 4 (circle dashed line) collects mAb 009. [Figure 6A] Activation of NK cells upon binding of purified anti-NKG2D mAbs (mAb001-mAb020) at 100 nM (left bar for each mAb) or 10 nM (right bar for each mAb) is shown. Figure 6A shows accumulation of CD107a activation marker. APC CD107A is anti-CD107A antibody staining without anti-NKG2D mAb clone binding, APC mIgG is a negative control mouse IgG antibody. IM-1060 is a positive control tribody and IM-1091 is a negative control tribody. [Figure 6B] NK cell activation upon binding of purified anti-NKG2D mAbs (mAb001-mAb020) at 100 nM (left bar for each mAb) or 10 nM (right bar for each mAb) is shown. Figure 6B shows IFN-γ secretion. IM-1091 is a negative control tribody. [Figure 7] FIG. 1 shows inhibition of NKG2D-MICA (MICA-MHC class I polypeptide-related sequence A) interaction by selected purified mAbs (mAb001-mAb020). [Figure 8A] 1 illustrates one embodiment of a tribody structure. [Figure 8B] 1 shows one embodiment of a Pro tribody structure. [Figure 8C] The identities of the various tribody molecules are listed. [Figure 9] Characterization of tribody proteins (IM1244-IM1251) by SDS-PAGE.

[0014] [Table 1] [Figure 10A] Characterization of tribody proteins (IM1244-IM1251) by analytical SEC. [Figure 10B] Characterization of tribody proteins (IM1244-IM1251) by analytical SEC. [Figure 10C] Characterization of tribody proteins (IM1244-IM1251) by analytical SEC. [Figure 10D] Characterization of tribody proteins (IM1244-IM1251) by analytical SEC. [Figure 10E] Characterization of tribody proteins (IM1244-IM1251) by analytical SEC. [Figure 10F] Characterization of tribody proteins (IM1244-IM1251) by analytical SEC. [Figure 10G] Characterization of tribody proteins (IM1244-IM1251) by analytical SEC. [Figure 10H] Characterization of tribody proteins (IM1244-IM1251) by analytical SEC. [Figure 11] Binding of tribody proteins (IM1244-IM1251, IM1060 and IM1091) to NK2G protein by ELISA is shown. IM-1060 is a positive control tribody and IM-1091 is a negative control tribody. [Figure 12A]Binding of tribody proteins (IM1244-IM1251, IM1060 and IM1091) to CHO cells overexpressing human NK2G protein (FIG. 12A) or cynoNKG2D (FIG. 12B) is shown. IM-1060 is a positive control tribody and IM-1091 is a negative control tribody. [Figure 12B] Binding of tribody proteins (IM1244-IM1251, IM1060 and IM1091) to CHO cells overexpressing human NK2G protein (FIG. 12A) or cynoNKG2D (FIG. 12B) is shown. IM-1060 is a positive control tribody and IM-1091 is a negative control tribody. [Figure 13] NK cell activation by tribody proteins (IM1245, IM1246, IM1249, IM1250, IM1060 and IM1091) at 300 nM, 60 nM, 12 nM and 2.4 nM (bars, left to right). E+T APC-CD107 is CD107a staining in the absence of NKG2D mAb. E APC-CD107 is CD107 staining in the absence of NKG2D mAb and target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present disclosure presents isolated anti-NKG2D antibodies, in which the unique CDR sequences of anti-NKG2D mAb are provided within a humanized framework (chimeric antibody; humanized antibody).In addition, the incorporation of the NKG2D antigen-binding region of these anti-NKG2D antibodies into a multivalent antibody construct is demonstrated.The anti-NKG2D antibodies disclosed herein can potentially be used as immunotherapy treatment for medical conditions, such as cancer.

[0016] As used herein, the term "antibody" may be used interchangeably with the term "immunoglobulin", all with the same properties and meanings. An antibody binding domain or antigen-binding site may be a fragment of an antibody, or a genetically engineered product of one or more fragments of an antibody, which fragment is responsible for specific binding to a target antigen. "Specifically binds" means that the binding is selective for the antigen of interest and can be distinguished from undesired or non-specific interactions. For example, an equilibrium dissociation constant of ≦10 -5 , 10 -6 , or 10 -7 An antibody is said to specifically bind to an NKG2D epitope when M is 0. In some embodiments, the equilibrium dissociation constant is ≦10 -8 M or 10 -9 In some further embodiments, the equilibrium dissociation constant is ≦10 -10 M, 10 -11 M or 10 -12 M. In some embodiments, the equilibrium dissociation constant is ≦10 -5 M~10 -12 The range may be M.

[0017] Half-maximal effective concentration (EC 50 ) refers to the concentration of a drug, antibody, or toxin that induces a response intermediate between the baseline and maximum response after a particular exposure time. In some embodiments, the response comprises binding affinity. In some embodiments, the response comprises a functional response, e.g., an agonist response. As used herein in certain embodiments, one of skill in the art will understand the EC of an anti-NKG2D antibody disclosed herein. 50 The measurement was performed using the half-maximal binding (EC 50 As used herein in certain embodiments, one of skill in the art will appreciate that the EC of the anti-NKG2D antibodies disclosed herein provides a measure of the EC 50 The measurements were taken as the agonist response (EC 50 It will be appreciated that the present invention provides a measure of the half-maximal effective concentration of an anti-NKG2D antibody that induces a potent agonistic effect (functional agonism).

[0018] In some embodiments, EC 50 comprises the concentration of antibody required to obtain a 50% agonist response that would be observed upon antibody binding. In certain embodiments, the EC 50 The nanomolar EC50 is commonly used as a measure of drug potency and, in some embodiments, may reflect the binding of an antibody to a receptor. 50 Anti-NKG2D antibodies having binding concentration measurements include strong binding anti-NKG2D antibodies. In some embodiments, the EC 50 Anti-NKG2D antibodies having functional agonism concentration measurements include functionally effective agonist antibodies. In certain embodiments, the anti-NKG2D antibodies disclosed herein comprise tight binders to NKG2D molecules. In certain embodiments, the anti-NKG2D antibodies disclosed herein comprise agonists of NKG2D molecules. In certain embodiments, the anti-NKG2D antibodies disclosed herein comprise tight binding agonists to NKG2D molecules.

[0019] In some embodiments, the binding EC of an anti-NKG2D antibody 50 is in the nanomolar range. In some embodiments, the binding EC 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.05 to 100 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.05 to 50 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.05 to 20 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.05 to 10 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.1 to 100 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.1 to 50 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.1 to 20 nM. 50In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.1 to 10 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 1 to 100 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 1 to 20 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 20 to 40 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 40 to 60 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 60 to 80 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 80 to 100 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 1 to 40 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 1 to 60 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 1 to 80 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 1 to 50 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.05 to 5 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody is in the range of about 0.1 to 5 nM. 50 includes a range of about 0.05 to 20 nM.

[0020] In some embodiments, the binding EC of an anti-NKG2D antibody 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.05 to 5 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody is in the range of about 0.1 to 5 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody is in the range of about 1 to 5 nM.50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.05 to 10 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.1 to 10 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 1 to 10 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody is in the range of about 5 to 10 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.05 to 15 nM. 50 In some embodiments, the binding EC of the anti-NKG2D antibody ranges from about 0.01 to 15 nM. 50 includes a range of about 1 to 15 nM.

[0021] In some embodiments, EC 50 are used herein as functions EC 50 In some embodiments, the functional EC of an anti-NKG2D antibody 50 is in the nanomolar range. In some embodiments, the functional EC 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.05 to 100 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.05 to 50 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.05 to 20 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.05 to 10 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.1 to 100 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.1 to 50 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.1 to 20 nM.50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.1 to 10 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 1 to 100 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 1 to 20 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 20 to 40 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 40 to 60 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 60 to 80 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 80 to 100 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 1 to 40 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 1 to 60 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 1 to 80 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 1 to 50 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.05 to 5 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.1 to 5 nM. 50 includes a range of about 0.05 to 20 nM.

[0022] In some embodiments, the functional EC of an anti-NKG2D antibody 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.05 to 5 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.1 to 5 nM. 50In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 1 to 5 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.05 to 10 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.1 to 10 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 1 to 10 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 5 to 10 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.05 to 15 nM. 50 In some embodiments, the functional EC of an anti-NKG2D antibody ranges from about 0.01 to 15 nM. 50 includes a range of about 1 to 15 nM.

