Anti-NKG2d antibodies and methods of use thereof

Abstract

The present disclosure provides antigen-binding sites that bind the Natural Killer group 2D (NKG2D) on natural killer cells, and pharmaceutical compositions comprising such antigen-binding sites.

Description

ANTLNKG2D ANTIBODIES AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 649,994, filed May 21, 2024, U.S. Provisional Patent Application No. 63 / 689,093, filed August 30, 2024, and U.S. Provisional Patent Application No. 63 / 767,081, filed March 5, 2025, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 2, 2025, is named DFY-136WO_SL.xml and is 32,100 bytes in size.FIELD OF THE DISCLOSURE

[0003] The present disclosure provides antigen-binding sites that bind the Natural Killer group 2D (NKG2D) on natural killer cells, and pharmaceutical compositions comprising such antigen-binding sites .BACKGROUND

[0004] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system and make up approximately 15% of circulating lymphocytes. NK cells are able to recognize target cells in the absence of Major Histocompatibility Complex (MHC) class I expression. Hence, they perform a critical role in the body's defense against diseases that evade T cell immunity by downregulating class I MHC expression. Like certain other immune effector cells, NK cells can induce target cell death by releasing cytolytic compounds or by binding receptors on target cell membranes and inducing apoptosis of the target cells. These mechanisms involve recruitment of NK cells to the site of the target cell and activation of the NK cells.

[0005] NK cells respond to signals through a variety of activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy self-cells, their activity is inhibited through killer-cell immunoglobulin-like receptors (KIRs) that recognizeMHC class I. Cancer cells and infected cells frequently downregulate their MHC class I expression, resulting in a reduction or loss of the inhibitory NK cell signaling. Moreover, activating receptors, such as Natural killer group 2 member D (NKG2D), natural cytotoxicity receptors (NCRs), and DNAX accessory molecule 1 (DNAM1), can be stimulated by their ligands expressed on cancer cells or infected cells, resulting in NK cell activation. NKG2D, for example, is encoded by Klrkl and is a C-type lectin-like type II transmembrane protein expressed on NK cells and subsets of T cells such as NKT cells. NKG2D recognizes a class of diverse MHC class I-like ligands that are upregulated on the surface of stressed cells. These ligands include MHC class I polypeptide related sequence A and B (MICA, MICB) and ULI 6 binding proteins 1-6 (ULBP1-6) in humans. They provide an activating signal to elicit cytotoxic activity of NKG2D-expressing lymphocytes and to release cytokines to recruit and activate other immune cells.

[0006] NK cells can also be activated by the constant region of some immunoglobulins through CD 16 on their surface, thereby killing the cells bound by the immunoglobulins via antibody dependent cell-mediated cytotoxicity (ADCC). The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals.

[0007] Despite the NK cell receptors known in the field, there remains a need for additional ligands and antigen binding sites that activate NK cells.SUMMARY OF THE DISCLOSURE

[0008] The present disclosure provides antigen-binding sites that bind an extracellular region of NKG2D (e.g., human NKG2D) and are capable of effectively engaging NK cells. For example, at least some of these antigen-binding sites bind an epitope that partially overlaps with the binding site of MHC class I polypeptide related sequence A (MICA) on NKG2D and competes with MICA for binding NKG2D.

[0009] Accordingly, in one aspect, the present disclosure provides an antigen-binding site that binds NKG2D, including a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH includes complementarity determining region (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) present in SEQ ID NO: 5, and the VL includes CDR1, CDR2, and CDR3 present in SEQ ID NO: 6.

[0010] In some embodiments, the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 7,8, 9, 14, 15, and 16, respectively, as defined according to Kabat. In some embodiments, the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 10, 11, 9, 14, 15, and 16, respectively, as defined according to Chothia. In some embodiments, the CDR1, CDR2, and CDR3 of the VH and the CDR1 and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 18, 12, 13, 17, and 16, respectively, and the CDR2 of the VL includes the amino acid sequence of NAK, as defined according to IMGT. In some embodiments, the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 19, 20, 13, 14, 21, and 16, respectively, as defined according to North. In some embodiments, the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 22, 20, 9, 14, 15, and 16, respectively, as defined according to AbM.

[0011] In some embodiments, the VH includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, and the VL includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In some embodiments, the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 5 and 6, respectively.

[0012] In some embodiments, the VH includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3, and the VL includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.

[0013] In another aspect, the present disclosure provides an antigen-binding site that binds NKG2D, including one or more paratopes that include amino acids S31, K50, D52, D55, E57, M99, and E101 of a VH comprising SEQ ID NO: 5, and amino acids Y30, N31, Y32, W92, S93, and 194 of a VL comprising SEQ ID NO: 6. In some embodiments, the one or more paratopes include amino acids T30, S31, W33, K50, D52, D55, E57, H59, M99, G100, E101, and F102 of a VH including SEQ ID NO: 5 and amino acids Y30, N31, Y32, F91, W92, S93, 194, and W96 of a VL including SEQ ID NO: 6. In some embodiments, the VH includes CDR1, CDR2, and CDR3 including the amino acid sequences set forth in SEQ ID NOs: 18, 20, and 9, respectively, and the VL includes CDR1 and CDR3 including the amino acid sequences set forth in SEQ ID NOs: 17 and 16, respectively. In someembodiments, the VL further includes a CDR2 including the amino acid sequence of NAK. In some embodiments, the VH includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, and the VL includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6.

[0014] In another aspect, the present disclosure provides an antigen-binding site that binds an epitope of NKG2D within the amino acid sequence of SEQ ID NO: 23. In some embodiments, the epitope of NKG2D comprises amino acids L174, S175, T180, 1181, and El 83 of SEQ ID NO: 1.

[0015] In another aspect, the present disclosure provides an antigen-binding site that competes with a reference antigen-binding site for binding NKG2D, wherein the reference antigen-binding site includes a VH including the amino acid sequence of SEQ ID NO: 5 and a VL including the amino acid sequence of SEQ ID NO: 6.

[0016] In some embodiments of any one of the aspects above, where applicable, the VH and VL are humanized.

[0017] In another aspect, the present disclosure provides an antibody that binds NKG2D, that includes any one of the antigen-binding sites disclosed herein and an antibody Fc region.

[0018] In another aspect, the present disclosure provides a pharmaceutical composition including any of the antigen-binding sites or the antibodies disclosed herein; and a pharmaceutically acceptable carrier or excipient.

[0019] In another aspect, the present disclosure provides one or more isolated nucleic acids encoding any of the antigen-binding sites or the antibodies disclosed herein.

[0020] In another aspect, the present disclosure provides a vector including the one or more nucleic acids disclosed herein.

