Antigen binding proteins

Abstract

The present disclosure relates to a ULBP6 binding protein that inhibits the interaction between ULBP6 and NKG2D, and a method of treating cancer with the ULBP6 binding protein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 336,336, filed on April 29, 2022, which is incorporated herein by reference.

[0003] Reference to a sequence listing

[0004] The official copy of the sequence listing is submitted with the specification in an xml file in ST26 format, with the file name "09402-014WO1.xml", the creation date is April 27, 2023, and the size is 171,756 bytes. The sequence listing submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety.

[0005] Names of the parties to the joint research agreement

[0006] The claimed subject matter of this application is filed by or on behalf of the parties to a joint research agreement that was in effect on or before the date the claimed subject matter was filed; the claimed subject matter is the result of activities conducted within the scope of the joint research agreement; and the parties to the joint research agreement include: GlaxoSmithKline Intellectual Property (No. 3) Limited and 23andMe, Inc.

[0007] A joint research agreement is a written contract, grant or cooperation agreement between the above parties for the purpose of conducting experimental, development or research work in the field of claimed subject matter. Technical Field

[0008] The present invention relates to cancer treatment. In particular, the present invention relates to ULBP6 binding proteins, including anti-ULBP6 antibodies and their use in treating cancer. Background of the Invention

[0010] Effective treatment of cancer is an ongoing goal in the field of oncology. Immunotherapy that enhances anti-tumor immune responses is a powerful approach to treating cancer. A variety of immuno-oncology antibodies are currently marketed, including monoclonal antibodies targeting PD-1 (e.g., OPDIVO / nivolumab and KEYTRUDA / pembrolizumab), PD-L1 (e.g., TECENTRIQ / atezolizumab, BAVENCIO / avelumab, IMFINZI / durvalumab), CTLA-4 (e.g., YERVOY / ipilimumab), and CD20 (e.g., RITUXAN / rituximab, ARZERRA / ofatumumab). These antibody-based cancer immunotherapies may each work via different mechanisms, such as increasing the immune reactivity of T cells, relieving inhibitory checkpoints on tumor-specific T cells, inducing apoptosis, inducing enhanced antibody-dependent cellular cytotoxicity (ADCC), and / or inducing complement-dependent cytotoxicity (CDC).

[0011] Although recent advances in the field of immunotherapy have improved the treatment of cancer, many patients do not respond to conventional therapies. Therefore, there remains a need for alternative or improved immunotherapeutic compositions and methods to more effectively treat cancer. Summary of the invention

[0012] Provided herein is a ULBP6 binding protein comprising: a) (i) any one or a combination of the CDRs selected from CDRH1, CDRH2, CDRH3 of SEQ ID NOs: 16 to 19 and / or CDRL1, CDRL2, CDRL3 of SEQ ID NOs: 26 to 29; or (ii) a CDR variant of (i), wherein the variant has 1, 2 or 3 amino acid modifications; or b) a VH region comprising a sequence that is at least 80% identical to the sequence of any one of SEQ ID NOs: 21 to 24; and / or a VL region comprising a sequence that is at least 80% identical to the sequence of any one of SEQ ID NOs: 31 to 34. The ULBP6 binding protein may comprise a CDRH1 of SEQ ID NO: 36, 39, 42 or 45; a CDRH2 of SEQ ID NO: 37, 40, 43 or 46; a CDRH3 of SEQ ID NO: 38, 41, 44 or 47; a CDRL1 of SEQ ID NO: 51, 54, 57 or 60; a CDRL2 of SEQ ID NO: 52, 55, 58 or 61; and / or a CDRL3 of SEQ ID NO: 53, 56, 59 or 62 or a variant thereof.

[0013] Also provided herein is a ULBP6 binding protein comprising 6 CDRs selected from the group consisting of: CDRH1 of SEQ ID NO:36, CDRH2 of SEQ ID NO:37, CDRH3 of SEQ ID NO:38, CDRL1 of SEQ ID NO:51, CDRL2 of SEQ ID NO:52, and CDRL3 of SEQ ID NO:53; CDRH1 of SEQ ID NO:39, CDRH2 of SEQ ID NO:40, CDRH3 of SEQ ID NO:41, CDRL1 of SEQ ID NO:54, CDRL2 of SEQ ID NO:55, and CDRL3 of SEQ ID NO:56; CDRH1 of SEQ ID NO:42, CDRH2 of SEQ ID NO:43, CDRH3 of SEQ ID NO:44, CDRL1 of SEQ ID NO:57, CDRL2 of SEQ ID NO:58, and CDRL3 of SEQ ID NO:59; or CDRH1 of SEQ ID NO:45, CDRH2 of SEQ ID NO:46, CDRH3 of SEQ ID NO:47, CDRL3 of SEQ ID NO:48, CDRL4 of SEQ ID NO:49; CDRH3 of SEQ ID NO:47, CDRL1 of SEQ ID NO:60, CDRL2 of SEQ ID NO:61 and CDRL3 of SEQ ID NO:62.

[0014] Also provided herein is a ULBP6 protein that binds to ULBP6 and ULBP2 with a KD less than or equal to 1 nM, and binds to ULBP5 with a KD greater than or equal to 1 nM.

[0015] The present invention also provides a ULBP6 binding protein that binds to human ULBP6 and competes with a reference ULBP6 binding protein for binding to the ULBP6, the binding protein comprising: a VH region sequence of SEQ ID NO:21 and a VL region sequence of SEQ ID NO:31; a VH region sequence of SEQ ID NO:22 and a VL region sequence of SEQ ID NO:32; a VH region sequence of SEQ ID NO:16 and a VL region sequence of SEQ ID NO:26; or a VH region sequence of SEQ ID NO:17 and a VL region sequence of SEQ ID NO:27.

[0016] Also provided herein is a nucleic acid sequence encoding one or both of the HC and LC of the ULBP6 binding protein provided herein. Also provided herein is an expression vector comprising the nucleic acid sequence provided herein. Also provided herein is a recombinant host cell comprising a nucleic acid sequence or an expression vector provided herein. Also provided herein is a method for producing a ULBP6 binding protein, the method comprising culturing a recombinant host cell provided herein under conditions suitable for expressing the nucleic acid sequence or vector, wherein a polypeptide comprising a ULBP6 binding protein provided herein is produced. Also provided herein is a cell line engineered to express the ULBP6 binding protein provided herein. Also provided herein is a pharmaceutical composition comprising a ULBP6 binding protein as defined herein and a pharmaceutically acceptable excipient.

[0017] Also provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a ULBP6 binding protein or pharmaceutical composition as defined herein. Also provided herein is a ULBP6 binding protein and pharmaceutical composition provided herein for therapy. Also provided herein is a ULBP6 binding protein and pharmaceutical composition provided herein for the treatment of cancer. Also provided herein is a ULBP6 antagonist for the treatment of basal cell carcinoma.

[0018] Also provided herein are ULBP6 antibodies and fragments thereof that: a) bind to one or more residues of SEQ ID NO: 125 and SEQ ID NO: 127; b) bind to one or more residues of SEQ ID NO: 126 and SEQ ID NO: 127; c) bind to a conformational epitope of SEQ ID NO: 125 and / or a conformational epitope of SEQ ID NO: 127; d) bind to a conformational epitope of SEQ ID NO: 126 and / or a conformational epitope of SEQ ID NO: 127; e) protect one or more residues of SEQ ID NO: 125, SEQ ID NO: 126 or SEQ ID NO: 127 from deuterium exchange in HDX-MS analysis of SEQ ID NO: 124; f) bind to human ULBP6 and result in a conformational epitope of SEQ ID NO: 125, SEQ ID NO: 126 or SEQ ID NO: 127 derived from human ULBP6. The peptide of NO:127 is more resistant to deuterium incorporation than the corresponding peptide derived from uncomplexed human ULBP6; g) binds to the NKG2D binding interface of human ULBP6; and / or h) disrupts the interaction of human ULBP6 with NKG2D by binding to one or more residues selected from E96, E154, Y184 and D189 of SEQ ID NO:124.

[0019] Also provided herein are ULBP6 antibodies and fragments thereof, which: a) bind to one or more residues of SEQ ID NO:128; b) bind to a conformational epitope of SEQ ID NO:128; c) protect one or more residues of SEQ ID NO:128 from deuterium exchange in HDX-MS analysis of SEQ ID NO:128; and / or bind to human ULBP6 and cause a peptide of SEQ ID NO:128 derived from human ULBP6 to be more resistant to deuterium incorporation than a corresponding peptide derived from uncomplexed human ULBP6. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Regional association curves of the ULBP6 locus on chromosome 6 depicting basal cell carcinoma. The x-axis shows the position on chromosome 6 when constructed using GRCh38 / hg38 as the human reference genome. The left y-axis is -log 10 (P value), from logistic regression test of association between phenotype of interest and genotype. Plus signs (+) indicate variants for which genotypes were imputed; circle symbols (o) indicate variants called using genotypes; x symbols (x) indicate imputed coding variants; diamond symbols (◇) indicate genotyped coding variants. Colors reflect linkage disequilibrium (calculated as r) between the index SNP (shown in gray) and other neighboring SNPs. 2 ). The gray horizontal line represents 5×10 -8 The genome-wide significance threshold of . The Confident Set trace shows the number and position of variants in the 99% credible set that likely contains the causal variant. The Gene trace shows the genes in the locus, with thick bars representing exons and thin lines representing introns.

[0021] Figure 2 Depicts validation of the NKG2D detection assay using tool antibodies (top) and the ability of anti-ULBP6 antibodies (h7B3 (E-) and h8E11 (E-)) to reverse sULBP6_02-induced NKG2D blockade / internalization / downregulation in the NKG2D detection assay of sensitized PBMCs from representative healthy donors compared to tool antibodies (1D4 and 6E1) and isotype controls (bottom).

[0022] Figure 3A Graphs depicting validation of the tool antibodies used (upper two panels).

[0023] Figure 3B Depicting an overview of the properties of the tool antibodies used.

[0024] Figure 3CDepicted are the ability of anti-ULBP6 antibodies (h7B3, h8E11 and isotype control) to block the immunosuppression conferred by sULBP6_02 as measured by IFNg secretion levels in a plate-bound MICA assay.

[0025] Figure 3D Depicted are the ability of anti-ULBP6 antibodies (h7B3, h1D4, h6E1 and isotype control) to block the immunosuppression conferred by sULBP6_02 as measured by IFNg secretion levels in a plate-bound MICA assay.

[0026] Figure 4 Depicted are the synergistic effects of NKG2D stimulation and FcgRIIIa assays on IFNg release from PBMCs by activation of NKG2D alone, FcgRIIIa alone, or both.

[0027] Figure 5A Depicts a mixed cell assay (PBMC and tumor cells) in which primed PBMCs are plated on COV644 tumor cells in the presence of 50 nM sULBP6_02 and IFNg release is measured to determine immune cell activation, and depicts the ability of effector-active (E+) anti-ULBP6 antibodies to increase IFNg secretion in a dose-dependent manner by neutralizing soluble ULBP6-mediated inhibition and driving ADCC from ULBP6 surface expression on the tumor surface.

[0028] Figure 5B Depicts a mixed cell assay (PBMC and tumor cells) in which primed PBMCs were plated on COV644 tumor cells in the presence of 50 nM sULBP6_02 and IFNg release was measured to determine immune cell activation, depicting a comparison of the maximal potency of E+, E- and isotype control antibodies in releasing IFNg from 200 nM antibody treatment.

[0029] Figure 6 Depicts IFNg release in a mixed cell assay (PBMCs and tumor cells) where pre-sensitized PBMCs are on COV644 tumor cells with 50 nM soluble ULBP6, where increased IFNg release can be driven by blocking the inhibitory effects of sULBP6_02 or by ADCC-mediated activation from E+ molecules bound to the surface of tumor cells; presents data for (upper panel) h7B3 E+ and h7B3 E- and (lower panel) h7B3 E+, h7B3 E-, h8E11 E+ and h8E11 E-. The upper panel depicts a dose titration of h7B3 E- to E+, where the antibodies showed similar EC 50, but E+ showed higher maximal potency. An isotype control was included at the maximum dose of the antibody for comparison. The figure below shows a comparison of four anti-ULBP6 antibodies (h7B3 E- and E+ and h8E11 E- and E+) in the same assay, where the antibodies all showed similar EC 50 , but the E+ antibody showed greater maximal efficacy. An isotype control was included at the maximum dose of the antibody for comparison.

[0030] Fig. 7A The ability of anti-ULBP6 antibodies (h7B3(E+) and h7B3(E-) to rescue the reduction of granzyme B in CD8 T cells (lower two panels) by soluble ULBP6 (measured using FACS from total PBMCs) in a mixed cell assay of primed PBMCs on COOV644 tumor cells with 50 nM sULBP6_02, where PBMC levels of granzyme B and Ki-67 were measured by FACS after co-incubation.

[0031] Figure 7B The ability of anti-ULBP6 antibodies (h7B3(E+) and h7B3(E-) to rescue the reduction of Ki-67 in CD8 T cells (measured using FACS from total PBMCs) by soluble ULBP6 is demonstrated in a mixed cell assay of primed PBMCs on COOV644 tumor cells with 50 nM sULBP6_02, where PBMC levels of granzyme B and Ki-67 were measured by FACS after co-incubation.

[0032] Figure 7C The ability of anti-ULBP6 antibodies (h7B3(E+) and h7B3(E-) to rescue the reduction of granzyme B in NK cells (measured using FACS from total PBMCs) by soluble ULBP6 is demonstrated in a mixed cell assay of primed PBMCs on COOV644 tumor cells with 50 nM sULBP6_02, where PBMC levels of granzyme B and Ki-67 were measured by FACS after co-incubation.

[0033] Fig.7D The ability of anti-ULBP6 antibodies (h7B3(E+) and h7B3(E-) to rescue the reduction of Ki-67 in NK cells (measured using FACS from total PBMCs) by soluble ULBP6 is demonstrated in a mixed cell assay of primed PBMCs on COOV644 tumor cells with 50 nM sULBP6_02, where PBMC levels of granzyme B and Ki-67 were measured by FACS after co-incubation.

[0034] Fig. 8AA mixed cell assay (PBMCs and tumor cells) with pre-sensitized PBMCs and COV644 tumor cells that have been treated with 50 nM sULBP6_02 is shown, where the assay looks for IFNg release as a measure of immune cell activation (which can be driven by blocking soluble ULBP6 with h7B3 E- or E+, or blocking ADCC with h7B3 E+). Dose titration of h7B3E+ relative to E- antibody is depicted, which showed similar EC 50 , but the maximum efficacy of the E+ antibody was higher. The maximum antibody dose for the isotype control is also shown.

[0035] Figure 8B A mixed cell assay (PBMCs and tumor cells) with pre-sensitized PBMCs and COV644 tumor cells that have been treated with 50 nM sULBP6_02 is shown, where the assay looks for IFNg release as a measure of immune cell activation (which can be driven by blocking soluble ULBP6 with h7B3 E- or E+, or blocking ADCC with h7B3 E+). Dose titration of h7B3E+ relative to E++DE antibody is depicted, which showed similar EC 50 , but the maximum efficacy of E++DE was increased. The maximum antibody dose for isotype control is also shown.

[0036] Figure 8C Shown is a mixed cell assay (PBMC and tumor cells) with pre-sensitized PBMC and COV644 tumor cells that have been treated with 50nM sULBP6_02, wherein the assay looks for IFNg release as a measure of immune cell activation (which can be driven by blocking soluble ULBP6 with h7B3 E- or E+, or blocking ADCC with h7B3 E+). The maximum dose (100nM) of all three h7B3 antibodies (E-, E+, E++DE) is depicted compared to isotype controls.

[0037] Fig. 9A Depicting a strongly responding donor in the INCUCYTE cell tumor killing assay setup. Demonstrating that GFP tumor cells were reduced in the case of h7B3 E-, further reduced in the case of h7B3 E+, and even more reduced in the case of h7B3 E++ compared to isotype control. The right panel provides the measurement of GFP at the final time point.

[0038] Fig. 9B Depicts a moderately reactive donor in an INCUCYTE assay setup. Shows a moderate reduction in GFP in the case of the h7B3E- antibody, and further reductions in GFP in both the E+ ("H7B3E+") and E++DE h7B3 ("h7B3 E++") antibodies compared to the isotype control. The right panel provides measurements of GFP at the final time point.

[0039] Fig. 9C Depicts a poorly reacting donor in the INCUCYTE assay setup. Only the h7B3 E++DE ("h7B3 E++") molecule showed a reduction in GFP compared to the isotype control. The right panel provides measurements of GFP at the final time point.

[0040] Fig.10 Depicted are IFNg levels from a mixed cell assay where primed PBMCs were incubated on COV644 cells for 24 h in the presence of 50 nM sULBP6_02 and isotype or anti-ULBP6 antibodies (h7B3 E++DE or h7B3 E+Afu in dose titration). Both antibodies showed similar EC values ​​with similar maximal potency for IFNg release. 50 curve.

[0041] Fig.11A Strong responding donors in the INCUCYTE assay setting are depicted and the observed GFP tumor cells were reduced in the case of h7B3 E+ and similarly further reduced in the case of both h7B3 E++DE ("h7B3 E++") and h7B3 E+Afu ("h7B3 Afu").

[0042] Fig. 11B Depicted are moderately reacting donors in the INCUCYTE assay setup and the observed modest reduction in GFP with the h7B3 E+ antibody, which was amplified with both the h7B3 E++DE ("h7B3 E++") and h7B3 E+Afu ("h7B3 Afu") antibodies compared to the isotype control.

[0043] Fig.12 Depicted are the concentration-time curves of h7B3(E+) and h8E11(E+) following administration of a 3 mg / kg intravenous dose by slow bolus in cynomolgus monkeys. Data were plotted on a semi-logarithmic graph as mean and standard deviation (N=3M / group) using GraphPad PRISM 9.0. LLOQ=lower limit of quantitation and SD=standard deviation.

[0044] Fig.13 Cartoon representation depicting the crystal structure of h7B3 Fab in complex with ULBP602. ULBP6 is colored in black (towards the top of the structure), the h7B3 Fab light chain is colored in light grey (towards the left side of the structure), and the heavy chain is colored in dark grey (towards the right side of the structure).

[0045] Fig.14Depicted (Panel A) Structural epitope of h7B3 on huULBP602, as determined by the change in solvent accessible surface area of ​​ULBP602 residues between the free and bound states. Epitope residues are colored in black and shown as bars on a cartoon representation of UBLP602. (Panel B) Surface representation of ULBP6 with residues comprising the epitope h7B3 epitope, colored in black. (Panel C) Structural binding site of NKG2D on ULBP6 determined from the crystal structure of ULBP6 in complex with NKG2D (PDB ID: 4s0u). On a cartoon representation of UBLP602, residues that interact with NKG2D promoter A (in the bars) are colored in black, and residues that interact with NKG2D promoter B are shown in gray. (Panel D) Surface representation of ULBP6, with residues comprising the NKG2D promoter A binding side colored in black, and residues that form the NKG2D promoter B binding site colored in gray.

[0046] Fig.15 Depicts ULBP6 phage binding to anti-HA antibody.

[0047] Fig.16 The binding affinity of ULBP6 to hu7B3, hu8E1, hu1D4, hu6E1, and hu8E8 is depicted.

[0048] Fig.17 Binding affinity to the ULBP6 receptor is depicted.

[0049] Fig.18 Depicted are the functional epitopes of 7B3 (Panels A and B), (Panels C and D), and 6E11 (Panels E and F), as identified herein.

[0050] Fig.19 131 peptides covering 87.7% of the ULBP6 construct sequence are depicted and were included in the evaluation of the data from the HDX experiments.

[0051] Fig. 20A Depicted are the different fraction uptakes of each detected peptide from amino acids 26 to 123 of ULBP6 from HDX experiments.

[0052] Fig. 20B Depicted are the different fraction uptakes of each detected peptide from amino acids 114 to 226 of ULBP6 from HDX experiments.

[0053] Fig.21 The amino acids of ULBP6 that were significantly protected by mAb1298, h7B3, h8E8, h1D4, h8E11, and h6E1 as determined by HDX experiments are depicted as underlined bold text.

[0054] Fig. 22 Depicted on the crystal structure of the NKG2D-ULBP6 complex, residues of the ULBP6 peptide have significant conservation by mAb1298 (Panel A), h7B3(E+Afu) (Panel B), h8E8(E+) (Panel C), h1D4(E+) (Panel D), h8E11(E+) (Panel E), and h6E1(E+) (Panel F), as determined by HDX and indicated in black. DETAILED DESCRIPTION

[0055] As used herein, "ULBP6" means any UL16 binding protein 6. Pseudonyms of ULBP6 include RAET1L, retinoic acid early transcript 1L, retinoic acid early transcript 1L protein and UL16 binding protein 6. ULBP6 is encoded by the RAET1L gene and is a UL16 binding protein family molecule. ULBP6 specifically binds to the activated receptor natural killer cell group 2D receptor (NKG2D). Like other NKG2D ligands, ULBP6 plays the role of a stress-inducible regulator of the NKG2D pathway. ULBP6 may be expressed as a cell surface membrane-bound form. ULBP6 may be a soluble molecule, which may be a cell surface or shed from the cell surface. As used herein, "sULBP6" refers to non-surface-bound, soluble or shed ULBP6. As used herein, "human ULBP6" refers to human ULBP6 or human sULBP6 bound to a cell surface membrane. Human ULBP6 is highly polymorphic, with two haplotypes (ULBP6_01 and ULBP6_02) being the most common haplotypes observed in the population (Antoun et al., 2010, Hum Immunol, 71:610-20; Eagle et al., 2009, Eur J Immunol, 39:3207-3216). The amino acid sequences of human sULBP6 haplotypes ULBP6_01 and ULBP6_02 are shown in Table 6.

[0056] As used herein, "NKG2D" refers to the natural killer cell group 2D receptor of ULBP6 and other NKG2DLs. NKG2D is encoded by the KLRK1 (killer cell lectin-like receptor K1) gene and is an activating receptor expressed by natural killer (NK) cells and T cells. It plays an important role in anti-pathogen and anti-tumor responses because the participation of NKG2D causes cell activation, cytokine and cytotoxic activity. In some cases, NKG2D is human NKG2D (hNKG2D).

[0057] As used herein, "NKG2DL" and "NKG2D ligand" are used interchangeably and refer to protein ligands of NKG2D. Human NKG2DL (hNKG2DL) includes MICA, MICB, ULBP1 (RAET1), ULBP2 (RAET1H), ULBP3 (RAET1N), ULBP4 (RAET1E), ULBP5 (RAET1G) and ULBP6 (RAET1L). Some NKG2DLs may be soluble molecules or shed from the cell surface. As used herein, "sNKG2DL" refers to non-surface-bound, soluble or shed NKG2DL.

[0058] ULBP6 and other NKG2DLs bind to the activating receptor NKG2D expressed on NK cells and T cells (e.g., CD8 T cells). The expression of these NKG2D ligands is usually lower than that under constant conditions, but can be highly induced under conditions of cell stress (e.g., by pathogen infection or malignant cells). When expressed on the cell surface of stress cells, ULBP6 can engage NKG2D, thereby enhancing NK cell / T cell activation immune monitoring. When ULBP6 is in a detached / soluble form, sULBP6 can act to block the interaction between NKG2D and other NKG2DLs, and therefore acts in an immunosuppressive manner by inhibiting NKG2D-mediated immune cell activation. Therefore, it is expected that the use of ULBP6 binding proteins to block the interaction of sULBP6 with NKG2D will increase NKG2D-mediated immune cell activation. ULBP6 binding proteins induced NKG2D-mediated immune cell activation can provide treatment for diseases such as cancer.

[0059] As used herein, the term "ULBP6 binding protein" refers to antibodies and other protein constructs, such as domains, that are capable of binding to ULBP6. In some cases, ULBP6 is human ULBP6. The terms "ULBP6 binding protein" and "antigen binding protein" are used interchangeably herein. This does not include natural cognate ligands or receptors (i.e., NKG2D).

[0060] The term "antibody" is used in a broad sense herein to refer to molecules with immunoglobulin-like domains (e.g., IgG, IgM, IgA, IgD or IgE), and includes monoclonal, recombinant, polyclonal, chimeric, human, humanized, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; single variable domains (such as, but not limited to, domain antibodies (DAB)), Fab, F(ab')2, Fv, disulfide-linked Fv, single-chain Fv, disulfide-linked scFv, diabodies, TANDABS, etc., as well as modified versions or antigen-binding fragments of any of the foregoing.

[0061] The terms whole, entire or intact antibody, used interchangeably herein, refer to heterotetrameric glycoproteins with an approximate molecular weight of 150,000 daltons. A complete antibody is composed of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. This H2L2 structure folds to form three functional domains, which include two antigen-binding fragments called 'Fab' fragments and an 'Fc' crystallizable fragment. The Fab fragment consists of the variable domain variable heavy chain (VH) or variable light chain (VL) at the amino terminus and the constant domains CH1 (heavy) and CL (light) at the carboxyl terminus. The Fc fragment consists of two domains formed by dimerization of paired CH2 and CH3 regions. Fc can cause effector functions by binding to receptors on immune cells or by binding to C1q (the first component of the classical complement pathway). The five classes of antibodies, IgM, IgA, IgG, IgE and IgD, are defined by different heavy chain amino acid sequences, which are called μ, α, γ, ε and δ, respectively, and each heavy chain can be paired with a kappa or lambda light chain. Most antibodies in serum belong to the IgG class and there are four isotypes of human IgG (IgGl, IgG2, IgG3 and IgG4) whose sequences differ mainly in their hinge regions.

[0062] Fully human antibodies can be obtained using a variety of methods, such as using yeast-based libraries or transgenic animals (e.g., mice) capable of producing a human antibody library. Yeasts that present human antibodies bound to an antigen of interest on their surface can be selected using methods based on FACS (fluorescence activated cell sorting) or by using labeled antigen capture beads. Transgenic animals that have been modified to express human immunoglobulin genes can be immunized with an antigen of interest and antigen-specific human antibodies separated using B cell sorting techniques. Then, the human antibodies produced using these techniques can be characterized by desired properties, such as affinity, developability, and selectivity.

[0063] Alternative antibody formats include alternative scaffolds, in which one or more CDRs of an antigen binding protein can be configured onto a suitable non-immunoglobulin protein scaffold or framework, such as an affibody, SpA scaffold, LDL receptor class A domain, avimer or EGF domain.

[0064] "Humanized antibody" refers to an engineered antibody whose CDR is derived from a non-human donor immunoglobulin, and the remaining immunoglobulin-derived portion of the molecule is derived from one or more human immunoglobulins. In addition, the framework support residues can be changed to retain binding affinity. Through the homology with the nucleotide and amino acid sequences of the donor antibody, suitable human acceptor antibodies can be antibodies selected from conventional databases (e.g., KABAT database, Los Alamos database, and Swiss Protein database). The acceptor antibody can be heterologous to the donor antibody, and in some embodiments, it can be a human antibody. The donor antibody can contribute the amino acid sequence, CDR, or other functional fragments or analogs of its variable region to the first immunoglobulin partner. The donor can provide the changed immunoglobulin coding region and the generated antibody with antigen-specific expression changes. Human antibodies characterized by homology with the donor antibody framework region (based on amino acids) may be suitable for providing heavy chain constant region and / or heavy chain variable framework region for insertion of donor CDR. Suitable acceptor antibodies capable of providing heavy chain and / or light chain constant or variable framework regions can be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains need not be derived from the same acceptor antibody.

[0065] The ULBP6 binding protein described herein may be an antibody or an antigen-binding fragment thereof. The ULBP6 binding protein may be a humanized antibody or an antigen-binding fragment thereof. The ULBP6 binding protein may comprise: a humanized VH region or a humanized heavy chain (HC) sequence; and / or a humanized VL region, or a humanized light chain (LC) sequence. The humanized HC may be humanized using the human germline sequence IGHV3-48, IGHV7-4-1, or IGHV1-2. The humanized HC may be humanized using the human germline sequence IGHV3-48. The humanized LC may be humanized using the human germline sequence IGKV1-33, IGKV3-11, or IGKV1-16. The humanized LC may be humanized using the human germline sequence IGKV1-33. The humanized LC may be humanized using the human germline sequence IGKV3-11.

[0066] The term "single variable domain" refers to a folded polypeptide domain comprising sequence characteristics of an antibody variable domain. It therefore comprises complete antibody variable domains, such as VH, VHH and VL, and modified antibody variable domains, for example, wherein one or more loops have been replaced by sequences that do not have antibody variable domain characteristics, or by sequences of antibody variable domains that have been truncated or comprise N-terminal or C-terminal extensions and folded fragments of variable domains that retain at least binding activity and specificity of the full-length domain. A single variable domain as defined herein is capable of binding antigens or epitopes independently of different variable regions or domains. "Domain antibodies" or "DABs" may be considered to be the same as human "single variable domains". A single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodents (e.g., as disclosed in WO 00 / 29004), nurse sharks and camelid VHHs. Camelid VHHs are immunoglobulin single variable domain polypeptides derived from species including camels, llamas, alpacas, dromedaries and alpacas that produce pure heavy chain antibodies that naturally lack light chains. Such VHH domains can be humanized according to standard techniques available in the art, and such domains are considered "single variable domains".