[0023] As used herein, the term "antibody" encompasses antibody fragment(s) that retain binding specificity, including, but not limited to, IgG, heavy chain variable region (VH), light chain variable region (VL), Fab fragment, F(ab')2 fragment, scFv fragment, Fv fragment, nanobody, minibody, diabody, triabody, tetrabody, and single domain antibody (see, e.g., Hudson and Souriau, Nature Med. 9;129:134-2003). Humanized antibodies, primatized antibodies, and chimeric antibodies are also encompassed as these terms are generally understood in the art. In some embodiments, the antibodies disclosed herein include precursor constructs in which the antigen binding site may be blocked by a regulatory domain, and exposure of the binding site includes regulated exposure based on environmental conditions, such as, but not limited to, exposure to the tumor microenvironment.

[0024] As used herein, the term "heavy chain variable region" may be used interchangeably with the term "VH domain" or the term "VH", all having the same meaning and properties. As used herein, the term "light chain variable region" may be used interchangeably with the term "VL domain" or the term "VL", all having the same meaning and properties. Those skilled in the art will recognize that a "heavy chain variable region" or "VH" in reference to an antibody encompasses a fragment of a heavy chain containing three complementarity determining regions (CDRs) inserted between adjacent stretches known as framework regions. The framework regions are more highly conserved than the CDRs and form a scaffold that supports the CDRs. Similarly, those skilled in the art will also recognize that a "light chain variable region" or "VL" in reference to an antibody encompasses a fragment of a light chain containing three CDRs inserted between framework regions.

[0025] As used herein, the term "complementarity determining region" or "CDR" refers to the hypervariable region(s) of a heavy or light chain variable region. Proceeding from the N-terminus, each heavy or light chain polypeptide has three CDRs, designated as "CDR1", "CDR2", and "CDR3". Crystallographic analysis of many antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen. Thus, the CDR regions are primarily responsible for the specificity of the antigen-binding site. In one embodiment, the antigen-binding site comprises six CDRs, including the CDRs from each of the heavy and light chain variable regions.

[0026] As used herein, the term "framework region" or "FR" refers to four adjacent amino acid sequences that frame the CDRs of a heavy or light chain variable region. Some FR residues may contact the bound antigen. However, FR residues are primarily involved in folding the variable region into the antigen-binding site. In some embodiments, the FR residues involved in folding the variable region include the residues directly adjacent to the CDR. Within the FR, certain amino acid residues and certain structural features are highly conserved. In this regard, all variable region sequences contain an internal disulfide loop of about 90 amino acid residues. When the variable region folds into the antigen-binding site, the CDRs are displayed as protruding loop motifs that form an antigen-binding surface. Regardless of the exact CDR amino acid sequence, it is generally recognized that there are conserved structural regions of the FR that affect the folding shape of the CDR loops into certain "canonical" structures. Furthermore, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction of antibody heavy and light chains.

[0027] Antibodies can exist in various formats or with various domains, including but not limited to, complementarity determining regions (CDRs), variable regions (Fv), VH domains, VL domains, single chain variable regions (scFv), and Fab fragments.

[0028] Those skilled in the art will appreciate that an scFv is a fusion polypeptide comprising the variable heavy (VH) and variable light (VL) regions of an immunoglobulin linked by a short linker peptide, which may have, for example, from 10 to about 25 amino acids.

[0029] Those skilled in the art will also understand that the term "Fab" with respect to an antibody generally encompasses the portion of an antibody consisting of a single light chain (both variable and constant regions) bound by disulfide bonds to the variable region and first constant region of a single heavy chain, whereas F(ab')2 includes a fragment of a heavy chain containing the VH domain and a light chain containing the VL domain.

[0030] In some embodiments, antibodies include whole antibody molecules, including monoclonal and polyclonal antibodies. In some embodiments, antibodies include antibody fragment(s) that retain binding specificity, including, but not limited to, variable heavy (VH) fragments, variable light (VL) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0031] In one embodiment, the anti-NKG2D antibody disclosed herein can be incorporated as part of a bispecific antibody.As is generally known in the art, a bispecific antibody is a recombinant protein that comprises antigen-binding fragments of two different monoclonal antibodies, and can thereby bind to two different antigens.In some embodiments, the anti-NKG2D antibody disclosed herein is bivalent to NKG2D.In some embodiments, the anti-NKG2D antibody disclosed herein is monovalent for binding to NKG2D.

[0032] In one embodiment, the anti-NKG2D antibodies disclosed herein can be incorporated as part of a multispecific antibody. In one embodiment, the multispecific antibody is a recombinant protein comprising antigen-binding fragments of at least two different monoclonal antibodies, for example, two, three or four different monoclonal antibodies. In one embodiment, the anti-NKG2D antibodies disclosed herein can be incorporated as part of a trispecific antibody.

[0033] In some embodiments, bispecific, trispecific, or multispecific antibodies are used in cancer immunotherapy by simultaneously targeting one or more antigen targets, e.g., cytotoxic T cells (CTLs) and tumor-associated antigens (TAAs), or by simultaneously targeting CTL receptor components such as CD3, effector natural killer (NK) cells, and tumor-associated antigens (TAAs).

[0034] Anti-NKG2D antibody The present disclosure provides several anti-NKG2D antibodies, in one embodiment, each of the anti-NKG2D antibodies comprises a set of three complementarity determining regions (CDRs) on the heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on the light chain (LCDR1, LCDR2, and LCDR3).

[0035] In one embodiment, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 12-14, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 16-18.

[0036] In one embodiment, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 20-22, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 24-26.

[0037] In one embodiment, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 28-30, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 32-34.

[0038] In one embodiment, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 36-38, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 40-42.

[0039] In another embodiment, the anti-NKG2D antibody comprises heavy and light chain CDR sequences that are at least 80% (eg, at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above.

[0040] In one embodiment, each of the anti-NKG2D antibodies presented herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the amino acid sequences of the heavy chain variable region and the light chain variable region can be one of the following pairs: SEQ ID NOs: 11 and 15, SEQ ID NOs: 19 and 23, SEQ ID NOs: 27 and 31, or SEQ ID NOs: 35 and 39. In another embodiment, the anti-NKG2D antibody comprises a VH and VL sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the above amino acid sequences.

[0041] In one embodiment, given the sequences of the heavy and light chain variable regions disclosed herein, one of skill in the art would readily use standard techniques known in the art to construct an anti-NKG2D scFv.

[0042] In certain embodiments, the present disclosure provides polypeptides comprising VH and VL domains that can be dimerized under suitable conditions. For example, the VH and VL domains can be combined in a suitable buffer and dimerized via suitable interactions, such as hydrophobic interactions. In another embodiment, the VH and VL domains can be combined in a suitable buffer containing enzymes and / or cofactors that can promote dimerization of the VH and VL domains. In another embodiment, the VH and VL domains can be combined in a suitable vehicle that allows them to react with each other in the presence of suitable reagents and / or catalysts.

[0043] In certain embodiments, the VH and VL domains may be contained within a longer polypeptide sequence, which may include, but is not limited to, a constant region, a hinge region, a linker region, an Fc region, or a disulfide bond region, or any combination thereof. A constant domain is an immunoglobulin folding unit of the constant portion of an immunoglobulin molecule, also referred to as a domain of the constant region (e.g., CH1, CH2, CH3, CH4, Ck, Cl). In some embodiments, the longer polypeptide may include multiple copies of one or both of the VH and VL domains generated according to the methods disclosed herein. For example, when the polypeptides generated herein are used to form diabodies or triabodies.

[0044] In one embodiment, the anti-NKG2D antibody provided herein can be an IgG, Fv, scFv, Fab, F(ab')2, minibody, diabody, triabody, nanobody, bispecific antibody, triaspecific antibody, multispecific antibody, or single domain antibody. For example, the anti-NKG2D antibody can be an IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the anti-NKG2D antibody comprises an IgG1. In some embodiments, the anti-NKG2D antibody comprises an IgG2. In some embodiments, the anti-NKG2D antibody comprises an IgG3. In some embodiments, the anti-NKG2D antibody comprises an IgG4.

[0045] In one embodiment, the present disclosure provides an antibody that binds to NKG2D with high affinity. In one embodiment, the binding affinity is calculated by a modified Scatchard method described by Frankel et al. (Mol.Immunol.,16:101-106,1979). In another embodiment, the binding affinity is measured by antigen / antibody dissociation rate. In another embodiment, the binding affinity is measured by competitive radioimmunoassay. In another embodiment, the binding affinity is measured by ELISA. In another embodiment, the antibody affinity is measured by flow cytometry.

[0046] In one embodiment, the present disclosure also provides isolated polynucleotide sequences encoding the heavy and light chain CDRs described herein. In another embodiment, the present disclosure also provides vectors comprising such polynucleotide sequences. In view of the amino acid sequences disclosed herein, one skilled in the art would readily construct a vector or plasmid encoding the amino acid sequence. In another embodiment, the present disclosure also provides host cells comprising the vectors provided herein. Depending on the use and experimental conditions, one skilled in the art would readily use suitable host cells to carry and / or express the above polynucleotide sequences.