[0021] In another aspect, the present disclosure provides an isolated cell including the one or more nucleic acids disclosed herein or a vector disclosed herein.

[0022] In another aspect, the present disclosure provides a method of producing a protein, the method including culturing a cell disclosed herein under conditions for expressing the antigen-binding site or antibody.

[0023] Other embodiments and details of the disclosure are presented herein below.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGs. 1A-1F are graphs of FACS analysis of TNFa staining of EL4-NKG2D cells treated with the indicated samples.

[0025] FIG. 2A shows a representation of a crystal structure of 2D3-CK Fab complexed with NKG2D solved by X-ray crystallography. Ribbon like structures are 2D3-CK Fab, middle structure is NKG2D. FIG. 2B shows a representation of NKG2D chain A (left of dashed line) and chain B (right of dashed line). The shaded regions represent the 2D3 epitope (dark grey), MICA epitope (medium gray), and the partial overlap between the two epitope regions (line pattern). FIG. 2C shows a representation of 2D3-CK Fab bound to NKG2D, where the paratope residues Y30, W92, E57, and 194 form non-covalent bonds with epitope residues 1181, E183, and S195, which also interact with MICA. FIG. 2D shows a representation of MICA bound to NKG2D, where the MICA residues K81, R64, and QI 66 form non-covalent bonds with 2D3-CK Fab residues 1181, E183, and S195.

[0026] FIG. 3A is a sensorgram showing the binding of MICA-His to NKG2D in the presence of 2D3 Fab. The top line shows the injection of 2D3 Fab followed by injection of a mixture of MICA-His and 2D3 Fab, and the bottom line shows the injection of 2D3 Fab followed by injection of only buffer. FIG. 3B is a sensorgram showing the binding of 2D3 Fab to NKG2D in the presence of MICA-His. The top line shows the injection of MICA-His followed by injection of a mixture of MICA-His and 2D3 Fab, and the bottom line shows the injection of MICA-His followed by injection of only buffer.

[0027] FIG. 4 is a human and mouse NKG2D peptide immunogen sequence analysis. Human NKG2D extracellular domain (ECD) sequence (UniProt accession no. P26718; SEQ ID NO: 26) was aligned to mouse NKG2D ECD (UniProt accession no. 054709; SEQ ID NO: 27). Secondary structure analysis is below the sequence alignment with alpha helices in oval bars and beta strands in arrows. Solvent accessibility is scored below secondary structure analysis, with a higher score indicating more solvent accessibility. Residues are highlighted according to MICA epitope residues on NKG2D chain A (white text with gray background), MICA epitope residues on NKG2D chain B (black text with dark gray background), or MICA residues overlapping with both NKG2D chains A and B (black text with light gray background). Residues in bold indicate residues predicted to form non-covalent bonds with MICA. Boxed sequences indicate peptide residues used for immunization.

[0028] FIG. 5 shows the sequence and structure of three peptides (SEQ ID NOs: 28-30) designed to produce antibodies that bind a similar epitope to MICA.DETAILED DESCRIPTION

[0029] The present disclosure provides antigen-binding sites that bind an extracellular region of NKG2D (e.g., human NKG2D) and are capable of effectively engaging NK cells. For example, at least some of these antigen-binding sites bind an epitope that partially overlaps with the binding site of MHC class I polypeptide related sequence A (MICA) on NKG2D and competes with MICA for binding NKG2D.Definitions

[0030] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0031] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0032] As used herein, the term “antigen-binding site” refers to the part of an immunoglobulin (a.k.a. antibody) molecule that participates in antigen binding. An antigenbinding site is also known as an antigen-binding fragment of an antibody. In certain embodiments (e.g., human antibodies), an antigen-binding site is formed by amino acid residues of variable domains of the heavy and light chains, which are also called “VH” and “VL,” respectively. Three highly divergent stretches within the variable domains of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions,” or “FR .” Thus, the term “FR” refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. The three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” Antigen-binding sites that include a VH and a VL can be incorporated in, for example, a Fragment antigen-binding region (Fab), which further includes a CHI domain and a CL domain, or a single-chain variable fragment (scFv), in which the VH is connected to the VL by a peptide linker. In certain embodiments, an antigen-binding site is formed by a single antibody chain providing a “single domain antibody” or “sdAb” (e.g., “VHH” in camelid or “VNAR” in cartilaginous fish). A “single domain antibody” is a small polypeptide or protein (generally -12-15 kDa) including a single monomeric heavy chain variable domain or a single monomeric or light chain variabledomain, but not both. For example, a VHH generally includes four framework regions flanking three complementarity determining regions (CDRs), represented as FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. A VNAR generally includes only two CDRs, namely, CDR1 and CDR3, but hypervariable regions HV2 and HV4 also contribute to antibody diversity. Single domain antibodies can also be synthetically produced using techniques well-known in the art. For example, methods of designing and producing single domain antibodies are described in Hoey et al., Exp. Biol. Med. 244: 1568- 76 (2019), Sonneson et al., Biochemistry 48:6693-95 (2009), Saerens et al. J. Mol. Biol. 352.59'1-60'1 (2005), Wagner et al. Int. J. Mol. Sci. 19:pii:E3444 (2018), Rizk et al. Nat. Struct. Mol. Biol. 18:437-42 (2011). A single domain antibody can also be synthetic, e.g., a synthetic fully human antibody. An exemplary method of generating synthetic fully human VH antibodies based on human germline sequences is described in Mindrebo et al., (2023) Proc. Natl. Acad. Sci. U.S.A. 120(24): e2216612120. An exemplary method of generating synthetic fully human VL antibodies based on human germline sequences is described in Kim et al., (2014) Mabs. 6(1): 219-35. As used herein, the term “single domain antibody” does not include a constant domain. To reduce immunogenicity in humans, antigen-binding sites can be humanized (see, e.g., Safdari et al., BiotechnoL Genet. Eng. Rev. (2013) 29: 175-86; Sulea, Methods Mol. Biol. (2022) 2446:299-312) or fully human. All the amino acid positions in heavy or light chain variable regions disclosed herein are numbered according to Kabat numbering, unless otherwise indicated.

