P2ry8 antibodies and methods of treating cancer

Abstract

The present disclosure provides antigen-binding proteins that bind P2RY8, bispecific antigen-binding proteins that bind P2RY8 and a second antigen, and conjugates thereof. Related polypeptides, nucleic acids, vectors, host cells, and conjugates are also provided. Further provided are kits and pharmaceutical compositions comprising such entities. Methods of making the antigen-binding proteins and methods of treating a subject having a cancer are also provided.

Description

Technical Field

[0001] This disclosure generally relates to antibodies specific to P2RY8 and their use in the treatment of cancer.

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 600,437, filed November 17, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0004] Antibodies constitute potent therapeutic agents characterized by limited side effects because they can specifically target different antigens on cells, bacteria, viruses, or toxins associated with P2RY8-related diseases such as lymphoma. Clinically, there is a need for new, additional antibodies, such as those described in this article, to meet the medical needs of patients with lymphoma-related conditions. Summary of the Invention

[0005] This document provides an antigen-binding protein that binds to the P2RY8 protein. In various aspects, the antigen-binding protein of this disclosure binds to the human P2RY8 protein (UniProt accession number: Q86VZ1; SEQ ID NO: 369) and optionally to the monkey P2RY8 protein, but substantially not to the dog P2RY8 protein (NCBI RefSeq accession number: XP_013967130.1; SEQ ID NO: 371). In various aspects, the antigen-binding protein binds to the extracellular domain (ECD) of the P2RY8 protein. In various embodiments, this disclosure provides an antigen-binding protein targeting the P2RY8 protein.

[0006] In various aspects, the antigen-binding protein binds to P2RY8, which is endogenously expressed by human cancer cells (e.g., lymphoma cells). In various cases, the antigen-binding protein of this disclosure inhibits tumor growth in a subject (e.g., a human) without requiring any other part to attach to the antigen-binding protein (e.g., a naked antigen-binding protein). In various cases, the naked antigen-binding protein inhibits tumor growth in a subject (e.g., a human).

[0007] Not bound by any particular theory, the antitumor activity of antigen-binding proteins presented herein allows such entities to be used in methods for reducing tumor growth and treating subjects with tumors or cancer. As further discussed herein, in various respects, antigen-binding proteins encompass antibodies, antibody fragments that bind antigens, or antibody protein products (e.g., engineered proteins containing antibodies or fragments thereof).

[0008] This disclosure also provides antigen-binding proteins comprising at least 3, 4, 5, or all of a specific set of amino acid sequences. In each respect, the antigen-binding protein comprises at least 3, 4, 5, or 6 complementarity-determining region (CDR) amino acid sequences of the P2RY8 antibody disclosed herein.

[0009] This disclosure also provides antigen-binding proteins comprising the amino acid sequences detailed herein.

[0010] This disclosure provides a bispecific antigen-binding protein that binds to a P2RY8 protein and a second antigen. The bispecific antigen-binding protein may include any of the antigen-binding proteins described herein. The second antigen may be a cell surface protein, optionally a protein that binds to regulate an immune response. The bispecific antigen-binding protein may take any structure, such as a biantibody, a tandem biantibody, a BiTE (bispecific T-cell connector), etc.

[0011] This disclosure also provides a conjugate comprising an antigen-binding protein or a bispecific antigen-binding protein and a heterologous moiety (e.g., a cytotoxic drug). The conjugate may contain a cleavable or non-cleavable linker. The conjugate may have a different number of heterologous moieties (pharmaceuticals) conjugated to the antigen-binding protein or bispecific antigen-binding protein described herein, preferably 1-8 agents / protein or 3-8 agents / protein. Alternatively, the conjugate may have a different number of two or more heterologous moieties conjugated to the antigen-binding protein or bispecific antigen-binding protein (e.g., two or more different agents). The conjugate may be a site-specific conjugate. The conjugate may be a homogeneous conjugate or a heterogeneous conjugate.

[0012] In some respects, the antigen-binding proteins of this disclosure may include tags (e.g., HA, myc, flag, GST) and / or may be detectably labeled (e.g., containing GFP, conjugated to a fluorescent agent, labeled with a radioisotope, attached to an enzyme (e.g., luciferase), etc.).

[0013] This article also provides information on related peptides, nucleic acids, vectors, host cells, and conjugates. Furthermore, it covers kits and pharmaceutical compositions containing such entities.

[0014] Methods for preparing antigen-binding proteins are also provided. In various embodiments, the method includes culturing host cells to express antigen-binding proteins or peptides, the host cells containing nucleic acids encoding antigen-binding proteins or peptides as described herein.

[0015] This document also provides a method for treating a subject suffering from cancer. In various embodiments, the method includes administering to the subject an amount of the disclosed pharmaceutical composition effective in treating the subject's cancer.

[0016] Methods for treating a subject with cancer expressing P2RY8 are also provided, comprising administering the pharmaceutical composition described herein to the subject. In various embodiments, the cancer expressing P2RY8 expresses the P2RY8 protein. Cancer cells expressing P2RY8 may overexpress wild-type P2RY8 protein, express mutant P2RY8 protein, or a fusion expressing P2RY8 protein. A method for inhibiting tumor growth in a subject is also covered, comprising administering the pharmaceutical composition described herein to the subject.

[0017] This article describes and provides a method for reducing tumor size or preventing cancer recurrence in a subject, comprising administering a pharmaceutical composition to the subject.

[0018] This article also provides a method for treating cancer in a subject diagnosed with P2RY8 protein overexpression, comprising administering the pharmaceutical composition described herein to the subject.

[0019] In various embodiments, the administration step induces apoptosis in tumor cells, such as cells expressing the P2RY8 protein. In various embodiments, administration induces antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), tumor necrosis and cell death or exhaustion, and / or disruption of tumor cell adhesion, each of which contributes to tumor regression or slows tumor growth. Attached Figure Description

[0020] Figure 1 This is a box plot of P2RY8 expression in normal human tissues or cells from GTEx data. The data was downloaded from the GTEx portal (gtexportal.org / home / datasets) as GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_tpm and plotted using the ggplot2 package in the R programming language.

[0021] Figure 2 This is a box plot of P2RY8 expression in human cancers from TCGA. P2RY8 RNA expression data from a total of 10,071 human cancer tissue samples were downloaded from cBioPortal (cbioportal.org / ) and plotted using the ggplot2 package in the R programming language.

[0022] Figure 3 This is a box plot showing P2RY8 expression in the TORL human cancer cell line group. P2RY8 RNA expression was analyzed in a total of 587 human cancer cell lines.

[0023] Figure 4The sequences of human P2RY8 protein (UniProt accession number: Q86VZ1; SEQ ID NO: 369), Macacafascicularis P2RY8 protein (NCI Ref Seq: XP_005595888.1; SEQ ID NO: 370), and dog P2RY8 protein (NCBI Ref Seq: XP_013967130.1; SEQ ID NO: 371) were aligned using the ClustalOmega multiple sequence alignment program (Sievers F. et al., (2011): Mol. Syst. Biol. 7:539).

[0024] Figures 5A-5C This study demonstrates the efficacy of P2RY8 mouse mAb in xenografts of P2RY8-positive lymphoma cell lines. Figures 5A-5C Indicates tumor volume (mm) 3 Changes over time (days) Figure 5A ) or tumor volume on day 24 (mm) 3 The average change () Figure 5B ) and percentage change in weight ( Figure 5C The figure shows the results in SCID mice carrying the human P2RY8-positive B-cell lymphoma cell line SU-DHL-5 (ATCC CRL-2958) after weekly treatment with either a control human IgG1 antibody (Hu-IgG) or one of five different chimeric anti-P2RY8 antibodies (P2RY8 mAb-690, P2RY8 mAb-716, P2RY8 mAb-720, P2RY8 mAb-724, or P2RY8 mAb-729) at 10 mg / kg. Each chimeric anti-P2RY8 antibody contains the variable regions of the mouse immunoglobulin (Ig) heavy and light chains (V). H and V L The study included the human Igγ1 (IgG1) heavy chain constant region and the human carpa (κ) light chain constant region. Treatment with rituximab (an anti-CD20 chimeric antibody against B-cell lymphoma) at 10 mg / kg every 4 days was provided for comparison.

[0025] Figures 6A-6C This study demonstrates the efficacy of P2RY8 mouse mAb in xenografts of P2RY8-positive lymphoma cell lines. Figures 6A-6C Indicates tumor volume (mm) 3 Changes over time (days) Figure 6A ) or tumor volume on day 15 (mm) 3 The average change () Figure 6B ) and percentage change in weight ( Figure 6C The figure shows the results in SCID mice carrying another human P2RY8-positive B-cell lymphoma cell line SU-DHL-10 (ATCC CRL-2963) after weekly treatment with a control human IgG1 antibody (Hu-IgG) or five different chimeric P2RY8 antibodies against the P2RY8 protein (P2RY8 mAb-690 (also referred to herein as 10-0690-m), P2RY8 mAb-716 (also referred to herein as 10-0716-m or P2RY8mAb-716), P2RY8 mAb-720 (also referred to herein as 10-0720-m), P2RY8 mAb-724 (also referred to herein as 10-0724-m), or P2RY8 mAb-729 (also referred to herein as 10-0729-m)) at 10 mg / kg. For comparison, treatment with rituximab (an anti-CD20 chimeric antibody against B-cell lymphoma) at 10 mg / kg every 4 days was provided.

[0026] Figures 7A-7C The results showed that P2RY8 mouse mAb was inactive in xenografts of P2RY8-negative lymphoma cell lines. Figures 7A-7C Indicates tumor volume (mm) 3 Changes over time (days) Figure 7A ) or tumor volume (mm) on day 23 3 The average change () Figure 7B ) and percentage change in weight ( Figure 7C The figure shows the results of weekly treatment with either a control human IgG1 antibody (Hu-IgG) or one of five different chimeric P2RY8 antibodies (P2RY8 mAb-690, P2RY8 mAb-716, P2RY8 mAb-720, P2RY8 mAb-724, or P2RY8 mAb-729) at 10 mg / kg in SCID mice carrying the human P2RY8-negative B-cell non-Hodgkin lymphoma cell line (SCC-3; RRID: CVCL_1683). Rituximab (an anti-CD20 chimeric antibody against B-cell lymphoma) at 10 mg / kg is provided for comparison.

[0027] Figure 8The study showed that the P2RY8-positive human large B-cell lymphoma cell line Karpas-1106P (RRID: CVCL_1821) was stained with four candidate anti-P2RY8 antibodies (716-h3 (also referred to in this paper as P2RY8 mAb-716-h3), 716-h4 (also referred to in this paper as P2RY8 mAb-716-h4), 716-h5 (also referred to in this paper as P2RY8 mAb-716-h5), or 716-h6 (also referred to in this paper as P2RY8 mAb-716-h6)) and the staining was performed using indirect immunofluorescence assays with Alexa Fluor. ® The top image shows the fluorescence staining results of rat anti-human IgG Fc secondary IgG2a antibody labeled with AF647 (AF647), while the bottom image shows the phase contrast image of cells in the same field of view. Scale bar = 50µm

[0028] Figure 9 The images show staining of P2RY8-positive human large B-cell lymphoma cell line Karpas-1106P (RRID: CVCL_1821) with three other candidate anti-P2RY8 antibodies, 716-h7, 716-h8 (also referred to herein as P2RY8 mAb-716-h8), and 716-h9, or a chimeric anti-P2RY8 antibody, 716-m (also referred to herein as P2RY8 mAb-716). Indirect immunofluorescence assays were performed using AF647-labeled rat anti-human IgG-Fc secondary IgG2a antibody. The upper panel shows the fluorescence staining results, and the lower panel shows phase-contrast images of the same cells. Scale bar = 50µm

[0029] Figures 10A-10C Indicates tumor volume (mm) 3 Changes over time (days) Figure 10A ) or tumor volume on day 20 (mm) 3 The average change () Figure 10B ) and percentage change in weight ( Figure 10CThe figure shows the results in SCID mice carrying the human P2RY8-positive large B-cell lymphoma cell line SU-DHL-5 (ATCC CRL-2958) after weekly treatment with four different humanized anti-P2RY8 antibodies (P2RY8 mAb-716-h3, P2RY8 mAb-716-h4, P2RY8 mAb-716-h5, or P2RY8 mAb-716-h6) at 10 mg / kg. The P2RY8 mAb-716-h3 to h6 humanized antibodies are human IgG1-kappa (IgG1κ) antibodies containing a CDR of the P2RY8 mAb-716 antibody and / or a CDR variant thereof. Treatment with rituximab at 10 mg / kg every 4 days was provided for comparison.

[0030] Figures 11A-11C Indicates tumor volume (mm) 3 Changes over time (days) Figure 11A ) or tumor volume (mm) on day 20 3 The average change () Figure 11B ) and percentage change in weight ( Figure 11C The figure shows the results in SCID mice carrying the human P2RY8-negative B-cell non-Hodgkin lymphoma cell line SCC-3 (RRID: CVCL_1683) after weekly treatment with four different humanized anti-P2RY8 antibodies (P2RY8 mAb-716-h3, P2RY8 mAb-716-h4, P2RY8 mAb-716-h5, or P2RY8 mAb-716-h6) at 10 mg / kg. Treatment with rituximab at 10 mg / kg every 4 days is provided for comparison.

[0031] Figure 12 This image shows the time progression of internalization of the AF647-labeled chimeric anti-P2RY8 antibody P2RY8 mAb-716 (also referred to herein as 0716m) in the P2RY8-positive human large B-cell lymphoma cell line Karpas-1106P (RRID: CVCL_1821) after staining with Hoechst 33342 DNA dye. Fluorescence microscopy images are superimposed on phase-contrast images, as shown in the two sets of images below. Scale bar = 10µm

[0032] Figure 13This image shows the time progression of internalization of the AF647-labeled chimeric anti-P2RY8 antibody P2RY8 mAb-716 (also referred to herein as 0716m) in the P2RY8-positive human large B-cell lymphoma cell line Karpas-1106P (RRID: CVCL_1821). Fluorescence microscopy images are superimposed on phase-contrast images, as shown at the last time point (below). Scale bar = 10µm.

[0033] Figure 14 This image shows the time progression of internalization of the AF647-labeled chimeric anti-P2RY8 antibody P2RY8 mAb-716 (also referred to herein as 0716m) in the P2RY8-positive human large B-cell lymphoma cell line SU-DHL-5 (ATCC CRL-2958). Fluorescence microscopy images are superimposed on phase-contrast images, as shown at the last time point (below). Scale bar = 10µm

[0034] Figure 15 This image shows the time-course progression of internalization of the AF647-labeled chimeric anti-P2RY8 antibody P2RY8 mAb-716 (also referred to herein as 0716m) in HEK293T cells transfected with the human P2RY8-mGFP expression vector and stained with Hoechst 33342 DNA dye. Fluorescence microscopy images are shown: anti-P2RY8 antibody (red), P2RY8-mGFP fusion protein (green), and cell nucleus (blue). The first image on the left shows only AF647 fluorescence; the AF647-labeled anti-P2RY8 antibody stained the cell membrane prior to internalization. Scale bar = 10µm

[0035] Figure 16 This image shows the time progression of internalization of the AF647-labeled humanized anti-P2RY8 antibody P2RY8 mAb-716-h3 (also referred to herein as 716-h3) in the P2RY8-positive human large B-cell lymphoma cell line Karpas-1106P (RRID: CVCL_1821). Fluorescence microscopy images are superimposed on phase-contrast images, as shown later at four time points. Scale bar = 10 µm

[0036] Figures 17A-17C This study demonstrates the efficacy of P2RY8-ADC in xenografts of P2RY8-positive SUDHL-10 lymphoma cell lines pretreated with IVIG. Figures 17A-17C Indicates tumor volume (mm) 3 Changes over time (days) Figure 17A ) or tumor volume (mm) on day 23 3 The average change () Figure 17B ) and percentage change in weight ( Figure 17CThe figure shows the results in SCID mice carrying the human P2RY8-positive B-cell lymphoma cell line SU-DHL-10 (ATCC CRL-2963) after weekly treatment with a non-targeting human IgG1 antibody (Hu-IgG control mAb), a Hu-IgG control mAb conjugated with MMAE (Hu-IgG control ADC), or a humanized P2RY8 antibody conjugated with MMAE (P2RY8-716-h3-ADC) at 2.5 mg / kg. Each mouse also received 10 mg / kg of human immunoglobulin (IVIG) 24 hours before each administration of the ADC or control mAb.

[0037] Figures 18A-18C This study demonstrates the efficacy of P2RY8-ADC in xenografts of P2RY8-positive SUDHL-5 lymphoma cell lines pretreated with IVIG. Figures 18A-18C Indicates tumor volume (mm) 3 Changes over time (days) Figure 18A ) or tumor volume on day 11 (mm) 3 The average change () Figure 18B ) and percentage change in weight ( Figure 18C The figure shows the results in SCID mice carrying the human P2RY8-positive B-cell lymphoma cell line SU-DHL-5 (ATCC CRL-2958) after weekly treatment with a non-targeting human IgG1 antibody (Hu-IgG control mAb), a Hu-IgG control mAb conjugated with MMAE (Hu-IgG control ADC), or a humanized P2RY8 antibody conjugated with MMAE (P2RY8-716-h3-ADC) at 2.5 mg / kg. Each mouse also received 10 mg / kg of human immunoglobulin (IVIG) 24 hours before each administration of the ADC or control mAb.

[0038] Figures 19A-19C This demonstrates the efficacy of P2RY8-ADC in xenografts of P2RY8-positive RL lymphoma cell lines. Figures 19A-19C Indicates tumor volume (mm) 3 Changes over time (days) Figure 19A ) or tumor volume on day 24 (mm) 3 The average change () Figure 19B ) and percentage change in weight ( Figure 19CThe figure shows the results in SCID mice carrying the human P2RY8-positive B-cell lymphoma cell line RL (ATCC CRL-2261) after weekly treatment with 10 mg / kg of non-targeted human IgG1 antibody (Hu-IgG control mAb), 5 mg / kg of MMAE-conjugated Hu-IgG control mAb (Hu-IgG control ADC), or 5 mg / kg of MMAE-conjugated humanized P2RY8 antibody (P2RY8-716-h3-ADC).