[0047] In one embodiment, the present disclosure also provides isolated polynucleotide sequences encoding the heavy and light chain variable regions described herein. In another embodiment, the present disclosure also provides vectors comprising such polynucleotide sequences. In view of the amino acid sequences disclosed herein, one skilled in the art would readily construct a vector or plasmid encoding the amino acid sequence. In another embodiment, the present disclosure also provides host cells comprising the vectors provided herein. Depending on the use and experimental conditions, one skilled in the art would readily use suitable host cells to carry and / or express the above polynucleotide sequences.

[0048] The following table (Table 1) lists the names of the anti-NKG2D antibody clones and the corresponding SEQ ID NOs for VH, VL and CDRs.

[0049] [Table 2]

[0050] Compositions for use In one embodiment, the present disclosure also provides a composition comprising an anti-NKG2D antibody disclosed herein and a pharma- ceutically acceptable carrier. Useful pharma- ceutically acceptable carriers are well known in the art. For example, Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the antibodies disclosed herein. In one embodiment, the composition comprises an anti-NKG2D antibody comprising a set of three complementarity determining regions (CDRs) on the heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on the light chain (LCDR1, LCDR2, and LCDR3).

[0051] In some embodiments of the composition, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 12-14, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 16-18.

[0052] In some embodiments of the composition, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs:20-22, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs:24-26.

[0053] In some embodiments of the composition, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 28-30, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 32-34.

[0054] In some embodiments of the composition, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 36-38, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 40-42.

[0055] In another embodiment, the composition comprises an anti-NKG2D antibody having heavy and light chain CDR sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above.

[0056] In another embodiment, the composition comprises an anti-NKG2D antibody having one of the following pairs of heavy chain variable regions and light chain variable regions: SEQ ID NOs: 11 and 15, SEQ ID NOs: 19 and 23, SEQ ID NOs: 27 and 31, or SEQ ID NOs: 35 and 39.

[0057] In another embodiment, the composition comprises an anti-NKG2D antibody having VH and VL sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above.

[0058] In some embodiments of the composition, the antibody disclosed herein may be in the form of a conjugate. As used herein, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently bound to an effector molecule or a second protein (such as a second antibody). The effector molecule may be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. An antibody conjugate may also be referred to as an "immunoconjugate." When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate may be referred to as an "antibody-drug conjugate." Other antibody conjugates include, for example, multispecific (e.g., bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).

[0059] Compositions comprising anti-NKG2D antibodies or antigen-binding fragments thereof can be administered to a subject (e.g., a human or animal) alone or in combination with a carrier, i.e., a pharma- ceutically acceptable carrier. Pharmaceutically acceptable means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. As is well known to those of skill in the art, the carrier will be selected to minimize any degradation of the polypeptides disclosed herein and to minimize any adverse side effects in the subject. The pharmaceutical compositions can be prepared by any method well known in the pharmaceutical art.

[0060] Pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein can be administered in any suitable manner (e.g., to a mammal, cell, or tissue) depending on whether local or systemic treatment is desired. For example, the compositions can be administered topically (e.g., ophthalmically, vaginally, rectally, intranasally, transdermally, etc.), orally, by inhalation, or parenterally (including by intravenous drip or subcutaneous, intracavity, intraperitoneal, intradermal, or intramuscular injection). Local intranasal administration refers to delivery of the composition to the nose and nasal cavity through one or both nostrils. The compositions can be delivered by a spray or droplet mechanism or via aerosolization. Alternatively, administration can be intratumoral, e.g., topically or by intravenous injection.

[0061] When the composition is administered parenterally, administration is generally by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for suspension in liquid prior to injection, or as emulsions. Furthermore, parenteral administration can include the preparation of sustained or sustained release systems to maintain a constant dosage.

[0062] How to use In one embodiment, the anti-NKG2D antibodies disclosed herein can be used to treat diseases such as cancer, autoimmune diseases, GvHD, viral infections, or bacterial infections. In another embodiment, the anti-NKG2D antibodies disclosed herein can be used to treat diseases associated with NKG2D. In another embodiment, the anti-NKG2D antibodies disclosed herein can be used to treat diseases associated with overexpression of NKG2D.

[0063] In some embodiments, the anti-NKG2D antibodies disclosed herein comprise cytotoxic activity, hi some embodiments, the anti-NKG2D antibodies disclosed herein are cytotoxic to cancer or tumor cells.

[0064] In some embodiments, the anti-NKG2D antibodies disclosed herein can be used in methods against cancer or tumors. In some embodiments, the cancer or tumor comprises a solid cancer or tumor. In some embodiments, the cancer or tumor comprises a non-solid (diffuse) cancer or tumor. In some embodiments, the cancer or tumor comprises a metastasis of a cancer or tumor.

[0065] As used herein, the term "method" refers to manner, means, techniques, and procedures for accomplishing a given task, and includes, but is not limited to, manner, means, techniques, and procedures known to or readily developed from known manner, means, techniques, and procedures by practitioners in the chemical, pharmaceutical, biological, biochemical, and medical fields.

[0066] As used herein, the terms "treat", "treatment", or "therapy" (as well as their different forms) refer to therapeutic treatment, including prophylactic or preventative measures, the purpose of which is to prevent or slow down (alleviate) undesirable physiological changes associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of a disease or condition, stabilization of a disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, improvement or alleviation of a disease or condition, and remission (whether partial or total) of a disease or condition, whether detectable or undetectable. Those in need of treatment include those already with a disease or condition, as well as those prone to having a disease or condition, or those in whom a disease or condition is to be prevented.

[0067] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a human or non-human animal to which treatment with the anti-NKG2D antibody compositions or formulations of the invention is provided. The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include all vertebrates, e.g., mammals such as non-human primates (e.g., higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, horses, or non-mammals such as reptiles, amphibians, chickens, and turkeys. The compositions described herein can be used to treat any suitable mammal, including primates such as monkeys and humans, horses, cows, cats, dogs, rabbits, and rodents such as rats and mice. In one embodiment, the mammal to be treated is a human. The human can be any human of any age. In one embodiment, the patient is an adult. In another embodiment, the human is a child. The human can be male, female, pregnant, middle-aged, adolescent, or elderly.

[0068] Pharmaceutical compositions suitable for use in the methods disclosed herein include compositions in which the active ingredient is contained in an amount effective to achieve the intended purpose. In one embodiment, a therapeutically effective amount refers to an amount of active ingredient effective to prevent, reduce or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art.

[0069] As used herein, "modulate" refers to "stimulating" or "inhibiting" the activity of a molecular target or pathway. For example, a composition modulates the activity of a molecular target or pathway if it stimulates or inhibits the activity of the molecular target or pathway by at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or about 99% or more, compared to the activity of the molecular target or pathway under the same conditions but lacking only the presence of the composition. In another example, a composition modulates the activity of a molecular target or pathway if it stimulates or inhibits the activity of the molecular target or pathway by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold, compared to the activity of the molecular target or pathway under the same conditions but lacking only the presence of the composition. The activity of a molecular target or pathway can be measured by any reproducible means. The activity of a molecular target or pathway can be measured in vitro or in vivo. For example, the activity of a molecular target or pathway can be measured in vitro or in vivo by a suitable assay known in the art for measuring activity. A control sample (not treated with the composition) can be assigned a relative activity value of 100%.

[0070] In one embodiment, the method comprises administering to a subject a composition comprising an effective amount of an anti-NKG2D antibody disclosed herein. In one embodiment, the composition comprises an anti-NKG2D antibody having heavy and light chain CDR sequences described herein. In another embodiment, the composition comprises an anti-NKG2D antibody having VH and VL sequences described herein.

[0071] One of skill in the art will understand that in some embodiments, modulating the immune response includes reducing or eliminating inflammation in situations where inflammation would have been the expected outcome without the use of an anti-NKG2D antibody described herein. One of skill in the art will also understand that in some embodiments, treating a tumor or cancer includes reducing tumor size, growth, and / or spread of the tumor or cancer compared to the outcome without the use of an anti-NKG2D antibody described herein.

[0072] In one embodiment, the disclosure provides a method of treating a disease in a subject comprising administering to the subject a composition comprising an effective amount of an anti-NKG2D antibody disclosed herein. In one embodiment, the composition comprises an anti-NKG2D antibody having heavy and light chain CDR sequences described herein. In another embodiment, the composition comprises an anti-NKG2D antibody having VH and VL sequences described herein.

[0073] In one embodiment, the present disclosure also provides for the use of a composition comprising an anti-NKG2D antibody to treat a disease in a subject. In one embodiment, the composition comprises an anti-NKG2D antibody having heavy and light chain CDR sequences described herein. In another embodiment, the composition comprises an anti-NKG2D antibody having VH and VL sequences described herein.