[0067] Antigen binding fragments can be provided by configuring one or more CDRs on a non-antibody protein scaffold. As used herein, "protein scaffold" includes but is not limited to immunoglobulin (Ig) scaffolds, such as IgG scaffolds, which can be four-chain or two-chain antibodies, or they can only contain the Fc region of an antibody, or they can contain one or more constant regions from an antibody, which constant region can be of human or primate origin, or it can be an artificial chimera of human and primate constant regions.

[0068] The protein scaffold may be an Ig scaffold, such as an IgG or IgA scaffold. The IgG scaffold may comprise some or all domains of a complete antibody (i.e., CH1, CH2, CH3, VH, VL). The antigen binding protein may comprise an IgG scaffold selected from IgG1, IgG2, IgG3, IgG4, or IgG4PE. For example, the scaffold may be IgG1. The scaffold may consist of or comprise the Fc region of an antibody or a portion thereof.

[0069] The protein scaffold may be a derivative of a scaffold selected from the group consisting of: CTLA-4, lipocalin, protein A derived molecules such as the Z domain (affine body, SpA), A domain (Avimer / Maxibody) of protein A; heat shock proteins such as GroE1 and GroES; transferrin (transmembrane antibody); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (tetranectin); human g-crystallin and human ubiquitin (avidin); PDZ domain; scorpion toxin kunitz-type domain of human protease inhibitor; and fibronectin / adenonectin (adnectin); proteins that have been engineered to obtain binding to an antigen (e.g., ULBP6) rather than a natural ligand.

[0070] The term multispecific antigen-binding protein refers to an antigen-binding protein comprising at least two different antigen-binding sites. Each of these antigen-binding sites is capable of binding to different epitopes that may be present on the same antigen or on different antigens. A multispecific antigen-binding protein may be specific for more than one antigen (e.g., two antigens or three antigens or four antigens).

[0071] Bispecifics can be broadly classified as having symmetrical or asymmetrical architectures. Bispecifics can have an Fc or can be fragment-based (lacking an Fc). Fragment-based bispecifics combine multiple antigen-binding antibody fragments in one molecule without an Fc region, such as Fab-scFv, Fab-scFv2, orthogonal Fab-Fab, Fab-Fv, tandem scFc (e.g., BiTE and BiKE molecules), diabodies, DARTs, TandAbs, scDiabodies, tandem dAbs, etc.

[0072] Symmetrical formats combine multiple binding specificities in a single polypeptide chain or single HL pair including Fc fusion proteins based on fragment formats and formats in which antibody fragments are fused to conventional antibody molecules. Examples of symmetric formats may include DVD-Ig, TVD-Ig, CODV-Ig, (scFv)4-Fc, IgG-(scFv)2, tetravalent ART-Fc, F(ab)4CrossMab, IgG-HC-scFv, IgG-LC-scFv, mAb-dAb, etc.

[0073] Asymmetric formats preserve the native architecture of native antibodies as closely as possible by forcing correct HL chain pairing and / or promoting H chain heterodimerization during co-expression of three (if common heavy or light chains are used) or four polypeptide chains (e.g. Triomab, Asymmetric Reengineered Technology Immunoglobulin (ART-Ig), CrossMab, Biclonics common light chain, ZW1 common light chain, DuoBody and knob-in-hole structure (KiH), DuetMab, κλ body, Xmab, YBODY, HET-mAb, HET-Fab, DART-Fc, SEEDbody, mouse / rat chimeric IgG).

[0074] Bispecific formats may also include antibodies fused to non-Ig scaffolds, such as Affimabs, Fynomabs, Zybodies, and Anticalin-IgG fusions, ImmTACs.

[0075] As used herein, the term "chimeric antigen receptor" ("CAR") refers to an engineered receptor consisting of an extracellular antigen binding domain (usually derived from a monoclonal antibody or a fragment thereof, such as a VH domain and a VL domain in the form of a scFv), optionally a spacer, a transmembrane region, and one or more intracellular effector domains. CAR is also referred to as a chimeric T cell receptor or chimeric immune receptor (CIR). CAR is genetically introduced into hematopoietic cells (eg, T cells) to redirect T cell specificity to a desired cell surface antigen to produce a CAR-T therapeutic agent.

[0076] As used herein, the term "spacer" refers to an oligopeptide or polypeptide that connects the transmembrane domain to the function of the target binding domain. This region may also be referred to as a "hinge region" or "stem region". The size of the spacer may vary depending on the location of the target epitope to maintain a set distance (e.g., 14 nm) when CAR: target is bound.

[0077] As used herein, the term "transmembrane domain" refers to the portion of the CAR molecule that passes through the cell membrane.

[0078] As used herein, the term "intracellular effector domain" (also referred to as "signaling domain") refers to the domain in the CAR that is responsible for intracellular signaling after the antigen binding domain binds to the target. The intracellular effector domain is responsible for the activation of at least one of the normal effector functions of the immune cell expressing the CAR. For example, the effector function of a T cell may be a cytolytic activity or an auxiliary activity including cytokine secretion.

[0079] Those skilled in the art will appreciate that the VH and / or VL domains disclosed herein can be incorporated into CAR-T therapeutic agents, for example in the form of scFv.

[0080] The antigen binding proteins described herein can show cross-reactivity between human ULBP6 and ULBP6 from another species (e.g., cynomolgus monkey ULBP2 / 6). It should be noted that cynomolgus monkey ULBP2 / 6 is so named because it is unclear whether a single cynomolgus monkey ortholog is evolutionarily related to human ULBP2 or ULBP6. Therefore, the cynomolgus monkey ortholog is referred to as cynomolgus monkey ULBP2 / 6 or cULBP2 / 6 herein. The antigen binding proteins described herein can specifically bind to human ULBP6 and cynomolgus monkey ULBP2 / 6. This is particularly useful because drug development often requires testing of lead candidate drugs in non-human primate systems before testing drugs in humans. A drug that can combine human and monkey species is provided so that people can test the results in these systems and compare the data side by side using the same drug. This avoids the complexity of needing to find drugs against cynomolgus monkey ULBP6 and separate drugs against human ULBP6, and also avoids the need to compare the results of people and cynomolgus monkeys using different drugs. Cross-reactivity between other species used in disease models (e.g., dog or monkey, particularly monkey) is also contemplated. Optionally, the antigen binding protein has a binding affinity for at least cynomolgus monkey ULBP2 / 6 that differs no more than 2, 5, 10, 50 or 100-fold from its binding affinity for human ULBP6.

[0081] Affinity, also known as "binding affinity" is the strength of binding at a single interaction site, i.e., one molecule (e.g., an antigen-binding protein of the invention) to another molecule (e.g., ULBP6) at a single binding site. The binding affinity of an antigen-binding protein to its target can be determined by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., BIACORE analysis). For example, the BIACORE method described in Example 7 can be used to measure binding affinity.

[0082] The equilibrium dissociation constant (KD) of the antigen binding protein-ULBP6 interaction may be 100 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. Alternatively, the KD may be between 5 nM and 10 nM; or between 1 nM and 2 nM. The KD may be between 1 pM and 500 pM; or between 500 pM and 1 nM. The KD may be between 100 pM and 500 pM; or between 100 pM and 1 nM. The KD may be between 100 pM and 2 nM; or between 100 pM and 5 nM. It will be appreciated by those skilled in the art that the smaller the KD value, the stronger the binding. The reciprocal of the KD (i.e., 1 / KD) is a number having units M. -1 The equilibrium association constant (KA) of a molecule is 1.0. It will be appreciated by those skilled in the art that the larger the KA value, the stronger the binding.

[0083] Human ULBP6 shows high sequence similarity to human ULBP2, but has lower sequence similarity to ULBP5. The ULBP6 binding protein described herein can bind to human ULBP6 and ULBP2 with a KD of less than or equal to 1 nM. Alternatively, the ULBP6 binding protein described herein can bind to human ULBP6 with a KD of less than or equal to 500 pM, and bind to ULBP2 with a KD of greater than or equal to 500 pM. Alternatively, the ULBP6 binding protein described herein can bind to human ULBP6 with a KD of less than or equal to 300 pM, and bind to ULBP2 with a KD of greater than or equal to 500 pM. The ULBP6 binding protein described herein can bind to human ULBP6 with a KD of less than or equal to 1 nM, and bind to ULBP5 with a KD of greater than or equal to 1 nM. Alternatively, the ULBP6 binding protein can bind to ULBP6 with a KD of less than or equal to 500 pM, and bind to ULBP5 with a KD of greater than or equal to 1 nM. Alternatively, the ULBP6 binding protein may bind to ULBP6 with a KD of less than or equal to 400 pM and bind to ULBP5 with a KD of greater than or equal to 1 nM. Alternatively, the ULBP6 binding protein may bind to ULBP6 with a KD of less than or equal to 300 pM and bind to ULBP5 with a KD of greater than or equal to 1 nM. Alternatively, the ULBP6 binding protein may bind to ULBP6 with a KD of less than or equal to 300 pM and bind to ULBP5 with a KD of greater than or equal to 2 nM.

[0084] The dissociation rate constant (kd) or "dissociation rate" describes the stability of the antigen binding protein-ULBP6 complex, i.e., the fraction of the complex that decays per second. For example, 0.01 s -1 The kd is equal to 1% decay of the complex per second. The dissociation rate constant (kd) of the antigen binding protein-ULBP6 interaction can be 1×10 -3 s -1 or smaller, 1×10 -4 s -1 or smaller, 1×10 -5 s -1 or smaller, or 1×10 -6 s -1 or less. Alternatively, kd may be between 1×10 -5 s -1 With 1×10 -4 s -1 or between 1×10 -4 s -1 With 1×10 -3 s -1 Alternatively, kd can be between 1×10 -3 s -1 With 1×10 -2 s-1 between.

[0085] The association rate constant (ka) or "binding rate" describes the rate at which the antigen binding protein-ULBP6 complex forms. The ka for an antigen binding protein-ULBP6 interaction may be about 1.5 x 10 6 M -1 s -1 Alternatively, ka can be between 1×10 6 M -1 s -1 With 1×10 5 M -1 s -1 Alternatively, ka can be between 1×10 6 M -1 s -1 With 5x10 6 M -1 s -1 or between 1×10 6 M -1 s -1 With 2x10 6 M -1 s -1 between.

[0086] "Isolated" means that a molecule (e.g., an antigen binding protein or nucleic acid) is removed from its previous environment. For example, the molecule can be purified from a substance that normally occurs in nature. For example, the mass of the molecule in a sample can be 95% of the total mass.

[0087] The ULBP6 binding protein described herein can be encoded by an isolatable nucleic acid. By delivering exogenous isolated nucleic acids encoding the heavy and light chains of the antibody, the production of ULBP6 binding proteins such as antibodies can be achieved in cells or living organisms. One or more nucleic acids encoding the heavy and light chains of the ULBP6 binding protein can be delivered to a subject in need. The heavy and light chains of the antibody can be delivered by the same or separate nucleic acids. The nucleic acid can be DNA or RNA. The nucleic acid can be mRNA. The nucleic acid encoding the ULBP6 binding protein can be modified or unmodified. The nucleic acid encoding the ULBP6 binding protein can include at least one chemical modification. The nucleic acid encoding the ULBP6 binding protein can be delivered to the subject naked (i.e., without encapsulation particles) or packaged (i.e., encapsulated in liposomes or polymer-based vehicles).

[0088] Also provided herein is a method for producing a ULBP6 binding protein in a cell, tissue or organism, the method comprising: contacting the cell, tissue or organism with a composition comprising an isolated nucleic acid, the nucleic acid comprising at least one chemical modification and encoding a ULBP6 binding protein. Also provided herein is a method for producing a ULBP6 binding protein in a cell, tissue or organism, the method comprising: contacting the cell, tissue or organism with a composition comprising a polynucleotide, the polynucleotide comprising at least one chemical modification and encoding a ULBP6 binding protein.

[0089] As used herein, the term "expression vector" means an isolated nucleic acid that can be used to introduce a nucleic acid of interest into a cell (e.g., a eukaryotic cell or a prokaryotic cell) or in a cell-free expression system in which a nucleic acid sequence of interest is expressed as a peptide chain (e.g., a protein). Such expression vectors may be, for example, cosmids, plasmids, viral sequences, transposons, and linear nucleic acids comprising a nucleic acid of interest. Once the expression vector is introduced into a cell or a cell-free expression system (e.g., a reticulocyte lysate), the protein encoded by the nucleic acid of interest is produced by a transcription / translation mechanism. Expression vectors within the scope of the present disclosure may provide the necessary elements for eukaryotic or prokaryotic expression, and include viral promoter-driven vectors, such as CMV promoter-driven vectors, such as pcDNA3.1, pCEP4 and its derivatives, baculovirus expression vectors, Drosophila expression vectors, and expression vectors driven by mammalian gene promoters such as human Ig gene promoters. Other examples include prokaryotic expression vectors, such as T7 promoter-driven vectors (e.g., pET41), lactose promoter-driven vectors, and arabinose gene promoter-driven vectors. Those of ordinary skill in the art will recognize many other suitable expression vectors and expression systems.

[0090] As used herein, the term "recombinant host cell" means a cell containing a nucleic acid sequence of interest separated before it is introduced into a cell. For example, the nucleic acid sequence of interest may be in an expression vector, and the cell may be prokaryotic or eukaryotic. Exemplary eukaryotic cells are mammalian cells, such as, but not limited to, COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2, 653, SP2 / 0, NS0, 293, HeLa, myeloma, lymphoma cells, or any derivatives thereof. Eukaryotic cells may be HEK293, NS0, SP2 / 0 or CHO cells. Escherichia coli is an exemplary prokaryotic cell. Recombinant cells according to the present disclosure may be produced by transfection, cell fusion, immortalization, or other procedures well known in the art. Nucleic acids of interest (e.g., expression vectors) transfected into cells may be extrachromosomally or stably integrated into the chromosomes of cells.

[0091] "CDR" is defined as the complementary determining region amino acid sequence of an antigen binding protein. These may be identical or overlapping with the hypervariable regions (HVR) of the heavy and light chains of immunoglobulins, depending on the method used to determine CDR and HDR. In the variable portion of an immunoglobulin, there are three heavy chain and three light chain CDRs (or CDR regions). Therefore, as used herein, "CDR" generally refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

[0092] As used herein, a hypervariable region may include the following extended or alternative hypervariable regions: V L 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the domain and V H 26-35 or 30-35 (H1), 50-61, 50-65 or 49-65 (H2), and 93-102, 94-102 or 95-102 (H3) in the variable domain. For each of these definitions, the variable domain residues are numbered according to Kabat et al., supra.

[0093] Throughout this specification, amino acid residues in the variable domain region within the variable domain sequence and the full-length antigen binding sequence (e.g., within the antibody heavy chain sequence or the antibody light chain sequence) are numbered according to the Kabat numbering convention. Similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" used in the examples follow the Kabat numbering convention. For further information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987).

[0094] It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, such as those described in Chothia et al., (1989) Nature 342:877-883. The structure and protein folding of the antigen binding protein may mean that other residues are considered part of the CDR sequence, and those skilled in the art will understand this.

[0095] Other numbering conventions for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) and "contact" (University College London) methods. The CDR regions of SEQ ID NOs: 16 to 19 and SEQ ID NOs: 26 to 29 may be defined by any numbering convention (e.g., Kabat, Chothia, AbM, and contact conventions).

[0096] Table 1 below shows one definition for each numbering convention used for each CDR or binding unit. The variable domain amino acid sequences are numbered using the Kabat numbering scheme in Table 1. It should be noted that some CDR definitions may differ depending on the individual publication used.

[0097] Table 1: CDR numbering convention

[0098] Kabat CDR Chothia CDR AbM CDR Contact CDR H1 31-35 / 35A / 35B 26-32 / 33 / 34 26-35 / 35A / 35B 30-35 / 35A / 35B H2 50-65 52-56 50-58 47-58 H3 95-102 95-102 95-102 93-101 L1 24-34 24-34 24-34 30-36 L2 50-56 50-56 50-56 46-55 L3 89-97 89-97 89-97 89-96

[0099] Provided herein is a ULBP6 binding protein comprising any one or a combination of CDRs selected from the group consisting of: a) a CDRH1 of SEQ ID NO: 16 or 21; a CDRH2 of SEQ ID NO: 16 or 21; a CDRH3 of SEQ ID NO: 16 or 21; a CDRL1 of SEQ ID NO: 26 or 31; a CDRL2 of SEQ ID NO: 26 or 31; a CDRL3 of SEQ ID NO: 26 or 31, or b) a CDRH1 of SEQ ID NO: 17 or 22; a CDRH2 of SEQ ID NO: 17 or 22; a CDRH3 of SEQ ID NO: 17 or 22; a CDRL1 of SEQ ID NO: 27 or 32; a CDRL2 of SEQ ID NO: 27 or 32; a CDRL3 of SEQ ID NO: 27 or 32, or c) a CDRH1 of SEQ ID NO: 18 or 23; a CDRH2 of SEQ ID NO: 18 or 23; a CDRH3 of SEQ ID NO: 18 or 23; a CDRH4 of SEQ ID NO: 18 or 23; a CDRH5 of SEQ ID NO: 18 or 23; a CDRH6 of SEQ ID NO: 18 or 23; a CDRH7 of SEQ ID NO: 18 or 23; a CDRH8 of SEQ ID NO: 18 or 23; a CDRH9 of SEQ ID NO: 18 or 23; a CDRH1 of SEQ ID NO: 18 or 23; a CDRH1 of SEQ ID NO: 18 or 23; a CDRH2 of SEQ ID NO: 18 or 23; a CDRH3 of SEQ ID NO: NO:28 or 33 CDRL1; SEQ ID NO:28 or 33 CDRL2; SEQ ID NO:28 or 33 CDRL3, or d) SEQ ID NO:19 or 24 CDRH1; SEQ ID NO:19 or 24 CDRH2; SEQ ID NO:19 or 24 CDRH3; SEQ ID NO:29 or 34 CDRL1; SEQ ID NO:29 or 34 CDRL2; SEQ ID NO:29 or 34 CDRL3.

[0100] Alternatively, the ULBP6 binding protein described herein may comprise all 6 CDRs selected from: a) CDRH1 of SEQ ID NO:36, CDRH2 of SEQ ID NO:37, CDRH3 of SEQ ID NO:38, CDRL1 of SEQ ID NO:51, CDRL2 of SEQ ID NO:52, and CDRL3 of SEQ ID NO:53; b) CDRH1 of SEQ ID NO:39, CDRH2 of SEQ ID NO:40, CDRH3 of SEQ ID NO:41, CDRL1 of SEQ ID NO:54, CDRL2 of SEQ ID NO:55, and CDRL3 of SEQ ID NO:56; c) CDRH1 of SEQ ID NO:42, CDRH2 of SEQ ID NO:43, CDRH3 of SEQ ID NO:44, CDRL1 of SEQ ID NO:57, CDRL2 of SEQ ID NO:58, and CDRL3 of SEQ ID NO:59; or d) CDRH1 of SEQ ID NO:45, CDRH2 of SEQ ID NO:46, CDRH3 of SEQ ID NO:47, CDRL1 of SEQ ID NO:48, and CDRL2 of SEQ ID NO:49. The CDRH2 of SEQ ID NO:46, the CDRH3 of SEQ ID NO:47, the CDRL1 of SEQ ID NO:60, the CDRL2 of SEQ ID NO:61 and the CDRL3 of SEQ ID NO:62.

[0101] The CDR can be modified by at least one amino acid substitution, deletion or addition, wherein the variant ULBP6 binding protein substantially retains one or more biological characteristics of the unmodified binding protein, which one or more biological characteristics may be the biological characteristics provided herein, such as inhibiting the binding of soluble ULBP6 to NKG2D.

[0102] It should be understood that each of CDR H1, H2, H3, L1, L2, L3 can be modified individually, or in any arrangement or combination with any other CDR combination to modify. CDR can be modified by replacing, lacking or adding up to 3 amino acids, such as 1 or 2 amino acids, such as 1 amino acid. Each modification of CDR, VH, VL or other protein provided herein can be a conservative substitution. Usually, the modification is a substitution, particularly a conservative substitution, such as shown in Table 2 below.

[0103] Table 2: Conservative substitutions

[0104] Sidechain member Hydrophobicity Met, Ala, Val, Leu, Ile Neutral hydrophilicity Cys, Ser, Thr Acidic Asp、Glu Alkaline Asn, Gln, His, Lys, Arg Residues that affect chain orientation Gly, Pro Aromatic Trp, Tyr, Phe

[0105] For example, in a variant CDR, the flanking residues comprising the CDR as part of an alternative definition, such as Kabat or Chothia, may be substituted with conservative amino acid residues.

[0106] As noted above, such antigen binding proteins comprising variant CDRs may be referred to herein as "functional CDR variants".

[0107] The ULBP6 binding protein described herein may comprise (i) any one or a combination of the CDRs selected from CDRH1, CDRH2, CDRH3 of SEQ ID NO: 16, and / or CDRL1, CDRL2, CDRL3 of SEQ ID NO: 26; or (ii) a CDR variant of (i), wherein the variant has 1, 2 or 3 amino acid modifications; or a VH region comprising a sequence that is at least 80% identical to the sequence of SEQ ID NO: 21; and / or a VL region comprising a sequence that is at least 80% identical to the sequence of SEQ ID NO: 31.

[0108] Alternatively, the ULBP6 binding protein described herein may comprise (i) any one or a combination of the CDRs selected from CDRH1, CDRH2, CDRH3 of SEQ ID NO: 17, and / or CDRL1, CDRL2, CDRL3 of SEQ ID NO: 27; or (ii) a CDR variant of (i), wherein the variant has 1, 2 or 3 amino acid modifications; or a VH region comprising a sequence that is at least 80% identical to the sequence of SEQ ID NO: 22; and / or a VL region comprising a sequence that is at least 80% identical to the sequence of SEQ ID NO: 32.

[0109] Alternatively, the ULBP6 binding protein described herein may comprise (i) any one or a combination of the CDRs selected from CDRH1, CDRH2, CDRH3 of SEQ ID NO: 18, and / or CDRL1, CDRL2, CDRL3 of SEQ ID NO: 28; or (ii) a CDR variant of (i), wherein the variant has 1, 2 or 3 amino acid modifications; or a VH region comprising a sequence that is at least 80% identical to the sequence of SEQ ID NO: 23; and / or a VL region comprising a sequence that is at least 80% identical to the sequence of SEQ ID NO: 33.

[0110] Alternatively, the ULBP6 binding protein described herein may comprise (i) any one or a combination of the CDRs selected from CDRH1, CDRH2, CDRH3 of SEQ ID NO: 19, and / or CDRL1, CDRL2, CDRL3 of SEQ ID NO: 29; or (ii) a CDR variant of (i), wherein the variant has 1, 2 or 3 amino acid modifications; or a VH region comprising a sequence that is at least 80% identical to the sequence of SEQ ID NO: 24; and / or a VL region comprising a sequence that is at least 80% identical to the sequence of SEQ ID NO: 34.

[0111] The ULBP6 binding protein described herein may include any one or a combination of CDRs selected from the group consisting of: CDRH1 of SEQ ID NO: 36, CDRH2 of SEQ ID NO: 37; and / or CDRH3 of SEQ ID NO: 38; CDRL1 of SEQ ID NO: 51; CDRL2 of SEQ ID NO: 52; and / or CDRL3 of SEQ ID NO: 53. Alternatively, the ULBP6 binding protein described herein may include any one or a combination of CDRs selected from the group consisting of: CDRH1 of SEQ ID NO: 39, CDRH2 of SEQ ID NO: 40; and / or CDRH3 of SEQ ID NO: 41; CDRL1 of SEQ ID NO: 54; CDRL2 of SEQ ID NO: 55; and / or CDRL3 of SEQ ID NO: 56. Alternatively, the ULBP6 binding protein described herein may comprise any one or a combination of CDRs selected from the group consisting of CDRH1 of SEQ ID NO: 42, CDRH2 of SEQ ID NO: 43; and / or CDRH3 of SEQ ID NO: 44; CDRL1 of SEQ ID NO: 57; CDRL2 of SEQ ID NO: 58; and / or CDRL3 of SEQ ID NO: 59. Alternatively, the ULBP6 binding protein described herein may comprise any one or a combination of CDRs selected from the group consisting of CDRH1 of SEQ ID NO: 45, CDRH2 of SEQ ID NO: 46; and / or CDRH3 of SEQ ID NO: 47; CDRL1 of SEQ ID NO: 60; CDRL2 of SEQ ID NO: 61; and / or CDRL3 of SEQ ID NO: 62.

[0112] CDR L1, L2, L3, H1 and H2 tend to structurally exhibit one of a limited number of main-chain conformations. The specific canonical structural category of CDR is defined jointly by the length and loop packing of CDR, and is determined by the residues at key positions in both CDR and framework regions (structure-determined residues or SDRs). Martin and Thornton (1996; J Mol Biol 263:800-815) have produced an automatic method for defining "key residue" canonical templates. Use cluster analysis to define the canonical category of CDR sets, and then identify the canonical template by analyzing hidden hydrophobes, hydrogen bond residues, and conserved glycine and proline. By comparing the sequence with the key residue template, and scoring each template using an identity or similarity matrix, the CDR of the antibody sequence can be classified into a canonical category.

[0113] Examples of CDR conventions are given below in Table 3. The amino acid numbering used is that of Kabat.

[0114] Table 3: CDR Specifications

[0115]

[0116]

[0117] Each CDR, each corresponding CDR, each binding unit, each heavy chain or light chain variable region, each heavy chain or light chain and each antigen binding protein can have multiple different CDR canonical positions, and therefore any combination of substitutions can exist in the antigen binding proteins described herein, provided that the canonical structure of the CDR is maintained so that the antigen binding protein can specifically bind to ULBP6.

[0118] As discussed above, specific canonical structural classes of CDRs are defined jointly by both the length of the CDRs and the loop packing, determined by residues located at key positions in both the CDRs and the framework regions.

[0119] As used herein, the term "epitope" refers to the part of the antigen that contacts the specific binding domain of the antigen binding protein, also known as the paratope. An epitope may be linear or conformational / discontinuous. A conformational or discontinuous epitope comprises amino acid residues separated by other sequences, i.e., not in a continuous sequence in the primary sequence of the antigen assembled by the tertiary folding of the polypeptide chain. Although the residues may come from different regions of the polypeptide chain, they are very close in the three-dimensional structure of the antigen. In the case of a multimeric antigen, a conformational or discontinuous epitope may include residues from different peptide chains. The specific residues contained in the epitope may be determined by a computer modeling program or by a three-dimensional structure obtained by methods known in the art such as X-ray crystallography. Epitope localization may be performed using various techniques known to those skilled in the art. Exemplary methods include peptide-based methods such as pepscan, in which a series of overlapping peptides are screened for binding using techniques such as ELISA or by in vitro display of a large library of peptide or protein mutants, such as on phage. Detailed epitope information may be determined by structural techniques including X-ray crystallography, solution nuclear magnetic resonance (NMR) spectroscopy, and cryogenic electron microscopy (cryo-EM). Mutagenesis (eg, alanine scanning) is an effective approach where loss of binding analysis is used for epitope localization. Another approach is hydrogen / deuterium exchange (HDX) combined with proteolysis and liquid chromatography-mass spectrometry (LC-MS) analysis to characterize discontinuous or conformational epitopes.

[0120] The term "antigen binding site" refers to a site on an antigen binding protein that is capable of specific binding to an antigen, which may be a single variable domain, such as a folded polypeptide domain containing sequence characteristics of an antibody variable domain, or it may be a paired VH / VL domain found on a standard antibody. Single-chain Fv (ScFv) domains may also provide an antigen binding site.

[0121] Competition between the ULBP6 binding proteins described herein and a reference ULBP6 binding protein (e.g., a reference antibody) can be determined by one or more techniques known to those skilled in the art, such as ELISA, FMAT, surface plasmon resonance (SPR), or FORTEBIO OCTET biolayer interferometry (BLI). Such techniques may also be referred to as epitope binning. Competition assays may be performed by BLI. There are several possible reasons for this competition: the two proteins may bind to the same or overlapping epitopes, there may be steric inhibition in binding, or binding of the first protein may induce a conformational change in the antigen, thereby preventing or reducing binding of the second protein. The ULBP6 binding protein may bind to human ULBP6 and compete with the reference ULBP6 binding protein for binding to human ULBP6. The reference ULBP6 binding protein may comprise (a) a VH region sequence of SEQ ID NO:21 and a VL region sequence of SEQ ID NO:31; or (b) a VH region sequence of SEQ ID NO:22 and a VL region sequence of SEQ ID NO:32; or (c) a VH region sequence of SEQ ID NO:16 and a VL region sequence of SEQ ID NO:26; or (d) a VH region sequence of SEQ ID NO:17 and a VL region sequence of SEQ ID NO:27.

[0122] The ULBP6 binding proteins described herein may share overlapping epitopes with a reference ULBP6 binding protein comprising a VH region comprising SEQ ID NO: 21 and a VL region comprising SEQ ID NO: 31; or a VH region comprising SEQ ID NO: 22 and a VL region comprising SEQ ID NO: 32. Alternatively, the ULBP6 binding proteins described herein may share overlapping epitopes with a reference ULBP6 binding protein comprising a HC sequence comprising SEQ ID NO: 75 and a LC sequence comprising SEQ ID NO: 105; or a HC sequence comprising SEQ ID NO: 76 and a LC sequence comprising SEQ ID NO: 106. Alternatively, the ULBP6 binding proteins described herein may not share overlapping epitopes with a reference ULBP6 binding protein comprising a VH region comprising SEQ ID NO: 25 and a VL region comprising SEQ ID NO: 35; or a HC sequence comprising SEQ ID NO: 79 and a LC sequence comprising SEQ ID NO: 109.