[0039] Figures 20A-20C The results showed that P2RY8-ADC lacked activity in xenografts of P2RY8-negative SCC-3 lymphoma cell lines. Figures 20A-20C Indicates tumor volume (mm) 3 Changes over time (days) Figure 20A ) or tumor volume on day 16 (mm) 3 The average change () Figure 20B ) and percentage change in weight ( Figure 20C The figure shows the results in SCID mice carrying the human P2RY8 negative B-cell lymphoma cell line SCC-3 (Cellosaurus CVCL_1683) after weekly treatment with 10 mg / kg of non-targeted human IgG1 antibody (Hu-IgG control mAb), 5 mg / kg of MMAE-conjugated Hu-IgG control mAb (Hu-IgG control ADC), or 5 mg / kg of MMAE-conjugated humanized P2RY8 antibody (P2RY8-716-h3-ADC).

[0040] Figures 21A-21C This study demonstrates the efficacy of P2RY8-ADC in xenografts of P2RY8-positive SUDHL-10 lymphoma cell lines that have not been pretreated with IVIG. Figures 21A-21C Indicates tumor volume (mm) 3 Changes over time (days) Figure 21A ) or tumor volume on day 20 (mm) 3 The average change () Figure 21B ) and percentage change in weight ( Figure 21C The figure shows the results in SCID mice carrying the human P2RY8-positive B-cell lymphoma cell line SU-DHL-10 (ATCC CRL-2963) after weekly treatment with 10 mg / kg of non-targeted human IgG1 antibody (Hu-IgG control mAb), 5 mg / kg of MMAE-conjugated Hu-IgG control mAb (Hu-IgG control ADC), or 5 mg / kg of MMAE-conjugated humanized P2RY8 antibody (P2RY8-716-h3-ADC).

[0041] Figures 22A-22C This study demonstrates the efficacy of P2RY8-ADC in xenografts of P2RY8-positive SUDHL-5 lymphoma cell lines that have not been pretreated with IVIG. Figures 22A-22C Indicates tumor volume (mm) 3 Changes over time (days) Figure 22A ) or tumor volume (mm) on day 20 3 The average change () Figure 22B ) and percentage change in weight ( Figure 22C The figure shows the results in SCID mice carrying the human P2RY8 positive B-cell lymphoma cell line SU-DHL-5 (ATCC CRL-2958) after weekly treatment with 10 mg / kg of non-targeted human IgG1 antibody (Hu-IgG control mAb), 5 mg / kg of MMAE-conjugated Hu-IgG control mAb (Hu-IgG control ADC), or 5 mg / kg of MMAE-conjugated humanized P2RY8 antibody (P2RY8-716-h3-ADC). Detailed Implementation

[0042] This disclosure describes an antigen-binding protein that targets the P2RY8 protein, for example, the P2RY8 protein (Gα). 13 The coupled receptor is specific to treat cancers expressing P2RY8.

[0043] antigen-binding proteins

[0044] This document provides antigen-binding proteins that bind to the P2RY8 protein. The antigen-binding proteins of this disclosure may be in any of a variety of forms known in the art. In various embodiments, the antigen-binding proteins of this disclosure are in the form of antibodies or antibody fragments or antibody protein products that bind antigens.

[0045] In various embodiments of this disclosure, the antigen-binding protein comprises, is substantially composed of, or is composed of antibodies. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin form, comprising heavy and light chains, and comprising variable and constant regions. For example, an antibody may be IgG, which is a "Y-shaped" structure formed by two pairs of identical polypeptide chains, each pair having one "light chain" (typically with a molecular weight of about 25 kDa) and one "heavy chain" (typically with a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG form, the variable region is generally about 100-110 or more amino acids, comprising three complementarity-determining regions (CDRs) primarily responsible for antigen recognition and varies considerably among other antibodies binding to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region is formed by the N-terminal region of each light and heavy chain, while the constant region is formed by the C-terminal portion of each heavy and light chain. (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4th Edition, Elsevier Science Ltd. / Garland Publishing, (1999)).

[0046] The general structure and properties of antibody CDRs have been described in the art. In short, in an antibody scaffold, CDRs are embedded within a framework of heavy and light chain variable regions, in which they constitute the regions primarily responsible for antigen binding and recognition. The variable region typically contains at least three heavy or light chain CDRs (named CDR regions 1-3, namely CDR1, CDR2, and CDR3, by Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342: 877-883), which are located within the framework regions (named framework regions 1-4, namely FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, ibid.). In relevant embodiments, the residues of the framework are altered. The modifiable heavy chain framework regions are located in regions designated H-FR1, H-FR2, H-FR3, and H-FR4, surrounding heavy chain CDR residues, and the modifiable light chain framework regions are located in regions designated L-FR1, L-FR2, L-FR3, and L-FR4, surrounding light chain CDR residues. Amino acids within the framework regions can be replaced by any suitable amino acid identified, for example, in the human framework or the human common framework. The variable regions of each light and heavy chain can be partitioned or assembled in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0047] Antibodies may contain any constant regions known in the art. Human light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including but not limited to IgM1 and IgM2. Embodiments of this disclosure include all such subclasses or isotypes of antibodies. Light chain constant regions may be, for example, κ or λ type light chain constant regions, such as human κ or λ type light chain constant regions. Heavy chain constant regions may be, for example, α, δ, ε, γ, or µ type heavy chain constant regions, such as human α, δ, ε, γ, or µ type heavy chain constant regions. Therefore, in various embodiments, the antibody is an isotype of IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In a preferred embodiment, the antibody is an antibody of isotype IgG. In a preferred embodiment, IgG1 is an antibody of subclass IgG1. In various aspects, the antibody includes a constant region containing one or more amino acid modifications relative to the naturally occurring counterpart to improve half-life / stability or to make the antibody more suitable for expression / manufacturability. In various cases, the antibody includes a constant region in which C-terminal Lys residues present in the naturally occurring counterpart are removed or cleaved.

[0048] Antibodies can be monoclonal antibodies. In some embodiments, antibodies contain sequences substantially similar to those of naturally occurring antibodies produced by mammals (e.g., mice, rabbits, goats, horses, hamsters, humans, etc.). In this respect, antibodies can be considered mammalian antibodies, such as mouse antibodies, rabbit antibodies, goat antibodies, horse antibodies, hamster antibodies, human antibodies, etc. In some respects, antigen-binding proteins are antibodies, such as human antibodies. In some respects, antigen-binding proteins are chimeric antibodies or humanized antibodies. The term "chimeric antibody" refers to an antibody containing domains from two or more different antibodies. Chimeric antibodies may, for example, contain a constant domain from one species and a variable domain from a second species, or more generally, may contain segments of amino acid sequences from at least two species. Chimeric antibodies may also contain domains from two or more different antibodies within the same species. When referring to the use of antibodies, the term "humanized" means that the antibody has at least a CDR region from a non-human source that has been engineered to have a structure and immune function more similar to a real human antibody than the original source antibody. For example, humanization may involve transplanting the CDR of a non-human antibody (such as a mouse antibody) into a human antibody. Humanization may also involve selecting amino acid substitutions to make the non-human sequence more similar to a human sequence. Information, including sequence information of the heavy and light chain constant regions of human antibodies, is publicly available from the UniProtKB database and other databases well known to those skilled in the art of antibody engineering and manufacturing. For example, the IgG1 constant region is available from the UniProtKB database as described below, which is incorporated herein by reference. In another instance, the human IgG2 constant region is available from the UniProtKB database with UniProt accession number P01859, which is also incorporated herein by reference.

[0049] By way of example only, the sequences of the constant region of the light chain of mouse immunoglobulin κ or the constant region of the heavy chain of immunoglobulin γ-2A include the following.

[0050]

[0051] By way of example only, the sequences of the human immunoglobulin κ light chain constant (IGKC) region, the human immunoglobulin λ constant 2 light chain (IGLC2) region, the human immunoglobulin γ1 heavy chain constant (IGHG1) region, and the human immunoglobulin γ2 heavy chain constant (IGHG2) region include the following.

[0052]

[0053] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves antibodies to produce two Fab fragments and a single Fc fragment. Pepsin cleaves antibodies to produce an F(ab')2 fragment and a pFc' fragment. In various aspects of this disclosure, the antigen-binding protein of this disclosure is an antigen-binding fragment of an antibody (also referred to as an antigen-binding antibody fragment, antigen-binding fragment, antigen-binding moiety). In various cases, the antigen-binding antibody fragment is either a Fab fragment or an F(ab')2 fragment.

[0054] Antibody structures have been used to create an increasing number of alternative antibody forms with molecular weights ranging from at least about 12–150 kDa and valences (n) ranging from monomers (n=1) to dimers (n=2), trimers (n=3), tetramers (n=4), and possibly higher; such alternative antibody forms are referred to herein as “antibody protein products.” Antibody protein products include products based on intact antibody structures and products that mimic antibody fragments that retain intact antigen-binding capabilities, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding fragment retaining an intact antigen-binding site is the Fv fragment, which consists entirely of a variable (V) region. The V region is linked to the scFv (single-chain variable fragment) fragment using soluble, flexible amino acid peptide linkers to stabilize the molecule, or a constant (C) domain is added to the V region to generate the Fab fragment [antigen-binding fragment]. In an exemplary aspect, the antigen-binding fragment is Fab. In an exemplary aspect, the antigen-binding fragment is scFv. Both scFv and Fab fragments can be readily generated in host cells, such as prokaryotic host cells. Other antibody protein products include disulfide-stabilized scFv (ds-scFv); single-chain Fab (scFab); and dimer and multimeric antibody forms, such as biantibodies, triantibodies, and tetraantibodies; or various forms of mini-abs consisting of scFv linked to oligomerized domains. The smallest fragments are the VHH / VH of camel heavy chain Abs and single-domain Abs (sdAbs). The most commonly used building blocks for creating new antibody forms are single-chain variable (V) domain antibody fragments (scFvs) containing V domains (VH and VL domains) of the heavy and light chains linked by peptide linkers of about 15 amino acid residues. Peptide antibodies or peptide-Fc fusions are another type of antibody protein product. The structure of a peptide antibody consists of a bioactive peptide grafted onto an Fc domain. Peptide antibodies are described in detail in the art. See, for example, Shimamoto et al., mAb 4(5): 586-591 (2012).

[0055] Other antibody protein products include single-chain antibodies (SCAs), biantibodies, triantibodies, tetraantibodies, bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five major classes: BsIgG, augmented IgG, bispecific antibody (BsAb) fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A: 97-106 (2015).

[0056] In all respects, the antigen-binding proteins of this disclosure comprise, are substantially composed of, or are composed of any of these antibody protein products. In all respects, the antigen-binding proteins of this disclosure comprise, are substantially composed of, or are composed of any of the following: scFv, Fab VHH / VH, Fv fragments, ds-scFv, scFab, dimer antibodies, polyantibodies (e.g., biantibodies, triantibodies, tetraantibodies), miniAbs, peptide antibodies of camelid heavy chain antibodies VHH / VH, sdAbs, biantibodies; triantibodies; tetraantibodies; bispecific or trispecific antibodies, BsIgG, augmented IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates.

[0057] In various cases, the antigen-binding protein of this disclosure is an antibody protein product in monomeric form or in polymeric, oligomeric, or multimeric form. In some embodiments where the antibody comprises two or more different antigen-binding domain fragments, the antibody is considered bispecific, trispecific, or multispecific, or divalent, trivalent, or multivalent, depending on the number of different epitopes that the antibody recognizes and binds.

[0058] In various embodiments, the anti-P2RY8 antibody or its antibody variants are selected from the group consisting of: human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, recombinant antibodies, antibody fragments that bind antigens, single-chain antibodies, monomeric antibodies, biantibodies, triantibodies, tetraantibodies, Fab fragments, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, and IgG4 antibodies.

[0059] In all respects, the antigen-binding protein of this disclosure is linked to a therapeutic agent. As described below, the therapeutic agent may be any therapeutic agent known in the art, including but not limited to chemotherapeutic agents, cytokines and growth factors, cytotoxic agents, etc. See “Conjugates” below.

[0060] Bispecific form

[0061] In an exemplary embodiment, the antigen-binding protein is bispecific and therefore capable of binding to two different and distinct antigens. In an exemplary embodiment, the antigen-binding protein is bispecific and binds to both the P2RY8 protein and a second antigen.

[0062] In an exemplary embodiment, the second antigen is a cell surface protein expressed by T cells. In an exemplary embodiment, the cell surface protein is a component of the T cell receptor (TCR), such as CD3. In an exemplary embodiment, the second antigen is a co-stimulatory molecule that assists in T cell activation, such as CD40 or 4-1BB (CD137). In an exemplary embodiment, the second antigen is an Fc receptor. In various aspects, the Fc receptor is an Fc γ receptor, an Fc-α receptor, or an Fc-ε receptor. In an exemplary embodiment, the Fc receptor is CD64 (Fc-γ RI), CD32 (Fc-γ RIIA), CD16A (Fc-γ RIIIIA), CD16b (Fc-γ RIIIIb), FcεRI, CD23 (Fc-εRII), CD89 (Fc-ε RI), Fcα / µR, or FcRn. In an exemplary embodiment, the Fc receptor is CD16A. In an exemplary embodiment, the second antigen is an immune checkpoint molecule, such as a protein involved in the immune checkpoint pathway. Immune checkpoint pathways and the molecules or proteins that function therein are known in the art. See, for example, Pardoll, Nat Rev Genet 12(4):252-264 (2012). In exemplary cases, immune checkpoint molecules are A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7. Optionally, immune checkpoint molecules are PD-1, LAG3, TIM3, or CTLA4.

[0063] More than fifty forms of bispecific antigen-binding proteins are known in the art, some of which are described in Kontermann and Brinkmann, Drug Discovery Today 20(7): 838-847 (2015); Brinkmann and Kontermann, mAb 9(2): 182-212 (2017); Zhang et al., Exp Hematol Oncol 6:12 (2017); Spiess et al., Mol Immunol.; 67(2 Pt A):95-106 (2015); Front Immunol 12:626616 (2021). In an exemplary aspect, the bispecific antigen-binding proteins of this disclosure are prepared by chemical engineering, genetic engineering, or tetrageneous hybridoma technology.

[0064] In an exemplary aspect, the bispecific antigen-binding protein is constructed from some or all of the constant domains of an antibody. In an exemplary aspect, the bispecific antigen-binding protein of this disclosure comprises an Fc polypeptide and retains Fc-mediated effector function. In various cases, the bispecific antigen-binding protein is a bispecific monoclonal antibody formed, for example, by chemical cross-linking of two monoclonal antibodies (MABs) or by a "mortar and pestle" technique. In an exemplary aspect, the bispecific antigen-binding protein is made by a "mortar and pestle" technique, wherein heterodimerization of the H chain is driven by introducing different mutations in the two CH3 domains, thereby producing an asymmetric antibody. A "mortar" mutation is generated in one HC and a "pestle" mutation is created in another HC to promote heterodimerization. In an exemplary aspect, the bispecific antigen-binding protein is a bispecific antibody produced by a tetrageneous hybridoma technique based on somatic cell fusion of two different hybridoma cells that produce monoclonal antibodies with the desired specificity (Zhang et al., 2017), ibid. In an exemplary aspect, bispecific antigen-binding proteins are crossMab, orthogonal Fab IgG, DVD-Ig, dual-antigen IgG, IgG-scFv, and scFv2-Fc (Kontermann and Brinkmann, 2015), ibid. In various aspects, bispecific antigen-binding proteins are Ig-scFv fusions, wherein a new antigen-binding moiety is added to full-length IgG to produce a fusion protein with tetravalent binding capacity for two different antigens, such as IgG C-terminal scFv fusions and IgG N-terminal scFv fusions. In an exemplary case, a bispecific antigen-binding protein is a dual variable domain IgG (DVD-IgG), wherein the LC and HC variable regions of IgG specific for one antigen are fused via linkers to the N-terminal LC and HC variable regions of IgG specific for a second antigen to form DVD-IgG. In an exemplary aspect, a bispecific antigen-binding protein is a biantibody-Fc fusion, which involves replacing the Fab fragment of IgG with a bispecific biantibody.

[0065] In alternative embodiments, the bispecific antigen-binding protein of this disclosure does not contain an Fc polypeptide. In an exemplary aspect, the bispecific antigen-binding protein comprises a variable domain of each parental monoclonal antibody, and a linker is cloned and linked to form a single-chain bispecific antibody. In an exemplary aspect, the bispecific antigen-binding protein is a tandem scFv, a biantibody form, a single-chain biantibody, a tandem biantibody (TandAb), an amphiphilic heavy targeting molecule (DART), a docking-locking (DNL), and a nanobody (Fan et al., J Hematol Oncol. 2015; 8:130). In various aspects, the bispecific antigen-binding protein is a bispecific F(ab) ’2. scFv, bispecific bispecific antibody (BsDb), single-chain bispecific bispecific antibody (scBsDb), single-chain bispecific tandem variable domain (scBsTaFv), docking-locked trivalent Fab (DNL-(Fab)3), single-domain antibody (sdAb), or bispecific single-domain antibody (BssdAb). In an exemplary aspect, the bispecific antigen-binding protein is a tandem scFv comprising two scFv fragments linked by an additional peptide linker such as a glycine-serine repeat motif. Optionally, the tandem scFv comprises the following structure: VL A -Connector 1–VH A -Connector 2–VH B -Connector 3–VL B (VL and VH are derived from single-chain antibody fragments; A and B represent parental monoclonal antibodies A and B). In an exemplary aspect, the bispecific antigen-binding protein is TandAb, which contains two pairs of VL and VH domains linked together as a single polypeptide chain (Reusch et al., MAb. 2015; 7(3):584-604). The two polypeptide products dimerize head-to-tail, forming a homodimer with a large molecular weight (approximately 105 kDa) after expression. In an exemplary aspect, the bispecific antigen-binding protein is a protein generated using crossMab technology, described in PNAS 108(27):11187-92 (2011). CrossMab has no chemical linkers or conjugates and is generated by a method that drives the correct light chain to associate in a bispecific heterodimeric IgG antibody. In an exemplary embodiment, CrossMab is a bivalent (1+1), trivalent (2+1), or tetravalent (2+2) bispecific crossMab, or a crossMab based on a non-Fc tandem antigen-binding fragment (Fab). In an exemplary case, crossMab is a crossMab. Fab crossMab VH-VL or crossMab CH1-CL .

[0066] In an exemplary aspect, the bispecific antigen-binding protein comprises a single-domain antibody or nanobody containing a single monomeric variable antibody domain. Optionally, the variable domain is based on a heavy chain variable domain. Alternatively, the variable domain is based on a light chain variable domain.