[0074] In one embodiment, the exact amount of the polypeptide or composition thereof required to induce the desired effect will vary from subject to subject, depending on the species, age, sex, weight, and general condition of the subject, the specific polypeptide, the route of administration, and whether other drugs are included in the regimen. Therefore, it is not possible to specify an exact amount for every composition. However, the appropriate amount can be determined by those skilled in the art using routine experimentation. Dosage can vary, and the polypeptide can be administered in one or more doses (e.g., two or more, three or more, four or more, or five or more) per day for one or more days. Guidance on selecting the appropriate dose of antibody can be easily found in the literature.

[0075] In one embodiment, the disease can be a viral infection, a bacterial infection, cancer, an autoimmune disease or immune disorder. In one embodiment, the disease can be an upper respiratory tract viral infection, an early stage pulmonary infection, or a late stage pulmonary infection. Many diseases and cancers are known to be caused by viruses. Examples of disease-causing viruses include, but are not limited to, norovirus, rotavirus; hepatitis virus A, B, C, D, or E; rabies virus, West Nile virus, enterovirus, echovirus, coxsackievirus, herpes simplex virus (HSV), HSV-2, varicella-zoster virus, mosquito-borne virus, arbovirus, St. Louis encephalitis virus, California encephalitis virus, lymphocytic choriomeningitis virus, human immunodeficiency virus (HIV), poliovirus, Zika virus, rubella virus, cytomegalovirus, human papillomavirus (HPV), enterovirus D68, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome coronavirus, SARS coronavirus 2, Epstein-Barr virus, influenza virus, respiratory syncytial virus, polyomavirus (such as JC virus, BK virus), Ebola virus, dengue virus, or any combination thereof.

[0076] In another embodiment, the disease is cancer, which may be, but is not limited to, carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, blastoma, chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone, osteosarcoma, rhabdomyosarcoma, heart cancer, brain cancer, astrocytoma, glioma, medulloblastoma, neuroblastoma, breast cancer, medullary carcinoma, adrenal cortical tumor, thyroid cancer, Merkel cell carcinoma, eye cancer, gastrointestinal cancer, colon cancer, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, hepatocellular carcinoma, pancreatic cancer, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, renal cell carcinoma, prostate cancer, testicular cancer, urethral cancer, uterine sarcoma, vaginal cancer, head cancer, neck cancer, nasopharyngeal cancer, hematopoietic cancer, non-Hodgkin's lymphoma, skin cancer, basal cell carcinoma, melanoma, small cell lung cancer, non-small cell lung cancer, or any combination thereof.

[0077] Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune-mediated arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, osteoarthritis, chronic primary polyarthritis, reactive arthritis, and rheumatoid arthritis such as ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis vulgaris, Psoriasis such as guttate psoriasis, pustular psoriasis, and nail psoriasis; Atopy, including atopic diseases such as hay fever and Job's syndrome; Dermatitis, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis; X-linked hyper-IgM syndrome, allergic intraocular inflammatory disease, urticaria, such as chronic idiopathic urticaria, including chronic allergic urticaria and chronic autoimmune urticaria; Myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, severe Dermatitis (including systemic sclerosis), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spinal optic MS, primary progressive MS (PPMS), relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal disease, ulcerative colitis, colitis, microscopic colitis, collagenous colitis, polyposal colitis, necrotizing colitis, transmural colitis, autoimmune inflammatory bowel disease), enterocolitis, pyoderma gangrenosum, erythema nodosum. plaques, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of the uvea in whole or in part, iritis, choroiditis, autoimmune blood diseases, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage such as meningitis, herpes of pregnancy, pemphigoid of pregnancy, scrotal pruritus, autoimmune premature ovarian failure, sudden hearing loss due to autoimmune disease, IgE-mediated diseases such as anaphylaxis, allergic rhinitis, atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic encephalitis and / or brainstem encephalitis, anterior uveitis, acute anterior uveitis,uveitis, such as granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis; glomerulonephritis (GN), with or without nephrotic syndrome, such as chronic or acute glomerulonephritis (primary GN, immune-mediated GN, etc.); membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranous proliferative GN (MPGN), includes types I and II, rapidly progressive GN, proliferative nephritis, autoimmune polyendocrine deficiency, plasma cell balanoposthitis, balanoposthitis, erythema annulare centrifugally, erythema psoriatica, erythema multiforme, granuloma annulare, lichen exudative, lichen sclerosus atrophicus, lichen simplex chronicus, lichen spinous, lichen planus, ichthyosis lamellar, epidermolytic keratosis, pyoderma gangrenosum, allergic symptoms and reactions, allergic reactions, allergies asthma such as eczema, bronchial asthma, bronchial asthma, autoimmune asthma, conditions with T cell infiltration and chronic inflammatory responses, immune responses to foreign antigens such as AB-0 blood type in the fetus during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus nephritis, lupus encephalitis, pediatric lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, lupus including alopecia, systemic lupus erythematosus (SLE), such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, juvenile onset (Type I) diabetes mellitus, including insulin-dependent diabetes mellitus (IDDM) of childhood and adult onset diabetes mellitus (Type II diabetes). Also, cytokines and T lymphocytes, granulomatous diseases including sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis including: vasculitis, large vasculitis (including polymyalgia rheumatica and giant T-cell (Takayasu) arteritis), medium vasculitis (including Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immune vasculitis, central nervous system vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis and ANCA-associated vasculitis, Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,Hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases associated with leukodialysis, CNS inflammatory diseases, Alzheimer's disease, Parkinson's disease, sepsis, trauma, multiple organ injury syndromes secondary to hemorrhage, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid and Pemphigoid such as cutaneous pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, mucomembranous pemphigus, and lupus erythematosus pemphigoid), autoimmune polyendocrinopathy, Reiter's disease or syndrome, burns, immune complex diseases such as pre-eclampsia, immune complex nephritis, antibody-mediated nephritis, polyneuropathy, chronic neuropathies such as IgM polyneuropathy and IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenias such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic keratoscleritis, episcleritis, autoimmune diseases of the testes and ovaries including autoimmune orchitis and oophoritis Autoimmune diseases, autoimmune endocrine disorders including thyroiditis such as primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyglandular syndromes such as autoimmune polyglandular syndrome (or polyendocrinopathy syndrome), paraneoplastic syndromes including neurological paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, allergic encephalomyelitis or allergic encephalomyelitis Encephalomyelitis such as myelitis and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS), sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant T-cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barre syndrome, Burger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis,Acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient epidermolytic dermatosis, liver cirrhosis such as primary biliary cirrhosis and pulmonary cirrhosis, autoimmune enteropathy syndrome, celiac disease or celiac disease, celiac disease (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, herpes zoster-associated pain, amyloidosis, non-cancerous lymphocytosis, primary lymphocytosis (including monoclonal B-cell lymphocytosis) (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndromes, epilepsy, migraine, cardiac arrhythmias, myopathy, hearing loss, blindness, channelopathies such as periodic paralysis, channelopathies of the CNS, autism, inflammatory myopathies, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinal inflammation, autoimmune liver disease, fibromyalgia, polyendocrine deficiency, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating disease and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia major, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, e.g. due to antisperm antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, habitual abortion, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, bird-lover's lung, allergic granulomatous vasculitis, Benign lymphocytic vasculitis, Alport syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, parasitic diseases such as leishmaniasis, cypanosomiasis, schistosomiasis, ascariasis, and aspergillosis, Sumpter syndrome, Kaplan syndrome, dengue fever, endocarditis, endocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema mediantidis, erythroblastosis fetalis, eosinophilic facies, Shulman syndrome, Felty syndrome, flariasis, chronic cyclitis, and ankyloglossia.Cyclitis such as iridocyclitis (acute or chronic), or Hook cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCK), acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroiditis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, ubiquitin thromboangiitis, thyrotoxicosis, dorsal tuberculosis, choroiditis, giant T Polymyalgia cellularis, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephrotic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplanted organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / lung disease, silicosis, aphtha, aphthous stomatitis, arteriosclerotic disease, aspergioneus, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, anaphylactic lenticular endophthalmopathy, allergic enteritis, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic febrile illness, Hammann-Rich disease, sensorineural hearing loss , paroxysmal hemoglobinuria, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, testicular granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Kervan thyroiditis, acquired spinal cord atrophy, nonmalignant thymoma, vitiligo, toxic shock syndrome, food poisoning, conditions with T cell infiltration, leukocyte adhesion deficiency, immune responses related to acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases related to leukocytosis, multiple organ injury syndrome, antigen-antibody complex mediated diseases, Anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine deficiency, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathies such as dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophilia,Eosinophil-related diseases such as pulmonary infiltrative eosinophilia, eosinophilia-myalgia syndrome, and Loeffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or eosinophil-containing granuloma, anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, scleral, episcleral, and chronic mucocutaneous candidiasis, Bruton's syndrome, and transient low-grade cancer of infancy. Maglobulinemia, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, vascular ectasia, autoimmune diseases associated with collagen diseases, rheumatism, neurological diseases, lymphadenitis, decreased blood pressure response, vascular insufficiency, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, diseases involving angiogenesis, allergic hypersensitivity, glomerulonephritis, reperfusion injury, ischemia-reperfusion injury, reperfusion injury of the myocardium or other tissues, lymphomatous tracheobronchitis, inflammatory Also contemplated are immune responses associated with acute and delayed hypersensitivity mediated by skin diseases, skin diseases with an acute inflammatory component, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory diseases, ocular and orbital inflammatory diseases, granulocyte transfusion associated syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelitis, intimal hyperplasia, peptic ulcers, valvular inflammation, and endometriosis.