[0123] As used in this specification, the term "neutralization" means that in vitro or in vivo, the biological activity of ULBP6 is reduced in the presence of the ULBP6 binding protein described herein, compared to the biological activity of ULBP6 in the absence of the ULBP6 binding protein. Neutralization can be attributed to blocking ULBP6 from binding to its receptor, preventing ULBP6 from activating its receptor, downregulating ULBP6 or its receptor, or affecting one or more of the effector functions. Neutralization can be determined or measured using one or more assays known to those skilled in the art or as described herein. For example, the blocking assay described in Example 7 can be used to assess the neutralizing ability of the ULBP6 binding protein.

[0124] The effect of ULBP6 binding protein on the interaction between ULBP6 and NKG2D may be partial or complete. Relative to the ULBP6-NKG2D interaction in the absence of ULBP6 binding protein, the neutralizing ULBP6 binding protein can neutralize the activity of ULBP6 by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The ULBP6 binding protein described herein can inhibit the interaction of human ULBP6 with human NKG2D by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0125] The ULBP6 binding protein described herein can inhibit the binding of sULBP6 to NKG2D. The ULBP6 binding protein can inhibit the binding of sULBP6 to NKG2D by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The ULBP6 binding protein described herein can block the binding of sULBP6 to NKG2D. In some cases, the ULBP6 binding protein can block the binding of sULBP6 to NKG2D by 70% or more, 75% or more, 80% or more, 90% or more, or 95% or more.

[0126] The "percent identity" or "% identity" between a query nucleic acid sequence and a target nucleic acid sequence is an "identity" value expressed as a percentage, calculated over the entire length of the query sequence using a suitable algorithm (e.g., BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoali gn) after pairwise global sequence alignment using a suitable algorithm (e.g., Needle man-Wunsch or GenePAST / KERR) or software (e.g., DNASTAR Lasergene or GenePAST / KERR). Importantly, the query nucleic acid sequence can be described by the nucleic acid sequences disclosed herein, in particular in one or more of the claims.

[0127] The "percent identity" or "% identity" between a query amino acid sequence and a subject amino acid sequence is an "identity" value expressed as a percentage, calculated over the entire length of the query sequence using a suitable algorithm (e.g., BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle or EMBOSS infoali gn), after pairwise global sequence alignment using a suitable algorithm (e.g., Needleman-Wunsch or GenePAST / KERR) or software (e.g., DNASTAR Lasergene or GenePAST / KERR). Importantly, the query amino acid sequence can be described by the amino acid sequences disclosed herein, in particular in one or more of the claims.

[0128] The query sequence may be 100% identical to the target sequence, or it may include up to a certain integer number of amino acid or nucleotide changes compared to the target sequence such that the % identity is less than 100%. For example, the query sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the target sequence. In the case of nucleic acid sequences, such changes include deletions, substitutions or insertions of at least one nucleotide residue, wherein the changes may occur at the 5'- or 3'-terminal position of the query sequence or at any position between those terminal positions, or are interspersed between the nucleotide residues in the query sequence alone, or are interspersed in one or more continuous groups within the query sequence. In the case of amino acid sequences, such changes include deletions, substitutions (including conservative and non-conservative substitutions) or insertions of at least one amino acid residue, wherein the changes may occur at the amino or carboxyl terminal position of the query sequence or at any position between those terminal positions, or are interspersed between the amino acid residues in the query sequence alone, or are interspersed in one or more continuous groups in the query sequence.

[0129] For antibody sequences, % identity can be determined over the entire query sequence length including the CDRs. Alternatively, % identity can exclude one or more or all CDRs. For example, all CDRs can be 100% identical to the target sequence, and the % identity variation can be in the remainder of the query sequence, such as the framework sequence, so that the CDR sequence is fixed and complete.

[0130] The ULBP6 binding protein described herein may comprise a CDRH3 that is 100% identical to any one of SEQ ID NO: 38, 41, 44, or 47. The ULBP6 binding protein described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 38. The ULBP6 binding protein described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 41. The ULBP6 binding protein described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 44. The ULBP6 binding protein described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 47.

[0131] The ULBP6 binding protein described herein may include a CDRH1 that is 100% identical to SEQ ID NO: 36; a CDRH2 that is 100% identical to SEQ ID NO: 37; and / or a CDRH3 that is 100% identical to SEQ ID NO: 38; a CDRL1 that is 100% identical to SEQ ID NO: 51; a CDRL2 that is 100% identical to SEQ ID NO: 52; and / or a CDRL3 that is 100% identical to SEQ ID NO: 53. Alternatively, the ULBP6 binding protein described herein may include a CDRH1 that is 100% identical to SEQ ID NO: 39; a CDRH2 that is 100% identical to SEQ ID NO: 40; and / or a CDRH3 that is 100% identical to SEQ ID NO: 41; a CDRL1 that is 100% identical to SEQ ID NO: 54; a CDRL2 that is 100% identical to SEQ ID NO: 55; and / or a CDRL3 that is 100% identical to SEQ ID NO: 56. Alternatively, the ULBP6 binding protein described herein may comprise a CDRH1 that is 100% identical to SEQ ID NO:42; a CDRH2 that is 100% identical to SEQ ID NO:43; and / or a CDRH3 that is 100% identical to SEQ ID NO:44; a CDRL1 that is 100% identical to SEQ ID NO:57; a CDRL2 that is 100% identical to SEQ ID NO:58; and / or a CDRL3 that is 100% identical to SEQ ID NO:59. Alternatively, the ULBP6 binding protein described herein may comprise a CDRH1 that is 100% identical to SEQ ID NO:45; a CDRH2 that is 100% identical to SEQ ID NO:46; and / or a CDRH3 that is 100% identical to SEQ ID NO:47; a CDRL1 that is 100% identical to SEQ ID NO:60; a CDRL2 that is 100% identical to SEQ ID NO:61; and / or a CDRL3 that is 100% identical to SEQ ID NO:62.

[0132] The ULBP6 binding protein described herein may include a CDRH1 that is 100% identical to SEQ ID NO: 36; a CDRH2 that is 100% identical to SEQ ID NO: 37; a CDRH3 that is 100% identical to SEQ ID NO: 38; a CDRL1 that is 100% identical to SEQ ID NO: 51; a CDRL2 that is 100% identical to SEQ ID NO: 52; and a CDRL3 that is 100% identical to SEQ ID NO: 53. Alternatively, the ULBP6 binding protein described herein may include a CDRH1 that is 100% identical to SEQ ID NO: 39; a CDRH2 that is 100% identical to SEQ ID NO: 40; a CDRH3 that is 100% identical to SEQ ID NO: 41; a CDRL1 that is 100% identical to SEQ ID NO: 54; a CDRL2 that is 100% identical to SEQ ID NO: 55; and a CDRL3 that is 100% identical to SEQ ID NO: 56. Alternatively, the ULBP6 binding protein described herein may comprise a CDRH1 that is 100% identical to SEQ ID NO:42; a CDRH2 that is 100% identical to SEQ ID NO:43; a CDRH3 that is 100% identical to SEQ ID NO:44; a CDRL1 that is 100% identical to SEQ ID NO:57; a CDRL2 that is 100% identical to SEQ ID NO:58; and a CDRL3 that is 100% identical to SEQ ID NO:59. Alternatively, the ULBP6 binding protein described herein may comprise a CDRH1 that is 100% identical to SEQ ID NO:45; a CDRH2 that is 100% identical to SEQ ID NO:46; a CDRH3 that is 100% identical to SEQ ID NO:47; a CDRL1 that is 100% identical to SEQ ID NO:60; a CDRL2 that is 100% identical to SEQ ID NO:61; and a CDRL3 that is 100% identical to SEQ ID NO:62.

[0133] The ULBP6 binding protein described herein may comprise a VH region at least 90% identical to SEQ ID NO: 21 and a VL region at least 90% identical to SEQ ID NO: 31. The ULBP6 binding protein described herein may comprise a VH region at least 95% identical to SEQ ID NO: 21 and a VL region at least 95% identical to SEQ ID NO: 31. The ULBP6 binding protein described herein may comprise a VH region at least 90% identical to SEQ ID NO: 22 and a VL region at least 90% identical to SEQ ID NO: 32. The ULBP6 binding protein described herein may comprise a VH region at least 95% identical to SEQ ID NO: 22 and a VL region at least 95% identical to SEQ ID NO: 32. The ULBP6 binding protein described herein may comprise a VH region at least 90% identical to SEQ ID NO: 23 and a VL region at least 90% identical to SEQ ID NO: 33. The ULBP6 binding protein described herein may comprise a VH region at least 95% identical to SEQ ID NO: 23 and a VL region at least 95% identical to SEQ ID NO: 33. The ULBP6 binding protein described herein may comprise a VH region at least 90% identical to SEQ ID NO: 24 and a VL region at least 90% identical to SEQ ID NO: 34. The ULBP6 binding protein described herein may comprise a VH region at least 95% identical to SEQ ID NO: 24 and a VL region at least 95% identical to SEQ ID NO: 34.

[0134] The ULBP6 binding protein described herein may comprise a VH region that is 100% identical to SEQ ID NO:21 and a VL region that is 100% identical to SEQ ID NO:31; a VH region that is 100% identical to SEQ ID NO:22 and a VL region that is 100% identical to SEQ ID NO:32; a VH region that is 100% identical to SEQ ID NO:23 and a VL region that is 100% identical to SEQ ID NO:33; or a VH region that is 100% identical to SEQ ID NO:24 and a VL region that is 100% identical to SEQ ID NO:34.

[0135] The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is at least 90% identical to SEQ ID NO: 75 and / or a light chain (LC) sequence that is at least 90% identical to SEQ ID NO: 105. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is at least 95% identical to SEQ ID NO: 75 and / or a light chain (LC) sequence that is at least 95% identical to SEQ ID NO: 105. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is at least 90% identical to SEQ ID NO: 76 and / or a light chain (LC) sequence that is at least 90% identical to SEQ ID NO: 106. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is at least 95% identical to SEQ ID NO: 76 and / or a light chain (LC) sequence that is at least 95% identical to SEQ ID NO: 106.

[0136] The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 75 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 105; a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 76 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 106; a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 77 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 107; or a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 78 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 108. The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 75 and a light chain (LC) sequence that is 100% identical to SEQ ID NO: 105. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 76 and a light chain (LC) sequence that is 100% identical to SEQ ID NO: 106. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 77 and a light chain (LC) sequence that is 100% identical to SEQ ID NO: 107. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 78 and a light chain (LC) sequence that is 100% identical to SEQ ID NO: 108.

[0137] The VH or VL (or HC or LC) sequence may be a variant sequence having up to 10 amino acid substitutions, additions or deletions. For example, the variant sequence may have up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions or deletions.

[0138] The HC sequence may be a variant sequence having up to 40 amino acid substitutions, additions or deletions. For example, the variant sequence may have up to 35, 30, 25, 20, 15 or 10 amino acid substitutions, additions or deletions.

[0139] The LC sequence may be a variant sequence having up to 20 amino acid substitutions, additions or deletions. For example, the variant sequence may have up to 15, 10 or 5 amino acid substitutions, additions or deletions.

[0140] The sequence changes may exclude, for example, one or more or all of the CDRs, e.g., the CDRs are identical to the VH or VL (or HC or LC) sequence, and the changes are in the remainder of the VH or VL (or HC or LC) sequence such that the CDR sequence is fixed and intact.

[0141] Typically, the changes will be substitutions, particularly conservative substitutions, for example as shown in Table 2.

[0142] The variant sequence substantially retains the biological characteristics of the unmodified protein, such as inhibiting the binding of ULBP6 to NKG2D.

[0143] Those skilled in the art will appreciate that when antigen binding proteins, such as antibodies, are produced in host cells, post-translational modifications may occur. For example, this may include cleavage of certain leader sequences, addition of various sugar moieties in various glycosylation patterns, non-enzymatic glycosylation, deamidation, oxidation, disulfide bond disruption and other cysteine ​​variants, such as free sulfhydryls, racemic disulfides, thioethers and trisulfide bonds, isomerization, C-terminal lysine shearing and N-terminal glutamine cyclization. The disclosure herein encompasses the use of antigen binding proteins that have been or have undergone one or more post-translational modifications. Therefore, the "antigen binding protein" or "antibody" of the present invention includes an "antigen binding protein" or "antibody" as previously defined that has undergone post-translational modifications as described herein, respectively.

[0144] Glycosylation is a post-translational non-enzymatic chemical reaction between reducing sugars (e.g., glucose) and free amino groups in proteins and is usually observed at the epsilon amine of lysine side chains or at the N-terminus of proteins. Glycosylation can occur during production and storage only in the presence of reducing sugars.

[0145] Deamidation can occur during production and storage, and is an enzymatic reaction that converts asparagine (N) primarily into isoaspartic acid (isoaspartate) and aspartic acid (aspartate) (D) at a ratio of about 3:1. Therefore, this deamidation reaction is related to the isomerization of aspartate (D) to isoaspartate. Both the deamidation of asparagine and the isomerization of asparagine involve the intermediate succinimide. To a much lesser extent, glutamine residues can be deamidated in a similar manner. Deamidation can occur in CDRs, in Fab (non-CDR regions), or in Fc regions.

[0146] Oxidation can occur during production and storage (i.e., in the presence of oxidizing conditions) and lead to covalent modifications of proteins, either directly induced by reactive oxygen species or indirectly induced by reaction with secondary byproducts of oxidative stress. Oxidation occurs primarily on methionine residues, but may also occur at tryptophan and free cysteine ​​residues. Oxidation can occur in CDRs, in Fab (non-CDR regions), or in the Fc region.

[0147] Disruption of disulfide bonds may occur during production and basic storage conditions. In some cases, disulfide bonds may be broken or incorrectly formed, resulting in unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryl groups (-SH) can promote reorganization.

[0148] Thioether formation and racemization of disulfide bonds can be performed under alkaline conditions, during production or storage, by β-elimination of disulfide bridges back to cysteine ​​residues via dehydroalanine and persulfide intermediates. Subsequent cross-linking of dehydroalanine and cysteine ​​results in the formation of thioether bonds, or free cysteine ​​residues can reform disulfide bonds with a mixture of D and L-cysteine.

[0149] Trisulfides result from the insertion of a sulfur atom into a disulfide bond (Cys-SSS-Cys) and are formed due to the presence of hydrogen sulfide in the production cell culture.

[0150] N-terminal glutamine (Q) and glutamate (glutamic acid) (E) in heavy chain and / or light chain can form pyroglutamic acid (pGlu) via cyclization. The formation of most pGlu occurs in production bioreactors, but it can be formed non-enzymatically, which depends on pH and temperature of processing and storage conditions. The cyclization of N-terminal Q or E is usually observed in natural human antibodies.

[0151] C-terminal lysine cleavage is an enzymatic reaction catalyzed by carboxypeptidases and is commonly observed in recombinant and natural human antibodies. Variants of this approach include the removal of lysine from one or both heavy chains due to cellular enzymes from recombinant host cells. This may result in the removal of any remaining C-terminal lysine after administration to a human subject / patient.

[0152] Pharmaceutical composition

[0153] The antigen binding proteins as described herein can be incorporated into pharmaceutical compositions for use in treating human diseases as described herein.A pharmaceutical composition can comprise an antigen binding protein optionally in combination with one or more pharmaceutically acceptable carriers and / or excipients.

[0154] Such compositions may contain pharmaceutically acceptable carriers as required by known and accepted pharmaceutical practice.

[0155] The pharmaceutical composition can be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, and intraportal). The pharmaceutical composition may be suitable for intravenous administration. The pharmaceutical composition may be suitable for subcutaneous administration. The pharmaceutical composition may be suitable for topical administration (which includes, but is not limited to epidermal, inhalation, intranasal or ocular administration) or parenteral administration (which includes, but is not limited to oral, vaginal or rectal administration).

[0156] The pharmaceutical composition may comprise 0.5 mg to 10 g of ULBP6 binding protein, such as 5 mg to 1 g of ULBP6 binding protein. Alternatively, the composition may comprise 5 mg to 500 mg of ULBP6 binding protein, such as 5 mg to 50 mg of ULBP6 binding protein.

[0157] Methods for preparing such pharmaceutical compositions are well known to those skilled in the art. Depending on the mode of administration and the specific protein used, other excipients may be added to the composition.

[0158] The effective dosage and treatment regimen for administering the antigen binding protein may depend on various factors, such as the age, weight and health status of the patient and the disease to be treated. Such factors are within the purview of the attending physician.

[0159] The pharmaceutical composition may be included in a kit containing the antigen binding protein together with other agents and / or instructions for use. For convenience, the kit may include predetermined amounts of agents and instructions for use. The kit may also include a device for administering the pharmaceutical composition.

[0160] The terms "individual", "subject" and "patient" are used herein. Generally, the individual of interest may be a subject, and the subject in need of treatment may be a patient. The individual, subject or patient may be an animal. The individual, subject or patient may be a mammal, such as a primate, such as a marmoset or monkey. The individual, subject or patient may be a human.

[0161] The antigen binding proteins described herein can also be used in therapeutic methods. It will be appreciated by those skilled in the art that reference to treatment herein refers to the treatment of established conditions. However, the antigen binding proteins disclosed herein can also be used to prevent certain diseases, depending on the condition. The antigen binding proteins described herein are used in an effective amount for therapeutic, prophylactic or preventive treatment. A therapeutically effective amount of the antigen binding proteins described herein is an amount that effectively improves or reduces one or more symptoms of a disease or prevents or cures the symptoms of a disease.

[0162] Antigen binding proteins can be prepared by any of a variety of conventional techniques. For example, antigen binding proteins can be purified from cells that naturally express them (eg, antibodies can be purified from hybridomas that produce them) or produced in recombinant expression systems.

[0163] Provided are ULBP6 binding proteins as described herein for use in therapy. Provided are ULBP6 binding proteins for use in treating cancer. Also provided are uses of ULBP6 binding proteins in the manufacture of medicaments for treating cancer. Also provided are uses of ULBP6 binding proteins in the manufacture of medicaments for treating cancer, wherein the ULBP6 binding protein comprises a VH region containing SEQ ID NO: 21 and a VL region containing SEQ ID NO: 31. Also provided are uses of ULBP6 binding proteins in the manufacture of medicaments for treating cancer, wherein the ULBP6 binding protein comprises a VH region containing SEQ ID NO: 22 and a VL region containing SEQ ID NO: 32. Also provided are pharmaceutical compositions comprising 100 to 1400 mg of ULBP6 binding proteins. Also provided are pharmaceutical compositions comprising 100 to 1400 mg of ULBP6 binding proteins, wherein the binding proteins comprise a VH region containing SEQ ID NO: 21 and a VL region containing SEQ ID NO: 31. Also provided herein is a pharmaceutical composition comprising 100 to 1400 mg of a ULBP6 binding protein, wherein the binding protein comprises a HC sequence comprising SEQ ID NO:75 and a LC sequence comprising SEQ ID NO:105.

[0164] Also provided herein is an isolated ULBP6 binding protein or a pharmaceutical composition comprising the isolated ULBP6 binding protein for use in treating cancer.

[0165] Production method

[0166] Antigen-binding proteins can be produced using a variety of different expression systems and purification schemes. Typically, host cells are transformed with recombinant expression vectors encoding desired antigen-binding proteins. Expression vectors can be maintained or integrated into host chromosomes as separate gene elements by the host, depending on the expression system. A wide range of host cells can be used, including prokaryotic cells (including gram-negative or gram-positive bacteria, such as Escherichia coli, Bacilli sp., Pseudomonas sp., Corynebacterium sp.), eukaryotic cells, including yeast (such as Saccharomyces cerevisiae, Pichia pastoris), fungi (such as Aspergillus sp.), or higher eukaryotic organisms, including insect cells and cell lines (such as CHO, NSO, PER.C6, HEK293, HeLa) of mammalian origin.

[0167] The host cell may be an isolated host cell. The host cell is not typically part of a multicellular organism such as a plant or animal. The host cell may be a non-human host cell.

[0168] Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are known in the art.

[0169] Cells can be cultured using various equipment such as shake flasks, spinner flasks, and bioreactors under conditions that promote the expression of antigen-binding proteins. Polypeptides can be recovered by conventional protein purification procedures. Protein purification procedures can generally consist of a series of unit operations that include, for example, one or more filtration and / or chromatographic processes developed to selectively concentrate and separate antigen-binding proteins. Purified antigen-binding proteins can be formulated into pharmaceutically acceptable compositions.

[0170] Fc engineering methods can be applied to modify the functional or pharmacokinetic properties of antibodies. Effector functions can be altered by making mutations in the Fc region that increase or decrease binding to C1q or Fcγ receptors and modify CDC or ADCC activity, respectively. The glycosylation pattern of the antibody can also be modified to alter effector functions. The in vivo half-life of an antibody can be altered by making mutations that affect Fc binding to FcRn (neonatal Fc receptor).

[0171] As used herein, the term "effector function" refers to one or more of the antibody-mediated effects, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated complement activation (including complement-dependent cytotoxicity (CDC)), complement-dependent cell-mediated phagocytosis (CDCP), antibody-dependent complement-mediated cell lysis (ADCML), and Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP).

[0172] It is believed that the interaction between the Fc region of an antigen binding protein or antibody and various Fc receptors (FcRs), including FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcRn, C1q, and type II Fc receptors, mediates the effector functions of the antigen binding protein or antibody. Significant biological effects can be the result of effector functions. In general, the ability to mediate effector functions requires that the antigen binding protein or antibody binds to the antigen, and not all antigen binding proteins or antibodies mediate every effector function.

[0173] Effector function can be assessed in a variety of ways, including, for example, assessing the ADCC effector function of antibodies applied to target cells mediated by natural killer (NK) cells via FcγRIII or by monocytes / macrophages via FcγRI, or assessing the CDC effector function of antibodies applied to target cells mediated by the complement cascade via C1q. For example, the antigen binding proteins described herein can be assessed for ADCC effector function in a natural killer cell assay.

[0174] The effect of mutations on effector functions (e.g., FcRn binding, FcγRs and C1q binding, CDC, ADCML, ADCC, ADCP) can be assessed.

[0175] Throughout this specification, the amino acid residues in the Fc region of an antibody sequence or a full-length antigen-binding protein sequence are numbered according to the EU index numbering convention.

[0176] Human IgG1 constant regions containing specific mutations have been shown to enhance binding to Fc receptors. In some cases, these mutations have also been shown to enhance effector functions, such as ADCC and CDC, as described below. The antigen binding proteins described herein may include any of the following mutations.

[0177] Enhanced CDC: Fc engineering can be used to enhance complement-based effector functions. For example (refer to IgG1), K326W / E333S; S267E / H268F / S324T; and IgG1 / IgG3 cross-subclassing can increase C1q binding; E345R and E345R / E430G / S440Y lead to preformed IgG hexamers.

[0178] Enhanced ADCC: Fc engineering can be used to enhance ADCC. For example (reference IgG1), F243L / R292P / Y300L / V305I / P396L; S239D / I332E; and S298A / E333A / K334A increase FcγRIIIa binding; S239D / I332E / A330L increase FcγRIIIa binding and reduce FcγRIIb binding; G236A / S239D / I332E improves binding to FcγRIIa, improves FcγRIIa / FcγRIIb binding ratio (activation / inhibition ratio), and enhances macrophage phagocytosis of antibody-coated target cells. The ULBP6 binding protein described herein is mutated at positions 239 and 332, such as S239D and I332E. The ULBP6 binding protein described herein may be mutated at three or more positions selected from 239, 332, and 330, such as S239D, I332E, and A330L (EU index number). The ULBP6 binding protein described herein may be an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a modified Fc region comprising an amino acid substitution selected from S239D / A330L / I332E, G236A / S239D / A330L / I332E, L235V / F243L / R292P / Y300L / P396L, or F243L / R292P / Y300L / V305I / P396L. An asymmetric Fc containing L234Y / L235Q / G236W / S239M / H268D / D270E / S298A mutations in one heavy chain and D270E / K326D / A330M / K334E in the opposing heavy chain increased affinity for FcγRIIIa F158 (lower affinity allele) and FcγRIIIa V158 (higher affinity allele), but did not increase binding affinity for the inhibitory FcγRIIb.

[0179] Enhanced ADCP: Fc engineering can be used to enhance ADCP. For example (refer to IgG1), G236A / S239D / I332E increases FcγRIIa binding and increases FcγRIIIa binding.

[0180] Increased co-engagement: Fc engineering can be used to increase co-engagement with FcRs. For example (refer to IgG1), S267E / L328F increases FcγRIIb binding; N325S / L328F increases FcγRIIa binding and decreases FcγRIIIa binding.

[0181] The antigen binding proteins described herein may comprise a heavy chain constant region with an altered glycosylation profile, such that the antigen binding proteins have enhanced effector functions, such as enhanced ADCC, enhanced CDC, or enhanced ADCC and CDC. Examples of suitable methods for producing antigen binding proteins with altered glycosylation profiles are described in WO2003011878, WO2006014679, and EP1229125, all of which are applicable to the antigen binding proteins described herein.

[0182] Deletion of the innermost α1,6-fucose residue on the Fc glycan portion of IgG1 antibody N297 can enhance affinity for FcγRIIIA. Therefore, non-fucosylated or hypofucosylated monoclonal antibodies may have increased therapeutic efficacy compared to monoclonal antibodies with standard fucosylation or higher fucosylation.

[0183] Provided herein are antigen-binding proteins comprising chimeric heavy chain constant regions. The antigen-binding proteins may comprise IgG1 / IgG3 chimeric heavy chain constant regions, so that the antigen-binding proteins have enhanced effector functions, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC functions. For example, the chimeric antigen-binding proteins of the present invention may comprise at least one CH2 domain from IgG3. The antigen-binding proteins may comprise a CH2 domain from IgG3, or both CH2 domains may be from IgG3. The chimeric antigen-binding proteins may comprise IgG1 CH1 domains, IgG3 CH2 domains, and IgG3 CH3 domains. The chimeric antigen-binding proteins may comprise IgG1 CH1 domains, IgG3 CH2 domains, and IgG3 CH3 domains, except that position 435 is H (histidine).

[0184] The antigen binding protein may comprise an IgG1 CH1 domain and at least one CH2 domain from IgG3. The chimeric antigen binding protein may comprise the following residues in the IgG1 CH1 domain and CH2 domain corresponding to IgG3 residues: 274Q, 276K, 296F, 300F, and 339T. The chimeric antigen binding protein may also comprise 356E in the CH3 domain, which corresponds to IgG3 residues. The antigen binding protein may also comprise one or more of the following residues, which correspond to IgG3 residues in the CH3 domain: 358M, 384S, 392N, 397M, 422I, 435R, and 436F.

[0185] Also provided is a method for producing an antigen binding protein, the method comprising: a) culturing a recombinant host cell comprising an expression vector comprising an isolated nucleic acid as described herein, wherein the expression vector comprises a nucleic acid sequence encoding a chimeric Fc region having IgG1 and IgG3 Fc region amino acid residues (e.g., as described above); and b) recovering the antigen binding protein.

[0186] Such methods of producing antigen-binding proteins with chimeric heavy chain constant regions can be performed using, for example, the COMPLEGENT technology system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kirin Co., Ltd. The COMPLEGENT system comprises a recombinant host cell comprising an expression vector in which a nucleic acid sequence encoding a chimeric Fc region with amino acid residues of IgG1 and IgG3 Fc regions is expressed to produce an antigen-binding protein with enhanced CDC activity, i.e., an increase in CDC activity relative to other identical antigen-binding proteins lacking such a chimeric Fc region, as described in WO2007011041 and US20070148165, both of which are incorporated herein by reference. Alternatively, CDC activity can be increased by introducing sequence-specific mutations into the Fc region of the IgG chain. One of ordinary skill in the art will also recognize other suitable systems.

[0187] Also provided is a method for producing the antigen binding protein described herein, the method comprising: a) culturing a recombinant host cell comprising an expression vector comprising an isolated nucleic acid as described herein, wherein the FUT8 gene encoding α-1,6-fucosyltransferase has been inactivated in the recombinant host cell; and b) recovering the antigen binding protein.

[0188] Such methods of producing antigen binding proteins can be performed, for example, using the POTELLIGENT technology system available from BioWa, Inc. (Princeton, NJ), wherein CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with enhanced ADCC activity relative to the same monoclonal antibody produced in cells with a functional FUT8 gene, as described in US7214775, US6946292, WO0061739 and WO0231240, all of which are incorporated herein by reference. Those of ordinary skill in the art will also recognize other appropriate systems.

[0189] The antigen binding proteins described herein can be produced in a host cell in which the FUT8 gene has been inactivated. The antigen binding proteins can be produced in - / - FUT8 host cells. The antigen binding proteins can be non-fucosylated at Asn297 (IgG1). The ULBP6 binding proteins described herein can be non-fucosylated. The ULBP6 binding protein can be an antibody comprising a heavy chain (HC) of SEQ ID NO: 75 and a light chain (LC) of SEQ ID NO: 105, wherein the antibody is non-fucosylated. The ULBP6 binding protein can be an antibody comprising a heavy chain (HC) of SEQ ID NO: 76 and a light chain (LC) of SEQ ID NO: 106, wherein the antibody is non-fucosylated.