[0067] In an exemplary aspect, the bispecific antigen-binding protein is a bispecific T-cell connector or BiTE®. BiTE is a bivalent small molecule that contains only the variable region of an antibody in the form of an scFv linked via a flexible peptide linker. In an exemplary aspect, the bispecific antigen-binding protein comprises an scFv containing the LC and HC variable regions of the currently disclosed P2RY8 antibody and the LC and HC variable regions of a second antibody specific to a second antigen. In some embodiments, BiTE comprises the LC and HC variable regions of a second antibody specific to CD3. In some embodiments, CD3 is CD3E.

[0068] In an exemplary case, the bispecific antigen-binding protein is a dual-affinity retargeting (DART) protein. Unlike BiTE®, the covalent link between the two chains of DART restricts the degrees of freedom of the antigen-binding site. Therefore, DART has a compact structure and is able to form a stable contact between target cells and effector cells. DART contains two engineered Fv fragments, whose own domain heads (VH) exchange with the VH of the other fragment. The exchanged Fv domains advantageously release the variant fragments from conformational constraints via short linker peptides.

[0069] In an exemplary aspect, the bispecific antigen-binding protein is HSABody, which comprises two scFvs fused to a modified HSA. HSABody is described in McDonagh et al., Mol Cancer Ther. 2012;11(3):582–93.

[0070] Therefore, in an exemplary aspect, the bispecific antigen-binding protein comprises an antigen-binding fragment of any of the currently disclosed P2RY8 antibodies. In an exemplary aspect, the bispecific antigen-binding protein comprises the Fab of any of the currently disclosed P2RY8 antibodies. In an exemplary aspect, the bispecific antigen-binding protein comprises the Fab of any of the currently disclosed P2RY8 antibodies and the Fab of a second antibody specific to a second antigen. In an exemplary aspect, the bispecific antigen-binding protein comprises F(ab)2 of any of the currently disclosed P2RY8 antibodies. In an exemplary aspect, the bispecific antigen-binding protein comprises F(ab') of any of the currently disclosed P2RY8 antibodies. In an exemplary aspect, the bispecific antigen-binding protein comprises F(ab') of any of the currently disclosed P2RY8 antibodies and F(ab') of a second antibody specific to a second antigen. In an exemplary aspect, the bispecific antigen-binding protein comprises F(ab')2 of any of the currently disclosed P2RY8 antibodies. In an exemplary aspect, the bispecific antigen-binding protein comprises scFv, which comprises the LC and HC variable regions of any of the currently disclosed P2RY8 antibodies. In various aspects, the antigen-binding fragment is based on a heavy chain variable region, and in other aspects, the antigen-binding fragment is based on a light chain variable region. In an exemplary aspect, the antigen-binding fragment comprises at least a portion of both an HC variable region and an LC variable region. In an exemplary aspect, the bispecific antigen-binding protein comprises at least one (possibly both) of the LC or HC variable region of a currently disclosed P2RY8 antibody and at least one (possibly both) of the LC and HC variable regions of a second antibody specific to a second antigen. In an exemplary case, the bispecific antigen-binding protein comprises an scFv that comprises the LC and HC variable regions of a currently disclosed P2RY8 antibody and the LC and HC variable regions of a second antibody specific to a second antigen.

[0071] Nucleic acid

[0072] This disclosure also provides nucleic acids comprising nucleotide sequences encoding antigen-binding proteins of this disclosure. As used herein, “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally refers to a polymer or modified form of DNA or RNA, which may be single-stranded or double-stranded, synthetic, or derived from a natural source (e.g., isolated and / or purified), and may contain natural, non-natural, or modified nucleotides; and may contain natural, non-natural, or modified internucleotide linkages, such as aminophosphate bonds or thiophosphate bonds, rather than phosphodiester bonds seen between nucleotides in unmodified oligonucleotides. Nucleic acids may comprise any nucleotide sequence encoding any of the antigen-binding proteins of this disclosure. Examples are shown in Tables 5 and 6 and in SEQ ID NO: 324-363.

[0073] The present invention also provides nucleic acid molecules encoding amino acid sequences corresponding to the antigen-binding proteins of the present invention. In one embodiment, the nucleic acid molecule is DNA (e.g., cDNA) or a hybrid thereof. Alternatively, the molecule is RNA or a hybrid thereof.

[0074] In some respects, the nucleic acids disclosed herein are recombinant. As used herein, the term “recombinant” means (i) a molecule constructed outside of living cells by conjugating a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in living cells, or (ii) a molecule produced by replication of the molecule described in (i) above. For the purposes of this document, replication may be in vitro or in vivo.

[0075] In some respects, nucleic acids are constructed using procedures known in the art, based on chemical synthesis and / or enzymatic ligation reactions. See, for example, Sambrook et al. (1989) *Molecular Cloning: A Laboratory Manual*, 2nd ed., Plainview, NY: Cold Spring Harbor Laboratory Press; and Ausubel et al. (1989) *Current Protocols in Molecular Biology*, New York: Greene Publishing and Wiley Interscience. For instance, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to enhance the biological stability of the molecule or the physical stability of the duplex formed during hybridization (e.g., phosphate thioester derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylpiperidine, inosine, N... 6 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl piracetamidine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-Isopentenyladenine, uracil-5-oxyacetic acid (v), wabimethoxyglycoside, pseudouracil, piracetamidine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetic acid, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids disclosed herein may be available from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0076] carrier

[0077] In some respects, the nucleic acids of this disclosure are incorporated into a vector. In this regard, this disclosure provides vectors comprising any of the nucleic acids currently disclosed. In various respects, the vector is a recombinant expression vector. For the purposes of this document, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, allows the host cell to express the mRNA, protein, polypeptide, or peptide. The vectors of this disclosure are not naturally occurring as a whole. However, portions of the vector may be naturally occurring. The vectors currently disclosed may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain natural, non-natural, or modified nucleotides. The vector may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In some respects, modified nucleotides or non-naturally occurring internucleotide linkages do not impede transcription or replication of the vector.

[0078] The vectors disclosed herein can be any suitable vector and can be used for transduction, transformation, or transfection of any suitable host. Suitable vectors include those designed for proliferation and amplification or for expression, or both, such as plasmids and viruses. The vectors can be plasmid-based expression vectors. In all respects, the vectors are selected from the group consisting of: pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, La Jolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Phage vectors such as λGTIO, λGTl 1, λZapII (Stratagene), λEMBL4, and λNMl 149 can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some respects, the vector is a viral vector, such as a retroviral vector. In other respects, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, a herpes simplex virus (HSV) vector, a vesicular stomatitis virus (VSV) vector, a vaccinia virus vector, or a lentiviral vector. See, for example, Howarth et al., Cell Biol. Toxicol. 26(1): 1-20 (2010). In other respects, the vector is a baculovirus vector that infects arthropods, such as insects. In other respects, the baculovirus vector is the alfalfa silver-striped moth multinucleovirus (AcMNPV) or the silkworm nucleopolyhedrovirus (BmNPV). See, for example, Khan, Adv Pharm Bull 3(2): 257-263 (2013); Miller, Bioessays 11(4): 91-96 (1989); Atkinson et al., Pestic Sci 28: 215-224 (1990).

[0079] Table 1 below provides a list of anti-P2YR8 antibody names, synonyms, and corresponding expression vector names that have distinctive features of anti-P2YR8 antibodies.

[0080] Table 1: Names of Exemplary Anti-P2YR8 Antibodies and Their Expression Vectors

[0081]

[0082]

[0083] The vectors disclosed herein can be prepared using standard recombinant DNA techniques, such as those described in Sambrook et al. (ibid.) and Ausubel et al. (ibid.). Circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0084] In some respects, the vector contains regulatory sequences, such as transcription and translation start and stop codons, which are specific to the host type to which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), depending on whether the vector is based on DNA or RNA.

[0085] Vectors may include one or more biomarker genes to select for transformed or transfected hosts. Biomarker genes include biocidal resistance, such as resistance to antibiotics, heavy metals, etc.; complementation in auxotrophic hosts to provide protrophic types, etc. Suitable biomarker genes for currently disclosed expression vectors include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0086] The vector may contain a natural or standard promoter operatively linked to a nucleotide sequence encoding a polypeptide (including its functional portion and functional variants), or to a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding the polypeptide. Selection of the promoter is within the general skill of a technician, e.g., strong, weak, inducible, tissue-specific, and development-specific promoters. Similarly, the combination of nucleotide sequences with promoters is also within the skill of a technician. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and promoters present in long terminal repeat sequences of mouse stem cell viruses.

[0087] host cells

[0088] This document provides host cells containing the nucleic acids or vectors disclosed herein. As used herein, the term "host cell" refers to any type of cell that may contain the vectors currently disclosed and is capable of producing expression products encoded by nucleic acids (e.g., mRNA, proteins). In some respects, host cells are adherent cells or suspension cells, i.e., cells that grow in suspension. In various respects, host cells are cultured cells or primary cells, i.e., cells isolated directly from an organism, such as the human body. Host cells can be any cell type, can originate from any type of tissue, and can be at any developmental stage.

[0089] In all respects, the antigen-binding protein is a glycosylated protein, and the host cell is a glycosylation-capable cell. In all respects, the glycosylation-capable cell is a eukaryotic cell, including but not limited to yeast cells, filamentous fungal cells, protozoan cells, algal cells, insect cells, or mammalian cells. Such host cells are described in the art. See, for example, Frenzel et al., Front Immunol 4: 217 (2013). In all respects, the eukaryotic cell is a mammalian cell. In all respects, the mammalian cell is a non-human mammalian cell. In some respects, the cells are Chinese hamster ovary (CHO) cells and their derivatives (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), engineered cells lacking dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or their derivatives (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic cancer cells P19, and mouse embryonic fibroblast cells NIH. 3T3, mouse fibroblasts L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells J558L, or young hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263 (2013)).

[0090] In order to amplify or replicate the vector, the host cell is, in some respects, a prokaryotic cell, such as a bacterial cell.

[0091] This disclosure also provides cell populations comprising at least one host cell described herein. In some aspects, the cell population is a heterogeneous population comprising host cells containing the described vector and at least one other cell not containing any of the vectors. Alternatively, in some aspects, the cell population is a substantially homogeneous population, wherein the population primarily comprises host cells containing the vector (e.g., is essentially composed of the host cells). In some aspects, the population is a clonal population of cells, wherein all cells in the population are clones of a single host cell containing the vector, such that all cells in the population contain the vector. In various embodiments of this disclosure, the cell population is a clonal population comprising host cells containing the vector as described herein.

[0092] Manufacturing method

[0093] This document also provides a method for producing an antigen-binding protein that binds to the P2RY8 protein. In various embodiments, the method includes culturing a host cell containing nucleic acid in a cell culture medium, the nucleic acid containing a nucleotide sequence encoding an antigen-binding protein as described herein; and harvesting the antigen-binding protein from the cell culture medium. The host cell can be any of the host cells described herein. In various aspects, the host cell is selected from the group consisting of CHO cells, NSO cells, COS cells, VERO cells, and BHK cells. In various aspects, the step of culturing the host cell includes culturing the host cell in a growth medium to support the growth and expansion of the host cell. In various aspects, the growth medium timely increases cell density, culture viability, and productivity. In various aspects, the growth medium contains amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and adherent factors. In various aspects, the growth medium is a fully chemically defined medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factor. In addition to providing nutrients, the growth medium also helps maintain pH and osmotic pressure. Various growth media are commercially available and are described in the art. See, for example, Arora, “Cell Culture Media: A Review” MATER METHODS 3:175 (2013).

[0094] In each respect, the method includes culturing host cells in a fed-batch culture medium. In each respect, the method includes culturing in a fed-batch culture medium. Methods for producing recombinant proteins are known in the art. See, for example, Li et al., “Cell culture processes for monoclonal antibody production” MAb 2(5): 466–477 (2010).

[0095] Methods for preparing antigen-binding proteins may include one or more steps for purifying the protein from a cell culture or its supernatant, and preferably recovering the purified protein. In various aspects, the method includes one or more chromatographic steps, such as affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, or hydrophobic interaction chromatography. In various aspects, the method includes purifying the protein using a protein A affinity chromatography resin.

[0096] In various embodiments, the method further includes the step of formulating the purified protein, thereby obtaining a formulation containing the purified protein. These steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0097] In all respects, the antigen-binding protein is linked to the polypeptide, and the antigen-binding protein is part of the fusion protein. Therefore, this disclosure also provides a method for producing a fusion protein comprising an antigen-binding protein that binds to the P2RY8 protein. In various embodiments, the method includes culturing host cells containing nucleic acids in a cell culture medium, the nucleic acids containing a nucleotide sequence encoding the fusion protein as described herein; and harvesting the fusion protein from the cell culture medium.

[0098] Conjugate

[0099] This disclosure also provides antigen-binding proteins that attach, link, or conjugate to a second part (e.g., a heterologous part, a conjugated part). Therefore, this disclosure provides a conjugate comprising an antigen-binding protein and a heterologous part. As used herein, the term "heterologous part" is synonymous with "conjugated part" and refers to any molecule (chemical or biochemical, naturally occurring or non-coding) that is different from the antigen-binding protein of this disclosure. Various heterologous parts include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents (e.g., cytotoxic agents, cytokines), or diagnostic agents.

[0100] In some embodiments, the heterosource portion is a polymer. The polymer may be branched or unbranched. The polymer may have any molecular weight. In some embodiments, the average molecular weight of the polymer is between about 2 kDa and about 100 kDa (the term "about" means that in a formulation of the water-soluble polymer, some molecules will weigh more than the specified molecular weight, while some molecules will weigh less than the specified molecular weight). In some aspects, the average molecular weight of the polymer is between about 5 kDa and about 50 kDa, between about 12 kDa and about 40 kDa, or between about 20 kDa and about 35 kDa.

[0101] In some embodiments, the polymer is modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, thereby allowing control over the degree of polymerization. In some embodiments, the polymer is water-soluble, so that the proteins to which it is attached will not precipitate in aqueous environments, such as physiological environments. In some embodiments, the polymer is pharmaceutically acceptable when, for example, the composition is used for therapeutic purposes. Additionally, in some aspects, the polymer is a mixture of polymers, such as copolymers or block copolymers.

[0102] In some embodiments, the polymer is selected from the group consisting of: polyamides; polycarbonates; polyolefins and their derivatives, including polyalkylene glycols, polyepoxides, and polyalkylene terephthalates; polymers of acrylates and methacrylates, including polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, polyisobutyl methacrylate, polyhexyl methacrylate, polyisodecyl methacrylate, polylauryl methacrylate, polyphenyl methacrylate, polymethyl methacrylate, polyisopropyl acrylate, polyisobutyl methacrylate, and polyoctadecyl acrylate; and polyvinyl polymers, including polyvinyl alcohol and polyvinyl ether. Polyvinyl acetate, polyethylene halides, polyvinyl acetate and polyvinylpyrrolidone; polyglycolic acid; polysiloxane; polyurethane and its copolymers; cellulose, including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate and sodium cellulose sulfate; polypropylene; polyethylene, including polyethylene glycol, polyethylene oxide and polyethylene terephthalate; and polystyrene.

[0103] The water-soluble polymer particularly preferred in this document is polyethylene glycol (PEG). As used herein, polyethylene glycol is intended to encompass any of the PEG forms that can be used to derive other proteins, such as mono(C1-C10)alkoxy polyethylene glycol or aryloxy polyethylene glycol. PEG is a linear or branched neutral polyether available in a wide range of molecular weights and is soluble in water and most organic solvents.

[0104] In some embodiments, the heterologous component is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), or a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan).

[0105] In some embodiments, the heterologous portion is a lipid. In some embodiments, the lipid is a fatty acid, eicosanoic acid, prostaglandin, leukotriene, thromboxane, N-acylethanolamine), glycerides (e.g., monosubstituted, disubstituted, trisubstituted glycerides), glycerophospholipids (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipids (e.g., sphingosine, ceramide), sterol lipids (e.g., steroids, cholesterol), isopentenyl alcohol lipids, glycolipids, polyketides, oils, waxes, cholesterol, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, or phospholipids.

[0106] In some embodiments, the heterologous portion is a therapeutic agent. The therapeutic agent may be any therapeutic agent known in the art. Examples of therapeutic agents covered in this article include, but are not limited to: natural enzymes, proteins of natural origin, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobulins, β-adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmic drugs, cardiac sympathomimetic drugs, angiotensin-converting enzyme (ACE) inhibitors, diuretics, cardiotonics, cholesterol and triglyceride lowering agents, bile acid sequestrants, fibrates, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, niacin derivatives, antiadrenergic agents, alpha-adrenergic blockers, centrally acting antiadrenergic agents, vasodilators, potassium sparganum, thiazides and related agents, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulin and analogues, alpha- Glucosidase inhibitors, biguanides, chloropicrin derivatives, sulfonylureas, thiazolidinediones, androgens, progestins, bone metabolism modifiers, anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin-releasing hormone antagonists, ovulation induction drugs, selective estrogen receptor modulators, antithyroid agents, thyroid hormones, bulk-forming agents, laxatives, anti-peristaltic agents, gut microbiota modulators, intestinal adsorbents, intestinal anti-infective agents, anti-anorexia agents, anti- Cachexia eliminators, anti-bulimia eliminators, appetite suppressants, anti-obesity eliminators, antacids, gastrointestinal tract eliminators, anticholinergics, aminosalicylic acid derivatives, biological response modifiers, corticosteroids, antispasmodics, 5-HT4 partial agonists, antihistamines, cannabinoids, dopamine antagonists, serotonin antagonists, cell protectants, histamine H2 receptor antagonists, mucosal protectants, proton pump inhibitors, Helicobacter pylori eradication therapy, erythropoiesis stimulants, hematopoietic agents.Anemia medications, heparin derivatives, antifibrinolytic agents, hemostatic agents, coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors, thromboxane A2 inhibitors, plasminogen activators, antithrombotic agents, glucocorticoids, mineralocorticoids, corticosteroids, selective immunosuppressants, antifungal agents, drugs involved in prophylactic treatment, drugs used to treat AIDS-related infections, cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, protease inhibitors, drugs used to treat or prevent anemia, drugs used to treat Kaposi's sarcoma, aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, Macrolides, oxazolidinones, penicillins, streptozotocins, sulfonamides, trimethoprim and its derivatives, tetracyclines, anthelmintics, amoeboids, biguanides, cinchona alkaloids, folic acid antagonists, quinoline derivatives, drugs used in the treatment of Pneumocystis carinii, acylhydrazides, imidazoles, triazoles, nitroimidazoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, drugs used in adjuvant therapy, barbiturates and their derivatives, benzodiazepines, gamma-aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids Anti-migraine preparations, biological response modifiers, carbamates, tricyclic derivatives, depolarizers, non-depolarizers, neuromuscular paralyzers, CNS stimulants, dopaminergic agents, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulfonates, aziridines, imidazotetraazines, nitrogen mustard analogs, nitrosoureas, platinum-containing compounds, antimetabolites, purine analogs, pyrimidine analogs, urea derivatives, anthracyclines, actinomycin, camptothecin derivatives, epipodophyllotoxin, taxanes, vinca alkaloids and analogs, antiandrogens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitors, antitumor drugs, azepam-decanedione derivatives, anxiolytics, stimulants, monoamine reuptake inhibitors Agents, selective serotonin reuptake inhibitors, antidepressants, benzisoxazole derivatives, butyrophenone derivatives, dibenzodiazepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperidine derivatives, phenothiazines, thienobenzodiazepine derivatives, thioxanol derivatives, allergen extracts, nonsteroidal drugs, leukotriene receptor antagonists, xanthine, endothelin receptor antagonists, prostaglandins, pulmonary surfactants, mucolytics, antimitotics, uricosuric agents, xanthine oxidase inhibitors, phosphodiesterase inhibitors, urotropine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants, parasympathomimetic drugs, halogenated hydrocarbons, esters of aminobenzoic acid, amides (e.g.,Lidocaine, articaine hydrochloride, bupivacaine hydrochloride), antipyretics, hypnotics and sedatives, cyclopyrrolidones, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, p-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase reactivators, nicotine replacement therapy, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, and vitamin K analogs and antagonists.