[0033] The CDRs of an antigen-binding site derived from a conventional antibody can be determined by various methods known in the art, which include but are not limited to:(i) the Kabat definition, as described in Kabat et al., J. Biol. Chem. 252, 6609-16 (1977) and Kabat et al., Sequences of protein of immunological interest (1991), NIH Publication No. 91- 3242;(ii) the Chothia definition, as described in Chothia et al., J. Mol. Biol. 196:901-17 (1987) and Chothia, et al., Nature 342:878-83 (1989);(iii) the MacCallum definition, as described in MacCallum et al., J. Mol. Biol. 262:732-45 (1996);(iv) the IMGT definition, as described in Lefranc, The Immunologist, 7, 132-36 (1999), Lefranc et al. Dev. Comp. Immunol., 27, 55-77 (2003), and Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005);(v) the AbM definition, as described in Martin, et al., Proc. Natl Acad. Sci. USA, 86, 9268-72 (1989), Pedersen et al., Immunomethods, 1, 126 (1992), and Rees et al., in Sternberg M.J.E.(ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-72 (1996);(vi) the North definition, as described in North B, et al., J. Mol. Biol. 406(2):228-56 (2011); or(vii) the Honegger definition, as described in Honegger et al., J. Mol. Biol. 309:657-70 (2001).As a result, once the heavy and / or light chain variable regions have been elucidated, then it is possible to identify the CDRs in such heavy and / or light chain variable regions using one of the foregoing approaches. The CDRs determined under these definitions typically include overlapping or subsets of amino acid residues when compared against each other.

[0034] In the context of a single domain antibody, CDR sequences can be determined using methods well-known in the art. For example, besides the methods available for conventional antibodies the disclosures of Vattekatte et al. PeerJ 8:e8408 (2020), Muyldermans et al. Protein Engineering 7 : 1129-35 (1994), Sircar et al. J. Immunol. 186:6357-67 (2011), Vu et al. Mol. Immunol. 34: 1121-31 (1997), and Zuo et al. BMC Genomics 18:797 (2017) provide details on the determination of CDR sequences in single domain antibodies.

[0035] As used herein, percent “identity” between a query sequence (e.g., a query amino acid sequence or nucleotide sequence) and a reference sequence is defined as the percentage of amino acid residues or nucleotides in the query sequence that are identical to the amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as Basic Local Alignment Search Tool (BLAST), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For example, BLAST analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (see Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264- 2268; Altschul, (1993) J. MO . EVOL. 36:290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25:3389-3402) are tailored for sequence similarity searching. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) NATURE GENETICS 6: 119- 129. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0036] As used herein, the term “pharmaceutical formulation” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0037] Throughout the description, where compositions are described as having, including, containing, incorporating, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps.

[0038] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.Antigen-binding Sites That Bind NKG2D

[0039] The antigen-binding sites disclosed herein, derived from the antibodies identified in Tables 1-6, can bind an extracellular region of NKG2D (e.g., human NKG2D). Human NKG2D is identified in the Uniprot database under Accession Number P26718. An exemplary wild-type human NKG2D protein has the amino acid sequence of: MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFC CFIAVAMGIRFIIMVTIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFD ESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWE DGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 1).

[0040] An exemplary wild-type human NKG2D extracellular region has the amino acid sequence of: IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMS QNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQ KGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 2).Table 1. Variable Domain Sequences of Exemplary Anti-NKG2D AntibodiesTable 2. Kabat CDR Sequences of Exemplary Anti-NKG2D AntibodiesTable 3. Chothia CDR Sequences of Exemplary Anti-NKG2D AntibodiesTable 4. IMGT CDR Sequences of Exemplary Anti-NKG2D AntibodiesTable 5. AbM CDR Sequences of Exemplary Anti-NKG2D AntibodiesTable 6. North CDR Sequences of Exemplary Anti-NKG2D Antibodies

[0041] In certain embodiments, the antigen-binding site that binds NKG2D of the present disclosure comprises an antibody heavy chain variable domain (VH) that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 1, and an antibody light chain variable domain (VL) that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the VL of the same antibody disclosed in Table 1. In certain embodiments, the antibody comprises the CDR1, CDR2, and CDR3 amino acid sequences of the VH and the CDR1, CDR2, and CDR3 amino acid sequences of the VL, as determined under any other CDR determination method known in the art, such as Kabat, Chothia, MacCallum, IMGT, AbM, North, or Honegger. In certain embodiments, the antigen-binding site comprises a set of CDR sequences of the same antibody as disclosed in Table 2, Table 3, Table 4, Table 5, or Table 6, defined according to Kabat, Chothia, IMGT, AbM, and North, respectively. It is also contemplated that the antigen-binding site may comprise CDR sequences under different definitions. In certain embodiments, the antigenbinding site comprises a set of VH and VL sequences of the same antibody as disclosed in Table 1

[0042] In certain embodiments, the antigen-binding site that binds NKG2D comprises a VH comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and a VL comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively, as defined according to Kabat. In certain embodiments, the antigen-binding site that binds NKG2D comprises a VH comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10, 11, and 9, respectively, and a VL comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively, as defined according to Chothia. In certainembodiments, the antigen-binding site that binds NKG2D comprises a VH comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 18, 12, and 13, respectively, and a VL comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NO: 17, NAK, and SEQ ID NO: 16, respectively, as defined according to IMGT. In certain embodiments, the antigen-binding site that binds NKG2D comprises a VH comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 22, 20, 9, respectively, and a VL comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively, as defined according to AbM. In certain embodiments, the antigen-binding site that binds NKG2D comprises a VH comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 19, 20, and 13, respectively, and a VL comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 14, 21, and 16, respectively, as defined according to North.

[0043] In certain embodiments, the antigen-binding site comprises a VH comprising an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 5, and a VL that comprising an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 6. In certain embodiments, the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 5 and 6, respectively.

[0044] In certain embodiments, the antigen-binding site comprises a VH comprising an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 3, and a VL that comprising an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 4. In certain embodiments, the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.

[0045] In certain embodiments, the antigen-binding site disclosed herein binds human NKG2D in a surface plasmon resonance (SPR) assay with a dissociation constant (KD) value smaller than or equal to (binding affinity greater than or equal to) 5 nM, 10 nM, 15 nM, or 20 nM. An exemplary method of SPR assay is described in Example 3 below.

[0046] The present disclosure also provides an antigen-binding site comprising a paratope comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or all 7amino acids selected from S31, K50, D52, D55, E57, M99, and E101 of a VH comprising SEQ ID NO: 5, and at least 1, at least 2, at least 3, at least 4, or all 5 amino acids selected from Y30, N31, Y32, W92, and 194 of a VL comprising SEQ ID NO: 6. In certain embodiments, the paratope comprises amino acids S31, D52, D55, E57, and E101 of a VH comprising SEQ ID NO: 5, and amino acids Y30, N31, Y32, W92, and 194 of a VL comprising SEQ ID NO: 6. In certain embodiments, the paratope comprises amino acids S31, K50, D52, D55, E57, M99, and E101 of a VH comprising SEQ ID NO: 5, and amino acids Y30, N31, Y32, W92, and 194 of a VL comprising SEQ ID NO: 6. In certain embodiments, this paratope binds one subunit of an NKG2D dimer.