[0190] Some isotypes of human constant regions (particularly IgG4 and IgG2 isotypes) may substantially lack the functions of a) activating complement by the classical pathway and / or b) ADCC. Various modifications may be made to the heavy chain constant region of the antigen binding protein to alter the effector function according to the desired effector properties. IgG1 constant regions comprising specific mutations have been described that reduce binding to Fc receptors and reduce effector functions, such as ADCC and CDC. Such modifications may be used alone, but may also be used alone or in combination with one another to alter effector functions.

[0191] Provided herein are antigen-binding proteins comprising a heavy chain constant region, the heavy chain constant region comprising a mutated and chimeric heavy chain constant region, as described above, respectively. For example, an antigen-binding protein comprising at least one CH2 domain from IgG3 and one CH2 domain from IgG1, and wherein the IgG1 CH2 domain has one or more mutations (e.g., the mutations may be selected from S239D, I332E, and A330L) at a position selected from 239, 332, and 330, such that the antigen-binding protein has an enhanced effector function compared to an equivalent antigen-binding protein having an IgG1 heavy chain constant region lacking the mutation, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC. The IgG1 CH2 domain may have mutations S239D and I332E. The IgG1 CH2 domain may have mutations S239D, A330L, and I332E.

[0192] The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is at least 90% identical to SEQ ID NO: 95 and / or a light chain (LC) sequence that is at least 90% identical to SEQ ID NO: 105. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is at least 95% identical to SEQ ID NO: 95 and / or a light chain (LC) sequence that is at least 95% identical to SEQ ID NO: 105. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is at least 90% identical to SEQ ID NO: 96 and / or a light chain (LC) sequence that is at least 90% identical to SEQ ID NO: 106. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is at least 95% identical to SEQ ID NO: 96 and / or a light chain (LC) sequence that is at least 95% identical to SEQ ID NO: 106.

[0193] The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 95 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 105; a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 96 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 106; a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 97 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 107; or a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 98 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 108. The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 95 and a light chain (LC) sequence that is 100% identical to SEQ ID NO: 105. The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO:96 and a light chain (LC) sequence that is 100% identical to SEQ ID NO:106.

[0194] Alternatively, an antigen binding protein is provided, comprising a chimeric heavy chain constant region and an altered glycosylation profile, as described above separately. The antigen binding protein may comprise an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less. The heavy chain constant region may comprise at least one CH2 domain from IgG3 and one CH2 domain from IgG1, and have an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less, for example, wherein the antigen binding protein is defucosylated. The antigen binding protein may have enhanced effector functions, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and enhanced CDC, compared to an equivalent antigen binding protein having an IgG1 heavy chain constant region lacking the glycosylation profile.

[0195] Alternatively, the antigen binding protein has at least one IgG3 heavy chain CH2 domain and at least one heavy chain constant domain from IgGl, wherein both IgG CH2 domains are mutated according to the restrictions described herein.

[0196] Provided herein are methods for producing an antigen binding protein described herein, the method comprising: a) culturing a recombinant host cell comprising an expression vector comprising an isolated nucleic acid described herein, wherein the expression vector comprises a nucleic acid sequence encoding a chimeric Fc domain having amino acid residues of IgG1 and IgG3 Fc domains (e.g., as described above); and wherein a FUT8 gene encoding α-1,6-fucosyltransferase has been inactivated in the recombinant host cell; and b) recovering the antigen binding protein.

[0197] Such methods of producing antigen binding proteins can be performed, for example, using the ACCRETAMAB technology system commercially available from BioWa, Inc. (Princeton, NJ), which combines the POTELLIGENT and COMPLEGENT technology systems to produce antigen binding proteins having enhanced ADCC and CDC activities relative to an otherwise identical monoclonal antibody that lacks a chimeric Fc domain and is fucosylated.

[0198] Also provided are antigen-binding proteins comprising a mutated and chimeric heavy chain constant region, wherein the antigen-binding protein has an altered glycosylation profile such that the antigen-binding protein has an enhanced effector function, such as enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC. The mutation may be selected from positions 239, 332, and 330, such as S239D, I332E, and A330L. The heavy chain constant region may comprise at least one CH2 domain from IgG3 and a CH1 domain from IgG1. The heavy chain constant region may have an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less, such as an antigen-binding protein that is defucosylated such that the antigen-binding protein has an enhanced effector function compared to an equivalent non-chimeric antigen-binding protein lacking the mutation and lacking the altered glycosylation profile.

[0199] Provided herein are antigen-binding proteins comprising constant regions, so that antigen-binding proteins have reduced effector functions, such as reduced ADCC and / or CDC. Heavy chain constant region may include the natural incapacitated constant region of IgG2 or IgG4 isotype or the mutated IgG1 constant region. Examples of suitable modifications are described in EP0307434. An example includes replacing with alanine at positions 235 and 237 (EU index numbering), i.e., L235A and G237A (commonly referred to as "LAGA" mutations). Another example includes replacing with alanine at positions 234 and 235 (EU index numbering), i.e., L234A and L235A (commonly referred to as "LALA" mutations). Other examples described in EP2691417 and US8969526 include the combination of P329G or P329R with the LALA mutation (EU index numbering) of IgG1 Fcs, and the combination of P329G or P329R with S228P and L235E (EU index numbering) of IgG4 Fcs.

[0200] Other changes and mutations that reduce effector function may include: (unless otherwise specified, reference is made to IgG1): N297A or N297Q or N297G without glycosylation; L235E; IgG4: F234A / L235A; and chimeric IgG2 / IgG4. IgG2: H268Q / V309L / A330S / P331S and IgG2: V234A / G237A / P238S / H268A / V309L / A330S / P331S can reduce FcγR binding to C1q (US8961967). The ULBP6 binding protein described herein may comprise a modified Fc region, wherein the modified Fc region comprises the amino acid substitutions N297A, N297Q or N297G.

[0201] Other mutations that reduce effector function include L234F / L235E / P331S; a chimeric antibody generated using the CH1 and hinge regions from human IgG2 and the CH2 and CH3 regions from human IgG4; IgG2m4, based on an IgG2 isotype derived from IgG4 with four key amino acid residue changes (H268Q, V309L, A330S, and P331S); IgG2σ containing V234A / G237A / P238S / H268A / V309L / A330S / P331S substitutions to eliminate affinity for Fcγ receptors and C1q complement proteins; IgG2m4 (H268Q / V309L / A330S / P331S, changed to IgG4); IgG4 (S228P / L234A / L235A); huIgG1L234A / L235A (AA); huIgG4 S228P / L234A / L235A; IgG1s (L234A / L235A / G237A / P238S / H268A / A330S / P331S); IgG4s1 (S228P / F234A / L235A / G237A / P238S); and IgG4s2 (S228P / F234A / L235A / DG236 / G237A / P238S, wherein D indicates deletion).

[0202] The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence at least 90% identical to SEQ ID NO: 85 and / or a light chain (LC) sequence at least 90% identical to SEQ ID NO: 105. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence at least 95% identical to SEQ ID NO: 85 and / or a light chain (LC) sequence at least 95% identical to SEQ ID NO: 105. The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence at least 90% identical to SEQ ID NO: 86 and / or a light chain (LC) sequence at least 90% identical to SEQ ID NO: 106. Alternatively, the ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence at least 95% identical to SEQ ID NO: 86 and / or a light chain (LC) sequence at least 95% identical to SEQ ID NO: 106.

[0203] The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 85 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 105; a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 86 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 106; a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 87 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 107; or a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 88 and / or a light chain (LC) sequence that is 100% identical to SEQ ID NO: 108. The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO: 85 and a light chain (LC) sequence that is 100% identical to SEQ ID NO: 105. The ULBP6 binding protein described herein may comprise a heavy chain (HC) sequence that is 100% identical to SEQ ID NO:86 and a light chain (LC) sequence that is 100% identical to SEQ ID NO:106.

[0204] Half-life (t 1 / 2 ) refers to the time required for the serum concentration of an antigen binding protein to reach half of its original value. The serum half-life of a protein can be measured by pharmacokinetic studies according to one method, wherein a radiolabeled protein is injected intravenously into mice and its plasma concentration is measured over time at regular intervals, such as from about 3 minutes to about 72 hours after injection. Those skilled in the art will be familiar with other methods for pharmacokinetic analysis and determination of molecular half-life.

[0205] It has been reported that the long half-life of IgG antibodies depends on their binding to FcRn. Therefore, substitutions that increase the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependence of the interaction with the target have been widely studied by engineering the constant region. The in vivo half-life of the antigen binding protein described herein can be altered by modifying the heavy chain constant domain or the FcRn binding domain therein.

[0206] In adult mammals, FcRn, also known as the neonatal Fc receptor, plays a key role in maintaining serum antibody levels by acting as a protective receptor that binds and rescues antibodies of the IgG isotype from degradation. IgG molecules are endocytosed by endothelial cells and, if they bind to FcRn, are recycled from the cells back into the circulation. In contrast, IgG molecules that enter the cell do not bind to FcRn, but are targeted to the lysosomal pathway where they are degraded.

[0207] FcRn may be involved in antibody clearance and transcytosis across tissues. Human IgG1 residues that have been determined to interact directly with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435. Mutations at any of these positions may increase the serum half-life and / or altered effector properties of the antigen-binding proteins of the invention.

[0208] The antigen binding proteins described herein may have amino acid modifications that increase the affinity of the constant region or its fragment to FcRn. Increasing the half-life (i.e., serum half-life) of therapeutic and diagnostic IgG antibodies and other bioactive molecules has many benefits, including reducing the dosage and / or frequency of these molecules. The antigen binding proteins may comprise all or a portion (FcRn binding portion) of the IgG constant domain having one or more of the following amino acid modifications.

[0209] For example, with reference to IgG1, M252Y / S254T / T256E (commonly referred to as "YTE" mutations) and / or M428L / N434S (commonly referred to as "LS" mutations) can increase FcRn binding at pH 6.0.

[0210] Half-life can also be enhanced by T250Q / M428L, V259I / V308F / M428L, N434A, and T307A / E380A / N434A mutations (eg, with reference to IgG1 and Kabat numbering).

[0211] Half-life and FcRn binding can also be extended by introducing H433K and N434F mutations (often referred to as "HN" or "enhancing" mutations) (cf. IgG1) (WO2006 / 130834).

[0212] WO00 / 42072 discloses polypeptides comprising variant Fc regions with altered FcRn binding affinity, the polypeptides comprising amino acid modifications at any one or more of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 and 447 (EU index numbering) of the Fc region.

[0213] WO02 / 060919 discloses a modified IgG comprising an IgG constant domain comprising one or more amino acid modifications relative to a wild-type IgG constant domain, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having a wild-type IgG constant domain, and wherein the one or more amino acid modifications are located at one or more of positions 251, 253, 255, 285-290, 308-314, 385-389 and 428-435.

[0214] Shields et al. (2001, J Biol Chem; 276: 6591-604) used alanine scanning mutagenesis to change residues in the Fc region of human IgG1 antibodies, and then evaluated binding to human FcRn. When changed to alanine, the positions that effectively abolished binding to FcRn included I253, S254, H435, and Y436. Other positions showed less significant reduction in binding, as shown below: E233-G236, R255, K288, L309, S415, and H433. When changed to alanine, several amino acid positions showed improvements in FcRn binding; of these, it is worth noting that P238, T256, E272, V305, T307, Q311, D312, K317, D376, E380, E382, S424, and N434. Many other amino acid positions showed either slight improvements in FcRn binding (D265, N286, V303, K360, Q 362, and A378), or no changes (S239, K246, K248, D249, M252, E258, T260, S267, H268, S269, D270, K274, N276, Y278, D280, V282, E283, H285, T289, K290, R292, E293, E294, Q295, Y296, N297, S298, R301, N315, E318, K320, K322, S324, K326, A327, P329, P331, E332, 3. K334, T335, S337, K338, K340, Q342, R344, E345, Q345, Q347, R356, M358, T359, K360, N361, Y373, S375, S383, N384, Q386, E388, N389, N390, K392, L398, S400, D401, K414, R416, Q418, Q419, N421, V422, E430, T437, K439, S440, S442, S444 and K447).

[0215] The combination variants were found to be most effective in improving FcRn binding. At pH 6.0, the E380A / N434A variant showed 8-fold better binding to FcRn relative to native IgG1, compared to 2-fold for E380A and 3.5-fold for N434A. Addition of T307A resulted in a 12-fold improvement in binding relative to native IgG1. The antigen binding proteins described herein may comprise the E380A / N434A mutations and have increased binding to FcRn.

[0216] Dall'Acqua et al. (2002, J Immunol.; 169: 5171-80) describe random mutagenesis and screening of a human IgG1 hinge-Fc fragment phage display library against mouse FcRn. They disclose random mutagenesis of positions 251, 252, 254-256, 308, 309, 311, 312, 314, 385-387, 389, 428, 433, 434 and 436. The major improvement in the stability of the IgG1-human FcRn complex occurs when replacing residues located in the band spanning the Fc-FcRn interface (M252, S254, T256, H433, N434 and Y436) and, to a lesser extent, at the periphery (e.g., V308, L309, Q311, G385, Q386, P387 and N389). The variant with the highest affinity for human FcRn was obtained by combining M252Y / S254T / T256E ("YTE") and H433K / N434F / Y436H mutations, and the variant showed a 57-fold increase in affinity relative to wild-type IgG1. The in vivo behavior of this mutant human IgG1 showed a nearly 4-fold increase in serum half-life in cynomolgus monkeys compared to wild-type IgG1.

[0217] Thus, antigen binding proteins with optimized binding to FcRn are also provided. The antigen binding protein may comprise at least one amino acid modification in the Fc region of the antigen binding protein, wherein the modification is located at an amino acid position selected from the group consisting of: 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325 ,307,308,309,311,315,317,320,322,325,327,330,332,334,335,338,340,342,343,345,347,350,352,354,355,356,359,360,361,362,369,370,371,375,378,380,382,384,385,386,387,389,390,392,393,394,395,396,397,398,399,400,401 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447.

[0218] In addition, various publications describe methods for obtaining physiologically active molecules with modified half-lives, either by introducing an FcRn binding polypeptide into the molecule (WO97 / 43316, US5869046, US5747035, WO96 / 32478 and WO91 / 14438), or by fusing the molecule to an antibody whose FcRn binding affinity is retained but whose affinity for other Fc receptors is greatly reduced (WO99 / 43713), or by fusing to the FcRn binding domain of an antibody (WO00 / 09560, US4703039).

[0219] Fc variants with enhanced FcRn affinity were identified in a screen at pH 6.0 to improve antibody cytotoxicity and half-life. The selected IgG variants can be produced as hypofucosylated molecules. The generated variants showed increased serum persistence in hFcRn mice, as well as conserved enhanced ADCC. Exemplary variants may include (with reference to IgG1 and Kabat numbering): P230T / V303A / K322R / N389T / F404L / N434S; P228R / N434S; Q311R / K334R / Q342E / N434Y; C226G / Q386R / N434Y; T307P / N389T / N434Y; P230S / N434S; P230T / V305A / T307A / A378V / L398P / N434S; P23OT / P387S / N434S; P230Q / E269D / N434S; N276S / A378V / N434S; T307A / N315D / A330V / 382 V / N389T / N434Y; T256N / A378V / S383N / N434Y; N315D / A330V / N361D / A387V / N4 34Y; V259I / N315D / M428L / N434Y; P230S / N315D / M428L / N434Y; F241L / V264E / T 307P / A378V / H433R; T250A / N389K / N434Y; V305A / N315D / A330V / P395A / N434Y; V264E / Q386R / P396L / N434S / K439R; and E294del / T307P / N434Y (where "del" indicates deletion).

[0220] Although substitutions in the constant region can significantly improve the function of therapeutic IgG antibodies, substitutions in strictly conserved constant regions have the risk of producing immunogenicity in humans, and substitutions in highly diverse variable region sequences may be less immunogenic. Reports on the variable region include engineering of CDR residues to improve binding affinity to antigens, and engineering of CDR and framework residues to improve stability and reduce the risk of immunogenicity. Affinity to antigens can be improved by affinity maturation using phage or ribosome display of random libraries.

[0221] Rational design based on sequence and structure can reasonably improve stability.Reducing the risk of immunogenicity (deimmunization) can be achieved by various humanization methods and removing potential T cell epitopes, which can be predicted using computer simulation technology or expected by in vitro assays.In addition, the variable region has been engineered to reduce pI.Although comparable to FcRn binding, compared with wild-type antibodies, it is observed that these antibodies have a longer half-life.Engineering or selecting antibodies with pH-dependent antigen binding can be used to modify antibodies and / or antigen half-life, for example, if antigen-mediated clearance mechanisms normally degrade antibodies when antibodies bind to antigens, then IgG2 antibody half-life can be shortened.Similarly, antigen: antibody complexes can affect the half-life of antigens, either by protecting antigens from typical degradation processes to extend the half-life, or by shortening the half-life via antibody-mediated degradation (target-mediated drug disposal). The antibody has a higher affinity for the antigen at pH 7.4 compared to endosomal pH (i.e., pH 5.5-6.0), such that the KD ratio at pH 5.5 / pH 7.4 or pH 6.0 / pH 7.4 is 2 or higher. For example, to enhance the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the antibody, pH sensitivity can be engineered into the antibody by introducing histidine residues into the CDRs.

[0222] Recirculating antibodies are engineered so that a single recycling antibody molecule can bind to an antigen multiple times (e.g., twice or more). Recirculating antibodies can dissociate from antigens under acidic conditions within cells. For example, recycling antibodies bound to membrane-bound antigens dissociate from antigens in a pH-dependent manner. Dissociated recycling antibodies are recycled by FcRn, and antigens are transferred to lysosomes and degraded. This mechanism enables recycling antibodies to repeatedly bind to antigens in plasma and can reduce antibody clearance.

[0223] Sweeping antibodies incorporate two antibody engineering techniques: one is variable region engineering ("pH switch" as described above) to enable the antibody to bind to antigens in plasma and dissociate from antigens in endosomes (after which the antigen undergoes lysosomal degradation), and the other is by engineering the constant region to increase endosome-mediated cellular uptake of the antibody-antigen complex, such as through FcRn, FcγRIIb or potentially other surface receptors. Sweeping antibodies can therapeutically target soluble antigens and enhance their elimination from circulation (Igawa T et al., Sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens from circulation. Immunol Rev. 2016 Mar; 270(1): 132-51). Igawa et al. developed a group of Fc variants with enhanced binding to FcRn, including M252Y, V308P, and N434Y, which have enhanced binding to FcRn compared to wild-type Fc antibody variants, which combined with pH-dependent binding to the target soluble IL-6 receptor enhanced the clearance of the target antigen (PLoS One.8(5):e63236, 2013). FcγRIIb can be used to accelerate the uptake rate of antibody-antigen complexes into cells. Igawa et al. developed an FcγRIIb sweeping antibody in which the Fc region of the pH-dependent antibody was engineered to selectively increase human FcγRIIb binding to increase the uptake rate of antibody-antigen complexes. The important role of this inhibitory receptor is to take up antibody-antigen complexes into liver endothelial cells (LSECs), so if the uptake of antibody-antigen complexes into cells is mediated by human FcγRIIb, it can reduce the concentration of antigens in the circulation. Comprehensive mutagenesis of the Fc domain of human IgG was used to identify an Fc variant (v12) containing the following mutations: E233D / G237D / P238D / H268D / P271G / A330R, which selectively increased binding affinity to human FcγRIIb. Studies in human FcγRIIb transgenic mice showed that the v12 variant accelerated the clearance of hsIL-6R, which exceeded that of a pH-dependent sweeping antibody with wild-type hIgG1, while maintaining comparable pharmacokinetics.

[0224] Also provided are immunoconjugates (interchangeably referred to as "antibody-drug conjugates," "ADCs," or "antigen binding protein-drug conjugates") comprising an antigen binding protein described herein conjugated to one or more drugs, such as a cytotoxic agent, a chemotherapeutic agent, an immunotherapeutic agent, a growth inhibitory agent, a toxin (e.g., a protein toxin such as an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), an antiviral agent, a radioactive isotope (e.g., a radioconjugate), an antibiotic, or a small interfering RNA (siRNA).

[0225] Immunoconjugates have been used to locally deliver cytotoxic agents, i.e., drugs that kill or inhibit cell growth or proliferation, in cancer therapy. Immunoconjugates allow, in particular, targeted delivery of drug moieties to tumors, where they accumulate intracellularly, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells. Compared to traditional chemotherapy, immunoconjugates can enable the selective delivery of potent cytotoxic payloads to target cancer cells, thereby improving efficacy, reducing systemic toxicity, and obtaining preferred pharmacokinetics (PK) / pharmacodynamics (PD) and biodistribution.

[0226] Both polyclonal antibodies and monoclonal antibodies can be used in these strategies. The drugs used in these methods include daunomycin, doxorubicin, methotrexate and vindesine. The toxins used in the antibody-toxin conjugates can include bacterial toxins (such as diphtheria toxin), plant toxins (such as ricin), small molecule toxins (such as geldanamycin), maytansines (EP 1391213) and calicheamicin.

[0227] An immunoconjugate can comprise an antigen binding protein (such as an antibody), and a drug (such as a toxin, e.g., a chemotherapeutic agent). The drug can be modified (e.g., via standard synthetic chemistry) to allow its chemical attachment (e.g., to include a reactive handle to allow its chemical attachment) to the reactive end of a linker that couples the drug to the antigen binding protein.

[0228] Also provided is a method of treating cancer in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of a ULBP6 binding protein or pharmaceutical composition as defined herein. The subject may be an animal or a human. The subject may be a human. In some embodiments, the subject may be a patient.

[0229] Cancers that can be treated include: adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, carcinosarcoma, melanoma, adrenal carcinoma, adrenocortical carcinoma, pheochromocytoma, breast cancer, ductal carcinoma in situ, lobular carcinoma, inflammatory breast cancer, invasive ductal carcinoma, Paget's disease of the nipple, papillary breast cancer, medullary carcinoma, breast cancer, anal cancer, cloacal carcinoma, anorectal melanoma, appendix cancer, appendix neuroendocrine tumor, appendix mucinous cystadenocarcinoma, colonic adenocarcinoma, signet ring cell adenocarcinoma, goblet cell carcinoma / adenoneuroendocrine carcinoma, bile duct cancer, intrahepatic bile duct cancer, extrahepatic bile duct cancer, Perihilar cholangiocarcinoma, distal extrahepatic cholangiocarcinoma, colorectal cancer, mucinous adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, esophageal cancer, esophageal squamous cell carcinoma, small cell carcinoma, leiomyoma, gallbladder cancer, nonpapillary adenocarcinoma, papillary adenocarcinoma, gastric cancer, gastric adenocarcinoma, liver cancer, hepatocellular carcinoma, fibrolamellar carcinoma, angiosarcoma, lymphangiosarcoma, angiosarcoma, hepatoblastoma, pancreatic cancer, pancreatic adenocarcinoma, ductal adenocarcinoma, acinar adenocarcinoma, acinar cell carcinoma, colloid carcinoma, giant cell tumor, hepatoid carcinoma, mucinous cystic neoplasm, pancreatic blastoma, serous cystadenoma, intraductal papillary mucinous neoplasm, pancreatic neuroendocrine tumor, gastrinoma, insulinoma, glucagonoma, vipoma, somatostatinoma, pancreatic polypeptide tumor, small intestine cancer, eye cancer, intraocular melanoma, intraocular lymphoma, intraocular retinoblastoma, conjunctival melanoma, eyelid cancer, sebaceous gland carcinoma, lacrimal gland tumor, malignant mixed epithelial tumor, adenoid cystic carcinoma, bladder cancer, urothelial carcinoma, kidney cancer, renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe cell RCC, collecting duct RCC, multilocular Cystic RCC, mucinous tubular and spindle cell carcinoma of the kidney, tubulocystic RCC, thyroidoid follicular RCC, acquired cystic kidney disease-associated RCC, RCC with t(6;11) translocation (TFEB), mixed large oncocytoma / chromophobe RCC, Wilms tumor, penile cancer, prostate cancer, castration-resistant prostate cancer, transitional cell carcinoma, testicular cancer, seminoma, classic seminoma, spermatocytic seminoma, nonseminoma, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, teratoma, Leydig cell tumor cell tumour), Sertoli cell tumour, rete testis cancer, urethral cancer, extracranial germ cell tumor, germ cell tumor, gonadoblastoma, mixed germ cell tumor, extragonadal germ cell tumor, endodermal sinus tumor, cervical cancer, cervical squamous cell carcinoma, endometrial cancer, ovarian cancer, fallopian tube cancer, epithelial cancer, dysgerminoma, sex cord-stromal tumor, gestational trophoblastic tumor, primary peritoneal cancer, uterine sarcoma, uterine papillary serous carcinoma, vaginal cancer, clear cell adenocarcinoma, vulvar cancer, verrucous carcinoma, head and neck cancer, head and neck squamous cell carcinoma, pharyngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, non-keratinizing squamous cell carcinoma, undifferentiated carcinoma, laryngeal cancer, oral cancer, oral cancer,Mucoepidermoid carcinoma, paranasal sinus and nasal cavity cancer, olfactory neuroblastoma, salivary gland cancer, epithelial-myoepithelial carcinoma, parathyroid cancer, thyroid cancer, papillary thyroid cancer, thyroid follicular carcinoma, Hurthle cell carcinoma, medullary thyroid cancer, anaplastic thyroid cancer, paraganglioma, carotid paraganglioma, cervical paraganglioma, vagal paraganglioma, leukemia, acute lymphoblastic leukemia, T-lymphoblastic leukemia, precursor B-cell lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute megakaryocytic leukemia, erythroleukemia, chronic lymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia leukemia, T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, T-cell large granular lymphocytic leukemia, NK-cell granular lymphocytic leukemia, hairy cell leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, plasma cell leukemia, lymphoma, Hodgkin's lymphoma, classical Hodgkin's lymphoma, nodular sclerosis classical Hodgkin's lymphoma, mixed cellularity classical Hodgkin's lymphoma, lymphocyte-rich classical Hodgkin's lymphoma, lymphocyte-depleted classical Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, primary effusion lymphoma lymphoma, T cell / histiocyte-rich large B cell lymphoma, lymphoplasmacytic lymphoma, lymphoblastic lymphoma, small lymphocytic lymphoma, double hit / triple hit lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma, small non-cleaved cell lymphoma, follicular lymphoma, follicular large cell lymphoma, immunoblastic lymphoma, intravascular large cell lymphoma, primary splenic lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, marginal zone lymphoma (MZL), extranodal MZL, intranodal MZL, splenic MZL, splenic MZL with villous lymphocytes, peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma, adult T cell lymphoma / leukemia, extranodal NK / T cell lymphoma, enteropathy-associated T cell lymphoma, liver Splenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, T-cell non-Hodgkin lymphoma not otherwise specified, gamma / delta T-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, post-transplant lymphoproliferative disorder, HIV-associated lymphoma, Langerhans cell histiocytosis, multiple myeloma, smoldering multiple myeloma, active multiple myeloma, plasmacytoma, solitary plasmacytoma of bone, extramedullary plasmacytoma, primary amyloidosis, myelodysplastic syndrome, refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia in transformation with excess blasts, myeloproliferative neoplasm, polycythemia vera, essential thrombocythemia, myelofibrosis,Systemic mastocytosis, bone cancer, Ewing's sarcoma, osteosarcoma, intramedullary osteosarcoma, juxtacortical osteosarcoma, extraskeletal osteosarcoma, malignant fibrous histiocytoma of bone, chordoma, typical chordoma, chondroid chordoma, dedifferentiated chordoma, chondrosarcoma, conventional chondrosarcoma, clear cell chondrosarcoma, myxoid chondrosarcoma, mesenchymal chondrosarcoma, dedifferentiated chondrosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, grape Grape-shaped rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, soft tissue sarcoma, extraskeletal sarcoma, dermatofibrosarcoma protuberans, epithelioid sarcoma, Kaposi's sarcoma, liposarcoma, malignant peripheral nerve sheath tumor, fibrosarcoma, myxosarcoma, synovioma, brain cancer, anaplastic astrocytoma, glioblastoma, glioblastoma multiforme, meningioma, pituitary adenoma, neurilemmoma, oligodendroglioma, ependymoma, medulloblastoma, astrocytoma , brainstem glioma, atypical teratoid / rhabdoid tumor, pinealoma, neuroblastoma, primary CNS lymphoma, primitive neuroectodermal tumor, diffuse intrinsic pontine glioma, lung cancer, non-small cell lung cancer (NSCLC), undifferentiated NSCLC, small cell lung cancer, squamous cell carcinoma of the lung, pleural pulmonary blastoma, bronchogenic carcinoma, malignant mesothelioma, malignant pleural mesothelioma, malignant peritoneal mesothelioma, thymoma, thymic carcinoma, skin cancer, keratoacanthoma, sebaceous gland carcinoma, sweat gland carcinoma, apocrine carcinoma, eccrine carcinoma, clear cell eccrine carcinoma, Merkel cell carcinoma, cutaneous T-cell lymphoma, mycosis fungoides, Sézary syndrome, chondroid syringoma, HPV-related carcinoma, tumors containing transformed cells, tumors containing cells in a premalignant state, premalignant hyperplasia, premalignant metaplasia, premalignant dysplasia, carcinoma in situ, mixed tumor, malignant mixed tumor, and combined carcinoma. ,

[0230] Treatable cancers include cancers with high tumor mutation burden (TMB), defective DNA mismatch repair system (dMMR), high microsatellite instability status (MSI-H), low microsatellite instability status (MSI-L), cancers with elevated microsatellite alterations at selected tetranucleotide repeat sequences (EMAST), microsatellite stable (MSS) cancers, cancers containing polymerase delta mutations (POLD), cancers containing polymerase epsilon mutations (POLE), or cancers with homologous recombination repair deficiency (HRD).