[0078] In some embodiments, the disease is a transplant-related disease, such as graft-versus-host disease (GvHD). According to one embodiment, the GVHD is acute GVHD. According to another embodiment, the GVHD is chronic GVHD.

[0079] In another embodiment, the disclosure provides a method of using a polynucleotide to treat the above-mentioned diseases or conditions, wherein the polynucleotide encodes an anti-NKG2D antibody described herein.

[0080] As used herein, the terms "comprise," "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0081] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an antibody" or "at least one antibody" can include a plurality of antibodies.

[0082] Throughout this application, various embodiments of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of anti-NKG2D antibodies and their uses. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0083] Whenever a numerical range is given herein, it is meant to include any recited numbers (fractional or integer) within the given range. The terms "ranging" between a first denoted number and a second denoted number and "ranging" from a first denoted number to a second denoted number are used interchangeably herein and are meant to include the first and second denoted numbers and all fractional and integer numbers therebetween.

[0084] When values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In one embodiment, the term "about" refers to a 0.1-5% deviation from the stated number or range of numbers. In another embodiment, the term "about" refers to a 1-10% deviation from the stated number or range of numbers. In another embodiment, the term "about" refers to up to a 20% deviation from the stated number or range of numbers. In one embodiment, the term "about" refers to a ±10% deviation from the stated number or range of numbers. In another embodiment, the term "about" refers to a ±5% deviation from the stated number or range of numbers.

[0085] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the anti-NKG2D antibodies and their uses pertain. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the anti-NKG2D antibodies and their uses, the methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are merely illustrative and are not necessarily intended to be limiting. Each reference or other citation mentioned herein is incorporated herein by reference in its entirety.

[0086] In the description provided herein, each of the steps for making and using the anti-NKG2D antibodies and variations thereof are described. This description is not intended to be limiting, and variations in the components, order of steps, and other variations will be understood to be within the scope of the present anti-NKG2D antibodies and their uses.

[0087] It is understood that certain features of the anti-NKG2D antibodies and their uses that are described in the context of separate embodiments for clarity can also be provided in combination in a single embodiment. Conversely, various features of the anti-NKG2D antibodies and their uses that are described in the context of a single embodiment for brevity, may be provided separately or in any suitable subcombination, or as appropriate, in other described embodiments of the anti-NKG2D antibodies and their uses. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment cannot function without those elements.

[0088] The various embodiments and aspects of the present anti-NKG2D antibodies as detailed above and claimed in the claims section below are found experimentally supported in the following examples. EXAMPLES

[0089] Example 1 Generation of mouse hybridoma monoclonal antibodies for NKG2D AIM: To generate monoclonal antibodies against NKG2D using mouse hybridoma technology.

[0090] Methods: Generation of mouse hybridoma anti-NKG2D antibodies. Anti-NKG2D antibodies were developed by immunizing Balb / c and SJL mice with NKG2D-ECD-hFc (extracellular domain of NKG2D fused to human Fc). Animals were bled and tested for antibody titers by ELISA (Test Bleed 1 and Test Bleed 2). Spleen cells from immunized mice with high titers were isolated and fused by standard fusion procedures to generate hybridomas producing specific antibodies. Supernatants containing antibodies produced by pools of these cells were screened primarily by ELISA for reactivity with NKG2D-ECD protein fused to human Fc (NKG2D-ECD-hFc) and secondarily by FACS for reactivity with NKG2D-overexpressing cells. For screening, a number of hybridoma supernatants were tested for target antibody activity by FACS.

[0091] Target-overexpressing cells were placed in 96-well round-bottom polystyrene plates and incubated with neat supernatants from hybridoma cultures. Cells were then washed and incubated with fluorescently labeled secondary antibodies. As a negative control, supernatants were tested with parental cells transfected with a non-target protein gene (found negative) to confirm that reactive antibodies specifically recognized the target protein. Positive pools were identified and cloned by limiting dilution. After one or two fusions, positive clones producing specific antibodies were identified and selected by ELISA and FACS.

[0092] Results: After immunization of animals, sera were tested for binding to recombinant human NKG2D-ECD-Fc (extracellular domain of NKG2D fused to human Fc) by ELISA and FACS (Figure 1). As shown in Figure 1A, test bleed (TB) 1 of Balb / c or SJL animals at various dilutions showed positive titers to human NKG2D-ECD-Fc by ELISA compared to pre-bleed (PB) samples. Figure 1B shows serial dilutions of test bleed 1 titers to human-NKG2D / Dap10 overexpressed in CHO cells and to cyno-NKG2D / Dap10 overexpressed in CHO cells, but no reactivity to CHO parental cells. SJL mice showed a stronger immune response compared to Balb / c mice when tested for binding to NKG2D / Dap10 CHO cells. Animals SJL#8748 and #8749 were selected for fusion after test bleed 2 with similar observations (data not shown). Primary screening was performed after electrofusion to identify positive clones by ELISA against recombinant human NKG2D-ECD-Fc, with human Fc as negative control. To identify cross-reactive clones, secondary screening was performed against CHO cells overexpressing human-NKG2D / Dap10 or cyno-NKG2D / Dap10, with CHO parent cells as negative control. In the primary screening, 103 clones were selected for secondary screening. Secondary screening revealed clones that were further subcloned, and finally, 18 clones were carried forward for 200 ml expression and Protein A purification. Figure 2 shows the identity of the selected clones.

[0093] Summary: Generation of mouse monoclonal hybridomas against human NKG2D protein yielded 20 clones that were identified as specific binders to human and cyno NKG2D after primary and secondary screening by ELISA and FACS. These clones were further expressed, purified, analyzed, and characterized for binding potency.

[0094] Example 2 Binding characterization of mouse hybridoma monoclonal antibodies against human NKG2D

[0095] Objective: To express, purify and characterize mouse monoclonal Ab against NKG2D by binding.

[0096] Methods: Expression and purification of hybridoma clones. Briefly, approximately 0.25-0.5 × 10 7 The cells were inoculated into roller bottles pre-filled with 100 mL of antibody production medium (Hybridoma-SFM+2.5% FBS (low IgG)) and incubated in a roller culture apparatus at a speed of 300 r / h for 14-16 days at 37 °C and in conditions without carbon dioxide. The cell suspension was then transferred to a 350 mL centrifuge bottle and centrifuged at 3,220 g and 4 °C for 15 min, followed by filtration through a 0.45 μm filtration capsule to remove cells and cell debris. The culture supernatant was then loaded onto a pre-equilibrated Protein A affinity column for affinity purification. The antibody was eluted from the column with 5 CV (column volume) of elution buffer (0.1 M sodium citrate buffer, pH 3.0), neutralized with Trizma base to a final pH of 7.0, then dialyzed overnight at 2-8 °C against 100 times the elution volume of PBS, pH 7.4, and sterile filtered through a 0.22 μm syringe filter in a biological safety cabinet. The purified antibody was then aliquoted and stored at -20°C or -80°C until use.

[0097] ELISA binding to target protein. Briefly, target protein NKG2D-ECD-Fc is diluted in PBS at a final concentration of 0.7 μg / mL and coated on ELISA plates (catalog 9018, supplier Corning) with 100 μL / well. Incubate O / N at 4°C. Plates are blocked with 250 μL 1% BSA in PBST for 1 h at 37°C and washed 4 times with PBST using a Biotek (Elx 405). All Abs were serially diluted and 100 μL / well of diluted antibody was added to the plate, incubated for 1 h at 37°C and washed 4 times with PBST. After adding 100 μL / well of goat anti-mouse IgG (Fab specific)-HRP (SIGMA A3682) 1:10000, cells were incubated for 0.5 h at 37°C. After washing 4 times with PBST, 100 μL / well of TMB substrate was added and incubated for 5 min at room temperature. 100 μL / well of 1N HCl was added to stop the reaction. Plates were read using an ELISA plate reader at 450 nM wavelength (instrument SpectraMax M5e). Data analysis was performed using Graphpad prism 5 software by using nonlinear regression (curve fitting): log(agonist) vs. response, where agonist is the antibody concentration (nM) and response is the OD value.