[0107] The antigen-binding protein disclosed herein can be conjugated to one or more cytokines and growth factors that effectively inhibit tumor metastasis, wherein the cytokine or growth factor has been shown to have an antiproliferative effect on at least one cell population. Such cytokines, lymphokines, growth factors, or other hematopoietic factors include, but are not limited to: M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factors, and erythropoietin. Other growth factors used in this study include: angiopoietin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil chemokine 1, cytokine-induced neutrophil chemokine 2 α, cytokine-induced neutrophil chemokine 2 β, β-endothelial growth factor, endothelin-1, epithelial-derived neutrophil attractant, and glial cell line-derived neurotrophic factor receptor α. 1. Glial cell line-derived neurotrophic factor receptor α 2. Growth-related proteins, growth-related protein α, growth-related protein β, growth-related protein γ, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β-binding protein I, transforming growth factor β-binding protein II, transforming growth factor β-binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase plasminogen activator receptor, and their chimeric proteins and biologically or immunologically active fragments.

[0108] In some embodiments, the conjugate comprises an antigen-binding protein and a cytotoxic agent as described herein. A cytotoxic agent is any molecule (chemical or biochemical) that is toxic to cells. In some aspects, when a cytotoxic agent is conjugated with an antigen-binding protein of this disclosure, the resulting effect is synergistic. That is, the effectiveness of the combination therapy of the antigen-binding protein and the cytotoxic agent is synergistic, i.e., its effectiveness is greater than the sum of the effects of the individual components. Therefore, the dosage of the cytotoxic agent can be reduced, and thus the risk of toxicity problems and other side effects is simultaneously reduced. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. Chemootherapeutic agents are known in the art and include, but are not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides, as described in U.S. Patent No. 6,630,124.

[0109] In some implementations, the chemotherapeutic agent is a platinum coordination compound. The term "platinum coordination compound" refers to any platinum coordination compound that provides platinum in ionic form to inhibit the growth of tumor cells.

[0110] In some embodiments, the platinum coordination compound is cis-diammine dihydrate platinum(II) ion, dichloro(diethylenetriamine)platinum(II), dichloro(ethylenediamine)platinum(II), diammine(1,1-cyclobutanedicarboxylic acid)platinum(II) (carboplatin), spiroplatin, isopropylplatinum, diammine(2-ethylmalonic acid)platinum(II), ethylenediaminemalonate platinum(II), hydrated (1,2-diaminocyclohexane)platinum sulfate(II), (1,2-diaminocyclohexane)malonate platinum(II), (4-carboxyphthalic acid)(1,2-diaminocyclohexane)platinum(II), (1,2-diaminocyclohexane)(isocitric acid)platinum(II), (1,2-diaminocyclohexane)cis(pyruvate)platinum(II), (1,2-diaminocyclohexane)platinum oxalate(II), omamarine, and tetraplatinum.

[0111] In some embodiments, cisplatin is a platinum coordination compound used in the compositions and methods of the present invention. Cisplatin is commercially available from Bristol Myers-Squibb Corporation under the name PLATINOL™ and is available in powder form for reconstitution with water, sterile saline, or other suitable mediators. Other platinum coordination compounds suitable for the present invention are known and are commercially available and / or can be prepared using conventional techniques. Cisplatin, or cis-dichlorodiamineplatin(II), has been successfully used for many years as a chemotherapeutic agent for the treatment of various human solid malignancies. Recently, other diaminoplatinum complexes have also shown efficacy as chemotherapeutic agents for the treatment of various human solid malignancies. Such diaminoplatinum complexes include, but are not limited to, spiroplatin and carboplatin. Although cisplatin and other diaminoplatinum complexes have been widely used as human chemotherapeutic agents, they must be delivered at high dose levels, which can lead to toxicity problems such as kidney injury.

[0112] In some implementations, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerases are enzymes that can alter the topological structure of DNA in eukaryotic cells. They are essential for cell function and cell proliferation. Generally, there are two types of topoisomerases in eukaryotic cells: type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of approximately 100,000. This enzyme binds to DNA and introduces a transient single-strand break, unwinding (or unwinding) the double helix, and then re-closes the break before dissociating from the DNA strand. Recently, various topoisomerase inhibitors have shown clinical efficacy in the treatment of patients with ovarian cancer, esophageal cancer, or non-small cell lung cancer.

[0113] In some respects, topoisomerase inhibitors are camptothecin or camptothecin analogues. Camptothecin is a water-insoluble cytotoxic alkaloid produced by *Camptotheca acuminata*, native to China, and *Nothapodytes foetidav*, native to India. Camptothecin exhibits tumor cell growth inhibitory activity against a variety of tumor cell types. Camptothecin analogues are generally specific inhibitors of DNA topoisomerase I. The term "topoisomerase inhibitor" refers to any tumor cell growth inhibitory compound associated with the structure of camptothecin. Camptothecin analogues include, but are not limited to, topotecan, irinotecan, and 9-aminocamptothecin.

[0114] In another embodiment, the cytotoxic agent is any camptothecin analogue that inhibits tumor cell growth claimed or described in the following documents: U.S. Patent No. 5,004,758, granted April 2, 1991, and European Patent Application No. 88311366.4, published on June 21, 1989, under EP 0 321 122; U.S. Patent No. 4,604,463, granted August 5, 1986, and European Patent Application No. EP 0 137 145, published April 17, 1985; U.S. Patent No. 4,473,692, granted September 25, 1984, and European Patent Application No. EP 0 074 256, published March 16, 1983; U.S. Patent No. 4,545,880, granted October 8, 1985, and European Patent Application No. EP 0 16, 1983, under EP 0 321 122. Patent application No. 0 074 256; European Patent Application Publication No. EP 0088 642 published on September 14, 1983; Wani et al., J. Med. Chem., 29, 2358-2363 (1986); Nitta et al., Proc. 14th International Congr. Chemotherapy, Kyoto, 1985, Tokyo Press, Anticancer Section 1, pp. 28-30, especially the compound called CPT-11. CPT-11 is a camptothecin analogue which has a 4-(piperidinyl)-piperidine side chain attached to the C-10 of 10-hydroxy-7-ethylcamptothecin via a carbamate bond. CPT-11 is currently undergoing human clinical trials and is also known as irinotecan; Wani et al., J. Med. Chem., 23, 554 (1980); Wani et al., J. Med. Chem., 30, 1774 (1987); U.S. Patent No. 4,342,776, issued August 3, 1982; U.S. Patent Application Serial No. 581,916, filed September 13, 1990, and European Patent Application Publication No. EP 418 099, published March 20, 1991; U.S. Patent No. 4,513,138, issued April 23, 1985, and European Patent Application Publication No. EP 0 074 770, published March 23, 1983; U.S. Patent No. 4,399,276, issued August 16, 1983, and European Patent Application Publication No. 0, published July 28, 1982. References 056 692; the full contents of each of these documents are hereby incorporated by reference. All of the camptothecin analogues listed above are commercially available and / or can be prepared using conventional techniques (including those described in the references listed above).Topoisomerase inhibitors can be selected from a group consisting of topotecan, irinotecan, and 9-aminocamptothecin.

[0115] In some embodiments, the camptothecin analog is the active metabolite of irinotecan (CPT-11). In some such embodiments, the camptothecin analog is 7-ethyl-10-hydroxycamptothecin (SN-38). As a metabolite, SN-38 is formed by the hydrolysis of irinotecan by a carboxylesterase. In some embodiments, SN-38 has one of the following structures:

[0116] or

[0117] SN-38 has been described in U.S. Patent Application Serial No. 7,999,083, U.S. Patent Application Serial No. 8,080,250, U.S. Patent Application Serial No. 8,759,496, U.S. Patent Application Serial No. 8,999,344, U.S. Patent Application Serial No. 10,195,288 and U.S. Patent Application Serial No. 9,808,537.

[0118] In some implementations, the camptothecin analogue is ecilecan mesylate. Ecinotecan mesylate (ecilecan mesylate) is a water-soluble camptothecin (CPT) that exhibits more potent topoisomerase I inhibitory activity and antitumor activity than other CPT analogues. Furthermore, ecilecan is effective against p-glycoprotein (P-gp)-mediated multidrug-resistant cells.

[0119] In some embodiments, the camptothecin analog is delutecan (Dxd), a potent derivative of ixenothecan, which has 10 times the topoisomerase I inhibitory potency of SN-38. In some embodiments, Dxd has the following structure:

[0120]

[0121] Dxd has been described in U.S. Patent Application Serial No. 6,407,115, U.S. Patent Application Serial No. 10,195,288, U.S. Patent Application Serial No. 9,808,537 and U.S. Patent Application Serial No. 6,407,115.

[0122] The preparation of various camptothecin analogues (including their pharmaceutically acceptable salts, hydrates, and solvates) and the preparation of oral and parenteral pharmaceutical compositions comprising such camptothecin analogues and pharmaceutically acceptable inert carriers or diluents are described in detail in U.S. Patent No. 5,004,758, granted April 2, 1991, and European Patent Application No. 88311366.4, published June 21, 1989, under Publication No. EP 0 321 122, the teachings of which are incorporated herein by reference.

[0123] In other embodiments of the invention, the chemotherapeutic agent is an antibiotic compound. Suitable antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozotocin.

[0124] In some embodiments, the chemotherapeutic agent is an antimitotic alkaloid. Generally, antimitotic alkaloids can be extracted from *Cantharanthus roseus* and have been shown to be effective anticancer chemotherapeutic agents. Many semi-synthetic derivatives have been studied in chemistry and pharmacology (see O. Van Tellingen et al., *Anticancer Research*, 12, 1699-1716 (1992)). The antimitotic alkaloids of the present invention include, but are not limited to, vincristine, vinblastine, vindesine, paclitaxel, and vinorelbine. The latter two antimitotic alkaloids are commercially available from Eli Lilly and Company and Pierre Fabre Laboratories, respectively (see U.S. Patent No. 5,620,985). In one embodiment, the antimitotic alkaloid is vinorelbine.

[0125] In other embodiments of the invention, the chemotherapeutic agent is a difluoronucleoside. 2'-Deoxy-2',2'-difluoronucleosides are known in the art to possess antiviral activity. Such compounds are disclosed and taught in U.S. Patent Nos. 4,526,988 and 4,808,614. European Patent Application Publication 184,365 discloses that these same difluoronucleosides possess oncolytic activity. In some aspects, the 2'-deoxy-2',2'-difluoronucleoside used in the compositions and methods of the present invention is 2'-deoxy-2',2'-difluorocytidine hydrochloride, also known as gemcitabine hydrochloride. Gemcitabine is commercially available or can be synthesized in a multi-step manner as disclosed and taught in U.S. Patent Nos. 4,526,988, 4,808,614, and 5,223,608, the teachings of which are incorporated herein by reference.

[0126] In various aspects, chemotherapeutic agents are antimitotic agents that inhibit cell division by blocking tubulin polymerization, destabilizing microtubules, or altering microtubule dynamics, such as maytansine or derivatives thereof (e.g., DM1 or DM4), auristatin or derivatives thereof. In various cases, the chemotherapeutic agent is auristatin. For example, in some aspects, auristatin is dolasstatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or PF-06380101. Auristatin has been described in the art. See, for example, Maderna, A. et al., Mol Pharmaceutics 12(6): 1798-1812 (2015). In various aspects, the conjugate comprises an antibody of the present disclosure in combination with MMAE. Optionally, the conjugate comprises a linker. In some aspects, the linker comprises a cleavable connecting portion. In various cases, the conjugate comprises an antibody of the present disclosure attached to an attachment group that is attached to a cathepsin-cleavable linker, which in turn is attached to a spacer that is attached to MMAE. In some aspects, the attachment group is attached to the antibody via Cys residues in the Fc region of the antibody. In an exemplary aspect, the attachment group comprises the structure of Formula I:

[0127]

[0128] [Formula I]

[0129] In an exemplary aspect, the cathepsin-cleavable adaptor comprises a structure of formula II:

[0130]

[0131] [Formula II].

[0132] In an exemplary aspect, the spacer sub-encompasses the structure of Formula III:

[0133]

[0134] [Formula III]

[0135] In some implementations, the MMAE has the following structure:

[0136]

[0137] This disclosure also provides conjugates comprising the antigen-binding protein of this disclosure linked to a peptide, thereby making the conjugate a fusion protein. Therefore, this disclosure provides fusion proteins comprising the antigen-binding protein of this disclosure linked to a peptide. In various embodiments, the peptide is a diagnostic marker, such as a fluorescent protein, like green fluorescent protein; or other tags, such as Myc tags. In various aspects, the peptide is one of the cytokines, lymphokines, growth factors, or other hematopoietic factors listed above.

[0138] The antigen-binding proteins disclosed herein can be labeled with radionuclides for diagnostic or therapeutic purposes. Radionuclides that can be used to label antigen-binding proteins include, but are not limited to, actinide-225. 225 Ac), Astatine-211 ( 211 At), Bismuth-213 ( 213 Bi), carbon-11 ( 11 C), Copper-64 ( 64 Cu), Fluorine-18 ( 18 F), Gallium-68 ( 68 Ga), indium-111 ( 111 In), Iodine-123 ( 123 I), iodine-124 ( 124 I), iodine-131 ( 131 I), lead-212 ( 212 Pb), lutetium-177 ( 177 Lu), Radium-223 ( 223 Ra), Technetium-99m ( 99m Tc), Thorium-227 227 Th), Yttrium-86 ( 86 Y), Yttrium-90 ( 90 Y) and Zirconium-89 89 Zr). Radionuclides that can be used for SPECT imaging include gamma (γ)-ray emitters, such as 123 I and 99m Tc. Radionuclides that can be used for PET imaging include positron emitters, such as... 11 C 18 F and 68 Ga. For therapeutic purposes, antigen-binding proteins labeled with alpha (α) and beta (β) particle emitters can be used. Alpha particle emitters include... 225 Ac、 213 Bi、 212 Pb and 227Th Beta particle emitters include 131 I, 177 Lu and 90 Y. Furthermore, both alpha and beta particle emitters can visualize fractional gamma-ray emission, allowing for simultaneous imaging and treatment. Antigen-binding proteins can be individually labeled with one radionuclide from a therapeutic radionuclide pair selected from the group consisting of: 43 / 44 Sc / 47 Sc、 64 Cu / 67 Cu、 83 Sr / 89 Sr、86 Y / 90 Y、 110 In / 111 In、 124 I / 131 I, 152 Tb / 161 Tb, 152 Tb / 149 Tb, 68 Ga / 177 Lu and 90 Y / 177 Lu, and radiolabeled antigen-binding protein pairs can be used for simultaneous imaging and therapy. Alternatively, a single antigen-binding protein can be labeled with two radionuclides from a diagnostic radionuclide pair and subsequently used for simultaneous imaging and therapy. Many radiolabeled antibodies used in clinical trials for cancer imaging and therapy are provided in Parakh S, Lee ST, Gan HK, Scott AM. Radiolabeled Antibodies for Cancer Imaging and Therapy. Cancers (Basel). 11 Mar 2022;14(6):1454. The use of radiolabeling for proteins and antibodies is well known in the field (Gupta S, Batra S, Jain M. Antibody labeling with radioiodine and radiometals. Methods Mol Biol. 2014;1141:147-57; Sugiura G, Kühn H, Sauter M, Haberkorn U, Mier W. Radiolabeling strategies for tumor-targeting proteinaceous drugs. Molecules. 2014 Feb 18;19(2):2135-65; Chont M, van Dongen GAMS, Vugts DJ. State of the Art in Radiolabeling of Antibodies with Common and Uncommon Radiometals for Preclinical and Clinical Immuno-PET. Bioconjugate Chemistry 2021;32 (7):1315-1330; P, Czarnecka K, Krolicki L, Mikiciuk-Olasik E, Szymanski P. Radiolabeled Peptides and Antibodies in Medicine. Bioconjugate Chem. 2021:32:25-42; and Holik HA, Ibrahim FM, Elaine AA, Putra BD, Achmad A, KartamihardjaAHS. The Chemical Scaffold of Theranostic Radiopharmaceuticals: Radionuclide, Bifunctional Chelator, and Pharmacokinetics Modifying Linker. Molecules. 2022;27(10):3062).

[0139] connector

[0140] In some embodiments, the conjugate is directly attached to the heterologous portion. In alternative embodiments, the conjugate includes a linker that attaches the compound of this disclosure to the heterologous portion. In some aspects, the linker comprises an atomic chain of 1 to about 60 atoms, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms in length. In some embodiments, all chain atoms are carbon atoms. In some embodiments, the chain atoms in the linker backbone are selected from the group consisting of C, O, N, and S. The chain atoms and the linker may be selected based on their desired solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the linker provides a functional group that is readily cleaved by enzymes or other catalysts or hydrolytic conditions present in the target tissue or organ or cell. In some embodiments, the linker is long enough to reduce the possibility of steric hindrance. In some embodiments, the linker is an amino acid or peptide linker. Such peptide linkers can be of any length. The lengths of various connectors range from approximately 1 to 50 amino acids, and from 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids.