[0231] Cancers that may be treated include breast cancer defined by expression profile (triple negative breast cancer, HER2 positive breast cancer, luminal A breast cancer, luminal B breast cancer, normal-like breast cancer) or breast cancer with a BRCA1 or BRCA2 mutation.

[0232] The cancer to be treated may express cell surface ULBP6 or sULBP6. sULBP6 shed from cancer cells can create an immunosuppressive environment. Therefore, the ULBP6 binding protein described herein can effectively treat cancers that express sULBP6. Therefore, treatable cancers include cancers that have been shown to express sULBP6. Treatable cancers include head and neck cancer, cervical cancer, bladder cancer, lung cancer, colorectal cancer, pancreatic cancer or glioblastoma. Treatable cancers include squamous cell carcinoma. The cancer to be treated may be head and neck squamous cell carcinoma (HNSCC). The cancer to be treated may be lung cancer. The cancer to be treated may be lung cancer. Lung cancer may be NSCLC.

[0233] The genetic data described in Example 1 show that single nucleotide polymorphisms at the ULBP6 locus are associated with sensitivity in the diagnosis of human basal cell carcinoma. Therefore, ULBP6 antagonists can be used to treat basal cell carcinoma. The ULBP6 binding proteins described herein can be used to treat basal cell carcinoma.

[0234] The ULBP6 binding proteins described herein can bind to cell surface ULBP6. For example, the ULBP6 binding protein can bind to ULBP6 (endogenous or otherwise bound) expressed on the surface of a tumor cell or cell line (such as a cell expressing CHO UBLP6, a COV644 cell, or a PANC02.13 cell). The half-maximal effective concentration (EC50) at which the ULBP6 binding protein binds to the cell surface ULBP6 can be determined using the FACS binding assay described in Example 7. The ULBP6 binding protein can bind to PANC02.13 cells and COV644 cells at an EC50 of less than or equal to 2 nM or less than or equal to 1 nM.

[0235] The ULBP6 binding proteins described herein can inhibit the binding of NKG2D-Fc to cell surface ULBP6. In some cases, the ULBP6 binding proteins can inhibit the binding of NKG2D-Fc to cell surface ULBP6 with an IC50 of less than or equal to 1 nM, less than or equal to 0.5 nM, less than or equal to 0.25 nM, or less than or equal to 0.2 nM.

[0236] Soluble ULBP6 can reduce NKG2D-mediated cell activation by binding to NKG2D, thereby inhibiting the binding of other NKG2DLs to NKG2D. sULBP6 may reduce NKG2D-mediated cell activation by binding to NKG2D, thereby inducing NKG2D internalization or blocking NKG2D-mediated activation. sULBP6 can reduce NKG2D-mediated cell activation by binding to NKG2D, thereby inducing NKG2D downregulation or blocking NKG2D-mediated activation. The ULBP6 binding protein described herein can increase NKG2D-mediated cell activation by inhibiting sULBP6-induced NKG2D blockade. Alternatively, the ULBP6 binding protein can increase NKG2D-mediated cell activation by inhibiting sULBP6-induced NKG2D internalization. Alternatively, the ULBP6 binding protein can increase NKG2D-mediated cell activation by inhibiting sULBP6-induced NKG2D downregulation. Alternatively, the ULBP6 binding protein can increase NKG2D-mediated cell activation by inhibiting sULBP6-induced NKG2D downregulation.

[0237] In some embodiments, the ULBP6 binding proteins provided herein can target tumor immune response escape mechanisms. Immune escape through MHC deletion can lead to vulnerability to NK-mediated killing. This can be driven by antigenic immunogenicity (e.g., new antigens derived from viral and / or tumor gene changes). Therefore, immune escape can occur through soluble NKG2D ligands (e.g., ULBP6). In some embodiments, the ULBP6 binding proteins provided herein can interfere with such immune escape. In some cases, tumor expression of ULBP2 / 5 / 6 plus a second NKG2D ligand can trigger drug activity in NK-infiltrated tumors (e.g., MICA / B prioritized by affinity). In some embodiments, soluble ULBP can be a readout of ULBP2 / 5 / 6+tumor burden, and is therefore a substitute for IHC in some cases.

[0238] The ULBP6 binding proteins described herein can inhibit the binding of sULBP6 to NKG2D, thereby increasing the activation of NKG2D through interaction with other NKG2DLs, such as MICA. The assay described in Example 9 can be used to determine MICA-mediated cell activation.

[0239] In addition to inhibiting the binding of sULBP6 to NKG2D, the ULBP6 binding protein described herein may also include a modified Fc region to enhance ADCC. The dual mechanisms of 1) inhibiting the binding of sULBP6 to NKG2D and 2) Fc receptor-mediated killing of tumor cells expressing ULBP6 can synergistically and effectively treat cancer. Compared with the ULBP6 binding protein lacking Fc effector function, the ADCC-enhanced ULBP6 binding protein can synergistically increase the levels of cytokines (e.g., IFNy) secreted by peripheral blood mononuclear cells (PBMCs) co-cultured with tumor cells expressing ULBP6. The ULBP6 binding protein can inhibit the binding of sULBP6 to NKG2D, thereby increasing the killing of tumor cells in vitro.

[0240] The ULBP6 binding protein described herein can bind to human ULBP6 and disrupt the interaction of ULBP6 with NKG2D. The NKG2D binding interface of ULBP6 includes ULBP6 residues that can interact with residues from NKG2D. By interacting with residues contained within or near the NKG2D binding interface of ULBP6, the ULBP6 binding protein can disrupt the interaction of ULBP6 with NKG2D. The HDX assay described in Example 17 can be used to characterize the interaction between ULBP6 and anti-ULBP6 antibodies. The ULBP6 binding protein can bind to one or more residues contained in alpha helix 1 (SEQ ID NO: 125) of human ULBP6 and / or one or more residues contained in alpha helix 2 (SEQ ID NO: 126). The ULBP6 binding protein can bind to one or more residues contained in SEQ ID NO: 128 and / or SEQ ID NO: 129 of human ULBP6. The ULBP6 binding protein can bind to a conformational epitope contained within alpha helix 1 (SEQ ID NO: 125) and / or alpha helix 2 (SEQ ID NO: 126) of human ULBP6. The ULBP6 binding protein can bind to a conformational epitope contained within SEQ ID NO: 128 and / or SEQ ID NO: 129 of human ULBP6. The ULBP6 binding protein can protect one or more residues contained within alpha helix 1 (SEQ ID NO: 125) and / or alpha helix 2 (SEQ ID NO: 126) of human ULBP6 from deuterium exchange in HDX-MS analysis. The ULBP6 binding protein can protect one or more residues contained within SEQ ID NO: 128 and / or SEQ ID NO: 129 of human ULBP6 from deuterium exchange in HDX-MS analysis. The ULBP6 binding protein can bind to human ULBP6 and cause peptides derived from alpha helix 1 (SEQ ID NO: 125) and / or alpha helix 2 (SEQ ID NO: 126) of human ULBP6 to be more resistant to deuterium incorporation than the corresponding peptides derived from uncomplexed human ULBP6. The ULBP6 binding protein can bind to human ULBP6 and cause peptides derived from SEQ ID NO: 128 and / or SEQ ID NO: 129 of human ULBP6 to be more resistant to deuterium incorporation than the corresponding peptides derived from uncomplexed human ULBP6. The ULBP6 binding protein can bind to the NKG2D binding interface of human ULBP6. The ULBP6 binding protein can disrupt the interaction of human ULBP6 with NKG2D by binding to one or more residues selected from E96, E154, Y184 and D189 located within the NKG2D binding interface of human ULBP6.

[0241] The ULBP6 binding proteins described herein may bind to one or more residues contained within alpha helix 1 (SEQ ID NO: 125), one or more residues contained within alpha helix 2 (SEQ ID NO: 126), and / or one or more residues contained within SEQ ID NO: 127 of human ULBP6. The ULBP6 binding protein may bind to one or more residues contained within SEQ ID NO: 128, SEQ ID NO: 129, and / or SEQ ID NO: 127 of human ULBP6. The ULBP6 binding protein may bind to a conformational epitope contained within alpha helix 1 (SEQ ID NO: 125), alpha helix 2 (SEQ ID NO: 126), and / or SEQ ID NO: 127 of human ULBP6. The ULBP6 binding protein may bind to a conformational epitope contained within SEQ ID NO: 128, SEQ ID NO: 129, and / or SEQ ID NO: 127 of human ULBP6. The ULBP6 binding protein can protect one or more residues contained within alpha helix 1 (SEQ ID NO: 125), alpha helix 2 (SEQ ID NO: 126) and / or SEQ ID NO: 127 of human ULBP6 from deuterium exchange in HDX-MS analysis. The ULBP6 binding protein can protect one or more residues contained within SEQ ID NO: 128, SEQ ID NO: 129 and / or SEQ ID NO: 127 of human ULBP6 from deuterium exchange in HDX-MS analysis. The ULBP6 binding protein can bind to human ULBP6 and cause peptides derived from alpha helix 1 (SEQ ID NO: 125), alpha helix 2 (SEQ ID NO: 126) and / or SEQ ID NO: 127 of human ULBP6 to be more resistant to deuterium incorporation than corresponding peptides derived from uncomplexed human ULBP6. The ULBP6 binding protein can bind to human ULBP6 and cause the peptides of SEQ ID NO: 128, SEQ ID NO: 129 and / or SEQ ID NO: 127 derived from human ULBP6 to be more resistant to deuterium incorporation than the corresponding peptides derived from uncomplexed human ULBP6.

[0242] Also provided herein are antibodies and antigen-binding fragments thereof that bind to UL-16 binding protein 6 (ULBP6), comprising: a variable heavy chain (VH) sequence having a heavy chain complementary determining region 1 (CDRH1) sequence, a heavy chain complementary determining region 2 (CDRH2) sequence, and a heavy chain complementary determining region 3 (CDRH3) sequence; and a variable light chain (VH) sequence having a light chain complementary determining region 1 (CDRL1) sequence, a light chain complementary determining region 2 (CDRL2) sequence, and a light chain complementary determining region 3 (CDRL3) sequence; wherein: CDRH1 is selected from the group consisting of SEQ ID NOs: 36, 39, 42, and 45; CDRH2 is selected from the group consisting of SEQ ID NOs: 37, 40, 43, and 46; CDRH3 is selected from the group consisting of SEQ ID NOs: 38, 41, 44, and 47; CDRL1 is selected from the group consisting of SEQ ID NOs: 51, 54, 57, and 60; CDRL2 is selected from the group consisting of SEQ ID NOs: 5 NO:52, 55, 58 and 61; or CDRL3 is selected from the group consisting of SEQ ID NO:53, 56, 59 or 62.

[0243] Also provided herein are antibodies and antigen-binding fragments thereof, wherein CDRH1 is SEQ ID NO:36, CDRH2 is SEQ ID NO:37, CDRH3 is SEQ ID NO:38, CDRL1 is SEQ ID NO:51, CDRL2 is SEQ ID NO:52, and CDRL3 is SEQ ID NO:53; CDRH1 is SEQ ID NO:39, CDRH2 is SEQ ID NO:40, CDRH3 is SEQ ID NO:41, CDRL1 is SEQ ID NO:54, CDRL2 is SEQ ID NO:55, and CDRL3 is SEQ ID NO:56; CDRH1 is SEQ ID NO:42, CDRH2 is SEQ ID NO:43, CDRH3 is SEQ ID NO:44, CDRL1 is SEQ ID NO:57, CDRL2 is SEQ ID NO:58, and CDRL3 is SEQ ID NO:59; or CDRH1 is SEQ ID NO:45, CDRH2 is SEQ ID NO:46, CDRH3 is SEQ ID NO:47. NO:47, CDRL1 is SEQ ID NO:60, CDRL2 is SEQ ID NO:61, and CDRL3 is SEQ ID NO:62.

[0244] Also provided herein are antibodies and antigen-binding fragments thereof, wherein: a VH region at least 80% identical to SEQ ID NO:21 and a VL region at least 80% identical to SEQ ID NO:31; a VH region at least 80% identical to SEQ ID NO:22 and a VL region at least 80% identical to SEQ ID NO:32; a VH region at least 80% identical to SEQ ID NO:23 and a VL region at least 80% identical to SEQ ID NO:33; or a VH region at least 80% identical to SEQ ID NO:24 and a VL region at least 80% identical to SEQ ID NO:34.

[0245] Also provided herein are antibodies and antigen-binding fragments thereof, wherein: a VH region at least 80% identical to SEQ ID NO: 16 and a VL region at least 80% identical to SEQ ID NO: 26; a VH region at least 80% identical to SEQ ID NO: 17 and a VL region at least 80% identical to SEQ ID NO: 27; a VH region at least 80% identical to SEQ ID NO: 18 and a VL region at least 80% identical to SEQ ID NO: 28; or a VH region at least 80% identical to SEQ ID NO: 19 and a VL region at least 80% identical to SEQ ID NO: 29.

[0246] Also provided herein are antibodies and antigen-binding fragments thereof, wherein the binding protein comprises: a heavy chain (HC) sequence that is at least 80% identical to any one of SEQ ID NOs: 75 to 78, 85 to 88, or 95 to 98; and / or a light chain (LC) sequence that is at least 80% identical to any one of SEQ ID NOs: 105 to 108.

[0247] Also provided herein are antibodies and antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof bind to ULBP6 and ULBP2 with a KD less than or equal to 1 nM, and bind to ULBP5 with a KD greater than or equal to 1 nM.

[0248] Also provided herein are antibodies and antigen-binding fragments thereof that inhibit the interaction of human ULBP6 with human NKG2D by 80% or more.

[0249] Also provided herein are antibodies and antigen-binding fragments thereof that bind to a human ULBP6 protein or a fragment thereof, and compete with a reference ULBP6 binding protein or a fragment thereof for binding to a human ULBP6 protein fragment, comprising: a VH region sequence of SEQ ID NO: 21 and a VL region sequence of SEQ ID NO: 31; or a VH region sequence of SEQ ID NO: 22 and a VL region sequence of SEQ ID NO: 32.

[0250] Also provided herein are antibodies and antigen-binding fragments thereof that inhibit the binding of soluble ULBP6 to NKG2D.

[0251] Also provided herein are antibodies and antigen-binding fragments thereof that inhibit the binding of soluble ULBP6 to NKG2D by 80% or more.

[0252] Also provided herein are antibodies and antigen-binding fragments thereof comprising a modified Fc region.

[0253] Also provided herein are antibodies and antigen-binding fragments thereof comprising a modified Fc region comprising the amino acid substitutions S239D and I332E (as numbered according to the EU index).

[0254] Also provided herein are non-fucosylated antigen-binding fragments and antigen-binding fragments thereof.

[0255] Also provided herein are antibodies and antigen-binding fragments thereof, wherein the modified Fc region comprises a set of amino acid substitutions selected from the group consisting of S239D / A330L / I332E, G236A / S239D / A330L / I332E, L235V / F243L / R292P / Y300L / P396L and F243L / R292P / Y300L / V305I / P396L (as numbered according to the EU index).

[0256] Also provided herein are nucleic acid sequences encoding one or both of the heavy chain (HC) and light chain (LC) of the antibodies or antigen-binding fragments thereof provided herein.

[0257] Also provided herein are expression vectors comprising nucleic acids of the antibodies and antigen-binding fragments thereof provided herein.

[0258] Also provided herein are recombinant host cells comprising the nucleic acid sequences of the antibodies and antigen-binding fragments thereof provided herein.

[0259] Also provided herein are methods for producing the antibodies or antigen-binding fragments thereof provided herein, the methods comprising culturing a recombinant host cell under conditions suitable for expression of a nucleic acid sequence or vector contained therein, wherein a polypeptide comprising the antibody or antigen-binding fragment thereof is produced.

[0260] Also provided herein are cell lines engineered to express the antibodies and / or antigen-binding fragments thereof provided herein.

[0261] Also provided herein are pharmaceutical compositions comprising one or more antibodies or antigen-binding fragments thereof provided herein and a pharmaceutically acceptable excipient.

[0262] Also provided herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein.

[0263] Also provided herein are methods for treating cancer, wherein the cancer is basal cell carcinoma, head and neck cancer, cervical cancer, bladder cancer, lung cancer, colorectal cancer, pancreatic cancer, or glioblastoma. In some such methods, the subject is a human.

[0264] Some methods provided herein may also include determining whether the subject expresses soluble ULBP6

[0265] Also provided herein are kits comprising one or more antibodies or antigen-binding fragments thereof provided herein and instructions for administering the one or more antibodies or antigen-binding fragments thereof to a subject having cancer.

[0266] Some such kits may be used in subjects having head and neck cancer, cervical cancer, bladder cancer, lung cancer, colorectal cancer, pancreatic cancer, or glioblastoma.

[0267] Also provided herein are ULBP6 antagonists for use in treating basal cell carcinoma.

[0268] Also provided herein are ULBP6 binding proteins and fragments thereof, which comprise a) HVRH1 of SEQ ID NO: 130, HVRH2 of SEQ ID NO: 131, HVRH3 of SEQ ID NO: 132, HVRL1 of SEQ ID NO: 145, HVRL2 of SEQ ID NO: 146, and HVRL3 of SEQ ID NO: 147; b) HVRH1 of SEQ ID NO: 133, HVRH2 of SEQ ID NO: 134, HVRH3 of SEQ ID NO: 135, HVRL1 of SEQ ID NO: 148, HVRL2 of SEQ ID NO: 149, and HVRL3 of SEQ ID NO: 150; c) HVRH1 of SEQ ID NO: 136, HVRH2 of SEQ ID NO: 137, HVRH3 of SEQ ID NO: 138, HVRL1 of SEQ ID NO: 151, HVRL2 of SEQ ID NO: 152, and HVRL3 of SEQ ID NO: 153. d) HVRH1 of SEQ ID NO:139, HVRH2 of SEQ ID NO:140, HVRH3 of SEQ ID NO:141, HVRL1 of SEQ ID NO:154, HVRL2 of SEQ ID NO:155, and HVRL3 of SEQ ID NO:156; or HVRH1 of SEQ ID NO:142, HVRH2 of SEQ ID NO:143, HVRH3 of SEQ ID NO:144, HVRL1 of SEQ ID NO:157, HVRL2 of SEQ ID NO:158, and HVRL3 of SEQ ID NO:159;

[0269] Example

[0270] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative, not limiting. It will be readily appreciated by those skilled in the art that the specific embodiments are merely illustrative of the present invention, as more fully described in the claims that follow. The embodiments and features described in this application should be understood to be at least interchangeable and combinable with other such embodiments and features contained therein.

[0271] Example 1: Genome-wide association study of basal cell carcinoma individuals

[0272] A genome-wide association study (GWAS) was performed on individuals diagnosed with basal cell carcinoma. A genetic region within the ULBP6 locus (6q25.1) was identified that met the accepted genome-wide statistical threshold of P < 5.0E10 -8 .

[0273] We identified a common genetic association between basal cell carcinoma and immune-related disease diagnosis in the 6q25.1 region, where multiple NKG2D ligands, ULBP6 (also known as RAET1L), ULBP1, ULBP2, ULBP3, ULBP4 (RAET1E), and ULBP5 (RAET1G) are present. The index variants were rs9478354 (dbSNP construct 153 identifier) ​​at position 150013230 on chromosome 6 (of human genome construct GRCh38 / hg38) (basal cell carcinoma; A / G, AF [allele frequency of allele G] = 0.55, OR = 0.965, P value = 7.2E-24), rs10666240 (dbSNP construct 153 identifier) ​​at position 150036968 on chromosome 6 (of human genome construct GRCH38 / hg38) (basal cell carcinoma; A / G, AF [allele frequency of allele G] = 0.55, OR = 0.965, P value = 7.2E-24), rs10666241 (dbSNP construct 153 identifier) ​​at position 150036968 on chromosome 6 (of human genome construct GRCh38 / hg38) (basal cell carcinoma; A / G, AF [allele frequency of allele G] = 0.55, OR = 0.965, P value = 7.2E-24), rs10666242 (dbSNP construct 153 identifier) ​​at position 150036968 on chromosome 6 (of human genome construct GRCh38 / hg38) (basal cell carcinoma; A / G, AF [allele frequency of allele G] = 0.55, OR = 0.965, P value = 7.2 rs5006620 (dbSNP construct 153 identifier) ​​at position 150015529 on chromosome 6 (of human genome construct GRCH38 / hg38) (alopecia areata; C / CAA, AF [allele frequency of allele CAA] = 0.45, OR = 0.930, P value = 9.9E-16) (dbSNP construct 153 identifier) ​​(alopecia universalis; C / T, AF [allele frequency of allele T] = 0.45, OR = 0.790, P value = 1.3E-21) Figure 1 ). These variants are located in the intergenic region at the end of ULBP6.

[0274] Figure 1 , is a regional association curve for the ULBP6 locus on chromosome 6 for basal cell carcinoma. The characteristic analyzed is basal cell carcinoma diagnosis, where "cases" are individuals with a basal cell carcinoma diagnosis and "controls" are individuals without a reported basal cell carcinoma diagnosis. The x-axis shows the physical position on the human genome construct (GRCh38 / hg38), and the y-axis on the left shows the -log10 of the P value associated with the phenotype of interest. Each point in the depicted graph represents a genetic variant tested for association in that region. The gray horizontal line represents 5X 10 -8 The genome-wide significance threshold of . The human genes in the region are depicted on the figure below. These GWAS data indicate that the indicated loci (gene regions) are associated with susceptibility to diagnosis of human basal cell carcinoma.

[0275] Example 2: ULBP6 expression in cancer patients

[0276] To determine whether human ULBP6 and other NKG2DLs are expressed by tumor cells and therefore may represent therapeutic targets for cancer, two complementary approaches were employed. The first approach investigated transcript expression by analyzing The Cancer Genome Atlas (TCGA) and single-cell RNA sequencing data, and the second approach measured levels of NKG2DL proteins in the serum of patients with various cancer types.

[0277] NKG2D ligand expression in tumor cells

[0278] Expression data from multiple tumor types from the TCGA project were analyzed for the expression of ULBP2 and ULBP6. In general, while ULBP2 appeared to be widely expressed in the cancer types analyzed, ULBP6 was found to be most highly expressed in squamous cell carcinomas, including HNSC, CESC, LUSC, and ESCA (squamous cell carcinoma pathology), compared to non-squamous cell carcinomas. Both the ULBP6_01 and ULBP6_02 haplotypes of ULBP6 were found to be expressed in tumor samples.

[0279] In addition, single cell RNA seq data from Puram et al. (PMID: 29198524) were analyzed for the expression of ULBP2 and ULBP6. This data showed that these ligands are specifically expressed on tumor cells.

[0280] Detection of NKG2DL in patient serum

[0281] To determine the levels of NKG2DL shed into serum (ie, shed / soluble NKG2DL, "sNKG2DL"), ELISA and Luminex assays were performed to measure the presence of NKG2DL in patient serum samples.

[0282] Materials and methods:

[0283] sNKG2DL in the serum of 158 patients and 9 cancer types was determined. Serum samples from individuals with cervical cancer, colorectal cancer, bladder cancer, lung cancer, melanoma, pancreatic cancer, head and neck cancer, esophageal cancer and glioblastoma, as well as cancer-free individuals (healthy donors) were obtained. The levels of ULBP1, ULBP2 / ULBP6, ULBP3 and MICB in serum samples were determined using ELISA kits from R&D systems according to the manufacturer's instructions. ELISA does not distinguish between ULBP2 and ULBP6 because the detection antibody binds to both ULBP6 and ULBP2. The MICA levels of serum samples were determined using the Luminex MICA assay according to the manufacturer's instructions. Each serum sample was screened twice (i.e., technical replicates).

[0284] The sNKG2DL concentrations from healthy donors were averaged and used to calculate the cutoff value for determining the sNKG2DL elevated in the serum samples from cancer patients. The cutoff value for each tested sNKG2DL was set to the mean value of the sNKG2DL concentration from healthy donors + 3 standard deviations. Each sNKG2DL level from the tumor sample was compared with the cutoff value. When the value in the two technical repetitions of the tested serum sample was higher than the calculated cutoff value of the sNKG2DL, it was defined as an elevated sNKG2DL level.

[0285] result:

[0286] As described in Table 4, shed ULBP2 / 6 (sULBP2 / 6) was elevated in 22.2% of patient sera compared to healthy donor control samples, while other sNKG2DLs (including ULBP1 and ULBP3 and MICA / B) were elevated in <5.2% of patients compared to healthy donor control samples. These results indicate that sULBP2 / 6 is the most upregulated sNKG2DL in the tested sera from cancer patients.

[0287] Table 4. sNKG2DL in serum from cancer patients

[0288]

[0289]

[0290] Example 3: Binding of human sNKG2DL to NKG2D

[0291] NKG2DLs exhibit variability in primary amino acid sequence, structure, expression profile, and binding affinity to NKG2D, which may result in functional differences between membrane-bound and soluble forms when interacting with NKG2D. Example 2 demonstrates that increased sULBP2 and / or sULBP6 protein is commonly observed in the serum of cancer patients. To rank the relative binding affinities of sULBP2, sULBP6, and other human sNKG2D ligands to the hNKG2D receptor, surface plasmon resonance (SPR) assays were performed using a BIACORE 8K (Cytiva) instrument as described below.

[0292] Materials and methods:

[0293] To illustrate the binding of human sNKG2D ligands to hNKG2D, SPR assays were performed at 37°C with various human sNKG2D ligands (including sMICA, sMICB, sULBP1, sULBP2, sULBP3, sULBP4, sULBP5, sULBP6_01 and sULBP6_02). Briefly, 0.5 μg / ml hNKG2D-Fc in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20) was applied to the CYTIVA sensor chip protein A at a flow rate of 30 μL / min to achieve approximately 50 response units in the second flow cell (FC2). FC1 was used for reference. For kinetic measurements, 3-fold serial dilutions of hNKG2D ligands from high concentration (50 nM for ULBP6_02 and 2 μM for other sNKG2DLs) to low concentration (0.07 nM for ULBP6_02 and 2.74 nM for other sNKG2DLs) were injected in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20) at 37°C (flow rate: 30 μL / min). Sensorgrams were recorded and referenced and buffer subtracted before evaluation by BIACORE 8K Evaluation Software (Version 1.1.1.7442). The association rate (k) was calculated using a simple one-to-one Langmuir binding model. on ) and dissociation rate (k off The equilibrium dissociation constant (KD) was calculated as k summarized in Table 5. off / k on ratio.

[0294] Table 5: Binding affinity of soluble ligands to hNKG2D-Fc fusion protein on BIACORE chip

[0295]

[0296] result:

[0297] No binding of sULBP4 to NKG2D was observed, while the binding affinities of the remaining sNKG2D ligands ranged from 594 nM to 4.8 nM. The strongest binding affinity of ULBP6 to NKG2D was observed. The binding affinity of haplotype 02 (sULBP6_02) was 4.8 nM, and the binding affinity of haplotype 01 (sULBP6_01) was 41 nM (Table 5). The much lower affinity of sULBP2 to NKG2D was observed to be 220 nM.

[0298] The relatively high affinity of soluble forms of ULBP6 for NKG2D shown herein and the prevalence of sULBP2 / ULBP6 in sera of cancer patients (Example 2) indicate that disruption of sULBP6-NKG2D interaction using anti-ULBP6 binding proteins may provide opportunities for the development of cancer therapeutics.

[0299] Example 4: Production of polypeptides

[0300] This example illustrates the preparation of ULBP6 antigen for producing anti-ULBP6 antibodies. The example also includes the production of other sNKG2DLs, including human sULBP1, sULBP2, sULBP3, sULBP4, sULBP5, sMICA and sMICB, used as antigens for screening anti-ULBP6 antibodies of the present disclosure. The example also includes the preparation of NKG2D and Fc tag polypeptides. Note that cULBP5 and cULBP2 / 6 refer to cynomolgus monkey soluble orthologs. Due to the high sequence similarity between human ULBP2 and ULBP6, it is unclear whether a single cynomolgus monkey ortholog is evolutionarily related to human ULBP2 or ULBP6. Therefore, the cynomolgus monkey ortholog is referred to as cULBP2 / 6 herein.

[0301] The sequences of human sULBP1-6, cULBP5 and cULBP2 / 6 polypeptide forms are provided in Table 6, and the sequences of human NKG2D (hNKG2D) or cynomolgus monkey NKG2D (cNKG2D), other sNKG2DLs (sMICA and sMICB) and Fc polypeptide forms are provided in Table 7.

[0302] Table 6: Human or cynomolgus monkey sULBP1-6 polypeptide sequences

[0303]

[0304]

[0305]

[0306] Table 7: Other peptide sequences

[0307]

[0308]

[0309]

[0310] Example 5: Generation and selection of anti-ULBP6 antibodies

[0311] This example illustrates the generation of anti-ULBP6 antibodies using mouse hybridoma technology.

[0312] immunity

[0313] Multi-strain mice were immunized with hULBP6-01 (SEQ ID NO: 6) and hULBP6-02 (SEQ ID NO: 7) produced in-house. SIGMA adjuvant system (MILLIPORE SIGMA St. Louis, MO), Freund's adjuvant, complete adjuvant (MILLIPORE SIGMA St. Louis, MO), TITERMAX gold adjuvant (MILLIPORE SIGMA St. Louis, MO), GERBU adjuvant (V-Biognostics), TLR cocktail (INVIVOGEN) were used for immunization. Antibody titers were determined by ELISA. Mice that showed sufficient antibody titers were given a final boost without adjuvant. Fusions were performed 3 days after the last boost. Hybridomas were generated, and mice were selected for antibody expression and cloned.