[0047] In another aspect, the present disclosure provides an antigen-binding site comprising a paratope comprising at least 1, at least 2, at least 3, at least 4, at least 5, or all 6 amino acids selected from S31, K50, D52, D55, E57, and E101 of a VH comprising SEQ ID NO: 5, and at least 1, at least 2, at least 3, or all 4 amino acids selected from Y32, W92, S93, and 194 of a VL comprising SEQ ID NO: 6. In certain embodiments, the paratope comprises amino acids S31, K50, D52, D55, and E57 of a VH comprising SEQ ID NO: 5, and amino acids Y32, W92, S93, and 194 of a VL comprising SEQ ID NO: 6. In certain embodiments, the paratope comprises amino acids K50, D52, D55, E57, and E101 of a VH comprising SEQ ID NO: 5, and amino acids Y32 and 194 of a VL comprising SEQ ID NO: 6. In certain embodiments, this paratope binds one subunit of an NKG2D dimer.

[0048] The present disclosure provides an antigen-binding site comprising one or more (e.g., two) paratopes comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or all 7 amino acids selected from S31, K50, D52, D55, E57, M99, and E101 of a VH comprising SEQ ID NO: 5, and at least 1, at least 2, at least 3, at least 4, at least 5, or all 6 amino acids selected from Y30, N31, Y32, W92, S93, and 194 of a VL comprising SEQ ID NO: 6. In certain embodiments, the one or more (e.g., two) paratopes comprise amino acids S31, K50, D52, D55, E57, and E101 of a VH comprising SEQ ID NO: 5, and amino acids Y30, N31, Y32, W92, S93, and 194 of a VL comprising SEQ ID NO: 6. In certain embodiments, the one or more (e.g., two) paratopes collectively bind both subunits of an NKG2D dimer.

[0049] The present disclosure also provides an antigen-binding site comprising a paratope comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 amino acids selected from T30, S31, W33, K50, D52, D55, E57, H59, M99, G100, E101, and F102 of a VH comprising SEQ ID NO: 5, and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 amino acids selectedfrom Y30, N31, Y32, F91, W92, S93, 194, and W96 of a VL comprising SEQ ID NO: 6. In certain embodiments, the paratope comprises amino acid E57 of a VH comprising SEQ ID NO: 5, and amino acids W92 and 194 of a VL comprising SEQ ID NO: 6. In certain embodiments, the paratope comprises amino acids T30, S31, W33, K50, D52, D55, E57, H59, M99, G100, E101, and F102 of a VH comprising SEQ ID NO: 5, and amino acids Y30, N31, Y32, F91, W92, S93, 194, and W96 of a VL comprising SEQ ID NO: 6. These amino acids fall within the VH CDR1 sequence of SEQ ID NO: 18, the VH CDR2 sequence of SEQ ID NO: 20, the VH CDR3 sequence of SEQ ID NO: 9, the VL CDR1 sequence of SEQ ID NO: 17, and the VL CDR3 sequence of SEQ ID NO: 16. Accordingly, in certain embodiments, the antigen-binding site of the present disclosure comprises a VH comprising CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 18, 20, and 9, respectively, and a VL comprising CDR1 and CDR3 amino acid sequences set forth in SEQ ID NOs: 17 and 16, respectively. In certain embodiments, the VL comprises a CDR2 amino acid sequence of NAK.

[0050] In certain embodiments, the paratope further comprises amino acids F29, H35, S54, and / or Y103 of a VH comprising SEQ ID NO: 5, and DI, 12, N28, 129, N50, K52, S67, G68, and / or P95 of a VL comprising SEQ ID NO: 6. In certain embodiments, the paratope comprises amino acids F29, T30, S31, W33, H35, K50, D52, S54, D55, E57, H59, M99, G100, E101, F102, and Y103 of a VH comprising SEQ ID NO: 5, and DI, 12, N28, 129, Y30, N31, Y32, N50, K52, S67, G68, F91, W92, S93, 194, P95, and W96 of a VL comprising SEQ ID NO: 6. In certain embodiments, the VH comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6.

[0051] The present disclosure also provides an antigen-binding site that binds to an epitope of human NKG2D within the amino acid sequence of ILSPNLLTIIE (SEQ ID NO: 23), corresponding to amino acids 173-183 of SEQ ID NO: 1. In certain embodiments, the epitope comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acids selected from L174, S175, T180, 1181, and E183 of SEQ ID NO: 1. In certain embodiments, the epitope comprises amino acids L174, S175, T180, 1181, and E183 of SEQ ID NO: 1. In certain embodiments, the epitope comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 amino acids selected from 1173, L174, S175, P176, N177, T180, 1181, and E183 of SEQ ID NO: 1. In certain embodiments, the epitope comprises aminoacids 1173, L174, S175, P176, N177, T180, 1181, and E183 of SEQ ID NO: 1. In certain embodiments, the epitope further comprises L178, L179, and / or 1182 of SEQ ID NO: 1. In certain embodiments, the epitope comprises SEQ ID NO: 23. In certain embodiments, the antigen-binding site binds or further binds amino acids K140, E141, K147, N163, G164, S165, W166, and / or S195 of SEQ ID NO: 1. In certain embodiments, the antigen-binding site binds or further binds amino acids K140, E141, K147, H159, N163, G164, S165, W166, S195, and / or 1200 of SEQ ID NO: 1. In certain embodiments, the antigen-binding site binds or further binds K140, E141, K147, L157, H159, N163, G164, S165, W166, Q167, G187, D188, S195, F196, K197, and / or 1200 of SEQ ID NO: 1. Such antigen-binding sites can be identified using, for example, a method described in Example 4 below.

[0052] The present disclosure also provides an antigen-binding site that competes for binding NKG2D (e.g., human NKG2D) with a reference antigen-binding site comprising the VH and VL sequences provided in Table 1. Such antigen-binding sites can be identified using, for example, a method described in Example 6 below. In certain embodiments, the antigen-binding site can block one or more natural ligands, such as MICA, from binding NKG2D. In certain embodiments, the antibody can bind human NKG2D and an NKG2D in other species such as cynomolgus monkey.