[0141] Various suitable adapters are known in the art. The adapter can be cleavable (cleavable adapter), for example, cleavable under physiological conditions, such as intracellular conditions, so that the adapter is cleaved to release a drug in the intracellular environment. Alternatively, the adapter can be cleavable under extracellular conditions, such as outside tumor cells or near tumor masses, so that the adapter is cleaved to release a drug that preferentially penetrates into tumor cells. In other embodiments, the adapter is uncleavable (uncleavable adapter), and the drug is released, for example, through antibody degradation.

[0142] The linker can bind to chemically reactive groups on the antibody moiety, such as free amino, imino, hydroxyl, thiol, or carboxyl groups (e.g., to the N-terminus or C-terminus, to the ε-amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic or aspartic acid residues, to the thioglycolic acid group of one or more cysteine ​​residues, or to the hydroxyl group of one or more serine or threonine residues). The binding site of the linker can be a native residue in the amino acid sequence of the antibody moiety, or it can be introduced into the antibody moiety, for example, through DNA recombination techniques (e.g., by introducing cysteine ​​or protease cleavage sites into the amino acid sequence) or through protein biochemistry (e.g., reduction, pH regulation, or proteolysis). The binding site of the linker can also be a non-natural amino acid. The binding site of the linker can also be a glycan on the antibody.

[0143] Typically, the linker is substantially inert under the conditions of the two groups it connects. The terms "bifunctional crosslinker," "bifunctional linker," or "crosslinker" refer to a modifier having two reactive groups at each end of the linker, thereby allowing one reactive group to react first with a cytotoxic compound to provide a compound with a linker portion and a second reactive group, which can then react with an antibody. Alternatively, one end of the bifunctional crosslinker can react first with an antibody to provide an antibody with a linker portion and a second reactive group, which can then react with a cytotoxic compound. The linker portion may contain chemical bonds that allow the cytotoxic portion to be released at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid-labile bonds, light-labile bonds, protease / peptidase-labile bonds, and esterase-labile bonds. See, for example, U.S. Patent Nos. 5,208,020, 5,475,092, 6,441,163, 6,716,821, 6,913,748, 7,276,497, 7,276,499, 7,368,565, 7,388,026, and 7,414,073. In some embodiments, the bond is a disulfide bond, a thioether bond, and / or a protease / peptidase unstable bond. Other connectors that can be used in this invention include uncut connectors (such as those described in detail in US20050169933), electrically conductive connectors, or hydrophilic connectors (such as those described in US 2009 / 0274713, US2010 / 0129314, and WO 2009 / 134976), each of which is expressly incorporated herein by reference.

[0144] In some embodiments, the connector is a hydrophilic connector that imparts hydrophilicity to the conjugate. In some embodiments, the hydrophilic connector comprises polyethylene glycol (PEG). In some embodiments, the hydrophilic connector comprises a reactive maleimide functional group that can react with a free thiohydrogen group, such as cysteine, to form a thioether bond. In some embodiments, the hydrophilic connector is CL2A. In some embodiments, the CL2A connector has the following structure:

[0145]

[0146] CL2A has been described in U.S. Patent Nos. 8,080,250, 8,759,496 and 10,195,288.

[0147] In some embodiments, the hydrophilic linker is CL2E. In some embodiments, CL2E contains a reactive maleimide functional group at one end and has the following structure:

[0148]

[0149] CL2E has been described in U.S. Patent Nos. 8,080,250, 8,759,496 and 10,195,288.

[0150] In some embodiments, the linker can be cleaved by a cleaving agent present in the intracellular environment (e.g., within lysosomes, endosomes, or cell membrane cisterns). The linker can be a peptide linker cleaved, for example, by intracellular or extracellular peptidases or proteases (including, but not limited to, lysosomal or endosomal proteases). In some embodiments, the peptide linker comprises at least two, at least three, at least four, or at least five amino acids in length.

[0151] In some embodiments, the peptide linker comprises VC-PAB, which contains valine and citrulline residues followed by a p-aminobenzyl group (valine-citrulline-p-aminobenzyl group). In some embodiments, VC-PAB also comprises a maleimide group (MC-VC-PAB) and may have the following structure:

[0152]

[0153] VC-PAB and MC-VC-PAB have been described in U.S. Patent Nos. 7,659,241, 7,829,531, 6,884,869, 6,214,345 and 6,214,345.

[0154] In some embodiments, the peptide linker comprises glycine-glycine-phenylalanine-glycine (GGFG). In some such embodiments, the linker is maleimide hexanoylglycine-glycine-phenylalanine-glycine (MC-GGFG). In some embodiments, the MC-GGFG linker has the following structure:

[0155]

[0156] GGFG and MC-GGFG have been described in U.S. Patent Nos. 9,808,537 and 10,195,288.

[0157] In other embodiments, the cleavable adapter is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. In some embodiments, the pH-sensitive adapter can be hydrolyzed under acidic conditions. For example, acid-labile adapters that can be hydrolyzed in lysosomes (e.g., hydrazones, thioureas, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used (see, for example, U.S. Patents 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661). Such adapters are relatively stable under neutral pH conditions, such as in blood, but unstable at pH values ​​below 5.5 or 5.0 (the approximate pH of lysosomes). In some embodiments, the hydrolyzable connector is a thioether connector, such as a thioether attached to the therapeutic agent via an acylhydrazone bond (see, for example, U.S. Patent No. 5,622,929).

[0158] In other embodiments, the linker can be cleaved under reducing conditions (e.g., a disulfide linker). Bifunctional crosslinkers capable of linking antibodies and cytotoxic compounds via disulfide bonds include, but are not limited to: N-succinimidyl-4-(4-nitropyridinyl-2-dithio)butyrate, N-succinimidyl-3-(2-pyridinyl dithio)propionate (SPDP), N-succinimidyl-4-(2-pyridinyl dithio)valerate (SPP), N-succinimidyl-4-(2-pyridinyl dithio)butyrate (SPDB), and N-succinimidyl-4-(2-pyridinyl dithio)-2-sulfonylbutyrate (sulfonyl-SPDB). Sulfonyl-SPDB is described, for example, in U.S. Patent 8,236,319, which is incorporated herein by reference. Alternatively, crosslinking agents incorporating thiol groups, such as 2-iminothiacyclopentane, homocysteine ​​thiolactone, or S-acetylsuccinic anhydride, can be used. In other embodiments, the linker may contain one or a combination of more of the previously described peptide linkers, pH-sensitive linkers, or disulfide linkers.

[0159] "Heterofunctional crosslinkers" are bifunctional crosslinkers having two different reactive groups. Heterofunctional crosslinkers containing an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link cytotoxic compounds to antibodies. Examples of such commercially available heterofunctional crosslinkers include succinimide-6-hydrazinoamide acetone hydrazone (SANH), succinimide-4-hydrazinophthalate hydrochloride (SHTH), and succinimide-hydrazinophthalate hydrochloride (SHNH). The hydrazine-containing benzodiazepine derivatives of the present invention can also be used to prepare conjugates with acid-unstable linkages. Examples of usable bifunctional crosslinkers include succinimide-paraformylbenzoate (SFB) and succinimide-paraformylphenoxyacetic acid ester (SFPA).

[0160] The linkers described herein can be used in any combination with the heterologous parts described herein. Furthermore, the linkers described herein may have any chemically reactive parts (e.g., maleimide, cysteine, etc.) that can react with any part of the antigen-binding protein of this disclosure (e.g., amino acids, disulfide bonds, carbohydrates (e.g., those from post-translational modifications)). Typically, lysine or cysteine ​​residues on the antibody or antigen-binding protein (e.g., cysteine ​​obtained by reducing disulfide bonds (e.g., inter- or intra-chain disulfide bonds from the antibody or antigen-binding protein), or engineered unpaired cysteine ​​residues) are used as conjugation sites. All the linkers and heterologous parts listed above described herein are commercially available and / or can be prepared using conventional techniques, including those described in the references listed above.

[0161] Adhesion

[0162] The heterologous portion to antigen-binding protein ratio (HAR) represents the number of heterologous portions attached to each antigen-binding molecule. In some embodiments, the HAR ranges from 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, the HAR ranges from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, the HAR is about 2, about 2.5, about 3, about 4, about 5, or about 6. In some embodiments, the HAR ranges from about 2 to about 4. HAR can be characterized by conventional methods, such as mass spectrometry, UV / Vis spectroscopy, ELISA assays, and / or HPLC.

[0163] In some embodiments, the conjugates are heterogeneous conjugates (also referred to as "conventional"), wherein the antigen-binding protein is conjugated with a varying number of heterologous moieties. In some embodiments, the heterogeneous conjugates follow a Gaussian or quasi-Gaussian distribution of the conjugate, wherein the distribution is centered around the average heterologous moiety loading value, with some antigen-binding proteins having conjugation values ​​above the average and others having conjugation values ​​below the average.

[0164] In some embodiments, the conjugate is a homogeneous conjugate in which a majority of the antigen-binding protein is conjugated to a defined number of heterologous moieties. In some embodiments, the homogeneous conjugate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HARs. In some embodiments, the homogeneous conjugate contains 2, 4, 6, or 8 HARs. In a preferred embodiment, the homogeneous conjugate contains 4 HARs. In other preferred embodiments, the homogeneous conjugate contains 2 HARs. In some embodiments, the homogeneous conjugate contains greater than or equal to 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the conjugate having a defined HAR. In some embodiments, the homogeneous conjugate comprises about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a conjugate having a defined HAR. In some embodiments, the homogeneous conjugate comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a conjugate having a defined HAR. In some embodiments, the homogeneous conjugate comprises a HAR distribution exhibiting a non-Gaussian or quasi-Gaussian distribution. In some embodiments, the homogeneity of the homogeneous conjugate is determined by chromatography, such as HPLC or any suitable chromatographic method. In some embodiments, the chromatogram is a HIC chromatogram. The homogeneous conjugate can be generated through site-specific conjugation.

[0165] In some implementations, the heterologous portion is conjugated to an antigen-binding protein (e.g., an antibody) in a site-specific manner.Various site-specific conjugation methods are known in the art, such as thiomab or TDC or conjugation at unpaired cysteine ​​residues (Junutula et al. (2008) Nat. Biotechnol. 26:925-932; Dimasi et al. (2017) Mol. Pharm. 14:1501-1516; Shen et al. (2012) Nat. Biotechnol. 30:184-9); thiol bridging (Behrens et al. (2015) Mol. Pharm. 12:3986-98); conjugation at glutamine using transglutaminase (Dennler et al. (2013) Methods Mol. Bio. 1045:205-15; Dennler et al. (2014) BioconjugChem. 25:569-78); and conjugation at engineered non-natural amino acid residues (Axup et al. (2012) Proc. Natl Acad Sci USA 104-16101-6; Tian et al. (2014) Proc Natl Acad Sci USA 111:1766-71; Van Brunt et al. (2015) Bioconjug Chem 26:2249-60; Zimmerman et al. (2014) Bioconjug Chem 25:351-61); selenocysteine ​​conjugation (Li et al. (2017) Cell Chem Biol 24:433-442); glycan-mediated conjugation (Okeley et al. (2013) Bioconjug Chem 24:1650-5); conjugation at galactose or GalNAc analogues (Ramakrishnan and Qasba (2002) J Biol Chem 277:20833-9; van Geel et al. (2015) Bioconjug Chem 26:2233-42); via glycan engineering (Zhou et al. (2014) Bioconjug Chem 25:510-20; Tang et al. (2017) Nat Protoc 12:1702-1721); via short peptide tags, such as glutamine tag engineering or sorting enzyme A-mediated transpeptidation (Strop et al. (2013) Chem Biol 20:161-7; Beerli et al. (2015) PLoS One 10:e0131177); and via aldehyde tags (Wu et al. (2009) Proc Natl Acad Sci USA 106:3000-5).

[0166] Compositions, pharmaceutical compositions and formulations

[0167] This document provides compositions comprising antigen-binding proteins, nucleic acids, vectors, host cells, or conjugates as currently disclosed. In some aspects, the composition comprises an antigen-binding protein in an isolated and / or purified form. In some aspects, the composition comprises a single type (e.g., structure) of an antigen-binding protein of this disclosure, or a combination of two or more antigen-binding proteins of this disclosure, wherein the combination comprises two or more different types (e.g., structures) of antigen-binding proteins.

[0168] In some aspects, the composition comprises an agent that enhances the chemical-physical characteristics of the antigen-binding protein, for example by stabilizing the antigen-binding protein at certain temperatures (e.g., room temperature), extending its shelf life, reducing degradation (e.g., oxidation, protease-mediated degradation), and prolonging its half-life. In some aspects, the composition comprises any of the agents disclosed herein as a heterologous or conjugated portion, optionally blended with or conjugated to the antigen-binding protein of this disclosure.

[0169] In various aspects of this disclosure, the compositions additionally comprise pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, antigen-binding proteins, nucleic acids, carriers, host cells, or conjugates (hereinafter referred to as "active agents") as disclosed herein are formulated into pharmaceutical compositions comprising an active agent and pharmaceutically acceptable carriers, diluents, or excipients. In this regard, this disclosure also provides pharmaceutical compositions comprising an active agent intended for administration to a subject, such as a mammal.

[0170] In some embodiments, the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition contains an active agent at a concentration of about 0.001 to about 30.0 mg / ml (w / v).

[0171] In all respects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, the term “pharmaceuticalally acceptable carrier” includes any of the standard drug carriers, such as phosphate-buffered saline solutions, water, emulsions (such as oil / water emulsions or water / oil emulsions); and various types of wetting agents. The term also covers any drug preparation approved by a U.S. federal regulatory agency or listed in the United States Pharmacopeia for use in animals (including humans).

[0172] Pharmaceutical compositions may contain any pharmaceutically acceptable ingredients, including, for example, acidifiers, additives, adsorbents, aerosol propellants, air displacement agents, alkalizers, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, bactericides, alkalis, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, solubilizers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film-forming agents, and flavor enhancers. Flavoring agents, flow aids, gelling agents, granulating agents, humectants, lubricants, mucosal adhesives, ointment bases, ointments, oily agents, organic bases, lozenge bases, pigments, plasticizers, polishing agents, preservatives, masking agents, skin penetrants, solubilizers, solvents, stabilizers, suppositories, surfactants, surface activators, suspending agents, sweeteners, therapeutic agents, thickeners, tension agents, toxic agents, viscous agents, water-miscible solubilizers, water softeners, or wetting agents. See, for example, Handbook of Pharmaceutical Excipients, 3rd edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated herein by reference in its entirety. Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated herein by reference in its entirety.

[0173] In each aspect, the pharmaceutical composition comprises formulation materials that are non-toxic to the recipient at the dosage and concentration used. In specific embodiments, the pharmaceutical composition comprises an active agent and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic balancers, tonics, antioxidants, antibiotics, antifungals, fillers, lyophilization protectants, antifoaming agents, chelating agents, preservatives, colorants, analgesics, or other agents. Optionally, in addition to one or more excipients, the pharmaceutical composition also comprises one or more polyols and / or one or more surfactants, including but not limited to pharmaceutically acceptable salts, osmotic balancers (tonics), antioxidants, antibiotics, antifungals, fillers, lyophilization protectants, antifoaming agents, chelating agents, preservatives, colorants, and analgesics.

[0174] In some embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. In such embodiments, suitable formulation materials include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); fillers (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; and hydrophilic polymers (such as poly… Vinylpyrrolidone; low molecular weight peptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as poloxamer, PEG, dehydrated sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triaton, tromethamine, lecithin, cholesterol, tyloxacillin); stability enhancers (such as sucrose or sorbitol); tension enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, and / or dextran); delivery mediators; diluents; excipients and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, (edited by AR Gennaro), 1990, Mack Publishing Company.

[0175] The pharmaceutical composition may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be, for example, between about 4 or about 5 and about 8.0, or between about 4.5 and about 7.5, or between about 5.0 and about 7.5. In various embodiments, the pH of the pharmaceutical composition is between 5.5 and 7.5.

[0176] This disclosure provides a method for producing a pharmaceutical composition. In various aspects, the method includes combining an antigen-binding protein, conjugate, fusion protein, nucleic acid, carrier, host cell, or a combination thereof with a pharmaceutically acceptable load, diluent, or excipient.

[0177] Application route

[0178] For the purposes of this disclosure, the active agent or pharmaceutical composition comprising the active agent may be administered to a subject via any suitable route of administration. For example, the active agent may be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following discussion of routes of administration is merely illustrative of various embodiments and should not be construed as limiting the scope in any way.

[0179] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The term "parenteral" means administered not through the digestive tract, but through other routes, such as subcutaneous, intramuscular, intraspinal, or intravenous administration. The active agents disclosed herein can be administered in a drug carrier, such as a sterile liquid or liquid mixture including water, saline, aqueous glucose solution and associated sugar solution, alcohol (such as ethanol or hexadecyl alcohol), glycol (such as propylene glycol or polyethylene glycol), dimethyl sulfoxide, glycerol, ketal (such as 2,2-dimethyl-1,3-dioxolane-4-methanol), ether, polyethylene glycol 400, oil, fatty acid, fatty acid ester or glycerol ester or acetylated fatty acid glycerol ester, with or without the addition of pharmaceutically acceptable surfactants (such as soap or detergent), suspending agents (such as pectin, carbomer, methylcellulose, hydroxypropyl methylcellulose or carboxymethylcellulose), or emulsifiers and other drug adjuvants.

[0180] Oils suitable for use in parenteral formulations include mineral oils, animal oils, vegetable oils, or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil. Fatty acids suitable for parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0181] Suitable soaps for parenteral preparations include fatty alkali metal, ammonium, and triethanolamine salts, while suitable detergents include: (a) cationic detergents, such as dimethyl dialkyl ammonium halides and alkylpyridine halides; (b) anionic detergents, such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates and sulfosuccinates; (c) nonionic detergents, such as fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene-polypropylene copolymers; (d) amphoteric detergents, such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0182] In some embodiments, the parenteral formulation contains about 0.5% to about 25% by weight of the active agent of this disclosure in solution. Preservatives and buffers may be used. To minimize or eliminate injection site irritation, such compositions may contain one or more nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is typically in the range of about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with a hydrophobic group formed by the condensation of propylene oxide and propylene glycol. In some aspects, the parenteral formulation is presented in single-dose or multi-dose sealed containers (such as ampoules and vials) and can be stored under lyophilized (freeze-dried) conditions, requiring only immediate addition of a sterile liquid excipient (e.g., water for injection) before use. In some respects, temporary injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the previously described categories.