[0314] Example 6: Screening and characterization of anti-ULBP6 antibodies

[0315] This example demonstrates a method for screening and selecting anti-ULBP6 antibodies that effectively block the interaction between soluble ULBP6 and NKG2D.

[0316] A. Enzyme-linked immunosorbent assay (ELISA)

[0317] To detect individual hybridoma clones secreting ULBP6-specific antibodies, direct ELISA was performed after culturing the selected hybridoma clones for 7 days.

[0318] Materials and methods:

[0319] In brief, 96-well MAXISORP flat-bottom plates (THERMO SCIENTIFIC, catalog number 439454) were coated with 1 μg / mL sULBP6_01, sULBP6_02 and sULBP2 (50 μL / well) in PBS at 4°C. The wells were washed twice with tap water and blocked at room temperature for 1 hour with a blocking solution (pH 7.4, 200 μL / well) containing 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Next, the wells were washed again, and 50 μL / well culture supernatants from the selected hybridomas were added and incubated at room temperature for 1 hour. After washing, a secondary anti-mouse IgG antibody (HRP, Jackson Immuno 115-035-164) coupled to horseradish peroxidase was used for detection. The plate was incubated at room temperature for 45 minutes. Tetramethylbenzidine (TMB) solution (0.12 mg / mL TMB in 25 mM NaH2PO4 containing 0.04% hydrogen peroxide) was used as substrate. After 5 minutes, the reaction was stopped with 2N H2SO4. The optical density (OD) was measured at 450 nm.

[0320] result:

[0321] From the primary direct ELISA, 191 clones (binders) were selected and subjected to a confirmatory indirect ELISA, resulting in a total of 90 binders that were further tested using FACS binding and single-point blocking ELISA assays as described below.

[0322] B. Flow cytometry cell binding assay

[0323] To determine whether the 90 ULBP6-specific binder antibody clones identified in the primary indirect ELISA assay also bind to cell surface expressed NKG2DL, a flow cytometry cell binding assay was performed.

[0324] Materials and methods:

[0325] CHO-S cells were transiently transfected to overexpress full-length membrane-bound human ULBP6_01, ULBP6_02, ULBP1, ULBP2, ULBP3, and ULBP5. CHO-S cells were harvested by centrifugation at 1200 RPM for 15 minutes. The cell pellets were resuspended in 10 mL FACS buffer (1× PBS containing 1% BSA) and counted. The cells were then plated at 1×10 6Cells / well were seeded into 96-well plates (Corning 3365). 50 μL of supernatant was transferred from a 96-well plate for culturing hybridoma cells to a 96-well plate containing target expression cells using a 96-head liquid handler. The supernatant was incubated with cells at 4 ° C for 1 hour. The cells were then washed twice with FACS buffer, incubated at 4 ° C for 45 minutes with a secondary anti-mouse IgG antibody (5 μg / mL, Jackson Immuno, 115-136-146) coupled to APC, and then washed twice with FACS buffer. After washing, the cells were resuspended in 100 μl FACS buffer for flow cytometry analysis.

[0326] result:

[0327] Of the 90 clones tested in the flow cytometry binding assay, 7 / 90 showed binding to CHO-S-ULBP1, 89 / 90 to CHO-S-ULBP2, 2 / 90 to CHO-S-ULBP3, 34 / 90 to CHO-S-ULBP5, 88 / 90 to CHO-S-ULBP6_01, and 87 / 90 to CHO-S-ULBP6_02. All 90 clones were then tested in a single-point biochemical blocking assay.

[0328] C. Single-site biochemical blocking assay

[0329] Biochemical blocking assays were performed to detect antibody clones that block binding of human soluble ULBP6 to human NKG2D. For these experiments, the ULBP6 haplotype sULBP6_02 with the highest affinity for NKG2D was used.

[0330] Materials and methods:

[0331] 96-well MAXISORP flat-bottom plates (THERMO SCIENTIFIC, catalog number 439454) were coated with NEUTRAVIDIN (1 μg / mL) in PBS, 100 μL / well, overnight at 4°C. The wells were washed twice with tap water and blocked at room temperature for 1 hour using 200 μL / well with a blocking solution (pH 7.4) containing 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). The wells were washed twice again, and 1 μg / ml biotinylated sULBP6_02 was added, and the plate was incubated at room temperature for 1 hour. 60 μl of pure hybridoma supernatant and 60 μl of NKG2D-Fc (10 nM) were pre-incubated, and the mixed solution was added to the plate for 1 hour at room temperature. After washing, a secondary anti-human IgG Fc antibody (HRP; Jackson Immuno. 109-035-098) coupled to horseradish peroxidase was used for detection. The secondary antibody solution was incubated at room temperature for 45 minutes. Tetramethylbenzidine (TMB) solution (0.12 mg / mL TMB containing 0.04% hydrogen peroxide in 25 mM NaH2PO4) was used as a substrate. After 5 minutes, the reaction was terminated with 2N H2SO4. The optical density (OD) was measured at 450 nm. Mouse anti-ULBP2 / 5 / 6 antibody (R&D SYSTEMS, MAB1298-100) was used as a positive control at 5 μg / ml. The culture medium was used as a negative control. The ELISA signal of the negative control was defined as 0% blocking activity. Mouse anti-hULBP2 / 5 / 6Ab (R&D SYSTEMS, MAB1298-100) showed 72% blocking activity.

[0332] result:

[0333] Of the 90 clones tested in a single-site biochemical blocking ELISA, 68 / 90 showed blocking of sULBP6_02 binding to NKG2D. The 7B3 and 8E11 clones showed 100% blocking activity. The 25 antibodies with the highest blocking activity were then tested for cynomolgus monkey cross-reactivity using cULBP2 / 6 from Ensembl v99.

[0334] D. Cynomolgus Monkey Cross-Reactivity

[0335] To determine cross-reactivity with cULBP2 / 6 proteins, binding to human and cynomolgus monkey sULBP6 proteins was tested by assessing binding of mouse hybridoma supernatants in the OCTET R-8BLI label-free detection system.

[0336] Materials and methods:

[0337] 300 μl of each hybridoma supernatant was pipetted into an Octet 96 well along with 300 nM human ULBP6 and cULBP2 / 6 proteins in 1X PBST (1X PBS, 0.05% Tween-20). A stable baseline of 60 seconds was measured using an anti-mouse Fc capture (AMC) biosensor in 1X PBST buffer, followed by capture of mouse antibodies from hybridoma supernatants for 150 to 300 seconds. After recording another stable baseline for 60 seconds, the biosensor was immersed in human soluble ULBP6 or cynomolgus ULBP2 / 6 to measure the interaction / association between mouse antibodies captured from hybridoma supernatants and sULBP6, followed by a 300 second dissociation step in 1X PBST.

[0338] result:

[0339] Twenty-five blocking antibodies were selected from the single-point blocking ELISA for cross-reactivity studies. Of these 25 antibodies, 10 antibodies showed good cross-reactivity to cynomolgus monkey ULBP6. 7B3 showed the strongest binding to cULBP2 / 6. Since 2 / 10 blocking antibodies (7B3 and 8E11) showed very strong blocking and good cross-reactivity in the single-point blocking assay, they were directly selected for testing in the functional assay, while the remaining 8 / 10 blocking antibodies were further tested in the three-point biochemical blocking assay.

[0340] E. Three-point biochemical blocking assay

[0341] Eight hybridoma clones were selected based on clone type, blocking activity using single-point pure supernatant, human / cynomolgus monkey cross-reactivity and binding affinity. These eight clones were tested in a three-point blocking assay for further ranking.

[0342] Materials and methods:

[0343] 96-well MAXISORP flat-bottom plates (THERMO SCIENTIFIC, catalog number 439454) were coated with hNKG2D-Fc (2 μg / mL) in PBS at 100 μL / well at 4°C. The wells were washed twice with tap water and blocked at room temperature for 1 hour with 200 μL / well of a blocking solution (pH 7.4) containing 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). The mouse IgG concentration of the supernatant was normalized at 1 nM, 0.1 nM, and 0.01 nM. 60 μl of normalized hybridoma supernatant and 60 μl of biotinylated ULBP6_02 (1 nM) were pre-incubated at room temperature for 1 hour, and the mixed solution was added to the coated plate for 15 minutes. After washing, secondary streptavidin poly-HRP (THERMO SCIENTIFIC, N200) was added to the wells and incubated at room temperature for 45 minutes. Tetramethylbenzidine (TMB) solution (0.12 mg / mL TMB with 0.04% hydrogen peroxide in 25 mM NaH2PO4) was used as substrate. After 5 minutes, the reaction was stopped with 2N H2SO4. The optical density (OD) was measured at 450 nm. In the assay, purified strong blocking mAb 7B3 was used as a positive control, and blank medium was used as a negative control.

[0344] result:

[0345] Of the eight clones tested in the three-point biochemical blocking assay, four clones (6E1, 1D4, 4B1, and 5B8) showed >50% blocking activity.

[0346] F. Down-selection of clones

[0347] Based on ELISA / FACS binding, cynomolgus monkey cross-reactivity, blocking efficiency in blocking ELISA and clonal type information, 4 clones (7B3, 8E11, 6E1, 1D4) were selected for cloning, expression and purification as recombinant humanized antibodies. A non-blocking antibody (8E8) was selected as a negative control.

[0348] These clones were subsequently humanized to generate four functional blocking human antibody clones (h7B3, h8E11, h6E1 and h1D4) and one strong binding but non-blocking human antibody clone (h8E8). The germline sequences (HC / LC) used for humanization were: h7B3 (HC: IGHV3-48, LC: IGKV1-33), h8E11 (HC: IGHV3-48, LC: IGKV3-11), h1D4 (HC: IGHV7-4-1, LC: IGKV1-33), h6E1 (HC: IGHV1-2, LC: IGKV1-16). These blocking antibodies were cloned into the pRK vector (in the hlgG1 and mlgG2a backbone), expressed in Expi293 cells, purified and characterized. The variable heavy chain (VH) and variable light chain (VL) sequences of the heavy chain (HC) and light chain (LC, kappa light chain, KC) of the non-humanized and humanized antibody clones herein are provided in Table 8. The complementary determining region (CDR) sequences of the VH and VL sequences of the HC and LC of the murine and humanized anti-ULBP6 antibody clones selected herein are provided in Table 9 (according to Kabat numbering). The sequences of the CH1 domains with fragment crystallizable (Fc) regions comprising the heavy chain domains CH2 and CH3 (Table 10) are provided, which can be modularly combined with the VH sequences cloned herein (Table 8). The provided Fc sequences include (a) an effector plus sequence (E+), which can be considered a wild-type (WT) Fc sequence and is fucosylated unless otherwise specified; (b) an effector minus sequence (E-), which comprises an N297G mutation (EU numbering) that eliminates effector function (mutations are in bold); and (c) an effector enhancing sequence that enhances effector function (referred to herein as "E++" or "E++DE" or "E++(DE)"), which comprises S239D and I332E mutations (abbreviated herein as DE) (mutations are in bold), and is fucosylated unless otherwise specified. In some examples, a wild-type Fc sequence (e.g., E+) may be non-fucosylated (Afu), such that the oligosaccharides in the Fc chain do not comprise a fucose unit. Such non-fucosylation in a wild-type Fc (e.g., E+Afu) results in enhanced effector function that is substantially similar to the effector function of the E++Fc sequence. Provided are constant light chain domain (CL) sequences (Table 11) that can be modularly combined with the VL sequences cloned herein (Table 8). The full-length sequences of the non-humanized and humanized cloned HCs and LCs contemplated and / or produced herein are provided in Table 12, wherein the variable chains are underlined and the CDR sequences are represented in bold. However, in some embodiments, the C-terminal lysine (Lys447) of the Fc region may or may not be present.Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. For example, an antibody lacking a C-terminal lysine may have a sequence of SEQ ID NO: 160 or SEQ ID NO: 161. The full-length nucleic acid sequences of the HC and LC of the humanized clones contemplated and / or produced herein are provided in Table 13.

[0349] Table 8: Variable chain sequences of selected non-humanized and humanized anti-ULBP6 antibodies

[0350]

[0351]

[0352]

[0353] Table 9: CDR and HVR sequences of selected non-humanized and humanized anti-ULBP6 antibodies

[0354]

[0355]

[0356] Table 10. Fc domain sequences of selected anti-ULBP6 antibodies

[0357]

[0358]

[0359] Table 11: CL domain sequences of selected anti-ULBP6 antibodies

[0360]

[0361] Table 12: Full-length sequences of selected non-humanized and humanized anti-ULBP6 antibodies

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371] Table 13. Full-length nucleic acid sequences of selected humanized anti-ULBP6 antibodies

[0372]

[0373]

[0374]

[0375]

[0376]

[0377] Example 7: Characterization of humanized candidate antibodies

[0378] A. SPR analysis of humanized antibodies

[0379] To determine the binding affinity of recombinant humanized h7B3, h8E11, h1D4 and h6E1 (blocking antibodies) and h8E8 (non-blocking antibody) to human and cynomolgus monkey soluble ULBP, SPR measurements were performed using a BIACORE 8K instrument as described below.

[0380] Materials and methods:

[0381] BIACORE was performed at 37°C with various human and cynomolgus NKG2D ligands, including soluble hULBP2, hULBP6_01, hULBP6_02, cULBP5, and cULBP2 / 6 (ENSEMBL v99). Briefly, 0.5 μg / ml recombinant h7B3, h8E11, h1D4, h6E1, and h8E8 in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20) were applied to the CYTIVA sensor chip protein A at a flow rate of 30 μL / min to reach approximately 50 response units in the second flow cell (FC2). The first flow cell (FC1) was retained as a reference. For kinetic measurements, 3-fold serial dilutions of hNKG2D and cNKG2D ligands from high concentration (10 nM or 50 nM) to low concentration (0.01 nM or 0.07 nM) were injected in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20) at 37°C (flow rate: 30 μL / min). Sensorgrams were recorded and referenced and buffer subtracted before evaluation using BIACORE 8K evaluation software (version 1.1.1.7442). The association rate (kon) and dissociation rate (koff) were calculated using a simple one-to-one Langmuir binding model. The equilibrium dissociation constant (KD) was calculated as the ratio of koff / kon as summarized in Table 14.

[0382] result:

[0383] Humanized h7B3, h8E11, h1D4 and h6E1 showed tight binding to human and cynomolgus ULBP (Table 14). h7B3 showed the greatest binding strength, followed by h8E11, h1D4, and then h6E1.

[0384] Table 14. Binding properties of humanized antibodies to human (h) and cynomolgus monkey (c) soluble ULBP proteins

[0385]

[0386] B. Blocking ELISA of humanized clones

[0387] To determine and compare the blocking efficacy of humanized h7B3, h8E11, h1D4 and h6E1 between human sULBP6 and hNKG2D, a blocking ELISA was performed.

[0388] Materials and methods:

[0389] 96-well plates were coated with 100 μl 1ug / ml hNKG2D per well and incubated overnight at 4°C. The plates were then blocked at room temperature for 1 hour using 300 μl / well blocking buffer (1X PBS, 1% BSA, and 0.05% Tween-20). The plates were then washed with washing buffer (1X PBS and 0.05% Tween-20). Next, each antibody was titrated 3 times from an initial concentration of 300 nM to a final concentration of 0.005 nM, and each titration was mixed with 1 μg / ml (pre-calculated EC80) biotinylated human sULBP6 at room temperature for 1 hour. The mixture was incubated on coated hNKG2D plates at room temperature for 15 minutes. The plates were washed and the binding of biotinylated human sULBP6 to hNKG2D was detected using 100 μl / well of streptavidin-HRP (1:10,000 dilution) for 1 hour at room temperature, followed by washing and addition of 100 μl TMB per well for 15 minutes at room temperature. The absorbance was measured at 450 nm.

[0390] result:

[0391] h7B3 and h8E11 were shown to be hNKG2D-hULBP6_01 interactors (IC 50 values ​​were 4.47 nM and 4.32 nM, respectively) and hNKG2D-hULBP6_02 interaction (IC 50 The strongest blocking antibody was the one with 400 ng / mL IgG and 1.52 nM IgG values, respectively (Table 15).

[0392] Table 15: Binding affinity of humanized anti-ULBP6 antibodies

[0393]

[0394]

[0395] C. Epitope Binning

[0396] To determine whether the humanized antibodies bind to the same or different epitopes on human sULBP6, epitope binning experiments were performed using Octet.

[0397] Materials and methods:

[0398] 100 μM of each antibody was diluted in 300 μl 1X PBST buffer (1X PBS and 0.05% Tween-20) and pipetted into different wells of the Octet plate. The anti-human IgG Fc capture (AHC) biosensor in 1X PBST was immersed in the first humanized antibody (e.g., h7B3) to capture it for 150 to 300 seconds. The biosensor was then immersed in 1X PBST buffer for 60 seconds to reach a stable baseline, followed by immersion of the same biosensor in human sULBP6 for another 150 to 300 seconds to promote the interaction between the first humanized antibody and sULBP6. The biosensor was then immersed in a second antibody (e.g., h8E11) to determine the blocking of the second antibody to sULBP6 by the first antibody. This process was repeated for each combination of two antibodies selected from h7B3, h8E8, h8E11, h1D4, and h6E1.

[0399] result:

[0400] The four blocking antibodies can be divided into two different epitope bins (Table 16). h7B3 shares an overlapping epitope with h8E11, and h1D4 shares an overlapping epitope with h6E1. h8E8 does not share an overlapping epitope with any other antibody tested.

[0401] Table 16: Summary of epitope binning of humanized anti-ULBP6 antibodies

[0402]

[0403] D. Characterization of humanized anti-ULBP6 antibodies

[0404] To determine the binding of h7B3, h8E11, h1D4 and h6E1 to surface ULBP6 expressed on tumor cells, flow cytometry analysis was performed. The binding of humanized antibodies to cell surface ULBP6 in PANC02.13 cells (which express ULPB6_02) and COV644 cells (which express ULPB6_01) was determined as summarized in Table 16. The blocking of humanized antibodies to NKG2D-Fc binding to cell surface ULBP6 on COV644 cells (which express ULBP6_01) was determined as summarized in Table 17.

[0405] Materials / Methods:

[0406] The binding of humanized anti-ULBP6 antibodies to cell surface ULBP6 on cancer cells with endogenous target expression was compared using a FACS assay. Cells were detached from the surface of the culture flask using ACCUTASE cell detachment solution, stained with LIVE / DEAD staining dye, and incubated with serially diluted antibodies h7B3, h8E11, h1D4, and h6E1 at concentrations ranging from 83.2 pM to 65 nM for 25 minutes at 4°C. Bound antibodies were detected with a 1:100 dilution of anti-hFc-APC antibody (BIOLEGEND catalog #40936). Binding of humanized anti-ULBP6 antibodies to cell surface ULBP6 was analyzed by FACS using a Cytoflex LX instrument. The geometric mean was determined and used for EC 50 calculate.

[0407] To determine the blockade of NKG2D-mFc binding to ULBP6, COV644 cells were incubated with hNKG2D-mFc alone or with serially diluted anti-ULBP6 blocking antibodies selected from h7B3, h8E11, h1D4, and h6E1. Binding of human NKG2D-mFc to surface ULBP6 was detected by flow cytometry using anti-mFc-APC antibody. The geometric mean was determined and the IC 50 value.

[0408] result

[0409] Binds to cell surface ULBP6 :h7B3, h8E11, h1D4 and h6E1 showed EC 50 The values ​​were 1.97 nM (PANC02.13) and 1.93 nM (COV644), 1.28 nM (PANC02.13) and 1.29 nM (COV644), 0.72 nM (PANC02.13) and 1.4 nM (COV644), 0.44 nM (PANC02.13) and 0.36 nM (COV644), respectively, calculated as the average of two technical replicates as described in Table 17.

[0410] Blocking the binding of NKG2D-Fc to ULBP6 on the surface of COV644 cells :h7B3, h8E11, h1D4 and h6E1 showed IC 50 The values ​​were 0.17 nM, 0.11 nM, 0.22 nM and 0.2 nM, respectively, calculated as the mean of two technical replicates as described in Table 17.

[0411] In summary, this example demonstrates the biochemical characterization of four humanized anti-ULBP6 antibodies that a) inhibit the interaction between soluble ULBP6 and NKG2D, and b) bind to ULBP6 expressed on the cell surface on tumor cell lines.

[0412] Table 17: Binding of humanized anti-ULBP6 antibodies to cell surface ULBP6

[0413]

[0414]

[0415] Example 8: Anti-ULBP6 antibody reverses sULBP6-induced NKG2D blockade / internalization

[0416] Preliminary data (not shown) showed that soluble ULBP6 competed with anti-NKG2D mAb for binding to NKG2D expressed by NKG2D expressing cells. In particular, haplotype sULBP6_02 was more able to compete with anti-NKG2D mAb for NKG2D binding than sULBP6_01. These data suggest that soluble ULBP6 may a) hinder the interaction of NKG2D with other NKG2DLs and / or b) induce the internalization of NKG2D on cells expressing NKG2D.

[0417] To determine the effect of anti-ULBP6 antibodies on soluble ULBP6-inhibited NKG2D blockade / internalization, NKG2D detection assays were performed on sensitized PBMCs from healthy donors. Soluble ULBP6_02 was used in the assay because it showed the highest binding affinity to NKG2D compared to ULBP6_01 (see Example 2) and more effectively inhibited the binding of anti-NKG2D mAbs.

[0418] Materials and methods:

[0419] In this assay, primed PBMCs (with 20 ng / ml of IL-2 and IL-15 for 36 hours in X-VIVO 15 medium) were incubated as follows: (a) without soluble ULBP6_02, (b) with soluble ULBP6_02 (50 nM), or (c) with soluble ULBP6_02 (50 nM) and anti-ULBP6 antibodies (h7B3(E-), h1D4(E-), h6E1(E-), h8E11(E-), or isotype control antibody (isotype(E-)); each in RPMI assay medium containing 10% FBS. After one hour of incubation, cells were collected and stained with anti-NKG2D-APC antibody to quantify the surface expression of NKG2D on various PBMC populations. 69D.12_315 is a tool antibody that binds weakly to ULBP6. 8E8 is a control antibody that binds but does not block the NKG2D ULBP6 interaction.

[0420] result:

[0421] The assay was first validated with tool antibodies ( Figure 2, upper figure). At high concentrations, the isotype antibodies did not alter NKG2D MFI in the presence of sULBP6. The weakly binding 69D.12_315 control antibody showed a modest reversal of sULBP6-induced NKG2D blockade / downregulation in a dose-dependent manner, while the higher affinity 7B3 antibody showed a stronger increase in NKG2D MFI. Relative to the isotype control, the non-blocking 8E8 antibody reduced the detectable NKG2D MFI by stabilizing the interaction between ULBP6 and NKG2D.

[0422] The tested anti-ULBP6 antibodies reversed sULBP6-induced NKG2D blockade / downregulation in a dose-dependent manner ( Figure 2 , lower panel). h7B3 (E-) and h8E11 (E-) showed restoration of NKG2D at lower doses compared to the other antibodies tested (curves shifted to the left). These results represent data from PBMCs from 3 different healthy donors.

[0423] These data indicate that humanized anti-ULBP6 antibodies, particularly 7B3 and 8E11, neutralize the inhibitory effects of soluble ULBP6, thus allowing NKG2D to interact with other NKG2DLs.

[0424] Example 9: MICA-mediated PBMC immune activation

[0425] The previous examples show that humanized anti-ULBP6 antibodies reduce the inhibitory effect of sULBP6 on cell surface NKG2D. By neutralizing sULBP6, anti-ULBP6 antibodies can activate NKG2D through alternative NKG2DLs (eg, MICA).

[0426] A PBMC interferon gamma (IFNg) secretion assay was performed to determine the restorative effect of anti-ULBP6 antibodies on MICA-mediated immune activation via neutralization of soluble ULBP6.

[0427] Materials and methods:

[0428] In this assay, primed PBMCs from healthy donors (using 20 ng / ml IL-2 and IL-15 for 36 hours in X-VIVO 15 medium) were incubated with anti-ULBP6 antibodies (h7B3(E-), h1D4(E-), h6E1(E-) and h8E11(E-)) at concentrations ranging from 0.39 nM to 200 nM or with an isotype control antibody (Iso(E-)) added at 200 nM in RPMI assay medium with 10% FBS on the surface of MICA-coated plates in the presence of soluble ULBP6_02 (50 nM). After 24 hours of incubation, the cell-free assay medium was collected and IFNg levels were analyzed using Luminex technology. 69D.12_315 is a tool antibody that binds weakly to ULBP6, and h8E8 is a control antibody that binds to ULBP6 but does not block its interaction with NKG2D.

[0429] result:

[0430] Activation of PBMCs was measured via IFNg secretion. The assay was first validated with tool antibodies ( Figure 3A and Figure 3B ). The h8E8 antibody failed to reverse the inhibitory effect of sULBP6 at all tested concentrations as expected. The h7B3 antibody reversed the sULBP6-induced blockade of MICA-mediated PBMC activation in a dose-dependent manner. The 69D.12_315 tool antibody showed a very modest contrast to the sULBP6-induced blockade of MICA-mediated PBMC activation, which is well correlated with its weak blocking and binding to ULBP6 ( Figure 3A and Figure 3B In summary, the sensitivity of the assay was able to distinguish the non-blocking antibody h8E8, which had no effect, from the weak ULBP6 binding antibody 69D12.315, which showed partial activity, and the strong ULBP6 binding and blocking antibody h7B3, which had the greatest effect ( Figure 3A and Figure 3B h7B3(E-), h1D4(E-), h6E1(E-), and h8E11(E-) showed comparable effects on blocking sULBP6 in the assay ( Figure 3C and Figure 3D ). The assay results from donor 1307 represent data from 3 independent healthy donors of PBMC.

[0431] Therefore, the anti-ULBP6 antibodies described herein can restore immune cell responses by neutralizing the inhibitory effects of soluble ULBP6 on NKG2D responses to NKG2DL.

[0432] Example 10: Synergistic effect of NKG2D and FcgRIIIa activation

[0433] In addition to the ULBP6 blocking effects described herein that enhance NKG2D activation, anti-ULBP6 antibodies can also engage immune cells expressing Fc receptors, for example by binding to surface ULBP6. An IFNg secretion assay was performed to determine whether NKG2D activation synergizes with FcgRIIIa activation by measuring IFNg secretion by PBMCs.

[0434] Materials and methods:

[0435] For IFNg secretion assay, PBMCs were pre-sensitized for 36 hours with IL-2 and IL-15 (20 ng / ml each) in X-VIVO 15. 96-well plates were coated overnight at 4°C with anti-lysozyme Fc E+ ("Ab E+") or anti-lysozyme Fc E- ("Ab E-") antibodies with or without 2 copies of ULBP6_02 (in Figure 4 These were plated in PBS and coated into assay wells at concentrations ranging from 25 nM to 400 nM coatings. This artificial fusion molecule is plate-bound. It can mimic cell surface NKG2D ligands and can lead to activation of NKG2D. On the day of the assay, the plates were washed, blocked with BSA, and pre-sensitized PBMCs were added to the plates and incubated at 37°C and 5% CO2 for 24 hours. This assay was run in the absence of sULBP6. Cell-free assay medium was collected and IFNg levels were determined using a Luminex IFNg kit or an Invitrogen ELISA kit (Catalog No. 88-7316-88).

[0436] result:

[0437] Plate-bound anti-lysozyme antibody negative effector (E-) ("Ab E-") had no effect on modulating IFNg secretion at any concentration and was therefore considered background ( Figure 4 Plate-bound anti-lysozyme antibody (E+) ("Ab E+") increased IFNg secretion above background levels in a dose-dependent manner ( Figure 4 NKG2D activation via ULBP6 fused to an anti-lysozyme antibody negative effector (E-) (ULBP6 / Ab E- in the figure) increased IFNg secretion above background levels in a dose-dependent manner ( Figure 4). The combination of ULBP6 and Fc positive effector anti-lysozyme antibody (E+) (ULBP6 / Ab E+) resulted in significantly higher IFNg secretion compared to the effects of ULBP6 / Ab E- or anti-lysozyme antibody E+ ("Ab E+") alone. Synergism of NKG2D and FcgRIIIa activation was observed, as simultaneous co-activation induced IFNg release greater than the sum of NKG2D activation alone and FcgRIIIa activation alone. This mechanistic insight supports the use of anti-ULBP6 antibodies with active Fc function when such synergy is desired.

[0438] Example 11: Tumor cell immune activation and PBMC mixing assay

[0439] To determine the ability of anti-ULBP6 antibodies to block soluble ULBP6-mediated inhibition of IFNg secretion, a mixed cell assay of tumor cells and PBMCs was performed. A comparison between the effects of the E+ and E- versions of the antibodies was also determined.