[0053] The antigen-binding sites disclosed herein can be incorporated in a bigger protein, such as an antibody. Accordingly, in certain embodiments, the present disclosure provides a protein, such as an anti-NKG2D antibody, comprising an antigen-binding site disclosed herein. In certain embodiments, the antibody further comprises an antibody Fc region, for example, a human IgGl, human IgG2, human IgG3, or human IgG4 Fc region. In certain embodiments, the antibody comprises two antigen-binding sites disclosed herein e.g., two identical antigen-binding sites) and a homodimer of antibody Fc regions.Production of Antibodies

[0054] The antigen-binding sites that bind NKG2D disclosed herein can be made using recombinant DNA technology well known to a skilled person in the art. For example, where an antigen-binding site includes a single polypeptide, a nucleic acid sequence encoding the polypeptide can be cloned into an expression vector. Where an antigen-binding site includes two polypeptides, a first nucleic acid sequence encoding the first polypeptide can be cloned into a first expression vector, and a second nucleic acid sequence encoding the second polypeptide can be cloned into a second expression vector. In some embodiments, the first and second expression vectors can be stably transfected together into host cells to produce themultimeric proteins. Alternatively, the first nucleic acid sequence and a second nucleic acid sequence encoding the first and second polypeptides, respectively, can be cloned into a single expression vector for co-expression of the two polypeptides.

[0055] To achieve a high yield of protein (e.g., antigen-binding site, or antibody,) encoded by two or more expression vectors, different ratios of the expression vectors can be explored to determine the optimal ratio for transfection into the host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.

[0056] Clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the protein. The protein can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freezethawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography. For example, a protein comprising an antibody Fc region (e.g., antibody) may be purified by affinity protein A capture chromatography, such as with a MabSelect SuRe resin (Cytiva), followed by a cation exchange chromatography polishing step, such as with a POROS XS strong cation exchange resin (Thermo Fisher). If higher purity is required, size exclusion chromatography may be used in addition to or in lieu of cation exchange chromatography to polish the protein further and remove unwanted homodimers of various molecular weights. Such size exclusion chromatography can, for example, be performed with a Superdex 200 prep grade resin (Cytiva).Pharmaceutical Compositions

[0057] The present disclosure provides pharmaceutical compositions or formulations that contain an antigen-binding site, or antibody, described herein. The pharmaceutical composition can be formulated for use in a variety of drug delivery systems. One or more pharmaceutically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23d ed. 2020).

[0058] In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine,arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see, Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23 d ed. 2020)).

[0059] Pharmaceutical compositions containing a protein disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0060] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof. An intravenous drug delivery formulation of the present disclosure may be contained in a bag, a pen, or a syringe. In certain embodiments, the bag may be connected to a channel including a tube and / or a needle.

[0061] In certain embodiments, the formulation is a liquid formulation. In certain embodiments, an aqueous formulation is prepared including the protein of the present disclosure in a pH-buffered solution. The pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide. In certain embodiments, a salt or buffer components may be added in an amount of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion. Intravenous formulations can be diluted with 0.9% Sodium Chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0062] In certain embodiments, the formulation is a lyophilized formulation including a protein disclosed herein and a lyoprotectant. The lyoprotectant may be sugar, e.g., disaccharides. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and / or a preservative. The amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1 :2 protein to sucrose or maltose. In certain embodiments, the protein to sucrose or maltose weight ratio may be from 1 :2 to 1 :5. Before lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted between 6 to 8. In certain embodiments, the pH range for the lyophilized drug product may be from 7 to 8. In certain embodiments, a “bulking agent” may be added. A “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol andsorbitol. The lyophilized formulations of the present disclosure may contain such bulking agents.

[0063] In certain embodiments, the lyophilized drug product may be constituted with an aqueous carrier. The aqueous carrier of interest herein is one which is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization. Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer’s solution or dextrose solution. In certain embodiments, the lyophilized protein product of the instant disclosure is constituted to about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0064] The pharmaceutical compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents. The composition in solid form can also be packaged in a container for a flexible quantity.

[0065] In certain embodiments, the present disclosure provides a formulation with an extended shelf life including the protein of the present disclosure, in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0066] A polyol, which acts as a tonicifier and may stabilize the protein, may also be included in the formulation. The polyol is added to the formulation in an amount which may vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also be altered with respect to the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g., mannitol) may be added, compared to a disaccharide (such as trehalose). In certain embodiments, the polyol which may be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg / mL. In certain embodiments, the concentration of mannitol may be about 7.5 to about 15 mg / mL. In certain embodiments, the concentration of mannitol may be about10 to about 14 mg / mL. In certain embodiments, the concentration of mannitol may be about 12 mg / mL. In certain embodiments, the polyol sorbitol may be included in the formulation.

[0067] A detergent or surfactant may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbates 20, 80 etc.) or pol oxamers (e.g., pol oxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated protein and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant which is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4thed., 1996). In certain embodiments, the formulation may contain polysorbate 80 between about 0.1 mg / mL and about 10 mg / mL, or between about 0.5 mg / mL and about 5 mg / mL. In certain embodiments, about 0.1% polysorbate 80 may be added in the formulation.

[0068] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of elastomer complying with USP and Ph Eur. In certain embodiments vials may be filled with 61.2 mL of the protein product solution in order to allow an extractable volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with 0.9% saline solution.

[0069] In certain embodiments, the liquid formulation of the disclosure may be prepared in combination with a sugar at stabilizing levels. In certain embodiments the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, a stabilizer may be added in an amount no greater than that which may result in a viscosity undesirable or unsuitable for intravenous administration. In certain embodiments, the sugar may be disaccharides, e.g., sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.

[0070] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.

[0071] In certain embodiments, a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1 : 10-29).

[0072] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, poly (2-hydroxyethyl -methacrylate), ethylene vinyl acetate, or poly-D(-)-3- hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0073] The description above describes multiple aspects and embodiments of the disclosure. The patent application specifically contemplates all combinations and permutations of the aspects and embodiments.EXAMPLES

[0074] The disclosure generally described herein will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and is not intended to limit the disclosure.Example 1: NKG2D ECD peptide design

[0075] This example describes an experiment using 3 peptides to antibodies that bind to a similar epitope as MICA.

[0076] The positional overlap with MICA, immunogenic potential, solvent exposure, and secondary structure prediction were all considered in the peptide design (FIG. 4). Peptide 1 (SEQ ID NO: 28) contains 7 NKG2D residues that are all solvent exposed with MICA non-covalent bond forming residues at the most interior position of the receptor. This peptide is aimed at generating antibodies at the cleft of the receptor, similar to other NKG2D ligands. Peptide 2 (SEQ ID NO: 29)contains 9 NKG2D residues forming a beta strand-turn - beta strand secondary structure, composed of a significant area of MICA epitope overlap, and with several non-identical residues compared to mouse NKG2D, improving immunogenicity. Peptide 3 (SEQ ID NO: 30) contains 9 NKG2D residues that are solvent exposed with 5 residues that form non-covalent bonds with MICA on one or both NKG2D chains, and with several residues that are not identical to mouse NKG2D, raising the peptide’simmunogenicity. Peptide 3 includes alanine as the second amino acid instead of isoleucine or leucine as in the human or mouse NKG2D sequence, respectively, to lower the hydrophobicity of the peptide. All peptides have a GGSG-mono human IgGl-Fc-6x-histidine tag for improving expression, solubility, ease of purification, and immunogenicity (SEQ ID NOs: 31-33). Sequence alignment was performed using ClustalOmega, secondary structure and solvent accessibility analysis was performed using JPred4, and NKG2D-MICA interface analysis was performed using PDBePISA with PDB: 1HYR used as input. The sequence and structure of the peptides, including tags for boosting immune response and improved expression, solubility and purification are shown in FIG. 5.