[0183] Injectable formulations conform to this disclosure. The requirements for effective drug delivery systems used in injectable compositions are well known to those skilled in the art (see, for example, Pharmaceuticals and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pp. 238-250 (1982) and ASHP Handbook on Injectable Drugs, Trissel, 4th edition, pp. 622-630 (1986)).

[0184] dose

[0185] The active agents disclosed herein are believed to be used in methods for inhibiting tumor growth, as well as other methods (including methods for treating or preventing cancer) as further described herein. For the purposes of this disclosure, the amount or dose of the active agent applied should be sufficient to produce, for example, a therapeutic or preventative response in a subject or animal within a reasonable timeframe. For example, the dose of the active agent disclosed herein should be sufficient to treat the cancer described herein for a period of about 1 to 4 minutes, 1 to 4 hours, or 1 to 4 weeks or longer, such as 5 to 20 weeks or longer, from the time of application. In some embodiments, the timeframe may be even longer. The dose will be determined by the efficacy of the particular active agent and the condition of the animal (e.g., a human) and the weight of the animal to be treated (e.g., a human).

[0186] Various assays for determining the dosage are known in the art. For the purposes of this document, one assay may be used to determine the initial dose to be administered to a mammal, comprising administering a dose of the active agent of this disclosure to a group of mammals, wherein each mammal in the group is administered a different dose of the active agent, and then comparing the degree of treatment of the cancer in each mammal. The degree of treatment of the cancer after administration of a dose can be represented, for example, by the degree of tumor regression achieved with the active agent in a mouse xenograft model. Methods for measuring tumor regression are known in the art and are described in the embodiments herein.

[0187] The dosage of the active agent disclosed herein will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of the specific active agent of this disclosure. Typically, the attending physician will consider a variety of factors, such as age, weight, general health condition, diet, sex, the active agent of this disclosure to be administered, route of administration, and severity of the condition being treated, to determine the dosage of the active agent of this disclosure for each individual patient. By way of example and not intended to limit the disclosure, the dosage of the active agent of this disclosure administered per dose may be from about 2 mg to about 10 mg / kg of the body weight of the treated subject. In embodiments, the dosage may be from about 2 mg to about 3 mg / kg of the body weight of the treated subject, from about 2 mg to about 4 mg / kg of body weight, from about 2 mg to about 5 mg / kg of body weight, from about 2 mg to about 6 mg / kg of body weight, from about 2 mg to about 7 mg / kg of body weight, from about 2 mg to about 8 mg / kg of body weight, or from about 2 mg to about 9 mg / kg of body weight. In a separate embodiment, the dosage may be from about 2 mg to about 3 mg / kg of the body weight of the treated subject. In different embodiments, the dosage may be from about 3 mg to about 4 mg / kg of body weight. In an embodiment, the dose may be from about 4 mg to about 5 mg / kg body weight. In an embodiment, the dose may be from about 5 mg to about 6 mg / kg body weight. In an embodiment, the dose may be from about 6 mg to about 7 mg / kg body weight. In an embodiment, the dose may be from about 7 mg to about 8 mg / kg body weight. In an embodiment, the dose may be from about 8 mg to about 9 mg / kg body weight. In an embodiment, the dose may be from about 9 mg to about 10 mg / kg body weight. In a preferred embodiment, the dose of the active agent of this disclosure administered each time may be from about 2.4 mg to 3 mg / kg body weight of the treated subject. In a preferred embodiment, the dose of the active agent administered each time may be selected from the group consisting of: about 2.4 mg / kg body weight, about 2.5 mg / kg body weight, about 2.6 mg / kg body weight, about 2.7 mg / kg body weight, about 2.8 mg / kg body weight, about 2.9 mg / kg body weight, and about 3.0 mg / kg body weight of the treated subject. By way of example and not intended to limit this disclosure, the intervals between doses may be about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or longer. The intervals between doses may be adjusted based on the subject's response to treatment. By way of example and not intended to limit this disclosure, the average dose of the active agent of this disclosure may be about 0.0001 to about 1 g / kg body weight / day, about 0.0001 to about 0.001 g / kg body weight / day, or about 0.01 mg to about 1 g / kg body weight / day. In some embodiments, the average dose of the active agent of this disclosure may be about 0.1 mg to about 0.01 mg to 1 mg / kg body weight / day.

[0188] Controlled release formulation

[0189] In some embodiments, the active agent described herein may be modified into a reservoir form, thereby controlling the manner in which the active agent of this disclosure is released into the body upon application, based on the time and location within the body (see, for example, U.S. Patent No. 4,450,150). The reservoir form of the active agent of this disclosure may be, for example, an implantable composition comprising the active agent and a porous or non-porous material (such as a polymer), wherein the active agent is encapsulated or diffused throughout the material and / or the non-porous material degrades. The reservoir is then implanted into the desired location within the subject's body, and the active agent is released from the implant at a predetermined rate.

[0190] In some aspects, pharmaceutical compositions containing active agents are modified to have any type of in vivo release characteristic. In some aspects, pharmaceutical compositions are formulations for immediate release, controlled release, sustained release, prolonged release, delayed release, or biphasic release. Methods for formulating peptides for controlled release are known in the art. See, for example, Qian et al., J Pharm 374:46-52 (2009) and International Patent Application Publications Nos. WO 2008 / 130158, WO2004 / 033036, WO2000 / 032218, and WO 1999 / 040942.

[0191] The compositions of the present invention may also contain, for example, micelles or liposomes, or some other encapsulation form, or may be administered in an extended release form to provide extended storage and / or delivery effects.

[0192] use

[0193] The antigen-binding proteins disclosed herein can be used to inhibit the proliferation of cancer cells and / or suppress tumor growth. Free from any particular theoretical constraints, the inhibitory effects of the antigen-binding proteins presented herein make such entities suitable for use in methods of treating cancer.

[0194] Therefore, this document provides methods for inhibiting cancer cell proliferation and / or inhibiting tumor growth in a subject, as well as methods for reducing tumor size in a subject. In various embodiments, the method includes administering to the subject an amount of the disclosed pharmaceutical composition that effectively inhibits tumor growth or reduces tumor size in the subject. In each embodiment, the growth of P2RY8-related or P2RY8-expressing tumors is inhibited. In each embodiment, the size of P2RY8-related or P2RY8-expressing tumors is reduced. In all respects, any tumor or cancer including B-cell tumors, thymic epithelial tumors, clear cell renal cell carcinoma, seminoma, other types of neuroepithelial tumors, thyroid cancer, pheochromocytoma, pancreatic cancer, hepatobiliary cancer, sarcoma, non-seminomatous germ cell tumors, pleural mesothelioma, non-small cell lung cancer, melanoma, non-clear cell renal cell carcinoma, breast cancer, esophageal and gastric cancer, bile duct cancer, prostate cancer, head and neck cancer, colorectal cancer, bladder cancer, cervical cancer, glioblastoma, ocular melanoma, ovarian epithelial tumors, endometrial cancer, glioma, lymphoma, leukemia, and myeloma, wherein P2RY8 is expressed or overexpressed. In all respects, the proliferation of P2RY8-associated or P2RY8-expressing lymphomas, leukemias, or myelomas is suppressed. In all respects, the proliferation of P2RY8-associated lymphomas is suppressed. Although leukemia, lymphoma, and myeloma are considered liquid tumors, in some cases, the aforementioned cancer indications develop into tumor masses. For example, acute myeloid leukemia can develop into an extramedullary tumor mass (called myeloid sarcoma) composed of medulloblasts, which disrupts the tissue structure. In some such cases, the antigen-binding protein and / or composition disclosed herein effectively reduces the size of such tumor masses.

[0195] As used herein, the terms “inhibition” or “reduction” and their derivatives may not represent 100% or complete inhibition or reduction. Rather, varying degrees of inhibition or reduction exist, which those skilled in the art will consider to have potential benefit or therapeutic effect. In this regard, the antigen-binding protein of this disclosure can inhibit tumor growth or reduce tumor size by any amount or level. In various embodiments, the inhibition provided by the methods of this disclosure is at least or about 10% inhibition (e.g., at least or about 20% inhibition, at least or about 30% inhibition, at least or about 40% inhibition, at least or about 50% inhibition, at least or about 60% inhibition, at least or about 70% inhibition, at least or about 80% inhibition, at least or about 90% inhibition, at least or about 95% inhibition, at least or about 98% inhibition). In various embodiments, the reduction provided by the method of this disclosure is at least or about 10% reduction (e.g., at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 95%, at least or about 98%).

[0196] This document also provides a method for treating a subject with cancer (e.g., cancer expressing P2RY8). In various embodiments, the method includes administering to the subject an amount of the disclosed pharmaceutical composition effective in treating the subject's cancer.

[0197] The “effective amount” of a substance (e.g., antibodies, cells, etc.) is the amount that can produce a medically desired outcome in a treated patient, such as reducing tumor burden, reducing tumor cell growth, or alleviating any cancer-related symptoms, with an acceptable benefit-risk ratio, preferably in humans or non-human mammals.

[0198] For the purposes of this document, the cancer described herein can be any cancer, such as any malignant growth or tumor caused by abnormal and uncontrolled cell division that can spread to other parts of the body via the lymphatic system or bloodstream, preferably wherein the cancer is associated with abnormal P2RY8 protein expression. In some embodiments, the cancer is associated with overexpression of the P2RY8 protein. In some embodiments, the cancer is associated with expression of a mutant P2RY8 protein. In some respects, cancer is a type of cancer selected from the following groups: acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), small bowel cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer and bladder cancer. In specific contexts, cancer includes lymphoma, leukemia, myeloma, mature B-cell tumors, thymic epithelial tumors, clear cell renal cell carcinoma, seminoma, other types of neuroepithelial tumors, thyroid cancer, pheochromocytoma, pancreatic cancer, hepatobiliary cancer, sarcoma, non-seminomatous germ cell tumors, pleural mesothelioma, non-small cell lung cancer, melanoma, non-clear cell renal cell carcinoma, breast cancer, esophageal and gastric cancer, bile duct cancer, prostate cancer, head and neck cancer, colorectal cancer, bladder cancer, cervical cancer, glioblastoma, ocular melanoma, ovarian epithelial tumors, endometrial cancer, or glioma. In all other contexts, cancer is any cancer characterized by moderate to high expression of P2RY8. See, for example... Figure 1 (A-100) and Figure 2(A-200). In various respects, cancer includes altered expression of the P2RY8 gene, overexpression of the P2RY8 gene, or expression of a mutant P2RY8 gene. In specific respects, myeloma is multiple myeloma. In specific respects, leukemia is acute myeloid leukemia (AML). In specific respects, AML is any of AML with a specific genetic abnormality, differentiated AML, myeloid sarcoma, and secondary myeloid neoplasms. In specific respects, leukemia is acute lymphoblastic leukemia. In specific respects, lymphoma is non-Hodgkin lymphoma. In specific respects, non-Hodgkin lymphoma is any of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), and lymphoplasmacytic lymphoma.

[0199] As used herein, the term "treatment" and related terms do not necessarily imply 100% or complete cure. Rather, different degrees of treatment exist, which those skilled in the art will consider to have potential benefit or therapeutic effect. In this respect, the methods for treating cancer disclosed herein can provide any amount or level of treatment. Furthermore, the treatment provided by the methods of this disclosure can include treatment of one or more symptoms or signs of the cancer being treated. Additionally, the treatment provided by the methods of this disclosure can include slowing the progression of cancer. For example, the method can treat cancer by enhancing T-cell activity or the immune response against cancer, reducing tumor or cancer growth, reducing tumor cell metastasis, increasing cell death of tumor or cancer cells, etc. In various aspects, the method achieves treatment by delaying the onset or recurrence of cancer, for example, at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, two months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, 4 years, or longer. In various aspects, the method achieves treatment by prolonging the survival of the subject.

[0200] The antigen-binding protein disclosed herein can also be used to detect P2RY8 in a sample or to diagnose P2RY8-positive cancer. Therefore, this disclosure provides a method for detecting P2RY8 in a sample. In various embodiments, the method includes contacting a sample with an antigen-binding protein, conjugate, or fusion protein as described herein, and measuring an immune complex containing the antigen-binding protein, conjugate, or fusion protein that binds to the P2RY8 protein. This disclosure also provides a method for diagnosing P2RY8-positive cancer in a subject. In various embodiments, the method includes contacting a biological sample containing cells or tissue obtained from a subject with an antigen-binding protein, conjugate, or fusion protein as described herein, and measuring an immune complex containing the antigen-binding protein, conjugate, or fusion protein that binds to the P2RY8 protein.

[0201] Subjects

[0202] In some embodiments of this disclosure, the subject is a mammal, including but not limited to: rodent mammals such as mice and hamsters; lagomorph mammals such as rabbits; carnivorous mammals, including felines (cats) and canines (dogs); artiodactyla mammals, including bovids (cattle) and suidae (pigs); or perissodactyla mammals, including equines (horses). In some aspects, the mammal belongs to the order Primates (Ceboids) or Simoids (monkeys) or Anthropoids (humans and apes). In some aspects, the mammal is a human.

[0203] Reagent test kit

[0204] In some embodiments, the antigen-binding protein of this disclosure is provided in the form of a kit. In various aspects, the kit contains a unit dose of the antigen-binding protein. For the purposes of this document, "unit dose" refers to a discrete amount dispersed in a suitable carrier. In various aspects, a unit dose is an amount sufficient to provide a subject with a desired effect, such as inhibiting cancer cell proliferation, inhibiting tumor growth, reducing tumor size, or treating cancer. Therefore, kits are provided herein that contain the antigen-binding protein of this disclosure, optionally provided in unit doses. In various aspects, the kit contains several unit doses, such as a weekly or monthly supply of unit doses, optionally each unit dose is individually packaged or otherwise separated from other unit doses. In some embodiments, the kit / unit dose components are packaged together with instructions for administration to a patient. In some embodiments, the kit includes one or more devices for administration to a patient, such as needles and syringes. In some aspects, the antigen-binding protein of this disclosure, its pharmaceutically acceptable salts, conjugates containing the antigen-binding protein, or polymers or dimers containing the antigen-binding protein are pre-packaged in a ready-to-use form, such as syringes, intravenous bags, etc. In some respects, the kit also contains other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of the carriers described herein. In certain respects, the kit contains the antigen-binding protein of this disclosure, as well as a pharmaceutical agent for chemotherapy or radiotherapy, such as a therapeutic agent.

[0205] Various implementation plans

[0206] In various embodiments of this disclosure, the antigen-binding protein binds to the human P2RY8 protein. In various embodiments of this disclosure, by way of example only, the antigen-binding protein binds to the cynomolgus monkey (Macaca fascicularis) P2RY8 protein. For example, the antigen-binding protein binds to both the human P2RY8 protein and the cynomolgus monkey (Macaca fascicularis) P2RY8 protein. For example, the antigen-binding protein binds to both the human P2RY8 protein and the cynomolgus monkey (Macaca fascicularis) P2RY8 protein with approximately the same affinity in the range of 2-3 times. For example, the antigen-binding protein does not bind to the canine P2RY8 protein or its binding to the canine P2RY8 protein is significantly weaker than its binding to the human P2RY8 protein. By way of example only, the binding affinity of antigen-binding proteins to human P2RY8 protein is approximately 10, 20, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, or 325 times that to dog P2RY8 protein. In a specific implementation, the antigen-binding protein binds to both human P2RY8 protein and cynomolgus monkey (cynomolgus monkey) P2RY8 protein, but does not bind to dog P2RY8 protein or has a binding affinity for dog P2RY8 protein that is significantly lower than that for human P2RY8 protein, being at most about 1 / 10, 1 / 20, 1 / 30, 1 / 50, 1 / 75, 1 / 100, 1 / 125, 1 / 150, 1 / 175, 1 / 200, 1 / 225, 1 / 250, 1 / 275, 1 / 300, or 1 / 325 of the latter.

[0207] In some embodiments, the antigen-binding protein comprises: (a) a heavy chain variable region amino acid sequence shown in any of Tables 2-3 and 14-31, or a variant sequence differing from the heavy chain by only one or two amino acids or having at least or about 70% sequence identity; (b) a light chain variable region amino acid sequence shown in this disclosure (e.g., shown in any of Tables 2-3 and 14-31), or a variant sequence differing from the light chain by only one or two amino acids or having at least or about 70% sequence identity; or (c) a combination of any of (a) and any of (b). In some embodiments, the variant sequence has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antigen-binding protein of this disclosure comprises an Fc polypeptide. In some embodiments, the antigen-binding protein of this disclosure comprises an Fc polypeptide containing unfucosylated glycan.

[0208] In some embodiments, the antigen-binding protein comprises: (a) heavy chain CDR1, (b) heavy chain CDR2, (c) heavy chain CDR3, (d) light chain CDR1, (e) light chain CDR2, (f) light chain CDR3, or (g) a combination of any two or more of (a)-(f), wherein each CDR comprises an amino acid sequence shown in this disclosure (e.g., shown in any of Tables 2-3 and 14-31), or a variant sequence differing from the present disclosure by only one or two amino acids or having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0209] In some embodiments, the antigen-binding protein comprises: (a) heavy chain FR1, (b) heavy chain FR2, (c) heavy chain FR3, (d) heavy chain FR4, (e) light chain FR1, (f) light chain FR2, (g) light chain FR3, (h) light chain FR4, or (i) any two or more of (a)-(h), wherein each FR comprises the amino acid sequence shown in any of Tables 2-3 and Tables 14-31, or a variant sequence differing by only one or two amino acids or having at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0210] In some embodiments, the antigen-binding protein of this disclosure is an antibody, such as a monoclonal antibody. In various aspects, the antibody is a chimeric antibody, a human antibody, or a humanized antibody. In various cases, the antigen-binding protein is IgG. In various cases, the antigen-binding protein is humanized IgG. In various cases, the antigen-binding protein is human IgG. In various aspects, the antigen-binding protein inhibits at least about 50% of P2RY8 in a soft agar 3D proliferation assay. + The colony growth of cancer cells, or the injection of human P2RY8 + In xenograft mice, P2RY8 inhibits tumor growth. In various aspects, antigen-binding proteins inhibit tumor growth upon injection of P2RY8. + In xenograft mice, P2RY8 inhibits tumor growth. Under various conditions, the antigen-binding protein inhibits tumor growth upon injection of P2RY8. +In xenograft mice, tumor growth was inhibited by at least 50%. In each respect, inhibition of tumor growth or cancer cell proliferation was observed with “naked” intact antibody (e.g., IgG1), wherein the “naked” intact antibody contains variable and constant regions of both heavy and light chains and does not contain drug conjugates (e.g., not an antibody-drug conjugate). In each respect, the antigen-binding protein of this disclosure induces antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity.