[0098] FACS binding to cells. Briefly, suspension culture cells are harvested directly or TrypLE Express Enzyme Cat: 12604-013, supplier Life technologies) digests adherent cells before harvesting. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Cells are harvested at 2 × 10 6Cells are suspended in FACS buffer (2% FBS in PBS) at a concentration of 1000 nM / mL and 100 μL / well of cell suspension is added to the plate (catalog 3799, supplier Corning). The plate is centrifuged at 2000 rpm for 5 min and the supernatant is discarded. The cells are then resuspended in 100 μL / well of tribody set antibody (400 nM starting, 4-fold dilution, 8 points including 0 point) and the plate is incubated at 4 °C for 60 min. The plate is centrifuged at 2000 rpm, 4 °C for 5 min and the supernatant is discarded. The cells are then washed 3 times with 170 μL of FACS buffer and resuspended with secondary antibody at 100 μL / well and incubated at 4 °C for 30 min in the dark. The plate is centrifuged at 2000 rpm, 4 °C for 5 min and the supernatant is discarded. The cells are then washed 3 times with FACS buffer and analyzed in a FACS bath.

[0099] Epitope binning by ELISA. Briefly, target antibodies are diluted in PBS to a final concentration of 1 μg / ml and coated on ELISA plates (catalog 9018, supplier Corning) at 100 μL / well. Incubate O / N at 4° C. After blocking with 250 μL 1% BSA in PBST for 1 h at 37° C., a series of concentrations of biotinylated antigen hNKG2D-ECD-Fc are added. After incubation at 37° C. for 1.5 h, the plate is washed 3 times with PBST and then 100 μL of streptavidin-HRP (catalog: S5512, supplier Sigma, 1:10000) is added to each well. After incubation at 37° C. for 1 h, the plate is washed 4 times with PBST. 100 μL / well TMB substrate is added and incubated at room temperature for 5 min. The reaction was stopped with 100 μL / well 1N HCL. Plates were read at 450 nM wavelength using an ELISA plate reader. Graphpad prism 5 software is used to find the EC80 of the antigen.

[0100] Below we describe the setup of a serial dilution of mAbs mixed with proteins before binding to mAb-coated plates. Anti-human NKG2D targeting antibodies are diluted in PBS to a final concentration of 1 μg / mL and coated with 100 μL / well on ELISA plates (catalog 9018, supplier Corning). Incubate O / N at 4 °C. After blocking with 250 μL 1% BSA in PBST for 1 h at 37 °C, a mixture of EC80 biotinylated antigen (2x preparation, add 50 μL) and competing antibody (40-80x preparation, add 50 μL) is added. After 1.5 h incubation at 37 °C, the plate is washed 3 times with PBST and then 100 μL of streptavidin-HRP (catalog: S5512, supplier Sigma, 1:10000) is added to each well. After 1 h incubation at 37 °C, the plate is washed 4 times with PBST. 100 μL / well TMB substrate was added and incubated for 5 minutes at room temperature. The reaction was stopped with 100 μL / well 1N HCl. Plates were read at 450 nM wavelength using an ELISA plate reader.

[0101] Results: Figure 3 presents the binding of purified mAbs to NKG2D-ECD-Fc by ELISA. EC80 values ​​ranging from 0.03 nM to 0.6 nM were observed among these antibodies, except for mAb 019.

[0102] Figure 4 presents FACS binding of selected purified mAbs to CHO overexpressing human / cyno NKG2D / Dap10 as well as primary NK cells purified from PBMCs of healthy donors. As shown in Figure 4, all purified mAbs showed binding activity to CHOK1-hNKG2D, CHOK1-cNKG2D and primary NK cells with various ranges of affinity and potency.

[0103] An EC50 value range of approximately 0.4-3.8 nM for binding to CHO cells overexpressing human NKG2D / Dap10 was observed (Figure 4A). Similarly, an EC50 value range of approximately 0.7-8 nM for binding to CHO cells overexpressing cynoNKG2D / Dap10 was observed (Figure 4B). The two concentrations tested for binding to primary NK cells showed dose-dependent binding for all mAbs with various MFIs (Figure 4C). No binding was observed to CHO parental cells (data not shown).

[0104] Figure 5 summarizes the epitope binning analysis performed by ELISA. The ELISA analysis suggests four groups that are distinct in their epitope bins as follows: Group 1 (continuous line with squares) collects mAbs 001, 002, 003, 004, 005, 006, 008, 011, 013, 015, 017, 018, 020; Group 2 (continuous circle) collects mAbs 007, 010, 014; Group 3 (dashed line with squares) collects mAbs 012, 016, 019; Group 4 (dashed circle) collects mAb 009. The positive control Ab is distinct from all groups.

[0105] Summary: Monoclonal antibodies (mAbs) against human NKG2D were successfully generated using hybridoma technology. 20 clones were identified and characterized. Selected mAbs were further expressed and produced by hybridoma clones. mAbs were further purified, analyzed by MS (Mass-Spec), characterized for ELISA binding to NKG2D-ECD-Fc antigen and FACS binding to cells expressing human and cyno NKG2D / Dap10, and finally tested for epitope binning by ELISA to obtain four bin groups.

[0106] Example 3 In vitro functional evaluation of mouse hybridoma monoclonal antibodies

[0107] Objective: To assess in vitro NK cell activation upon binding of anti-NKG2D mAb to primary NK cells by CD107a and IFNg upregulation.

[0108] Methods: Briefly, NK cells are isolated from PBMCs collected from healthy donors using EasySep™ Human NK Cell Isolation Kit (STEMCELL, Cat. No. 17955). NK cells are suspended in RPMI 1640 (Gibco, Cat.-A 10491-01) containing 10% FBS and 100 ng / mL hIL-2 (R&D SYSTEMS, Cat.-219-IL) for 2 days. Antibodies are cross-linked by coating onto 96-well plates (Cat-3599, Corning) and then incubated overnight at 4°C. The supernatant is removed from the coated plates and the plates are then washed twice with 200 μL of PBS. Harvest stimulated NK cells by centrifugation at 500 g for 5 min, adjust the concentration of cells to 5x105 / mL with NK cell stimulation medium (RPMI 1640+10%FBS+20ng / mL hIL-2), then add 200μL / well of NK cells to Ab-coated plate. Add APC anti-CD 107 a (Biolegend, Cat: 328620) and control (0.5μL / test) separately to NK cells. Incubate plates at 37°C in a humidified 5% CO2 atmosphere for 4 hours. Collect supernatants for IFN-γ measurement and cells for CD107a measurement. CD107a measurement: Transfer cells to FACS plate (Cat-3799, Corning). Wash cells twice with 200μL of FACS buffer (2% FBS in PBS). APC-anti-CD 107 a, FITC-anti-CD 3 (Invitrogen, Cat: 17-0037-41) and BV 421-anti-CD 56 (BioLegend, Cat: 318328) and controls are added to the NK cell suspension, respectively, and the cells are incubated for 1 h at 4°C. After washing twice with 200 μL of FACS buffer, the cells are resuspended in 100 μL of cold PBS. Keep the cells in the dark and subject the cells to FACS (FACS Canto II, BD Biosciences) analysis. IFN-γ measurement: IFN-γ was measured according to the protocol of the Human IFNγ DuoSet ELISA Kit (R&D, DY285).Briefly, working concentrations of capture antibodies in PBS are coated onto 96-well microplates (Cat-9018, Corning) and incubated overnight at room temperature. After washing three times, the plate is blocked with 300 μL of reagent diluent (1% BSA in PBS) for at least 1 h at room temperature. After washing three times, 100 μL of appropriately diluted samples or standards in reagent diluent are added and incubated for 2 h at room temperature. After washing three times, 100 μL of diluted detection antibody is added and incubated for 2 h at room temperature. After washing three times, 100 μL of working dilution of streptavidin-HRP B is added and incubated for 20 min at room temperature. After washing three times, 100 μL of substrate solution is added and incubated for 20 min at room temperature. Add 50 μL of stop solution. Read OD450 using a microplate reader (Molecular Device, Cat: Spectra Max M5e).