[0077] Immunization and hybridoma production

[0078] 3 NZBWF1 / J mice were initially immunized with 100 pg of recombinant humanIgGl -Fc-human NKG2D, boosted with 50 pg, and boosted 2 more times with 50 pg of 1 of 3 human IgGl-fused human NKG2D peptides (SEQ ID NOs: 31-33) (FIG. 5) for the purpose of obtaining agonistic anti-NKG2D antibodies. Test bleeds were taken, and anti-NKG2D antibody titers were measured by binding to recombinant human NKG2D by ELISA. The spleens and lymph nodes were extracted, and cells fused to myeloma cells to create immortal hybridoma cell lines. The secreted antibodies were screened for binding to recombinant human NKG2D by ELISA, and positive clones were subcloned and rescreened to obtain clonal, anti-NKG2D hybridomas. Clonal anti-NKG2D hybridoma antibodies were sequenced and the variable domains were cloned into human IgGl backbone vectors for subsequent expression and purification. All immunization and hybridoma screening work was performed by Green Mountain Antibodies in Burlington, VT and sequencing performed at SynBuild in Tempe, AZ.

[0079] Reporter cell line screening

[0080] 100 pL of goat anti-mouse Fc antibody (Novex, catalog no. A16092) was coated to each well of a 96-well plate and 100 pL of hybridoma was added to the anti -mouse Fc antibody wells in duplicate and incubated overnight at 4°C. PBS (Gibco) and cell activation cocktail (phorbol 12-myristate 13-acetate and ionomycin, Biolegend, catalog no. 423301) were prepared separately as negative and positive controls, respectively. 100 pL of 106 / mL EL4-NKG2D cells and 100 pL of Brefeldin A / monensin solution (BioLegend) were added to each well. The plate was incubated at 37°C and 5% CO2 for 4 hours and EL4-NKG2D cells were then transferred to a new 96-well plate for fluorescence-activated cell sorting (FACS) staining. Cells were pelleted, washed, and incubated with 100 pL of zombie live / dead dye (BioLegend, 1 :2000 dilution) for 15 minutes in the dark. The cells were washed with FACSbuffer (PBS with 2% heat-inactivated-fetal bovine serum (Gibco, catalog no. 16140-071)) and then incubated with 100 pL of fixation buffer (4% paraformaldehyde) for 15 minutes in the dark. The cells were washed with FACS buffer, pelleted, and resuspended in 200 pL permeabilization wash buffer (BioLegend). After washing the cells, 50 pL of anti-TNF-PE (BioLegend, catalog no. 506306) was added to cells and incubated at room temperature in the dark for 30 minutes. Cells were washed twice with permeability wash buffer and resuspended in 200 pL of FACS buffer for analysis. Cells were analyzed using an Attune NxT flow cytometer and gated for size, single and live cells, and TNFa staining. All three mice produced anti-NKG2D antibodies as determined by test bleed ELISAs, and a determination of agonism was assessed by TNFa upregulation in the NKG2D reporter cell line, with peptide 3 eliciting the most agonistic clones.Table 7. Sequences of constructs used in Example 1Example 2: Activation of NKG2D by 2D3

[0081] This example characterizes the activity of 2D3 and a humanized antibody thereof. 2D3 is a murine antibody that binds NKG2D identified from a mouse hybridoma. It includes a heavy chain variable domain (VH) and a light chain variable domain (VL) having the amino acids sequences of SEQ ID NOs: 3 and 4, respectively. 2D3 was humanized to produce a VH and a VL having the amino acid sequences of SEQ ID NOs: 5 and 6.

[0082] The activity of 2D3 was measured using a reporter cell line called “EL4- NKG2D,” a T lymphoblast cell line that expresses a fusion protein of human NKG2D extracellular domain and transmembrane domain, and a human CD3(^ intracellular domain. Briefly, the humanized 2D3 antibody, MHC class I polypeptide related sequence A (MICA)- His (monovalent), MICA-Fc (bivalent), and an unrelated IgGl antibody (trastuzumab) were diluted to 20 pg / mL in sterile PBS. Wells in a multi-well plate were coated with 100 pL of each sample in quadruplicate and incubated overnight at 4 °C. PBS and a cell activation cocktail (Phorbol 12-myristate 13-acetate (PMA) and ionomycin) were prepared separately to be used as the negative and positive controls, respectively. 100 pL of 106 / mL EL4-NKG2D cells and 100 pL of Brefeldin A / monensin solution were added to each well. The plate was incubated at 37°C and 5% CO2 for 4 hours and EL4-NKG2D cells were then transferred to a new 96 well plate for staining. The cells were pelleted, washed, and incubated with 100 pLof zombie live / dead dye (1 :2000 dilution) for 15 minutes in the dark. The cells were washed with FACS buffer (PBS with 2% HI-FBS) and then incubated with 100 pL of fixation buffer (4% paraformaldehyde) for 15 minutes in the dark. The cells were washed with FACS buffer, pelleted, and resuspended in 200 pL permeabilization wash buffer. After washing, 50 pL of anti-TNFa-PE was added to cells and incubated at room temperature in the dark for 30 minutes. The cells were washed twice with permeability wash buffer and resuspended in 200 pL of FACS buffer for analysis. The cells were analyzed using a flow cytometer (Attune NxT flow cytometer), and gated for size, single and live cells, and TNFa staining. Humanized 2D3 showed comparable levels of intracellular TNFa in the EL4-NKG2D reporter cell line compared to MICA-Fc (bivalent) or MICA-His (monovalent) (FIGs. 1A- 1F).Example 3: Binding affinity of 2D3 to NKG2D

[0083] This example characterizes the binding affinity of humanized 2D3 to NKG2D.