[0211] This invention includes a bispecific antigen-binding protein that binds to a P2RY8 protein and a second antigen, wherein the antigen-binding protein binding to P2RY8 is any of the antigen-binding proteins or fragments thereof described herein. In some embodiments, the bispecific antigen-binding protein comprises: (a) a heavy chain variable region amino acid sequence shown in any of Tables 2-3 and 14-31, or a variant sequence differing from the heavy chain variable region by only one or two amino acids or having at least or about 70% sequence identity; (b) a light chain variable region amino acid sequence shown in any of Tables 2-3 and 14-31, or a variant sequence differing from the light chain variable region by only one or two amino acids or having at least or about 70% sequence identity; or (c) a combination of any of (a) and any of (b). In some embodiments, the variant sequence has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the bispecific antigen-binding protein comprises an Fc polypeptide. In some embodiments, the bispecific antigen-binding protein comprises an Fc polypeptide containing unfucosylated glycan.

[0212] In various embodiments, the bispecific antigen-binding protein binds to the P2RY8 protein and the second antigen. In some embodiments, the bispecific antigen-binding protein comprises an antigen-binding fragment of an antibody specific to the second antigen. In various embodiments, the second antigen is a cell surface protein expressed by T cells, optionally a component of the T cell receptor (TCR), such as CD3. In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is CD3E.

[0213] In various embodiments, the second antigen is a co-stimulatory molecule that assists in T cell activation, such as CD40 or 4-1BB (CD137). In various embodiments, the second antigen is an Fc receptor, optionally an Fc γ receptor, an Fc-α receptor, or an Fc-ε receptor. In some embodiments, the Fc receptor is CD64 (Fc-γ RI), CD32 (Fc-γ RIIA), CD16A (Fc-γ RIIIA), CD16b (Fc-γ RIIIb), FcεRI, CD23 (Fc-ε RII), CD89 (Fc-ε RI), Fcα / µR, or FcRn. In some embodiments, the Fc receptor is CD16A.

[0214] In various embodiments, the second antigen is an immune checkpoint molecule, such as a protein involved in the immune checkpoint pathway, optionally A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7. In some embodiments, the immune checkpoint molecule is PD-1, LAG3, TIM3, or CTLA4. In various embodiments, the bispecific antigen-binding protein comprises any of the currently disclosed P2RY8 antibodies, scFv, Fab, Fab', or F(ab')2.

[0215] In various embodiments, the bispecific antigen-binding protein comprises the antigen-binding protein of this disclosure. In some embodiments, the bispecific antigen-binding protein comprises the structure of a nanobody, a bibody, BiTE®, DART, TandAb, CrossMab, or HSAbody.

[0216] This disclosure provides a conjugate comprising an antigen-binding protein or bispecific antigen-binding protein as described herein and a heterologous moiety. In some embodiments, the conjugate comprises a cytotoxic agent or a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking microtubule polymerization. In some embodiments, the antimitotic agent is aurestatin. In some embodiments, aurestatin is MMAE.

[0217] In various embodiments, the conjugates of this disclosure are conjugated to antigen-binding proteins via cleavable linkers. In some embodiments, the cleavable linker is VC-PAB.

[0218] In some embodiments, the conjugate comprises an antigen-binding protein as an antibody. The antibody is a monoclonal antibody, optionally an IgG antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0219] In various embodiments, the average number of pharmaceutical units conjugated to each antigen-binding protein in the conjugates of this disclosure ranges from 1 to 8, preferably wherein the average number of pharmaceutical units conjugated to each antigen-binding protein ranges from 3 to 8. In some embodiments, the conjugates are heterogeneous conjugates. In other embodiments, the conjugates are homogeneous conjugates. In some embodiments, the conjugates comprise a heterologous moiety or pharmaceutical agent, wherein the pharmaceutical agent is conjugated at a specific site on the antigen-binding protein. In some embodiments, the specific site is an unpaired cysteine ​​residue. In some embodiments, the unpaired cysteine ​​residue can be obtained from the reduction of disulfide bonds. In some embodiments, the unpaired cysteine ​​residue can be obtained by reducing interchain disulfide bonds in the hinge region of the antibody. In some embodiments, the antibody having reduced interchain disulfide bonds in the hinge region is an IgG isotype. In some embodiments, the IgG isotype is IgG1 or IgG2. In some embodiments, the IgG isotype is humanized. In some embodiments, the IgG isotype is humanized IgG1. In some embodiments, the conjugates comprise humanized IgG and a heterologous moiety or pharmaceutical agent. In some embodiments, the conjugate comprises humanized IgG1 or humanized IgG2 and a heterologous portion or agent. In some embodiments, the conjugate comprises humanized IgG1 and a heterologous portion or agent. In some embodiments, the conjugate comprises humanized IgG1 or humanized IgG2 and 3-8 units of a heterologous portion or agent. In some embodiments, the average amount of the heterologous portion or agent is in the range of about 3-8 units per humanized IgG1 or humanized IgG2. In some embodiments, the conjugate comprises humanized IgG1 and 4 units of a heterologous portion or agent. In some embodiments, the average amount of the heterologous portion or agent is in the range of about 4 units per humanized IgG. In a preferred embodiment, the homogeneous conjugate comprises 4 HARs. In other preferred embodiments, the homogeneous conjugate comprises 2 HARs. In some embodiments, the homogeneous conjugate comprises 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a conjugate with a defined HAR. In some embodiments, the homogeneous conjugate comprises about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a conjugate with a defined HAR.In some embodiments, the homogeneous conjugate comprises at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a conjugate having a defined HAR. In some embodiments, the homogeneous conjugate comprises a non-Gaussian or quasi-Gaussian HAR distribution. In some embodiments, the homogeneity of the homogeneous conjugate is determined by chromatography, such as HPLC or any suitable chromatographic method. In some embodiments, the chromatogram is a HIC chromatogram. The homogeneous conjugate can be generated by site-specific conjugation.

[0220] In some implementations, antigen-binding proteins, bispecific antigen-binding proteins, or conjugates may be detectably labeled. Examples of detectable components include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, luciferin, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin (PE); examples of luminescent substances include luminol; examples of bioluminescent substances include luciferase, luciferin, and amylopectin; and examples of suitable radioactive substances include... 125 I, 131 I, 35 S or 3 H. As used herein, the term "labeled" in relation to antigen-binding proteins, bispecific antigen-binding proteins, or conjugates is intended to include direct labeling by conjugating (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), or indocyanine (Cy5)) to a protein or conjugate, as well as indirect labeling of antibodies by reactivity with the detectable substance. For example, antigen-binding proteins, bispecific antigen-binding proteins, or conjugates may be labeled with amplifiable and detectable nucleic acid sequences, or with antisense oligonucleotides to reduce the expression of a specific gene, making subsequent detection and measurement of expression possible. In some embodiments, antigen-binding proteins, bispecific antigen-binding proteins, or conjugates may be detectably labeled as fusion proteins comprising peptides (HA, myc, GST, etc.) or polypeptides (GFP, etc.).

[0221] This disclosure also provides a fusion protein comprising the antigen-binding protein or bispecific antigen-binding protein described herein. This disclosure also provides a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein, bispecific antigen-binding protein, conjugate, or fusion protein of this disclosure. In various aspects, the conjugate may be a fusion protein of an antigen-binding protein and a second protein or peptide. This disclosure provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein, conjugate, or fusion protein of this disclosure. This disclosure further provides a host cell comprising the nucleic acid or vector of this disclosure.

[0222] This disclosure provides a method for producing an antigen-binding protein or a bispecific antigen-binding protein that binds to the P2RY8 protein, comprising: (i) culturing a host cell of the present disclosure in a cell culture medium, wherein the host cell contains a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein or bispecific antigen-binding protein described herein; and (ii) harvesting the antigen-binding protein or bispecific antigen-binding protein from the cell culture medium. Furthermore, a method for producing a fusion protein comprising an antigen-binding protein or a bispecific antigen-binding protein that binds to the P2RY8 protein is provided, comprising: (i) culturing a host cell of the present disclosure in a cell culture medium, wherein the host cell contains a nucleic acid comprising a nucleotide sequence encoding the fusion protein of the present disclosure; and (ii) harvesting the fusion protein from the cell culture medium.

[0223] This disclosure also provides a method for producing a pharmaceutical composition comprising combining the antigen-binding protein of the present invention, the bispecific antigen-binding protein of the present disclosure, a conjugate, a fusion protein, a nucleic acid, a carrier, a host cell, or a combination thereof with a pharmaceutically acceptable carrier, diluent, or excipient. A pharmaceutical composition comprising the antigen-binding protein of the present disclosure, the bispecific antigen-binding protein, the conjugate, the fusion protein, the nucleic acid, the carrier, a host cell, or a combination thereof, and a pharmaceutically acceptable carrier, diluent, or excipient is also provided.

[0224] This document provides a method for treating a subject with cancer expressing P2RY8, comprising administering to the subject an amount of the pharmaceutical composition described herein that is effective in treating the cancer. It also provides a method for inhibiting tumor growth in a subject, comprising administering to the subject an amount of the pharmaceutical composition described herein that is effective in inhibiting tumor growth, wherein the tumor expresses P2RY8. This disclosure provides a method for reducing tumor size in a subject, comprising administering to the subject an amount of the pharmaceutical composition described herein that is effective in reducing tumor size, wherein the tumor expresses P2RY8. It also provides a method for preventing cancer recurrence in a subject, comprising administering to the subject an amount of the pharmaceutical composition described herein that is effective in preventing cancer recurrence, wherein the tumor expresses P2RY8.

[0225] This disclosure provides a method for detecting P2RY8 protein in a sample, comprising contacting the sample with the antigen-binding protein of the present invention, a bispecific antigen-binding protein of the present disclosure, a conjugate, or a fusion protein, and measuring an immune complex comprising the antigen-binding protein, conjugate, or fusion protein binding to P2RY8 protein. This document also provides a method for diagnosing P2RY8-positive cancer in a subject, comprising contacting a biological sample comprising cells or tissue obtained from the subject with the antigen-binding protein, a bispecific antigen-binding protein, a conjugate, or a fusion protein of the present disclosure, and measuring an immune complex comprising the antigen-binding protein, conjugate, or fusion protein binding to P2RY8 protein.

[0226] Another exemplary implementation is given below.

[0227] 1. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0228] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 177) or a variant sequence differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0229] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 185) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0230] 2. The antigen-binding protein as described in Embodiment 1, comprising: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 178) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 179) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 180) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 186) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (e) a light chain CDR2 comprising the amino acid sequence: RAS (SEQ ID NO: 186) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity. (a) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) a light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO: 188) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f).

[0231] 3. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0232] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 145) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0233] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 153) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0234] 4. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0235] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 161) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0236] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 169) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0237] 5. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0238] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 193) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0239] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 201) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0240] 6. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0241] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 209) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0242] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 217) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0243] 7. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0244] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 225) or a variant sequence differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0245] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 233) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0246] 8. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0247] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 241) or a variant sequence differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0248] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 249) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0249] 9. An antigen-binding protein comprising: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 146) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 147) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 148) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 154) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (e) a light chain CDR2 comprising the amino acid sequence: RAS (SEQ ID NO: 147) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity. (155) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) a light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO:156) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or a combination of any two or more of (g) (a)-(f).

[0250] 10. An antigen-binding protein comprising: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 162) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 163) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 164) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 170) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (e) a light chain CDR2 comprising the amino acid sequence: RAS (SEQ ID NO: 162). 171) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO:172) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or a combination of any two or more of (g) (a)-(f).

[0251] 11. An antigen-binding protein comprising: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 194) or a variant sequence differing from GYSITSDYA by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 195) or a variant sequence differing from GYSITSDYA by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 196) or a variant sequence differing from ARGYWYFDV by only one or two amino acids or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 202) or a variant sequence differing from ESVDSYGYTF by only one or two amino acids or having at least or about 70% sequence identity; and (e) a light chain CDR2 comprising the amino acid sequence: RAS (SEQ ID NO: 194). 203) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO:204) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or a combination of any two or more of (g) (a)-(f).

[0252] 12. An antigen-binding protein comprising: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 210) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 211) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 212) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 218) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (e) a light chain CDR2 comprising the amino acid sequence: RAS (SEQ ID NO: 210). 219) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) a light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO:220) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or a combination of any two or more of (g) (a)-(f).

[0253] 13. An antigen-binding protein comprising: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 226) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 227) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 228) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 234) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (e) a light chain CDR2 comprising the amino acid sequence: RAS (SEQ ID NO: 226). (235) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) a light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO:236) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or a combination of any two or more of (g) (a)-(f).

[0254] 14. An antigen-binding protein comprising: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 242) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 243) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 244) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 250) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (e) a light chain CDR2 comprising the amino acid sequence: RAS (SEQ ID NO: 250) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity. 251) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO:252) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or a combination of any two or more of (g) (a)-(f).

[0255] 15. The antigen-binding protein as described in Embodiment 9, further comprising: (a) a heavy chain FR1 comprising the amino acid sequence:

[0256] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 149) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWVSY (SEQ ID NO: 150) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYC (SEQ ID NO: 151) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 149) (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 157) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 158) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 157) 159) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 160) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0257] 16. The antigen-binding protein as described in Embodiment 10, further comprising: (a) a heavy chain FR1 comprising the amino acid sequence:

[0258] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 165) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWVSY (SEQ ID NO: 166) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYC (SEQ ID NO: 167) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 167) (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 173) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 174) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYC (SEQ ID NO: 173) 175) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 176) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0259] 17. The antigen-binding protein as described in Embodiment 2, further comprising: (a) a heavy chain FR1 comprising the amino acid sequence:

[0260] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 181) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 182) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 183) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 181) (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 189) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 190) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 189) 191) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 192) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0261] 18. The antigen-binding protein as described in Embodiment 11, further comprising: (a) a heavy chain FR1 comprising the amino acid sequence:

[0262] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 197) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 198) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 199) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 199) (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 205) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 206) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYC (SEQ ID NO: 205) 207) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 208) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0263] 19. The antigen-binding protein as described in Embodiment 12, further comprising: (a) a heavy chain FR1 comprising the amino acid sequence:

[0264] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 213) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 214) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 215) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 215) 216) or variant sequences differing from the amino acid sequence by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 221) or variant sequences differing from the amino acid sequence by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 222) or variant sequences differing from the amino acid sequence by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) light chain FR3, comprising the amino acid sequence: NVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYC (SEQ ID NO: 222) 223) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 224) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0265] 20. The antigen-binding protein as described in Embodiment 13, further comprising: (a) a heavy chain FR1 comprising the amino acid sequence:

[0266] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 229) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 230) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 231) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 229) (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 237) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 238) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYC (SEQ ID NO: 237) 239) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 240) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0267] 21. The antigen-binding protein as described in embodiment 14, further comprising: (a) a heavy chain FR1 comprising the amino acid sequence:

[0268] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 245) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 246) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 247) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 245) (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 253) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 254) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 254) 255) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 256) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0269] 22. An antigen-binding protein comprising: (a) a heavy chain variable region having an amino acid sequence:

[0270] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 145) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0271] (b) Light chain variable region, which has an amino acid sequence:

[0272] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 153) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0273] 23. An antigen-binding protein comprising:

[0274] (a) Heavy chain variable region having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 161) or a variant sequence differing from the sequence by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0275] (b) Light chain variable region, which has an amino acid sequence:

[0276] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 169) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0277] 24. An antigen-binding protein comprising:

[0278] (a) Heavy chain variable region having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 177) or a variant sequence differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0279] (b) Light chain variable region, which has an amino acid sequence:

[0280] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 185) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0281] 25. An antigen-binding protein comprising:

[0282] (a) Heavy chain variable region having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 193) or a variant sequence differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0283] (b) Light chain variable region, which has an amino acid sequence:

[0284] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 201) or a variant sequence differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0285] 26. An antigen-binding protein comprising:

[0286] (a) Heavy chain variable region having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 209) or a variant sequence differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0287] (b) Light chain variable region, which has an amino acid sequence:

[0288] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 217) or variant sequences differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0289] 27. An antigen-binding protein comprising:

[0290] (a) Heavy chain variable region, which has an amino acid sequence:

[0291] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 225) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0292] (b) Light chain variable region, which has an amino acid sequence:

[0293] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 233) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0294] 28. An antigen-binding protein comprising:

[0295] (a) Heavy chain variable region, which has an amino acid sequence:

[0296] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 241) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0297] (b) Light chain variable region, which has an amino acid sequence:

[0298] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 249) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0299] 29. The antigen-binding protein as described in any of the preceding embodiments, wherein the variant sequence has at least about 80% or at least or about 85% sequence identity.

[0300] 30. The antigen-binding protein as described in any of the preceding embodiments, wherein the variant sequence has at least about 90% sequence identity or at least or about 95% sequence identity.

[0301] 31. The antigen-binding protein as described in any of the foregoing embodiments, wherein:

[0302] (a) The antigen-binding protein binds to human P2Y receptor family member 8 (P2RY8) protein (SEQ ID NO: 369); (b) The antigen-binding protein has a dissociation constant (Ki) of approximately less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, or 0.25 nM. D (c) The antigen-binding protein binds to the extracellular domain of human P2RY8; (d) The antigen-binding protein binds to human P2RY8 preferentially compared to dog P2RY8 (SEQ ID NO: 371); or (e) The antigen-binding protein does not bind to dog P2RY8; or any combination thereof.

[0303] 32. The antigen-binding protein of embodiment 31, wherein the human P2RY8 (SEQ ID NO: 369) is expressed on the cell surface; the canine P2RY8 (SEQ ID NO: 371) is expressed on the cell surface; and the antigen-binding protein preferentially binds to the human P2RY8 compared to the canine P2RY8, with the former binding at least about 10, 20, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, or 325 times more strongly than the latter.