[0440] A. Antibody neutralization of sULBP6-mediated inhibition of IFNg secretion in a mixed cell assay of tumor cells and PBMCs

[0441] Materials and methods:

[0442] For mixed cell assays using COV644 cancer cells (expressing shed and surface ULBP6), PBMCs from healthy donors were pre-sensitized for 36 hours with IL-2 and IL-15 (20 ng / ml each) in X-VIVO 15 culture medium. 24 hours before the experiment, 60,000 COV644 cells were plated in a 96-well plate format. On the day of the assay, floating sensitized PBMCs were collected, counted and added to COV644 cells at a ratio of 2: 1. In addition, antibodies (dose titration of h7B3, h8E11, h1D4 and h6E1 from 200nM to 0.5nM) and 50nM soluble recombinant sULBP6_02 were added to the plate. The assay was run in RPMI culture medium containing 10% FBS. After 24 hours of co-incubation, supernatants were collected and IFNg levels were measured using LUMINEX. Dose titration curves and maximum efficacy values ​​were established for each antibody.

[0443] result:

[0444] All anti-ULBP6 antibodies neutralized sULBP6-mediated inhibition of IFNg secretion in both tumor cell and PBMC mixed cell assays, as Figure 5A The antibodies varied in their ability to increase IFNg in the assay, but all responded in a dose-dependent manner. Based on the highest dose of antibody used (200 nM) (maximum potency), the E+ molecule was determined to be superior to the E- and isotype controls ( Figure 5B ).

[0445] B. Complete titration of antibodies (E+ vs. E-) revealed similar EC 50 , but the maximum activity of E+ molecules is higher than that of E- big

[0446] A second PBMC mixed cell assay was performed as described in Part A of the Examples of the present invention to determine the EC of the h7B3 molecule. 50 and maximum activity.

[0447] Materials and methods:

[0448] For mixed cell assays using COV644 cancer cells (expressing shed and surface ULBP6), PBMCs from healthy donors were pre-sensitized for 36 hours with IL-2 and IL-15 (20 ng / ml each) in X-VIVO 15 medium. 24 hours before the experiment, 60,000 COV644 cells were plated in a 96-well plate format. On the day of the assay, floating sensitized PBMCs were collected, counted and added to COV644 cells at a ratio of 2: 1. In addition, antibodies (h7B3 (E+ and E-), h8E11 (E+ and E-) titrated from 0.05nM to 200nM doses) and 50nM recombinant sULBP6_02 were added to the plate. The assay was run in RPMI medium containing 10% FBS. After 24 hours of co-incubation, the supernatant was collected and IFNg levels were measured using LUMINEX. Establishment of ECs of h7B3(E-) and h7B3(E+) 50 curve.

[0449] result:

[0450] Compared with isotype control ( Figure 6 , upper panel), both h7B3 E- and h8E11 E- antibodies showed dose-dependent increases in IFNg levels due to neutralization of the inhibitory effects of soluble ULBP6. Both E+ antibodies showed higher maximal potency, which may be attributed to tumor surface-bound ADCC of the E+ antibodies compared to the E- versions of the same antibodies. For the comparison of the E- and E+ antibodies, h7B3 showed greater maximal potency than h8E11. In the case of the E+ versions of these antibodies, IFNg maximal potency levels were further increased ( Figure 6 , lower panel). H7B3 E- has a similar EC as h7B3 E+ 50 value.

[0451] Example 12: Cytotoxicity and proliferation in tumor immunity mixed cell assay

[0452] In addition to IFNg secretion, in the presence of tumor cells and anti-ULBP6 antibodies described herein, the level of immune cell activation can also be determined by measuring the immune cell surface expression of cytotoxic and proliferation markers. Tumor immune mixed cell assays were performed using PBMCs from healthy donors. PBMCs were incubated with tumor cells and 500nM sULBP6_02 (with or without anti-ULBP6 antibodies), and staining of granzyme B and Ki-67 was measured by flow cytometry. The results show the effect of anti-ULBP6 antibodies on the cytotoxicity and proliferation of NK cells and T cells in the presence of soluble ULBP6.

[0453] Materials and methods:

[0454] PBMCs from healthy donors were sensitized for 36 hours in IL-2 and IL-15 (20 ng / ml each) containing X-VIVO 15 culture medium. Floating cells were collected and re-plated (100,000 cells per well) on COV644 cancer cells (60,000 cells per well). 50 nM of sULBP6-02 was added to each well together with 100 nM of 7B3 (E-), 7B3 (E+) or one of the isotype (E+) (isotype control) antibodies. The assay was run in RPMI culture medium containing 10% FBS. Cells were collected from the wells 24, 48, or 72 hours after incubation and stained with anti-granzyme B or anti-Ki-67 antibodies and gated on CD8 T cells and NK cell populations (as defined by FACS antibodies CD3, CD8, and CD56).

[0455] result:

[0456] Granzyme B and Ki-67 in CD8 and NK cell populations generally decreased over time in the presence of 50 nM sULBP6_02 (isotype antibody). Anti-ULBP6 antibodies (h7B3(E+) and h7B3(E-)) were able to rescue these decreases, with the E+ antibody generally showing greater efficacy than the E- antibody ( Fig. 7A , Figure 7B , Figure 7C and Fig.7D ).

[0457] Example 13: Blocking activity of positive effector antibodies

[0458] To increase ADCC, anti-ULBP6 antibodies with ADCC enhancing mutations S239D / I332E were prepared.In this example, the ULBP6 blocking activity and FcG receptor activation of E-, E+ and E++ candidate anti-ULBP6 antibodies were compared in a mixed cell assay using COV644 cancer cells and PBMCs.

[0459] Materials and methods:

[0460] For mixed cell assays using COV644 cancer cells, PBMCs from healthy donors were pre-sensitized for 36 hours with IL-2 and IL-15 (20 ng / ml each) in X-VIVO 15 culture medium. 24 hours before the experiment, 60,000 COV644 cells were plated in a 96-well plate format. On the day of the assay, floating sensitized PBMCs were collected, counted and added to COV644 cells at a ratio of 2: 1. In addition, antibodies (various concentrations of h7B3 (E-), h7B3 (E+) and h7B3 (E++) (DE)) and 50 nM soluble recombinant sULBP6_02 were added to the plate. The assay was run in RPMI culture medium containing 10% FBS. After 24 hours of co-incubation, the assay supernatant was collected and IFNg levels were measured using luminex.

[0461] result:

[0462] Due to the inhibition of soluble ULBP6, h7B3(E-), h7B3(E+), and h7B3(E++)DE all showed a dose-dependent increase in IFNg in this assay ( Fig. 8A and Figure 8B ). The maximum potency of IFNg in this assay was increased in the case of h7B3(E++)(DE) compared to h7B3(E-) and h7B3(E+) ( Figure 8C ).

[0463] Example 14: Immune cell killing assay (E-, E+, E++ antibodies)

[0464] To test whether the anti-ULBP6 antibodies described herein could enhance tumor cell killing, the INCUCYTE Immune Cell Killing Assay was performed using PBMCs from eight healthy donors to detect immune cell-mediated tumor cell killing.

[0465] Materials and methods:

[0466] PBMCs from 8 healthy donors were sensitized in IL-2 and IL-15 (20ng / ml) in X-VIVO 15 culture medium for 36 hours. One day before the assay was set up, 3,000 COV644-GFP cancer cells (COV644 cancer cell lines engineered to express GFP) were plated in 96-well plates suitable for INCUCYTE assays. On the day of the assay, PBMCs were plated on top of COV644 cells at a ratio of 25:1 in the presence of 50nM human sULBP6_02 protein. In addition, isotype or anti-ULBP6 antibodies (100nM) were also included in this experiment. The humanized antibodies tested in this assay include h7B3 (E-) (ULBP6 neutralizers without FcgR engagement), h7B3 (E+) (ULBP6 neutralizers with FcgR engagement) and h7B3 (E++) (ULBP6 neutralizers with enhanced FcgR engagement). The assay was run in RPMI medium containing 10% FBS. The assay plate was placed in the INCUCYTE machine and GFP levels (per well) were measured over 5 days of the assay. The lower GFP signal between conditions was interpreted as indicating the presence of fewer tumor cells, possibly due in part to tumor cell killing via CD8 T cells and NK cells activated by the ULBP6 antibody.

[0467] result:

[0468] The h7B3 antibody effectively blocked the immunosuppressive activity of soluble ULBP6 and thereby increased immune cell activation and tumor cell killing. Compared with h7B3 E-, h7B3E+ further enhanced tumor cell killing efficacy. Compared with h7B3 E+, h7B3 E++(DE) further enhanced tumor cell killing efficacy. A total of 8 healthy donors were screened in this assay. 4 out of 8 responded strongly to h7B3, such as Fig. 9A Three of the eight donors were defined as moderate responders to h7B3, as shown in Fig. 9B One of the eight was identified as a low responder to h7B3, Fig. 9C Table 18 summarizes the strength of response for each donor and shows all eight donors evaluated in this INCUCYTE assay, as well as the extent to which they responded to the ability of each anti-ULBP6 antibody to kill tumor cells.

[0469] Table 18: Response to anti-ULBP6 antibody treatment of healthy donor PBMCs in the INCUCYTE immune cell killing assay.

[0470]

[0471] Example 15: Effector-enhanced and non-fucosylated antibodies

[0472] This example shows the testing of non-fucosylated anti-ULBP6 antibodies in the immune cell-tumor cell hybrid assay and the INCUCY TE killing assay described above.

[0473] A. IFNg expression in mixed cell assay

[0474] To compare the effects of h7B3(E+), h7B3(E++)(DE) and H7B3(E+) (non-fucosylated, Afu; "h7B3 E+Afu") antibodies on IFNg expression in the previously described mixed cell assay.

[0475] Materials and methods:

[0476] PBMCs were sensitized in X-VIVO 15 medium containing IL-2 and IL-15 (20 ng / ml each) for 36 hours. Floating cells were collected and re-plated (100,000 cells per well) on COV644 cancer cells (60,000 cells per well). 50 nM of sULBP6-02 was added to each well with h7B3 (E+), h7B3 (E++) (DE mutation), or H7B3 (E+) (Afu) ("h7B3 E+Afu") or isotype E+ (isotype control) antibodies at different doses from 0.05 nM to 200 nM. The analysis was run in RPMI medium containing 10% FBS. The assay supernatant was collected after 24 hours and IFNg levels were evaluated.

[0477] result:

[0478] The 7B3(E+) antibody showed a significant increase in IFNg compared to the isotype control, and the h7B3(E++)DE and h7B3(E+) afucosylated ("h7B3 E+Afu") molecules further increased IFNg ( Fig.10 ). In this assay, there was little difference in maximum potency and curve shape between the h7B3(E++)DE and H7B3(E+) non-fucosylated ("h7B3 E+Afu") molecules. The H7B3(E+) non-fucosylated ("h7B3 E+Afu") molecule was equivalent to the 7B3(E++)DE molecule in promoting IFNg secretion.

[0479] B. Tumor cell killing

[0480] An INCUCYTE killing assay was performed on 4 healthy donors to compare the ability of h7B3 E+, h7B3E++(DE), and h7B3 E+Afu to increase tumor cell killing.

[0481] Materials and methods:

[0482] PBMCs from 4 healthy donors were sensitized in IL-2 and IL-15 (20 ng / ml each) in X_VIVO 15 medium for 36 hours. One day before the assay was set up, 3,000 COV644-GFP cancer cells (COV644 cancer cell line engineered to express GFP) were plated in a 96-well plate suitable for INCUCYTE assay. On the day of the assay, PBMCs were plated on top of COV644 cells at a ratio of 25: 1 in the presence of 50 nM sULBP6_02 protein. In addition, isotype or anti-ULBP6 antibodies were also included in this experiment. The assay was run in RPMI medium containing 10% FBS. Humanized antibodies tested in this assay included h7B3 (E+) (ULBP6 neutralizer without FcgR engagement), h7B3 E++ (DE) (ULBP6 neutralizer with FcgR enhanced engagement), and h7B3 Afu (E++) (ULBP6 neutralizer with enhanced FcgR engagement by afucosylation). The assay plates were placed in the INCUCYTE machine and GFP levels were measured over the 5 days of the assay. The lower GFP signal between conditions may be due to the presence of fewer tumor cells, due in part to increased tumor cell killing via CD8 T cells and NK cells due to increased immune activity driven by the ULBP6 antibody.

[0483] result:

[0484] In the tumor killing assay, the h7B3 E+ afucosylated (Afu) antibody was comparable to h7B3E++(DE), with some variation between donors. The h7B3 E++(DE) and h7B3E+Afu ("h7B3 Afu") molecules showed less average GFP per well, which may suggest less tumor per well, which may be attributed to increased tumor cell killing compared to h7B3 E+ and isotype controls. Two of the four donors showed strong responses to the h7B3 E+_Afu ("h7B3 Afu") and h7B3E++DE antibodies ( Fig.11A ), and two showed moderate responses ( Fig. 11B ).

[0485] Example 16: Pharmacokinetic properties of anti-ULBP6 antibodies in cynomolgus monkeys

[0486] The objectives of this study were to determine the pharmacokinetic properties of anti-ULBP6 antibodies in a pharmacologically relevant species, cynomolgus macaques (Macaca fascicularis, Mf), and to screen the variable regions of h7B3(E+) and h8E11(E+) to identify sequence propensities that could cause adverse pharmacokinetic behavior.

[0487] Materials and methods:

[0488] Male (M) cynomolgus monkeys (primitive, 3-4 years old, 3.0 to 3.3 kg, Chinese origin) were administered a single 3 mg / kg dose of h7B3 (E+) (PUR4377) or h8E11 (E+) (PUR4379) by slow intravenous push (5 minutes infusion). Approximately 12 whole blood samples were collected from each animal starting before dosing (pre-dose) and ending 28 days after dosing (post-dose) and then processed into serum. Serum samples were analyzed for the amount of h7B3 (E+) or h8E11 (E+) using a qualified ELISA with a lower limit of quantification (LLOQ) of 0.015 μg / mL. The group mean (N=3) concentration-time curves of h7B3 (E+) and h8E11 (E+) are shown on a semi-logarithmic graph (GraphPad PRISM 9.0). The average PK parameters estimated by non-compartmental analysis (NCA) (Phoenix WinNonlin8.2) are also listed.

[0489] result:

[0490] The anti-ULBP6 antibodies h7B3(E+) and h8E11(E+) have a typical IgG PK profile with linear elimination ( Fig.12 ). h7B3(E+) and h8E11(E+) had long mean half-lives of 11.8 to 13.3 days, respectively (Table 19), consistent with other IgG therapies with linear PK in monkeys (Betts et al., 2018, mAbs, 10(5); 751-764). Other PK properties, including clearance and volume of distribution, were within the typical range for therapeutic IgGs in monkeys (Table 19). There was no difference in exposure between h7B3(E+) and h8E11(E+), as assessed by Cmax and AUClast, confirming that there was no significant sequence bias between the two clones (Table 19). There was no evidence of target-mediated drug disposition (TMDD) at the dose levels tested, suggesting that 3 mg / kg may be a target saturation dose for healthy monkeys ( Fig.12 ).

[0491] Table 19: Mean PK parameters of h7B3(E+) and h8E11(E+) in cynomolgus monkeys estimated by non-compartmental analysis (NCA)

[0492]

[0493]

[0494] Example 17: Structural localization of the h7B3 binding site on ULBP602

[0495] Materials and methods:

[0496] For crystallization, a human ULBP602 construct covering residue positions 26 to 218 was generated. The construct included five His tags at the C-terminus of the protein for purification purposes. Subsequent complex formation, crystallization, data collection, model solving, and model building and improvement were performed by Proteros Biostructures GmbH, Germany. The complex between ULBP6 and h7B3 Fab was formed by mixing the two proteins in a ratio of 1.2: 1 and incubating at room temperature for 2.5 hours. The complex was purified by size exclusion chromatography using a HiLoad 16 / 60 Superdex 75 preparative column pre-equilibrated in 20 mM HEPES pH 7.0, 100 mM NaCl.

[0497] The h7B3-Fab ULBP602 co-crystals were obtained under the following crystal conditions: 0.1 ul H7B3-Fab ULBP602 complex at 26.72 mg / ml in 20 mM HEPES pH 7.0, 100 mM NaCl was mixed with a buffer containing 0.1 ul 10% glycerol (v / v), 0.1 M MES pH 6.9, 5% PEG 1000 w / v, 30% PEG 600 w / v. The resulting droplets were incubated at 4°C.

[0498] The obtained co-crystals were flash frozen in liquid nitrogen. Diffraction data of h7B3-Fab and ULBP602 co-crystals were obtained at the beamline PXI-X10SA of the Swiss Light Source (SLS, Villigen, Switzerland). Diffraction data were collected at a wavelength of 100 Kelvin using a Dectris EIGNER2Si 16M detector. Data were processed using the autoROC XDS and autoPROC AIMLESS programs (Table 20).

[0499] Table 20: Data collection statistics

[0500]

[0501] Phaser and ULB P6 (4S0U) from CCP4 (https: / / www.ccp4.ac.uk / ) software package were used as templates to obtain phase information for determining the structure by molecular replacement. Subsequently, COOT (https: / / www2.mrc-lmb.cam.ac.uk / personal / pemsley / coot / ) and software package CCP4 were used to build and improve the model according to standard protocols (Table 21).

[0502] Table 21: Improvements and model statistics

[0503]

[0504] For the free R-factor calculation, approximately 5.0% of the measured reflections were excluded from the refinement procedure.

[0505] result

[0506] The crystal structure of h7B3 Fab Resolution Fig.13 ). The structure of ULBP602 is highly similar to the previously solved crystal structure of the complex of UBLP6 and NKG2D (PDB entry: 4s0u), with an RMSD of 1098 atoms. h7B3 Fab binding to ULBP6 buried on ULBP6 Using the change in solvent accessible surface area (SASA) between the free ULBP6 structure and the structure bound in the complex, we identified 32 residues that comprise the structural epitope (Table 22). NKG2D is a dimeric protein containing two promoters (A and B) (Zuo et al., 2017). The h7B3 epitope on ULBP602 overlaps with most of the binding site of the NKG2D A promoter and some of the binding site of the NKG2D B promoter ( Fig.14 ).

[0507] In addition to the SASA-based epitope, we also identified an extended structural epitope consisting of the presence of any residue in ULBP602 at h7B3. This epitope contains 54 residues (Table 22).

[0508] Table 22: Structural epitopes of h7B3 for binding to ULBP602

[0509]

[0510]

[0511]

[0512] Example 18: Identification of hULBP6 epitope residues involved in h7B3, h8E11 and h6E1 binding

[0513] To identify the epitope residues involved in the binding of all human clones h7B3, h8E11, and h6E1 to hULBP6, functional epitope mapping with phage display was used. Briefly, human ULBP602 (residues R26-G218) carrying a mouse IgG signal peptide and a C-terminal His tag was generated, followed by functional epitope mapping with phage display, including displaying ULBP6 on phage, generating a ULBP6 phage mutagenesis library, sorting the ULBP6 phage mutagenesis library, and NGS sequencing and data analysis.

[0514] Materials and methods:

[0515] Human ULBP602 (residues R26-G218) carrying a mouse IgG signal peptide and a C-terminal His tag was transiently expressed in 293 cells. The secreted protein was purified from the cell culture supernatant by Ni-NTA chromatography, Ni Excel, 10 mL, XK16 / 5 column was balanced with PBS pH 7.4 as the loading buffer. Elution was performed using a step gradient of 4%, 50%, 100% PBS pH 7.4, 500 mM imidazole. The eluted protein was further purified by size exclusion chromatography (Superdex, 60L XK15 / 30, using PBSk pH 7.4 as buffer). The heavy chain with a C-terminal His tag and the light chain of Fab 7B3 were cloned into two independent vectors and co-transfected into ExpiCHO cells. The Fab was formed from the heavy and light chains and purified from the cell culture supernatant by Ni-NTA chromatography. For purification, the clarified supernatant medium from the expression culture was loaded on a Ni-NTA column balanced with PBS. After loading, the column was washed with PBS and 20 mM imidazole. The eluted protein was further purified by size exclusion chromatography (16 / 600 HiLoad Superdex200, using 2X PBS, pH 6.5). In order to prepare the ULBP6-Fab h7B3 complex, a molar excess of ULBP6 (molar ratio 1.2: 1) was incubated with Fab h7B3 in PBS at pH 7.4 for 2.5 hours, and then size exclusion chromatography was performed on a HiLoad 16 / 60 Superdex 75 pre-classification column pre-balanced with 20 mM HEPES pH 7.0, 100 nM NaCl.

[0516] To display ULBP6 on phage, the production of ULBP6 phage was followed by phage purification, ELISA assay, display of ULBP6 on phage, and folding of ULBP6 on phage as described below.

[0517] The phagemid encoding human ULBP6-HA-pIII is designed to display the ULBP6 protein on the phage surface. The vector is constructed by fusing the ULBP6 gene to the N-terminal domain of the M13 minor coat protein (gIII). An epitope tag (HA tag, amino acid sequence: YPYDVPDYA) is added between the ULBP6 and pIII genes. The phagemid is chemically transformed into XL-1blue competent cells (Agilent Technologies, catalog #200249) and spread on carbenicillin-50 agar plates and incubated overnight at 37°C. Then one colony is inoculated in 5mL 2YT broth (TEKNOVA, catalog #Y0167) for amplification. The culture is incubated with shaking at 37°C until the OD600 reaches 0.3 / mL. 50μL M13K07 helper phage (New England Biolabs, catalog #N0315S) is added to the culture. After shaking for 1 hour at 37° C., 5 mL of the culture was expanded into 50 mL of 2YT broth supplemented with 50 μg / mL carbenicillin and 50 μg / mL kanamycin. The expanded culture was incubated at 37° C. for 4 hours and then at 30° C. overnight.

[0518] The phage cultures were harvested by spinning at 8000 g for 10 minutes at 4°C. The supernatant was collected for phage purification. The phages were precipitated from the supernatant by 2.5M NaCl, 20% PEG solution (TEKNOVA, catalog # P4137) and resuspended by 1xPBS buffer (137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.47mM KH2PO4, pH 7.4). The PEG precipitation process was repeated to increase the purity. The final phage pellet was resuspended in 1xPBS buffer to a concentration of 10.0OD / mL. The OD was read at 268nm on a Nanodrop (ThermoFisher Scientific). The purified phages were stored at 4°C.

[0519] MaxiSorp plates (ThermoFisher Scientific, catalog #437915) were coated with 2 μg / mL antibodies or recombinant protein receptors (e.g., anti-HA antibodies, anti-ULBP6 antibodies, and ULBP6 receptors) overnight and then blocked with 1% bovine serum albumin (BSA, ThermoFisher Scientific, catalog #PI37520) in 1×PBS buffer for 30 minutes. Phages were diluted to 1.0 OD / mL in 120 μL ELISA buffer (0.5% BSA, 0.05% Tween-20 in 1×PBS) in a 96-well plate (ThermoFisher Scientific, catalog #156545). Serial dilutions were performed 3-fold. 65 μL of the diluted phage solution was loaded into each corresponding well in the blocked MaxiSorp plate and incubated for 15 minutes at room temperature with shaking. Then, 100 μL / well of horseradish peroxidase (HRP)-labeled anti-M13 antibody (Sino Biological, catalog #11973-MM05T-H) diluted 1:2500 in ELISA buffer was added as the second reagent. ELISA plates were developed using TMB solution (ThermoFisherScientific, catalog #34022), and the reaction was terminated by adding 50 μL of 2M H2SO4 (VWR, catalog #BDH7259-1). The absorbance was read at 450 nm on a VersaMax microplate reader (Molecular Devices).

[0520] The display of ULBP6 on phage was verified by studying the HA tag expression using ELISA assay. Fig.15 As shown, the binding of ULBP6 phage to anti-HA antibody demonstrated the expression of HA tag and the display of ULBP6 on phage. Negative control using irrelevant phage further excluded nonspecific phage binding to anti-HA antibody.

[0521] The folding of ULBP6 on phage was verified by studying phage binding to ULBP6 lead antibodies discovered using recombinant ULBP6 or the ULBP6 receptor NKG2D (Table 23) using ELISA assays. The ULBP6 phage showed binding affinity to hu7B3, hu8E1, hu1D4, hu6E1 and hu8E8 and the ULBP6 receptor ( Fig.16 and Fig.17 ). The results demonstrated that ULBP6 was correctly folded on the phage.

[0522] Table 23: Anti-ULBP6 Antibodies and ULBP6 Receptors

[0523] Anti-ULBP6 Antibody PUR# 7B PUR3922 h8E11 PUR3926 HkDJ PUR3987 h6E PUR3993 8E8 PUR3924 NKG2D PUR4928 Isotype antibody control PUR3678

[0524] The ULBP6 phage library was engineered for functional epitope mapping by generating random mutagenesis in the gene encoding ULBP6. Mutagenesis was performed using error-prone PCR. The error-prone PCR products were subcloned into phagemids and transformed into XL-1 blue cells to generate a mutagenesis-induced ULBP6 phage library.

[0525] Use GeneMorph II random mutagenesis kit (Aglient, catalog #200550) to implement error-prone PCR. Control the mutation frequency by adjusting the initial target DNA amount and the PCR cycle number. In order to determine the functional residues in antibody binding, the desired mutation frequency is one amino acid change (1-2 nucleotide changes) for each gene. In order to achieve the desired mutation frequency, the error-prone PCR mixture is set as shown in Table 24. The forward and reverse primers used are provided in Table 25.

[0526] Table 24: Error-prone PCR reaction setup

[0527] Components Total 50 μL reaction Mutazyme II Reaction Buffer 5μL 40mM dNTP mix 1μL (200M final concentration) 100ng / μL primer 1.25μL Mutazyme II DNA polymerase (2.5U / μL) 1μL Template 100ng / μL 5μL water 35.5μL

[0528] Table 25: Primers for error-prone PCR

[0529]

[0530] To amplify DNA in a thermal cycler, set up the program as shown in Table 26.

[0531] Table 26: Error-prone PCR thermal cycling settings

[0532]

[0533] The error-prone PCR product was purified using the QIAquick gel extraction kit (Qiagen, catalog #28706X4). The purified PCR product and phagemid vector were digested with restriction enzymes Mlul (New England Biolabs, catalog #R3198) and Xhol (New England Biolabs, catalog #r0146S), and then ligated. The ligated product was transformed into XL-1Blue competent cells. After transformation, XL-1Blue cells were plated on carbenicillin-50 agar plates and incubated overnight at 37°C. 20-30 colonies were selected for Sanger sequencing (ELIM Biopharmaceuticals) and mutation frequency was analyzed. Unbiased mutations were randomly generated in the amplified region using the above-mentioned error-prone PCR protocol. The mutation frequency was 1.2bp / gene.

[0534] In order to produce the phage library induced by mutation, the error-prone PCR product was transformed into XL-1blue competent cells by electroporation. Cells were recovered in 25mL SoC culture medium (ThermoFisher Scientific, catalog #15544034) at 37°C for 40 minutes. 250μL M13K07 helper phage was added to the culture. After incubation at 37°C for 1 hour, the 25mL culture was expanded to 60mL 2YT broth with 50μg / mL carbenicillin and 50μg / mL kanamycin. The amplified culture was incubated at 37°C for 4 hours and then incubated at 30°C overnight. The phage library was harvested by rotating the culture at 8000g for 10 minutes at 4°C, and purified according to the above-mentioned phage purification method. The purified ULBP6 phage library was stored at 4°C. Cell pellets were stored for NGS sequencing.

[0535] The ULBP6 phage library was sorted twice with lead antibodies (7B3, 6E1 and 8E11) by a first round of plate panning and a second round of solution panning. Enrichment was determined by phage titration to evaluate panning performance.

[0536] MaxiSorp plates were coated with 5 μg / mL lead antibodies (7B3, 6E1, and 8E11) in 1xPBS at 100 μL / well overnight at 4°C, 8 wells for each antibody. Two wells were coated with 1xPBS buffer as background. The next day, the plates were washed 6 times with 1xPBST (1×PBS, 0.1% Tween20 detergent) and blocked with 1% BSA (65 μL / well) in 1×PBS buffer for 30 minutes at room temperature. Then, 40 μL of 1xPBS containing 1% Tween-20 was added to each well and incubated for another 30 minutes at room temperature. The plates were washed 6 times with 1xPBST. The mutation-induced phage library was diluted to 2.5 OD / mL and blocked with 1xPBS buffer containing 1% BSA and 0.05% Tween-20 for 30 minutes, shaking at room temperature. The closed phage library is added to the closed plate with 100 μL / well and incubated at room temperature with shaking for 2 hours. The plate is gently washed 10 times with 1xPBST buffer. The bound phage is eluted from the plate using 0.1M HCl (100 μL / well) and neutralized with 1M Tris alkali and 1% BSA. The eluted phage is titrated to determine enrichment (Table 27) and amplified using XL-1Blue cells. The sorted phage library is purified and stored at 4°C. Cell pellets are stored for NGS sequencing.

[0537] Table 27: Plate Panning Enrichment

[0538] Antibody Phage enrichment 7B 75x h6E1 220x h8E11 145x

[0539] MaxiSorp plates were coated with 100 μL / well of 5 μg / mL Neutravidin (ThermoFisher Scientific, catalog #31000) in 1xPBS overnight at 4°C. TM The lead antibodies (7B3, 6E1 and 8E11) were biotinylated using the Sulfo-NHS-LC biotinylation kit (ThermoFisher Scientific, catalog #21435). The phage library was diluted in 600 μL SuperBlock (ThermoFisherScientific, catalog #37515) supplemented with 0.05% Tween-20 and incubated at room temperature for 30 minutes. The blocked phage library was then incubated with the biotinylated lead antibody at 100 nM, 20 nM or 0 nM (background), followed by shaking and incubation at room temperature for 2 hours. The MaxiSorp plate was washed 6 times with 1xPBS T and blocked with 65 μL / well SuperBlock for 30 minutes at room temperature. Then, 40 μL of 1xPBS containing 1% Tween-20 was added to each well and incubated for another 30 minutes at room temperature. The plate was washed 6 times with 1xPBST. The phage / antigen solution was diluted 6 times with Superblock, loaded onto a 100 μL / mL Maxisorp plate, and incubated at room temperature with shaking for 15 minutes. The plate was gently washed 10 times with 1xPBST buffer. The bound phage was eluted from the plate using 0.1M HCl (100 μL / well) and neutralized with 1M Tris alkali and 1% BSA. The eluted phage was titrated to determine enrichment (Table 28) and amplified using XL-1Blue cells. The sorted phage library was purified and stored at 4°C. The cell pellets were stored for NGS sequencing.