[0084] The equilibrium dissociation constant (KD) and kinetic parameters of 2D3 was measured by surface plasmon resonance (SPR). Briefly, the 2D3-hIgGl mAb was expressed in Expi293, a mammalian suspension cell line, and purified by Protein A affinity chromatography (MabSelect SuRe) and cation exchange chromatography (POROS XS) to produce 95.9% monomer as determined by analytical size exclusion chromatography (SEC). 2D3 Fab was prepared from the mAb using a Fab preparation kit and purified by Protein A affinity chromatography and semi-preparative size exclusion chromatography (Superdex 200) to produce 99.8% monomer, as determined by analytical size exclusion chromatography. SPR assay was conducted using a Biacore 8K instrument (Cytiva) equipped with a series S sensor chip CM5. Mouse IgG2a Fc fused with human NKG2D extracellular domain, a bivalent protein, was captured on the chip, and 2D3 Fab or MICA fused with a His tag (ACRO Biosystems), both monovalent proteins, were injected as analyte. This assay setup was designed to avoid avidity effects from the system.

[0085] 2D3 Fab and MICA-His both bound recombinant human NKG2D with low nM affinities at 25°C (Table 8). 2D3 Fab had a calculated Rmax value about 2.4-fold higher than MICA-His, which was not fully explained by the difference in molecular weight of the analyte nor capture level. After adjusting for differences in molecular weight and capture level of the analyte, 2D3 Fab demonstrated a 1.7-fold higher binding response than MICA- His (Table 9, in which stoichiometry was calculated using the following equation: Stoichiometry = (MWcapture * Rmax) / (MWanaiyte * capture level)). MICA is known to binddimeric NKG2D in a 1 : 1 stoichiometry and this near 2-fold increase in normalized stoichiometry suggested that 2D3 Fab has a 2: 1 stoichiometry of Fab to dimeric NKG2D.Table 8. Binding Affinity MeasurementsTable 9. Calculated and normalized stoichiometry of 2D3 or MICA to NKG2DS.D. = Standard deviation; Calculated stoich. = Calculated stoichiometry; Norm. Stoich. = Normalized Stoichiometry.Example 4: Identification of 2D3 Epitope and Paratope

[0086] This example identifies the epitope and paratope of 2D3 by solving a crystal structure of 2D3 complexed with NKG2D.

[0087] Briefly, 2D3 Fab or a variant thereof called 2D3 -Cysteine Kappa (CK) Fab was expressed in Expi293 cells, a mammalian suspension cell line, and purified via an antibody affinity resin (KanCap G) and size exclusion chromatography (Superdex 200). Human NKG2D extracellular domain fused to a human IgGl Fc with an intervening Factor Xa cleavage site was expressed in Expi293 GnTi-, a mammalian suspension cell line, and purified using protein A resin and by size exclusion chromatography (Superdex 200). The fusion construct was cleaved by Factor Xa enzyme, and the human IgGl Fc portion was removed by protein A resin. The NKG2D protein was then treated with EndoH enzyme tohydrolyze glycans down to terminal GlcNAc. 2D3 Fab or 2D3-CK Fab and human NKG2D were mixed in a 1.0 to 0.8 molar ratio, and the complex was purified by size exclusion chromatography in 25 mM HEPES, 150 mM NaCl, pH 7.4. The complex was concentrated to 12.3 mg / mL for crystallography studies.

[0088] To crystallize the complex, an initial screening was performed using the sitting drop method, and the diffracted crystal was grown from 0.2 M ammonium tartrate dibasic, 20% w / v PEG 3,350. The crystals were harvested with oil cryoprotectant, and flash frozen in liquid nitrogen. Data were collected using 1 A wavelength. An exposure of 20 ms was used to acquire 720 0.25° rotation images and processed using autoPROC.

[0089] To solve the crystal structure, initial phases were obtained by molecular replacement using chains E and D of Protein Data Bank (PDB) deposition 6IAP for 2D3 Fab or 2D3-CK Fab, and chains A and B of PDB deposition 6HYR for NKG2D using the CCP4i2 program PHASER. The model was further refined through iterative cycles using REFMAC5 and COOT. All structural figures were prepared in PyMOL. Interaction surface areas and hydrogen / salt-bridge bonds were calculated using the PDBePISA online tool. The initial structure of the 2D3 and NKG2D complex was solved to 3.9 A. To improve crystal quality, the kappa constant domain FG loop (HQGLSSP, SEQ ID NO: 24) was replaced by a shorter loop found in rabbits (QGTTS, SEQ ID NO: 25) that has been demonstrated to improve Fab packing, thereby to generate the 2D3-CK Fab construct. This effort resulted in a 2.98 A model of the 2D3-CK-NKG2D complex (FIG. 2A), which confirms the 2: 1 stoichiometry of 2D3 Fab to NKG2D homodimer.

[0090] As shown by FIG. 2C, each 2D3 Fab interacted with a single NKG2D monomer, positioned away from the NKG2D dimer interface. The 2D3 Fab bound the NKG2D dimer at an angle approximately 45 degrees to the membrane, resulting in a complex that exhibits a 2-fold rotational symmetry. In contrast, NKG2D natural ligand MICA bound the NKG2D dimer in a 1 : 1 stoichiometry (FIG. 2D), where MICA vertically interacted with a concave surface formed by the NKG2D dimer at its midpoint. Of note, all the other NKG2D natural ligands reported engage the NKG2D dimer in a manner similar to MICA, whereas 2D3 has a different binding mode compared to NKG2D natural ligands.2D3-Fab Epitope and Paratope Information

[0091] Each 2D3-Fab bound about 981 A2 of surface area of NKG2D, compared to about 2,257 A2 occupied by MICA. Thirteen amino acid residues of NKG2D, namely,K140, E141, K147, N163, G164, S 165, W166, L174, S175, T180, 1181, E183 and S195, were identified as interacting with 2D3 (Table 10). Among the NKG2D residues interacting with a 2D3 residue, three overlapped with the residues bound by MICA: El 83 on NKG2D chain A and 1181 and SI 95 on NKG2D chain B, all of which formed hydrogen bonds with 2D3 and MICA (FIG. 2B-2D, Table 10). Among these residues, the linear sequence of SEQ ID NO: 23, corresponding to amino acids 173-183 in SEQ ID NO: 1, was found to primarily bind the heavy chain CDR3 of 2D3. In addition, every residue in SEQ ID NO: 23 is located within 6 A from a 2D3 residue.

[0092] Table 10 below summarizes the paratope and amino acid positions of the epitope and the amino acids further bound by the antigen-binding site disclosed herein.Table 10. 2D3 Fab, human NKG2D epitope / paratope non-covalent bond residues2D3-CK Epitope and Paratope Information

[0093] Each 2D3-CK bound about 950 A2of surface area of NKG2D, compared to about 846 A2occupied by MICA. Among these residues, the linear sequence of SEQ ID NO: 23, corresponding to amino acids 173-183 in SEQ ID NO: 1, was found to primarily bind the heavy chain CDR3 of 2D3. In addition, every residue in SEQ ID NO: 23 is located within 6 A from a 2D3 residue. Other NKG2D residues located within 6 A from a 2D3 residue included L157, Q167, G187, D188, F196, H159, S165, 1173, P176, 1200, and K197. Table 11 below summarizes the paratope and amino acid positions of the epitope and the amino acids further bound by the antigen-binding site disclosed herein.Table 11. 2D3-CK, human NKG2D epitope / paratope non-covalent bond residuesExample 5: Competition of 2D3 for NKG2D Binding with MICA

[0094] This example characterizes competition between 2D3 and MICA for binding NKG2D.