[0109] Results: Figure 6 shows the activation of NK cells upon binding of purified anti-NKG2D mAb (10 nM or 100 nM) as determined by accumulation of the CD107a activation marker or IFN-γ secretion. The validity of the assay was confirmed by the window determined by the IM-1060 positive control tribody and the IM-1091 negative control tribody. Dose-dependent CD107a + NK cell accumulation was determined upon mAb binding by FACS compared to a mouse IgG control (Figure 6A). Ten of the mAbs tested also stimulated IFN-γ secretion measured in the supernatants by ELISA (Figure 6B).

[0110] Summary: Twenty purified anti-NKG2D mAbs were characterized by binding and activation. The Abs were ranked based on their binding activity as well as their NK activation activity. Four mAbs (mAb 001, mAb 009, mAb 010, and mAb 019), one from each bin, were confirmed by Mass-Spec (data not shown) and further selected based on their demonstrated characteristics for incorporation into tribody constructs and evaluation.

[0111] Example 4 Assessment of blockade of NKG2D-MICA interaction by mouse hybridoma monoclonal antibodies in vitro Objective: To evaluate the blocking activity of purified mAbs against the receptor-ligand NKG2D-MICA interaction in vitro.

[0112] Method: RBA based on competitive ELISA: Dilute target protein (NKG2D-ECD-hFc, lot: 20200413002, CP) is added to PBS at a final concentration of 1 μg / ml and coated on an ELISA plate (catalog 9018, supplier Corning) with 100 μL / well. Incubate O / N at 4°C. After blocking with 250 μL 1% BSA in PBST for 1 h at 37°C, a mixture of serial concentrations of Mab diluted 1:5 from 300 nM is added (600 nM preparation, add 50 μL) and human MICA-His is added at EC80 (2x preparation, add 50 μL). After 1.5 h incubation at 37°C, the plate is washed 3 times with PBST and then 100 μL of anti-His-HRP (1:5000) is added to each well. After 1 hour incubation at 37°C, plates are washed 4 times with PBST. 100 μL / well TMB substrate is added and incubated for 5 minutes at room temperature. The reaction is stopped with 100 μL / well 1N HCl. Plates are read at 450 nM wavelength using an ELISA plate reader.

[0113] Results: The mAbs were analyzed for their ability to block the NKG2D-MICA interaction. As shown in Figure 7, the range of inhibition rates, expressed as IC50 values, was 1.4 nM to 8 nM, while no inhibitory activity was observed with the mIgG1 negative control.

[0114] Conclusion: All tested mAbs demonstrated blocking activity against the NKG2A-MICA receptor-ligand interaction.

[0115] Example 5 Recombinant production of chimeric tribodies bearing anti-human NKG2D derived from a mouse hybridoma clone against human NKG2D Objective: To introduce the murine CDR sequences of selected anti-human NKG2D mAbs (mAb 001, mAb009, mAb010 and mAb019) into a tribody / Pro tribody construct to generate a trispecific chimeric Ab containing an anti-CD3 Fab, a single chain of an anti-NKG2D mAb and a single chain of anti-5T4, as shown in Figure 8A and Figure 8B, respectively, and previously described in WO2020 / 225805, the entire contents of which are incorporated herein by reference.

[0116] Methods: Hybridoma sequencing. Briefly, selected positive monoclonal hybridoma cells (~1 × 10 7 ) were collected for total RNA isolation. Total RNA was used for cDNA synthesis according to the SMARTer® RACE 5' / 3' kit manual, and random primers were used for the synthesis of first strand cDNA. To amplify the heavy and light chain variable regions by PCR, the synthetic cDNA was used as a template and primers from the mouse Ig-primer set (Novagen, 69831-3) were used as gene-specific primers (GSPs). PCR products of the correct size were collected and purified using NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel, 740609.250) according to the kit manual, and subjected to TA cloning and sequencing. The heavy and light chain variable regions (VH and VL) of NKG2D-mAb were cloned into tribody constructs as described below, which were then characterized for binding to NKG2D by ELISA and FACS.

[0117] Gene synthesis and plasmid construction. The coding sequences of the heavy (HC) and light (LC) chains of the triabody were generated by DNA synthesis and PCR, and then subcloned into pCDNA3.4-based plasmids (Invitrogen) for protein expression in mammalian cell systems. Finally, the gene sequences in the expression vectors were confirmed by DNA sequencing.

[0118] Expression of trispecific antibody constructs. Transient expression of tribody / Pro-tribody antibodies was performed by co-transfection of paired HC and LC constructs (1:1 HC / LC ratio for tribody format or 2.5:1 HC / LC ratio for Pro-tribody format) into CHO cells using the PEI method. Briefly, approximately 5.5×10 6 CHO cells at 100 μg / ml were used as hosts. Transfection was initiated by adding a mixture of 1 mg total DNA and 4 mg PEI in 100 mL OptiMEM medium (Invitrogen) to the cells and gently mixing. The cells were then cultured for 8–10 days in an incubator shaker at 120 rpm, 37°C, and 8% CO2. After 24 h, peptone and glucose feeding was performed every 2–3 days thereafter depending on cell density and viability. Cell culture was terminated on days 8–10 when cell viability dropped to <80%. Conditioned medium was collected for protein purification.

[0119] Purification of trispecific antibody constructs. Protein purification by affinity chromatography and SEC was performed using an AKTA pure instrument (GE Lifesciences). Affinity capture of tribodies was achieved by passing the harvested supernatant through a column of CaptureSelect™ CH1-XL Affinity Matrix (Thermo Scientific). After washing the column with Buffer A (25 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5), proteins were eluted with Buffer B (50 mM sodium citrate, 150 mM NaCl, pH 3.0) and immediately neutralized with 1 / 6 volume of Buffer D (1 M arginine, 400 mM succinic acid, pH 9.0). The affinity purified protein was then concentrated to 5-10 mg / ml using an Amicon 30 kD concentrator (Merck Millipore) and subjected to SEC purification on a Superdex200 column (GE Lifesciences) equilibrated with SEC buffer: 200 mM arginine, 137 mM succinic acid, 0.05% Tween-80, 150 mM NaCl, pH 5.0. The target tribody fraction was collected and then 5% trehalose (146 mM) was added. The target tribody was analyzed using SDS-PAGE and HPLC-SEC.

[0120] SDS-PAGE analysis of tribody constructs. SDS-PAGE analysis of tribodies was performed under reducing and non-reducing conditions in precast polyacrylamide gels. Briefly, 2 μg of tribody sample was mixed with NuPAGE™ LDS sample buffer (thermofisher-NP0008) with or without 70 mM DTT. After 10 min incubation at 25° C. or 90° C., samples and unstained protein standards (BIO RAD-161-0363) were loaded onto the gel. Electrophoresis was performed at a constant voltage of 120 V with 1× Tris-glycine SDS running buffer. After electrophoresis, the gel was stained overnight with Coomassie blue and destained with destaining solution (10% acetic acid, 40% methanol and 50% water). The destained gel was scanned with a gel imaging system (Tanon-2500R).

[0121] SEC-HPLC analysis of trispecific antibody constructs. Analytical SEC-HPLC was performed using a Shimadzu LC-10 HPLC instrument (Shimadzu Corp.). 20 μl samples of 1 mg / ml are loaded onto a Superdex 200 Increase 5 / 150GL column (GE Lifesciences). The mobile phase was 2×PBS and the flow rate was 0.3 mL / min for 15 min.

[0122] LC-MS analysis of tribody constructs. Tribodies were separated at room temperature using an ACQUITY UPLC BEH200 A, SEC column (water 1.7 μm, 4.6 × 300 mm) and detected by ESI-MS (Thermo, MS-B20-03). The mobile phase was 0.1% formic acid:acetonitrile (75:25, v / v) and the flow rate was 0.2 mL / min. Mass spectrometry was performed in positive ion mode. Other parameters for mass spectrometry were a resolution of 17500, a scan range of 1000-5000 m / z, in-source CID of 60 eV, sheath gas flow rate of 30 L / min, capillary temperature of 350 °C, and spray voltage of 2.5 KV.

[0123] Results: Eight tribody constructs displaying two variants of each of the selected mAb clones (mAb001 designated IM-1244 / 5, mAb009 designated IM-1246 / 7, mAb010 designated IM-1248 / 9, and mAb019 designated IM-1250 / 1) were designed to generate trispecific chimeric Abs comprising an anti-CD3 Fab, a single chain of anti-NKG2D, and a single chain of anti-5T4 either without (FIG. 8A) or with (FIG. 8B) a regulatory domain as shown in FIG 8. FIG 8C details the Ab identities generated, where LH indicates the anti-NKG2D variable light chain fused upstream of the anti-NKG2D variable heavy chain and HL indicates the anti-NKG2D variable light chain fused downstream of the anti-NKG2D variable heavy chain. The expressed HC and LC tribody constructs of each tribody molecule associate to form a single molecule as indicated by a single major 100 kDa band observed in SDS-PAGE under non-reducing (NR) conditions and a double band of 50 kDa under reducing (R) conditions (Figure 9). Figure 9 presents SDA-PAGE analysis of IM-1244-IM1251, respectively, where the left lane represents markers in kDa (M), the center lane represents reducing conditions (R), and the right lane represents non-reducing conditions (NR). Figures 10A-H present SEC-HPLC analysis of IM-1244-IM 1251, respectively, demonstrating a single peak at a retention time of approximately 6 minutes, consistent with the expected retention time of the predicted Mw based on the mass calibration curve.