[0540] Table 28: Solution Panning Enrichment

[0541] Antibody Phage enrichment, 100nM Phage enrichment, 20nM 7B 176x 222x h6E 286x 77x h8E11 48x 234x

[0542] For sequencing, the ULBP6 mutation-induced phagemid library was split into two amplicons: amplicon 1 spanned the N-terminus of the protein, from positions 29 to 142, while amplicon 2 covered the C-terminus of the protein, including amino acids 101 to 218. All amino acid positions were numbered according to the uniport entry Q5VY80ULBP6_HUMAN. Phagemid libraries were extracted from phage pellets using the Plasmid Miniprep Kit (New England Biolabs, catalog # T1010S). For each amplicon, NGS samples were prepared into adapters and barcodes by two PCR cycles. Using the primers provided in Table 29, Amplicon PCR was performed using High-Fidelity 2X Master Mix (New England Biolabs, catalog #M0492S).

[0543] Table 29: Amplicon PCR Primers

[0544]

[0545] The amplicon PCR reaction settings are shown in Table 30, and the thermal cycle settings are shown in Table 31. The amplicon PCR product was purified with 2% E-Gel EXSize Select (Invitrogen, catalog # G661012). Sequencing adapters were further added using the Nextera XT DNA library preparation kit (Illumina, catalog # FC-131-1024), and the reaction products were purified using AMPure XP beads (ThermoFisher Scientific, catalog # NC9933872). The prepared samples were extracted with 25% PhiX control (Illumina, FC-110-3001) and sequenced using an Illumina MiSe q NGS sequencer.

[0546] Table 30: Amplicon PCR reaction setup

[0547] Components Total 50 μL reaction 2x Q5 Master Mix 25μL Forward primer (10 μM) 2.5μL Reverse primer (10 μM) 2.5μL Template (10 ng / μL) 1μL PCR grade water 19μL

[0548] Table 31: Thermal Cycling Settings

[0549]

[0550] First, the sequencing results obtained from the panning of each of 6E1, 7B3 and 8E11 were analyzed separately (see Table 4 for sample details). In the first step, paired reads were aligned using a flash pairwise aligner. The samples sequenced in sequencing operation 1 (Table 4) were aligned, and the alignment was limited to reads with 100% identical overlap to minimize the impact of sequencing errors in subsequent analysis. Due to the slightly lower quality of the reads in sequencing operation 2, 1% mismatches were allowed in the overlapping regions. Sequencing samples and their statistics are provided in Table 32.

[0551] Table 32: Sequencing samples and statistics

[0552]

[0553]

[0554] Next, a position weight matrix (PWM) was generated for each amino acid sample, which contained the frequency of each of the 20 amino acids at each analyzed position. The assembled paired end reads were translated into amino acid sequences. The occurrences of all 20 amino acids were then counted for each protein position in all reads. The analysis was limited to the segments of each amplicon covered by both R1 and R2. The analysis strategy was selected to minimize the impact of sequencing errors on the position weight matrix. Therefore, PWM covers the following regions of ULBP6: Amplicon 1: 43-122, Amplicon 2: 118-202. Using PWM, the mutation frequency of each position in the PWM is calculated by dividing the sum of all mutation counts at a given position by the total read count covering the position:

[0555] F x =m i x / r x

[0556] where F x is the mutation frequency at position x, m i x is the mutation count of mutation i at position x, and r x is the total number of reads at position x. The mutation frequency is then used to calculate the log2 enrichment ratio (ER):

[0557] ER=log2(S x / P x )

[0558] Where S x is the mutation frequency at amino acid position x in the sorted library, and P x is the mutation frequency at amino acid position x in the pre-sorted library.

[0559] To identify residues of functional epitopes, linear regression analysis was performed between ERs from different epitope bins, comparing the ER values ​​obtained for h7B3 to those obtained for h6E1 and comparing the ER values ​​for h8E11 to those obtained for h6E1. The idea is that positions where mutations have no effect or an indirect effect on antibody binding (e.g., because they often destabilize the antigen fold) should have a small number of residues, whereas mutations that directly affect binding of one of the antibodies will have a large number of residues. Positions with a residue greater than 0.5 were considered part of a functional epitope.

[0560] result:

[0561] For h7B3, four ULBP6 positions were identified with residual differences greater than 0.5 when comparing ER to h6E1 (Table 33):

[0562] Table 33: Positions in the functional epitope of h7B3

[0563]

[0564] For h8E11, two ULBP6 positions were identified with residual differences greater than 0.5 when comparing ER to h6E1 (Table 34).

[0565] Table 34: Positions in the functional epitope of h8E11

[0566]

[0567] For h6E1, seven ULBP6 positions were identified with residual differences greater than 0.5 when comparing ER to h7B3 and h8E11 (Table 35).

[0568] Table 35: Positions in the functional epitope of 6E1

[0569]

[0570]

[0571] Three of the four residues identified as the 7B3 functional epitope (183H, 184Y, and 189D) are surface exposed and located in the NKG2D promoter A binding site, as shown in Fig.18 These three residues are also located within the structural epitope identified by the crystal structure of 7B3 in complex with ULBP6.

[0572] The two residues identified as the functional epitope of 8E11 are also located within the NKG2D promoter A binding site on ULBP6, as Fig.18 The functional epitope of 8E11 overlaps with that of 7B3 because they share two residues 189D in the epitope.

[0573] In contrast to the functional epitopes of 7B3 and 8E1, the residues of the functional epitope of 6E11 are different. Two of the seven residues are located in the NKG2D promoter B binding site, such as Fig.18 As shown in Figures E and F of the .

[0574] Example 19: ULBP6-antibody interactions based on HDX

[0575] The interaction between human ULBP6 and six different monoclonal antibodies (mAbs) was evaluated using hydrogen-deuterium exchange mass spectrometry (HDX).

[0576] Materials and methods:

[0577] The ULBP6 protein used in this example had the following sequence before treatment, as provided in Table 36.

[0578] Table 36: ULBP6 amino acid sequences used in HDX experiments

[0579]

[0580] Residues 20-212 of SEQ ID 123 correspond to residues 26-218 of the native human ULBP6_02 sequence provided herein as SEQ ID NO: 7 (Uniprot: Q5VY80).

[0581] h7B3 (E+Afu), h8E11 (E+), h1D4 (E+), h6E1 (E+), h8E8 (E+), and mab1298 (mIgG2a, R&D SYSTEMS, clone #165903) were tested and Fig.21 A. Fig.21 B and Fig. 22 They are called 7B3, 8E11, 1D4, 6E1, 8E8 and 1298 respectively.

[0582] Buffers and solvents used for the HDX experiments described herein are provided in Table 36.

[0583] Table 36: HDX buffers and solvents

[0584]

[0585] Liquid handling, deuterium labeling, online digestion, and LCMS analysis were performed on an automated HDX-MS system containing the LEAP H / DX PAL TM ROBOTICS (LEAP-TRAJAN), ACQUITY M-Class UPLC (WATERS), HDX MANAGER (WATER S), and CYCLIC mass spectrometer (WATERS). Software packages used for data acquisition and interpretation included MASSLYNX, v4.2 (WATERS), Chronos, v4.11.0-beta.24 (AXE LSEMRAU), ProteinLynxGlobal SERVER (PLGS), v3.0.2 (WATER S), and HDEXAMINER, v3.3.0 (LEAP-TRAJAN).

[0586] All samples were kept at 0°C prior to HDX analysis. An automated LEAP-TRAJAN liquid handling workstation was used to prepare samples for HDX. Samples for peptide mapping were prepared as 40 pmol of ULBP6 protein stock in 5 μl MOPS-H2O buffer, diluted to a final volume of 60 μl with 55 μl MOPS-H2O. The prepared samples were labeled as 40 pmol of ULBP6 protein (apo reference) or 40 pmol of ULBP6 protein + 0.75 molar equivalents of mAb (complex of ULBP6 with each of mAb1298, h7B3, h8E8, h1D4, h8E11 and h6E1) in 5 μl MOPS-H2O buffer, diluted to a final volume of 60 μl with 55 μl MOPS-D2O, and incubated at 20°C for a period of 20, 200, 2000 or 20000 seconds.

[0587] For peptide mapping, 55 μl of the reaction mixture was quenched 1:1 with pre-cooled quench buffer for 1 min (labeling experiments) or quenched at 0°C for 1 to 20 min (peptide mapping). 105 μl of the reaction mixture was then injected onto an immobilized digestion column (Nepenthesin 2, Affipro model AP-PC-004) and maintained at 15°C.

[0588] During digestion, the released peptides were trapped on a guard column (2.1 x 5 mm WATERSVANGUARD BEH C18 1.8 μm) maintained at 0.5 °C. Digestion / retention was performed in solvent A for 4 minutes at a flow rate as shown in Table 37.

[0589] Table 37: Flow rate of solvent A during sample digestion / retention

[0590] # Time (minutes) Flow rate (μl / min) 1 initial 100.000 3 3.00 100.000 4 3.20 200.000 5 3.90 200.000 6 4.00 100.000

[0591] The retained peptides were then eluted from the guard column onto an analytical column (1.0 x 50 mm ULP BEH C18, Model: 186002344) in solvent A at 0.5°C according to the initial conditions and analytical gradient provided in Table 38.

[0592] Table 38: Elution gradients for peptide mapping and labeling experiments

[0593] # Time (minutes) Flow rate (μl / min) B％ 1 initial 20.000 11.0 2 0.80 20.000 16.0 3 10.00 20.000 34.0 4 12.00 20.000 46.0 5 12.50 20.000 98.0 6 14.90 20.000 98.0 7 15.00 20.000 11.0

[0594] During elution, mass spectra were recorded from 2.0 to 15.0 min in either HDMSE ion mobility acquisition mode (for peptide mapping experiments) or HDMS ion mobility mode (for labeling experiments). Four replicates were recorded for each peptide mapping and labeling time course.

[0595] The peptide map raw data were processed with PLGS. The PLGS output from multiple replicates was further optimized for sequence coverage, data quality, and reproducibility by implementing the selection algorithm LARS (Sorensen & Salbo, 2018) in-house. The LARS program was applied after hard filtering conditions including PLGS peptide score > 6.5, peptide length ≥ 25, and mass error ≥ 7 ppm.

[0596] Raw data from each labeling time course were analyzed against LARS peptide maps using HDEXAMINER to determine the renewal (ΔFU) values ​​for all peptides. Peptides that showed possible errors (e.g., overlap, poor signal-to-noise ratio, bimodal isotope distribution, or high residues) were removed after visual inspection. The final HDEXAMINER state file was uploaded to the in-house HDX database and visualization tool for interpretation.

[0597] result:

[0598] A final set of 131 peptides was included in the evaluation, which covered 87.7% of the total ULBP6 construct sequence provided herein as SEQ ID NO: 123 (96.3% of the native portion of the ULBP6_02 sequence provided herein as SEQ ID NO: 7), as Fig.19 shown.

[0599] A cutoff of 1 standard deviation was applied to distinguish mAb-induced site-specific protection from global protection at the peptide level. All residues of any peptide exceeding this threshold were considered significantly protected. Fig. 20A (amino acids 26 to 123 of ULBP6) and Fig. 20B (amino acids 114 to 226 of ULBP6) depicts different fraction uptake for each peptide tested. Fig.21 In Figure 2, ULBP6 peptides identified as significantly protected by mAb1298, h7B3, h8E8, h1D4, h8E11, and h6E1 are identified as underlined bold text. The crystal structure of the NKG2D-ULBP6 complex is shown in Fig. 22 Figures 1 to 2 are provided in Figures A to F of , in which residues of the ULBP6 peptide are indicated in black, which have significant protection by mAb1298 (Figure A), h7B3(E+Afu) (Figure B), h8E8(E+) (Figure C), h1D4(E+) (Figure D), h8E11(E+) (Figure E), and h6E1(E+) (Figure F), as determined by HDX.

[0600] Based on the significantly protected residues, the antibodies were identified into three categories of HDX protection patterns, as provided in Table 39.

[0601] Table 39: Pattern categories of antibody protection of ULBP6 residues determined by HDX

[0602] category Antibody 1 mAb1298, h1D4(E+), h6E1(E+) 2 H7B3(E+Afu), h8E11(E+) 3 H8E8(E+)

[0603] Antibodies in class 1 provided significant protection of residues 35 / 36 to 58 / 59 of ULBP6. Antibodies in class 2 protected residues 35 / 36 to 58 / 59 of ULBP6 at a low but significant level, while providing stronger protection of residues 81 / 82 to 96, 150 to 157, and 181 to 190 of ULBP6. Antibodies in class 3 provided significant protection of residues 150 to 169 of ULBP6.

[0604] Considering the position of the above modes in the crystal structure of the NKG3D / ULBP6 complex, the HDX data are compatible with the following binding site.

[0605] The HDX protection pattern of class 1 antibodies is restricted to β-strands 1 and 2, including the middle connecting loop, e.g. Fig. 22 This is consistent with mAbs that target β-sheets away from the NKG2D binding site without disrupting the NDG2D / ULBP6 complex.

[0606] Amino acid segments with strong protection patterns described herein are provided in Table 40.

[0607] Table 40: Protected amino acids of hULBP6 determined by HDX

[0608]

[0609] Strong protection patterns were observed for Class 2 antibodies on the first α-helix (SEQ ID NO: 126 and / or SEQ ID NO: 127), the second α-helix (SEQ ID NO: 127), and the middle loop (SEQ ID NO: 128). The additional weak protection pattern on β-strand 2 (residues 36-58 of ULBP6), which is opposite to the above α-helix, is of marginal significance. Fig. 20A and Fig. 20B It was observed that the peptide covering residues 35 / 36 to 58 / 59 is likely allosteric and does not necessarily indicate direct contacts of the antibodies on these residues. Overall, the protection pattern of class 2 antibodies is consistent with α-helical binding located in the interface of the NKG2D-ULBP6 complex, suggesting that class 2 antibodies can disrupt the interaction between NKG2D and ULBP6.

[0610] The class 3 antibody showed a very strong HDX protection pattern for residues 150 to 169. As the major protection feature is on one side of the NKG2D-ULBP6 complex, there was no prediction as to whether this antibody would disrupt this complex.

[0611] Example 20: In vivo tumor model for evaluating the activity of anti-ULBP6 antibodies

[0612] This example illustrates selected in vivo tumor model studies of the functional activity of one or more anti-ULBP6 antibodies provided herein and may not include all in vivo studies and / or results that may inform on the functional activity or another property of a ULBP6 binding protein provided herein.

[0613] Materials and methods

[0614] The anti-ULBP6 antibodies provided herein can be prepared for administration to a subject. For example, the anti-ULBP6 antibody can be prepared to be approximately or precisely 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg or 1000 mg / kg, or a dose within a range between any of the above two doses. In some embodiments, the anti-ULBP6 antibody can be prepared so that a predetermined volume (e.g., at least about 1 mL / kg, no more than about 40 mL / kg, or between 1 mL / kg and 40 mL / kg) can be administered at one or more doses, such as a dose provided herein or another dose determined to be effective. In some embodiments, a dose of unknown effectiveness can be administered. A control formulation can be prepared that can substantially match the buffer and concentration of the test article, or its effect. In some embodiments, the control formulation can be the same as a formulation comprising an anti-ULBP6 antibody, wherein an antibody having no physiologically significant binding to ULBP6, no physiologically significant binding to FcyR, or no physiologically significant binding to both ULBP6 and FcyR is added, or no antibody is present.

[0615] Murine tumor models can be used to probe the functional activity of one or more anti-ULBP6 antibodies. Briefly, murine tumor models can be engineered to express human ULBP6 or injected with tumor cells expressing either murine or human ULBP6. Murine tumor models can host human immune cells or retain their own immune system.

[0616] Anti-ULBP6 antibody preparations or control preparations can be administered to a mouse tumor model. For example, anti-ULBP6 can be administered in one or more doses, such as the doses provided above. The preparation can be administered to the animal, for example, once a week or more than once. Animals can be sacrificed at different time points, for example, with different tumor sizes, at which time tumors, lymphoid tissues, blood and / or other samples can be collected. In some embodiments, tumor size can be monitored, for example, using a caliper or via one or more appropriate imaging-based techniques.

[0617] Collected blood and / or tissue samples can be processed to isolate phenotypic readout information of biomarkers to prepare slides for IHC or other pathological assessment of, for example, DNA and / or RNA.

[0618] The processed samples can be assessed to determine the presence, absence, or levels of immune cells, as well as their relative status (eg, activation, proliferation, and / or cytotoxicity).

[0619] result:

[0620] We expect to restore CD8 T cell and NK cell cytotoxicity against tumor cells after anti-ULBP6 treatment in vivo. Therefore, phenotypic changes of CD8 T cells and NK cells should be detectable and / or tumor growth inhibited.

[0621] This section describes the results for mice provided herein, but contemplates results for other subjects (e.g., rats, rabbits, monkeys, cats, dogs, hamsters, humans) having tumor burden and / or metastases that express ULBP6 and receiving formulations comprising anti-ULBP6 antibodies.

[0622] Compared to tumor growth in mice or groups of mice receiving a formulation comprising a control antibody, tumor growth (e.g., measured by an acceptable method as a change in tumor volume over time) in mice or groups of mice receiving a formulation comprising an anti-ULBP6 antibody can be at least about 25% slower, at least about 50% slower, at least about 60% slower, at least about 70% slower, at least about 80% slower, at least about 90% slower, at least about 95% slower, or at least about 99% slower, or a range between any two of the foregoing values. For example, for the dose ranges provided herein, the reduction in tumor growth can be dose-dependent.

[0623] Tumor size can be reduced by at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, or a range between any two of the foregoing values, compared to the start of treatment of the mouse or group of mice receiving the formulation comprising the anti-ULBP6 antibody. For example, the dose range provided herein, the reduction in tumor size can be dose-dependent.

[0624] In a mouse or group of mice receiving a formulation comprising an anti-ULBP6 antibody, metastasis may be reduced by at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or a range between any two of the foregoing values, compared to metastasis in the same mouse or group of mice before receiving the formulation.

[0625] In mice or groups of mice receiving a formulation comprising an anti-ULBP6 antibody, the activation of one or more types of immune cells in mice receiving a formulation comprising an anti-ULBP6 antibody may be increased by at least about 20%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or a range between any two of the foregoing values, compared to control mice or groups of mice.

Claims

1. A ULBP6 binding protein, comprising: a) (i) any one or combination of the CDRs selected from CDRH1, CDRH2, CDRH3 of SEQ ID NOs: 16 to 19 and / or CDRL1, CDRL2, CDRL3 of SEQ ID NOs: 26 to 29; or (ii) a CDR variant of (i), wherein the variant has 1, 2 or 3 amino acid modifications; or b) a VH region comprising a sequence that is at least 80% identical to the sequence of any one of SEQ ID NOs: 21 to 24; and / or a VL region comprising a sequence that is at least 80% identical to the sequence of any one of SEQ ID NOs: 31 to 34.

2. The ULBP6 binding protein according to claim 1, wherein the CDR of a) (i) is selected from the group consisting of: CDRH1 of SEQ ID NO:36, 39, 42 or 45; CDRH2 of SEQ ID NO:37, 40, 43 or 46; CDRH3 of SEQ ID NO:38, 41, 44 or 47; CDRL1 of SEQ ID NO:51, 54, 57 or 60; CDRL2 of SEQ ID NO: 52, 55, 58 or 61; and CDRL3 of SEQ ID NO: 53, 56, 59 or 62.

3. The ULBP6 binding protein according to claim 1 or 2, wherein all 6 CDRs are present in the binding protein and are selected from: CDRH1 of SEQ ID NO:36, CDRH2 of SEQ ID NO:37, CDRH3 of SEQ ID NO:38, CDRL1 of SEQ ID NO:51, CDRL2 of SEQ ID NO:52, and CDRL3 of SEQ ID NO:53; CDRH1 of SEQ ID NO:39, CDRH2 of SEQ ID NO:40, CDRH3 of SEQ ID NO:41, CDRL1 of SEQ ID NO:54, CDRL2 of SEQ ID NO:55, and CDRL3 of SEQ ID NO:56; CDRH1 of SEQ ID NO:42, CDRH2 of SEQ ID NO:43, CDRH3 of SEQ ID NO:44, CDRL1 of SEQ ID NO:57, CDRL2 of SEQ ID NO:58, and CDRL3 of SEQ ID NO:59; or CDRH1 of SEQ ID NO:45, CDRH2 of SEQ ID NO:46, CDRH3 of SEQ ID NO:47, CDRL1 of SEQ ID NO:60, CDRL2 of SEQ ID NO:61, and CDRL3 of SEQ ID NO:

62.

4. A ULBP6 binding protein, comprising 6 CDRs selected from the following: CDRH1 of SEQ ID NO:36, CDRH2 of SEQ ID NO:37, CDRH3 of SEQ ID NO:38, CDRL1 of SEQ ID NO:51, CDRL2 of SEQ ID NO:52, and CDRL3 of SEQ ID NO:53; CDRH1 of SEQ ID NO:39, CDRH2 of SEQ ID NO:40, CDRH3 of SEQ ID NO:41, CDRL1 of SEQ ID NO:54, CDRL2 of SEQ ID NO:55, and CDRL3 of SEQ ID NO:56; CDRH1 of SEQ ID NO:42, CDRH2 of SEQ ID NO:43, CDRH3 of SEQ ID NO:44, CDRL1 of SEQ ID NO:57, CDRL2 of SEQ ID NO:58, and CDRL3 of SEQ ID NO:59; or CDRH1 of SEQ ID NO:45, CDRH2 of SEQ ID NO:46, CDRH3 of SEQ ID NO:47, CDRL1 of SEQ ID NO:60, CDRL2 of SEQ ID NO:61, and CDRL3 of SEQ ID NO:

62.

5. The ULBP6 binding protein according to claim 4, wherein the binding protein comprises: a VH region at least 80% identical to SEQ ID NO:21 and a VL region at least 80% identical to SEQ ID NO:31; a VH region at least 80% identical to SEQ ID NO:22 and a VL region at least 80% identical to SEQ ID NO:32; A VH region that is at least 80% identical to SEQ ID NO:23 and a VL region that is at least 80% identical to SEQ ID NO:33; or A VH region at least 80% identical to SEQ ID NO:24 and a VL region at least 80% identical to SEQ ID NO:

34.

6. The ULBP6 binding protein according to any one of the preceding claims, wherein the binding protein comprises: a) a heavy chain (HC) sequence that is at least 80% identical to any one of SEQ ID NOs: 75 to 78, 85 to 88, or 95 to 98; and / or b) a light chain (LC) sequence that is at least 80% identical to any one of SEQ ID NOs: 105-108.

7. A ULBP6 protein that binds to ULBP6 and ULBP2 with a KD of less than or equal to 1 nM, and binds to ULBP5 with a KD of greater than or equal to 1 nM.

8. The ULBP6 binding protein according to any one of the preceding claims, wherein the binding protein inhibits the interaction of human ULBP6 with human NKG2D by 80% or more.

9. A ULBP6 binding protein that binds to human ULBP6 and competes with a reference ULBP6 binding protein for binding to human ULBP6, the ULBP6 binding protein comprising: The VH region sequence of SEQ ID NO:21 and the VL region sequence of SEQ ID NO:31; The VH region sequence of SEQ ID NO:22 and the VL region sequence of SEQ ID NO:32; The VH region sequence of SEQ ID NO: 16 and the VL region sequence of SEQ ID NO: 26; or The VH region sequence of SEQ ID NO:17 and the VL region sequence of SEQ ID NO:

27.

10. The ULBP6 binding protein of any one of the preceding claims, wherein the binding protein is an antibody or an antigen-binding fragment thereof. The ULBP6 binding protein according to claim 10 , wherein the antibody or antigen-binding fragment thereof inhibits the binding of soluble ULBP6 to NKG2D. 12 . The ULBP6 binding protein according to claim 11 , wherein the antibody or antigen-binding fragment thereof inhibits the binding of soluble ULBP6 to NKG2D by 80% or more. 13 . The ULBP6 binding protein according to any one of claims 10 to 12, wherein the antibody or antigen-binding fragment thereof comprises a modified Fc region. 14 . The ULBP6 binding protein of claim 13 , wherein the modified Fc region comprises amino acid substitutions S239D and I332E (as numbered according to the EU index).

15. The ULBP6 binding protein according to any one of claims 10 to 13, wherein the antibody or antigen-binding fragment thereof is non-fucosylated.

16. The ULBP6 binding protein of claim 13, wherein the modified Fc region comprises amino acid substitutions selected from S239D / A330L / I332E, G236A / S239D / A330L / I332E, L235V / F243L / R292P / Y300L / P396L or F243L / R292P / Y300L / V305I / P396L (as numbered according to the EU index).

17. A nucleic acid sequence encoding one or both of the HC and LC of the ULBP6 binding protein according to any one of the preceding claims. An expression vector comprising the nucleic acid sequence of claim 17 .

19. A recombinant host cell, comprising the nucleic acid sequence of claim 17 or the expression vector of claim 18.

20. A method for producing a ULBP6 binding protein, the method comprising culturing the recombinant host cell of claim 19 under conditions suitable for expressing the nucleic acid sequence or vector, thereby producing a polypeptide comprising the ULBP6 binding protein.

21. A ULBP6 binding protein produced by the method of claim 20.

22. A cell line engineered to express the ULBP6 binding protein of any one of claims 1 to 16 and 21.

23. A pharmaceutical composition comprising the ULBP6 binding protein of any one of claims 1 to 16 and 21 and a pharmaceutically acceptable excipient.

24. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the ULBP6 binding protein of any one of claims 1 to 16 and 21, or the pharmaceutical composition of claim 23.

25. The method of claim 24, wherein the cancer is head and neck cancer, cervical cancer, bladder cancer, lung cancer, colorectal cancer, pancreatic cancer, or glioblastoma.

26. The method of claim 24 or 25, wherein the subject is a human.

27. The method of claim 24, 25 or 26, further comprising determining whether the subject expresses soluble ULBP6.

28. The ULBP6 binding protein of any one of claims 1 to 16 and 21 or the pharmaceutical composition of claim 23 for use in therapy.

29. The ULBP6 binding protein of any one of claims 1 to 16 and 21 or the pharmaceutical composition of claim 23, for use in treating cancer.

30. The ULBP6 binding protein of claim 29, wherein the cancer is head and neck cancer, cervical cancer, bladder cancer, lung cancer, colorectal cancer, pancreatic cancer, or glioblastoma.

31. A ULBP6 binding protein for use in treating basal cell carcinoma.

32. A ULBP6 antibody or a fragment thereof, wherein the antibody or the fragment thereof: a) binds to one or more residues of SEQ ID NO: 125 and / or SEQ ID NO: 126; b) binds to a conformational epitope of SEQ ID NO: 125 and / or c) protecting one or more residues of SEQ ID NO: 125 or SEQ ID NO: 126 from deuterium exchange in HDX-MS analysis of SEQ ID NO: 124; d) binds to human ULBP6 and causes the peptide of SEQ ID NO: 125 or SEQ ID NO: 126 derived from human ULBP6 to be more resistant to deuterium incorporation than the corresponding peptide derived from uncomplexed human ULBP6; e) binds to the NKG2D binding interface of human ULBP6; and / or f) disrupting the interaction of human ULBP6 with NKG2D by binding to one or more residues selected from E96, E154, Y184 and D189 of SEQ ID NO:

124.

33. A ULBP6 antibody or a fragment thereof, wherein the antibody or the fragment thereof: a) binds to one or more residues of SEQ ID NO: 128 and / or SEQ ID NO: 129; b) binds to a conformational epitope of SEQ ID NO: 128 and / or SEQ ID NO: 129; c) protecting one or more residues of SEQ ID NO: 128 and / or SEQ ID NO: 129 from deuterium exchange in HDX-MS analysis; and / or d) binds to human ULBP6 and causes the peptide of SEQ ID NO: 128 and / or SEQ ID NO: 129 derived from human ULBP6 to be more resistant to deuterium incorporation than the corresponding peptide derived from uncomplexed human ULBP6; e) binds to the NKG2D binding interface of human ULBP6; and / or f) disrupting the interaction of human ULBP6 with NKG2D by binding to one or more residues selected from E96, E154, Y184 and D189 of SEQ ID NO:

124.

34. The ULBP6 binding protein of claim 32 or claim 33, wherein the binding protein: a) binds to one or more residues of SEQ ID NO: 127; b) binds to a conformational epitope of SEQ ID NO: 127; c) protecting one or more residues of SEQ ID NO: 127 from deuterium exchange during HDX-MS analysis; and / or d) The peptide of SEQ ID NO: 127 that binds to human ULBP6 and causes it to be more resistant to deuterium incorporation than the corresponding peptide derived from uncomplexed human ULBP6.

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