[0095] 2D3 Fab was prepared using the same method as described in Example 3. To determine 2D3 and MICA cross-blocking profiles, mouse IgG2a Fc fused human NKG2D was diluted to 0.5 pg / mL in running buffer (10 mM HEPES, 150 mM NaCl, 0.005% P20, pH 7.4) and Fc captured across flow cell 2 for 1 min at 10 pL / min to a density of 41 - 46 RU. In channel 1, 100 nM 2D3 Fab was injected as dual 1 solution for 120 sec and was followed by 100 nM 2D3 Fab and 500 nM MICA-His as dual 2 solution for 300 sec at 30 pL / min. The complex was allowed to dissociate for 120 sec. In channel 2, 500 nM MICA-His was injected as dual 1 solution and was followed by 100 nM 2D3 Fab and 500 nM MICA-His as dual 2 solution, using the same association and dissociation times as described above. The surfaces were regenerated with 3 x 20 second injections of 10 mM glycine, pH 1.7, at 100 pL / min between cycles. The data were double referenced and visualized using Biacore Insight software.

[0096] A saturating concentration of 2D3 Fab injected over human NKG2D prevented co-engagement of MICA to NKG2D (FIG. 3A), whereas a saturating concentration of MICA-His injected over human NKG2D allowed for apparent additional binding of 2D3 to NKG2D (FIG. 3B). These results suggest that the partial overlap in epitopes (Table 10) allowed 2D3 Fab to block MICA from binding NKG2D (FIG. 3A), but MICA did not fully block 2D3 Fab from binding NKG2D (FIG. 3B). The overall response suggests that onlyone Fab is co-engaging NKG2D pre-bound by MICA, presumably NKG2D chain A due to less epitope overlap. It is also possible that 2D3 Fab outcompeted MICA during the second injection due to having a faster association rate for NKG2D as MICA dissociates from the receptor.Example 6: Screen for anti-NKG2D antibodies Competing with 2D3

[0097] This example describes an epitope binning experiment using an SPR instrument as an assay to screen for antibodies that bind the same epitope.

[0098] Briefly, NKG2D is immobilized on a chip surface active flow cell via amine coupling chemistry. 2D3 is injected over the immobilized NKG2D for 300 seconds at 30 pL / min at a saturating concentration of 300 nM. A second injection of 300 nM 2D3 and saturating concentration of a test anti-NKG2D antibody is immediately injected after for 300 second at 30 pL / min. Dissociation is monitored for 300 seconds. If an additional SPR signal is observed after the second injection, the test antibody does not compete with 2D3. If there is no additional SPR signal, the test antibody competes with 2D3. The chip can be regenerated with a solution that fully dissociates NKG2D binders from the receptor without compromising the immobilized NKG2D, for assessing another test antibody.INCORPORATION BY REFERENCE

[0099] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0100] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein. Various structural elements of the different embodiments and various disclosed method steps may be utilized in various combinations and permutations, and all such variants are to be considered forms of the disclosure. The scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. An antigen-binding site that binds NKG2D, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises complementarity determining region (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) present in SEQ ID NO: 5, and the VL comprises CDR1, CDR2, and CDR3 present in SEQ ID NO: 6.

2. The antigen-binding site of claim 1, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 7, 8, 9, 14, 15, and 16, respectively, as defined according to Kabat.

3. The antigen-binding site of claim 1, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 10, 11, 9, 14, 15, and 16, respectively, as defined according to Chothia.

4. The antigen-binding site of claim 1, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1 and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 18, 12, 13, 17, and 16, respectively, and the CDR2 of the VL comprises the amino acid sequence of NAK, as defined according to IMGT.

5. The antigen-binding site of claim 1, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 19, 20, 13, 14, 21, and 16, respectively, as defined according to North.

6. The antigen-binding site of claim 1, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 22, 20, 9, 14, 15, and 16, respectively, as defined according to AbM.

7. The antigen-binding site of any one of claims 1-6, wherein the VH comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6.

8. The antigen-binding site of claim 7, wherein the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 5 and 6, respectively.

9. The antigen-binding site of any one of claims 1-6, wherein the VH comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.

10. The antigen-binding site of claim 9, wherein the VH and the VL comprise the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.

11. An antigen -binding site that binds NKG2D, comprising one or more paratopes comprising amino acids S31, K50, D52, D55, E57, M99, and E101 of a VH comprising SEQ ID NO: 5, and amino acids Y30, N31, Y32, W92, S93, and 194 of a VL comprising SEQ ID NO: 6.

12. The antigen-binding site of claim 1 or 11, wherein the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 18, 20, and 9, respectively, and the VL comprises CDR1 and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 17 and 16, respectively.

13. The antigen-binding site of claim 12, wherein the VL further comprises a CDR2 comprising the amino acid sequence of NAK.

14. The antigen-binding site of any one of claims 11-13, wherein the VH comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6.

15. An antigen -binding site that binds an epitope of NKG2D within the amino acid sequence of SEQ ID NO: 23.

16. The antigen-binding site of claim 15, wherein the epitope of NKG2D comprises amino acids L 174, SI 75, T180, 1181, and El 83 of SEQ ID NO: 1.

17. An antigen-binding site that competes with a reference antigen-binding site for binding NKG2D, wherein the reference antigen-binding site comprises a VH comprising theamino acid sequence of SEQ ID NO: 5 and a VL comprising the amino acid sequence of SEQ ID NO: 6.

18. The antigen-binding site of any one of claims 1-8 and 11-17, wherein the VH and VL are humanized.

19. An antibody that binds NKG2D, comprising the antigen-binding site of any one of claims 1-18 and an antibody Fc region.

20. A pharmaceutical composition comprising: the antigen -binding site of any one of claims 1-18 or the antibody of claim 19; and a pharmaceutically acceptable carrier or excipient.

21. One or more isolated nucleic acids encoding the antigen-binding site of any one of claims 1-18 or the antibody of claim 19.

22. A vector comprising the one or more nucleic acids of claim 21.

23. An isolated cell comprising the one or more nucleic acids of claim 21 or the vector of claim 22.

24. A method of producing a protein, the method comprising culturing the cell of claim 23 under conditions for expressing the antigen-binding site or antibody.

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