[0124] Conclusion: The trispecific tribody variant was successfully expressed and purified.

[0125] Example 6 Binding of tribody antibody constructs to NKG2D recombinant protein Objective: To study the binding potency of different tribody antibody constructs composed of anti-5T4ScFv domain, anti-CD3εFab domain, and anti-human NKG2D from mouse hybridoma clones to NKG2D recombinant protein by ELISA and to cells expressing membrane-bound NKG2D protein by FACS. The different formats can be composed of CAP masking sequences, cleavable linkers, non-cleavable linkers, and point mutated engager sequences that lack binding activity for a particular engager and serve as negative controls for the tribody / Pro tribody formats.

[0126] Methods: ELISA and FACS binding of tribody-antibody constructs to antigens or cells as described in Example 2 above.

[0127] Results: Figure 11 demonstrates the analysis of the expressed trispecific constructs for their binding to NKG2D-ECD-Fc protein by ELISA. The binding EC50 to NKG2D-ECD-Fc protein was, as expected, 2.6 nM for IM-1244 (small circle), 1.1 nM for IM-1245 (small square), 1.7 nM for IM-1248 (small diamond), and 0.7 nM for IM-1249 (large circle), while no binding was observed for the negative control tribody IM-1091 NKG2D mutant variant (large diamond) (Figure 11).

[0128] Figure 12 demonstrates the analysis of the expressed trispecific constructs for their binding to cells expressing NKG2D protein by FACS. The binding EC50 to CHO cells overexpressing human NKG2D was 8.6, 7, 12, 2.5, 4.9, 4.7 nM for IM-1244 (small circle), IM-1245 (small square), IM-1246 (small up triangle), IM-1247 (small down triangle), IM-1248 (small diamond), IM-1249 (large circle) and IM-1250 (large square), respectively (Figure 12A). The binding EC50 to CHO cells overexpressing cynoNKG2D was 21, 13, 15, 30, 5.9, 5.3 and 2.3 nM for IM-1244 (small circle), IM-1245 (small square), IM-1246 (upper small triangle), IM-1247 (lower small triangle), IM-1248 (small diamond), IM-1249 (large circle) and IM-1250 (large square), respectively (Figure 12B). The IM-1251 EC50 value (large triangle) was not applicable, but as expected, no binding was observed for the negative control tribody IM-1091 NKG2D mutant variant (large diamond). IM-1060 (large triangle) was used as a positive control tribody. No binding to CHO parental cells was detectable (data not shown).

[0129] Conclusion: Mouse variable light and heavy chains sequenced from selected hybridoma clones were identified, converted to scFv, and introduced into tribody constructs to form chimeric tribody molecules. These molecules were expressed, purified, and further characterized by binding assays, showing that they retained their binding properties to human NKG2D.

[0130] Binding of chimeric tribodies consisting of anti-NKG2D in a tribody format retained binding to the NKG2D protein, as shown in Figure 11. Similarly, binding was also confirmed on cells overexpressing both human and cynoNKG2D on the cell surface, as shown in Figure 12. Tribodies IM-1245, IM-1246, IM-1249 and IM-1250, representing mAb 001, mAb 009, mAb 010 and mAb 019, respectively, were further selected for functional activity in vitro.

[0131] Example 7 Functional evaluation of in vitro tribody-antibody constructs Objective: To evaluate in vitro dose-dependent NK cell activation by tribody variants against primary PBMCs from healthy donors in the presence of cancer cells.

[0132] Methods: NK cell activation in the presence of target cells. PBMCs are suspended in RPMI 1640 (Gibco, Cat-A 10491-01) containing 10% FBS and 100 ng / mL hIL-2 (R&D SYSTEMS, Cat-219-IL) for 2 days. After stimulation, PBMCs and target cells NCI-H226 are harvested by centrifugation at 500 g for 5 min and the cells are diluted to a concentration of 4 × 10 each using assay buffer (RPMI 1640 + 5% FBS + 10 ng / mL hIL-2). 6 / mL and 2 × 10 5Adjust the volume to 100 µL / mL, then add 50 µL of PBMC suspension and 50 µL of NCI-H226 suspension to a round-bottom 96-well plate, respectively, with an ET ratio of 20:1. Prepare diluted antibody (2x) in assay buffer, add 100 µL / well and mix thoroughly. Add APC anti-CD 107 a (Biolegend, Cat: 328620) and control (0.5 µL / test) separately to the samples. Incubate the plate for 4 hours at 37°C in a humidified 5% CO2 atmosphere. After incubation, centrifuge the plate at 250g for 4 minutes at 4°C, discard the supernatant and transfer all cells to the FACS plate using FACS buffer (PBS + 2% FBS). After washing twice with FCAS buffer, FITC-anti-CD3, BV 421-anti-CD56 and APC-CD107a are added in 100 μL of FACS buffer and the plate is incubated for 45 min at 4° C. in the dark. After washing twice with FCAS buffer, the cells are resuspended in 100 μL of cold PBS. The cells are subjected to multicolor FACS analysis.

[0133] Results: After co-culture of PBMCs from healthy donors, 60-70% activation as measured by CD107a MFI on the CD56+ gated NK cell population was observed with NCI-H226 targeting cells expressing the 5T4 protein in the presence of various tribodies (IM-1245, IM-1246, IM-1249, IM-1250) at 300 nM, 60 nM, 12 nM, and 2.4 nM (left to right), while 30% basal activation occurs in the absence of tribodies. A lower activation of 35-40% was observed in the negative control tribody IM-1091 NKG2D mutant variant lacking binding to NKG2D. IM-1060 served as a positive control tribody (Figure 13).

[0134] Conclusion: Four selected tribodies composed of mouse anti-human NKG2D mAb clone sequences from four bins were shown to activate NK cells in vitro.

Claims

1. 1. An isolated anti-NKG2D antibody comprising three complementarity determining regions (CDRs) on a heavy chain (HCDR1, HCDR2, and HCDR3) and three CDRs on a light chain (LCDR1, LCDR2, and LCDR3), (i) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 12-14, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 16-18; or (ii) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 20-22, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 24-26; or (iii) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 28-30, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 32-34; or (iv) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 36-38, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 40-42; Isolated anti-NKG2D antibodies.

2. 2. The anti-NKG2D antibody of claim 1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising the amino acid sequences of SEQ ID NOs: 11 and 15, SEQ ID NOs: 19 and 23, SEQ ID NOs: 27 and 31, or SEQ ID NOs: 35 and 39.

3. The antibody is an IgG, an Fv, an scFv, an Fab, an F(ab') ２ 2. The anti-NKG2D antibody of claim 1, which is a minibody, a diabody, a triabody, a nanobody, a bispecific antibody, or a single domain antibody.

4. The anti-NKG2D antibody of claim 3 , wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

5. A composition comprising an anti-NKG2D antibody according to any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

6. the anti-NKG2D antibody comprises three complementarity determining regions (CDRs) on the heavy chain (HCDR1, HCDR2, and HCDR3) and three CDRs on the light chain (LCDR1, LCDR2, and LCDR3); (i) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 12-14, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 16-18; or (ii) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 20-22, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 24-26; or (iii) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 28-30, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 32-34; or (iv) the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 36-38, and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 40-42; The composition of claim 5.

7. The composition of claim 6, wherein the anti-NKG2D antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and light chain variable region comprise the amino acid sequences of SEQ ID NOs: 11 and 15, SEQ ID NOs: 19 and 23, SEQ ID NOs: 27 and 31, or SEQ ID NOs: 35 and 39.

8. 3. An isolated polynucleotide sequence encoding an anti-NKG2D antibody according to claim 1 or 2.

9. A vector comprising the polynucleotide sequence of claim 8.

10. A host cell comprising the vector of claim 9.

11. 10. A composition for use in treating a disease in a subject, comprising an effective amount of an anti-NKG2D antibody according to claim 1 or 2.

12. The composition of claim 11, wherein the disease is cancer, an autoimmune disease, GvHD, a viral infection, a bacterial infection, or a disease associated with NKG2D.

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