[0304] 33. The antigen-binding protein as described in any of the preceding embodiments is an antibody or an antibody fragment that binds to an antigen.

[0305] 34. The antigen-binding protein as described in embodiment 33, wherein the antibody is a monoclonal antibody.

[0306] 35. The antigen-binding protein as described in embodiment 33 or 34, wherein the antibody is a human antibody or a humanized antibody.

[0307] 36. The antigen-binding protein as described in any one of embodiments 33-35, wherein it is IgG.

[0308] 37. The antigen-binding protein as described in embodiment 36, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

[0309] 38. The antigen-binding protein as described in embodiment 36 or 37, wherein the IgG is IgG1.

[0310] 39. The antigen-binding protein as described in embodiment 33, wherein the antibody fragment binding the antigen is selected from the group consisting of scFv, F(ab')2, Fab, Fab' and Fv.

[0311] 40. The antigen-binding protein as described in any of the preceding embodiments, which inhibits tumor growth in xenograft mice injected with human cancer cells.

[0312] 41. The antigen-binding protein as described in any of the preceding embodiments, wherein the antigen-binding protein comprises: (a) an Fc polypeptide, or (b) an Fc polypeptide comprising fucosylated glycans.

[0313] 42. A conjugate comprising an antigen-binding protein as described in any of the foregoing embodiments or the antigen-binding proteins or antigen-binding fragments thereof described herein.

[0314] 43. The conjugate as described in embodiment 42, comprising a cytotoxic agent or a chemotherapeutic agent.

[0315] 44. The conjugate as described in embodiment 43, wherein the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking microtubule polymerization.

[0316] 45. The conjugate as described in embodiment 44, wherein the antimitotic agent is aurestatin.

[0317] 46. ​​The conjugate as described in embodiment 45, wherein the auristatin is monomethyl auristatin E (MMAE).

[0318] 47. The conjugate as described in any one of embodiments 43-46, wherein the agent is conjugated to the antigen-binding protein via a cleavable linker.

[0319] 48. The conjugate as described in embodiment 47, wherein the cuttable connector is VC-PAB.

[0320] 49. The conjugate as described in any one of embodiments 42-48, wherein the antigen-binding protein is an antibody.

[0321] 50. The conjugate as described in embodiment 49, wherein the antibody is a monoclonal antibody.

[0322] 51. The conjugate as described in embodiment 49 or 50, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0323] 52. The conjugate as described in any one of embodiments 49-51, wherein the antibody is an IgG antibody, optionally wherein the IgG is IgG1.

[0324] 53. The conjugate as described in any one of embodiments 42-52, wherein the average number of pharmaceutical units conjugated to each antigen-binding protein is in the range of 1 to 8, preferably wherein the average number of pharmaceutical units conjugated to each antigen-binding protein is in the range of 3 to 8.

[0325] 54. The conjugate as described in any one of embodiments 42-53, wherein the conjugate is a heteroconjugate.

[0326] 55. The conjugate as described in any one of embodiments 42-53, wherein the conjugate is a homogeneous conjugate.

[0327] 56. The conjugate as described in any one of embodiments 42-55, wherein the pharmaceutical agent is conjugated at a specific site of the antigen-binding protein.

[0328] 57. The conjugate as described in embodiment 56, wherein the specific site is an unpaired cysteine ​​residue.

[0329] 58. The conjugate of any one of embodiments 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 145 and SEQ ID NO: 153.

[0330] 59. The conjugate of any one of embodiments 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 161 and SEQ ID NO: 169.

[0331] 60. The conjugate of any one of embodiments 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 177 and SEQ ID NO: 185.

[0332] 61. The conjugate of any one of embodiments 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 193 and SEQ ID NO: 201.

[0333] 62. The conjugate of any one of embodiments 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 209 and SEQ ID NO: 217.

[0334] 63. The conjugate of any one of embodiments 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 225 and SEQ ID NO: 233.

[0335] 64. The conjugate of any one of embodiments 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 241 and SEQ ID NO: 249.

[0336] 65. A bispecific antigen-binding protein comprising: an antigen-binding protein as described in any one of embodiments 1-41, an antigen-binding protein of Table 3 or a fragment thereof, said antigen-binding protein or fragment thereof binding to human P2Y receptor family member 8 (P2RY8) protein (SEQ ID NO: 369); and a second antigen-binding protein binding to a second antigen.

[0337] 66. The bispecific antigen-binding protein as described in embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises:

[0338] (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence:

[0339] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 177) or a variant sequence differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0340] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 185) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0341] 67. The bispecific antigen-binding protein of embodiment 66, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 178) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 179) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 180) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 180). (a) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) light chain CDR2 containing the amino acid sequence: RAS (SEQ ID NO: 187) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO: 188) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f).

[0342] 68. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding to the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence:

[0343] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 145) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0344] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 153) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0345] 69. The bispecific antigen-binding protein of embodiment 68, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 146) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 147) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 148) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 148). (a) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) light chain CDR2 containing the amino acid sequence: RAS (SEQ ID NO: 155) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO: 156) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f).

[0346] 70. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence:

[0347] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 161) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0348] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 169) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0349] 71. The bispecific antigen-binding protein of embodiment 70, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 162) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 163) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 164) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 164). (a) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) light chain CDR2 containing the amino acid sequence: RAS (SEQ ID NO: 171) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO: 172) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f).

[0350] 72. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence:

[0351] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 193) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0352] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 201) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0353] 73. The bispecific antigen-binding protein of embodiment 72, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 194) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 195) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 196) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 194). (a) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) light chain CDR2 containing the amino acid sequence: RAS (SEQ ID NO: 203) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO: 204) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f).

[0354] 74. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding to the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence:

[0355] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 209) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0356] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 217) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0357] 75. The bispecific antigen-binding protein of embodiment 74, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 210) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 211) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 212) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 210). (a) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) light chain CDR2 containing the amino acid sequence: RAS (SEQ ID NO: 219) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO: 220) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f).

[0358] 76. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence:

[0359] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 225) or a variant sequence differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0360] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 233) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0361] 77. The bispecific antigen-binding protein of embodiment 76, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 226) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 227) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 228) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 228). (a) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) light chain CDR2 containing the amino acid sequence: RAS (SEQ ID NO: 235) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO: 236) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f).

[0362] 78. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence:

[0363] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 241) or a variant sequence differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (b) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0364] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 249) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0365] 79. The bispecific antigen-binding protein of embodiment 78, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain CDR1 comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 242) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) a heavy chain CDR2 comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 243) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) a heavy chain CDR3 comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 244) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; and (d) a light chain CDR1 comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 244). (a) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) light chain CDR2 containing the amino acid sequence: RAS (SEQ ID NO: 251) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) light chain CDR3 containing the amino acid sequence: QQSNEDPFT (SEQ ID NO: 252) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (g) a combination of any two or more of (a)-(f).

[0366] 80. The bispecific antigen-binding protein of embodiment 66, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) a heavy chain FR1 comprising the amino acid sequence:

[0367] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 181) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 182) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 183) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 181) (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 189) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 190) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 189) 191) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 192) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0368] 81. The bispecific antigen-binding protein as described in embodiment 68, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) a heavy chain FR1 comprising the amino acid sequence:

[0369] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 149) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWVSY (SEQ ID NO: 150) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYC (SEQ ID NO: 151) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 149) (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 157) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 158) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 157) 159) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 160) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0370] 82. The bispecific antigen-binding protein as described in embodiment 70, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) a heavy chain FR1 comprising the amino acid sequence:

[0371] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 165) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWVSY (SEQ ID NO: 166) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYC (SEQ ID NO: 167) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 167) (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 173) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 174) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYC (SEQ ID NO: 173) 175) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 176) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0372] 83. The bispecific antigen-binding protein as described in embodiment 72, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) a heavy chain FR1 comprising the amino acid sequence:

[0373] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 197) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 198) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 199) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 199) (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 205) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 206) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYC (SEQ ID NO: 205) 207) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 208) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0374] 84. The bispecific antigen-binding protein as described in embodiment 74, wherein the antigen-binding protein binding the P2RY8 protein further comprises:

[0375] (a) Heavy chain FR1, comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 213) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 214) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) Heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 215) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 215) 216) or variant sequences differing from the amino acid sequence by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 221) or variant sequences differing from the amino acid sequence by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 222) or variant sequences differing from the amino acid sequence by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) light chain FR3, comprising the amino acid sequence: NVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYC (SEQ ID NO: 222) 223) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 224) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0376] 85. The bispecific antigen-binding protein as described in embodiment 76, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) a heavy chain FR1 comprising the amino acid sequence:

[0377] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 229) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 230) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 231) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 229) (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 237) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 238) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYC (SEQ ID NO: 237) 239) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 240) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0378] 86. The bispecific antigen-binding protein as described in embodiment 78, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) a heavy chain FR1 comprising the amino acid sequence:

[0379] (a) EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 245) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (b) heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 246) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (c) heavy chain FR3, comprising the amino acid sequence: TYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 247) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (d) heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 245) (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 253) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (f) Light chain FR2, comprising the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 254) or a variant sequence differing from only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; (g) Light chain FR3, comprising the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 254) 255) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 256) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or a combination of any two or more of (i) (a)-(h).

[0380] 87. The bispecific antigen-binding protein as described in embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain variable region having an amino acid sequence of:

[0381] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 177) or variant sequences differing from the SEQ ID NO by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) a light chain variable region having an amino acid sequence:

[0382] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 185) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0383] 88. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain variable region having the following amino acid sequence:

[0384] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 145) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) a light chain variable region having an amino acid sequence:

[0385] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 153) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0386] 89. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain variable region having an amino acid sequence:

[0387] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 161) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0388] (b) Light chain variable region, which has an amino acid sequence:

[0389] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 169) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0390] 90. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain variable region having an amino acid sequence of:

[0391] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 193) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0392] (b) Light chain variable region, which has an amino acid sequence:

[0393] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 201) or a variant sequence differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0394] 91. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain variable region having an amino acid sequence of:

[0395] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 209) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0396] (b) Light chain variable region, which has an amino acid sequence:

[0397] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 217) or variant sequences differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0398] 92. The bispecific antigen-binding protein as described in embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain variable region having an amino acid sequence of:

[0399] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 225) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0400] (b) Light chain variable region, which has an amino acid sequence:

[0401] DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 233) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0402] 93. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) a heavy chain variable region having an amino acid sequence:

[0403] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 241) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or

[0404] (b) Light chain variable region, which has an amino acid sequence:

[0405] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 249) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

[0406] 94. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein is selected from the group consisting of: (a) antigen-binding proteins comprising: (i) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising an amino acid sequence:

[0407] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 257) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0408] DIQMTQSPSSLSASVGDRVTITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 265) or a variant sequence differing from only one or two amino acids or having at least or about 85% sequence identity; (b) an antigen-binding protein comprising: (i) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0409] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 273) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0410] DIQLTQSPSSLSASVGDRATITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 281) or a variant sequence differing from only one or two amino acids or having at least or about 85% sequence identity; (c) an antigen-binding protein comprising: (i) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0411] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 289) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0412] DIQMTQSPSSLSASVGDRVTITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 297) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and (d) an antigen-binding protein comprising: (i) CDR1-3 derived from the heavy chain variable region, said heavy chain variable region comprising an amino acid sequence:

[0413] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 305) or variant sequences differing from only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) derived from CDR1-3 of the light chain variable region, the light chain variable region comprising the amino acid sequence:

[0414] DIQLTQSPSSLSASVGDRATITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 313) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0415] 95. The bispecific antigen-binding protein of embodiment 65, wherein the antigen-binding protein binding the P2RY8 protein is selected from the group consisting of: (a) an antigen-binding protein comprising: (i) a heavy chain variable region having an amino acid sequence:

[0416] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 257) or variant sequences differing from the SEQ ID NO by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (ii) a light chain variable region having an amino acid sequence:

[0417] DIQMTQSPSSLSASVGDRVTITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 265) or a variant sequence differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; (b) an antigen-binding protein comprising: (i) a heavy chain variable region having an amino acid sequence:

[0418] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 273) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) a light chain variable region having an amino acid sequence:

[0419] DIQLTQSPSSLSASVGDRATITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 281) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; (c) an antigen-binding protein comprising:

[0420] (i) Heavy chain variable region, which has an amino acid sequence:

[0421] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 289) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) a light chain variable region having an amino acid sequence:

[0422] DIQMTQSPSSLSASVGDRVTITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 297) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and (d) an antigen-binding protein comprising: (i) a heavy chain variable region having an amino acid sequence:

[0423] EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 305) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) a light chain variable region having an amino acid sequence:

[0424] DIQLTQSPSSLSASVGDRATITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 313) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

[0425] 96. A bispecific antigen-binding protein as described in any one of embodiments 65-95, wherein the second antigen is a cell surface protein expressed by a T cell, optionally a component of a T cell receptor (TCR), such as CD3.

[0426] 97. A bispecific antigen-binding protein as described in any one of embodiments 65-95, wherein the second antigen is a co-stimulatory molecule that assists in T cell activation, such as CD40 or 4-1BB (CD137).

[0427] 98. A bispecific antigen-binding protein as described in any one of embodiments 65-95, wherein the second antigen is an Fc receptor, optionally an Fc γ receptor, an Fc-α receptor, or an Fc-ε receptor.

[0428] 99. The bispecific antigen-binding protein as described in embodiment 98, wherein the Fc receptor is CD64 (Fc-γ RI), CD32 (Fc-γ RIIA), CD16A (Fc-γ RIIIA), CD16b (Fc-γ RIIIb), FcεRI, CD23 (Fc-ε RII), CD89 (Fc-ε RI), Fc α / µR, or FcRn.

[0429] 100. A bispecific antigen-binding protein as described in embodiment 98 or 99, wherein the Fc receptor is CD16A.

[0430] 101. A bispecific antigen-binding protein as described in any one of embodiments 65-95, wherein the second antigen is an immune checkpoint molecule, such as a protein involved in the immune checkpoint pathway, optionally A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7.

[0431] 102. The bispecific antigen-binding protein as described in embodiment 101, wherein the immune checkpoint molecule is PD-1, LAG3, TIM3, or CTLA4.

[0432] 103. The bispecific antigen-binding protein as described in any one of embodiments 65-95, wherein the second antigen is CD3.

[0433] 104. The bispecific antigen-binding protein as described in any one of embodiments 65-95, wherein the second antigen is CD3E.

[0434] 105. A bispecific antigen-binding protein as described in any one of embodiments 65-95, wherein the second antigen is CD16A.

[0435] 106. A bispecific antigen-binding protein as described in any one of embodiments 65-105, comprising scFv, Fab, or F(ab')2 of any of the currently disclosed P2RY8 antibodies.

[0436] 107. A bispecific antigen-binding protein as described in any one of embodiments 65-106, comprising the structure of a nanobody, BiTE®, DART, TandAb, CrossMab, or HSAbody.

[0437] 108. A fusion protein comprising an antigen-binding protein as described in any of the foregoing embodiments or the antigen-binding proteins or antigen-binding fragments thereof described herein.

[0438] 109. A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein as described in any one of embodiments 1-41, a bispecific antigen-binding protein as described in any one of embodiments 65-107, or a fusion protein as described in embodiment 108.

[0439] 110. The nucleic acid as described in embodiment 109, wherein the nucleic acid is complementary DNA (cDNA).

[0440] 111. A vector comprising nucleic acid as described in embodiment 109 or 110.

[0441] 112. The vector as described in embodiment 111, further comprising an internal ribosome entry site (IRES).

[0442] 113. A host cell comprising a nucleic acid as described in embodiment 109, cDNA as described in embodiment 110, or a vector as described in embodiment 111 or 112.

[0443] 114. The host cell as described in embodiment 113, wherein the host cell is a bacterial cell.

[0444] 115. The host cell as described in embodiment 113, wherein the host cell is a eukaryotic cell.

[0445] 116. The host cell as described in embodiment 113 or 115, wherein the host cell is a mammalian cell.

[0446] 117. The host cell as described in embodiment 116, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0447] 118. A method for producing an antigen-binding protein that binds to a member of the P2Y receptor family 8 (P2RY8) protein, comprising: (i) culturing host cells as described in any one of embodiments 113-117 in a cell culture medium, and (ii) harvesting the antigen-binding protein from the cell culture medium.

[0448] 119. A method for producing a bispecific antigen-binding protein comprising an antigen-binding protein that binds to a member of the P2Y receptor family 8 (P2RY8) protein, the method comprising: (i) culturing host cells in a cell culture medium as described in any one of embodiments 113-117, and (ii) harvesting the bispecific antigen-binding protein from the cell culture medium.

[0449] 120. A method for producing a fusion protein comprising an antigen-binding protein that binds to a member of the P2Y receptor family 8 (P2RY8) protein, the method comprising: (i) culturing host cells as described in any one of embodiments 113-117 in a cell culture medium, and (ii) harvesting the fusion protein from the cell culture medium.

[0450] 121. A method for preparing a pharmaceutical composition comprising combining an antigen-binding protein as described in any one of embodiments 1-41, a conjugate as described in any one of embodiments 42-64, a bispecific antigen-binding protein as described in any one of embodiments 65-107, a fusion protein as described in embodiment 108, a nucleic acid as described in embodiment 109 or 110, a carrier as described in embodiment 111 or 112, a host cell as described in any one of embodiments 113-117, or a combination thereof, with a pharmaceutically acceptable carrier, diluent, or excipient.

[0451] 122. A pharmaceutical composition comprising an antigen-binding protein as described in any one of embodiments 1-41, a conjugate as described in any one of embodiments 42-64, a bispecific antigen-binding protein as described in any one of embodiments 65-107, a fusion protein as described in embodiment 108, a nucleic acid as described in embodiment 109 or 110, a carrier as described in embodiment 111 or 112, a host cell or a combination thereof as described in any one of embodiments 113-117; and a pharmaceutically acceptable carrier, diluent, or excipient.

[0452] 123. A method of treating a subject with cancer expressing P2RY8, comprising administering to the subject an amount of a pharmaceutical composition as described in embodiment 122 that is effective in treating the cancer.

[0453] 124. A method for inhibiting tumor growth in a subject, comprising administering to the subject an amount of a pharmaceutical composition as described in embodiment 122 that effectively inhibits tumor growth.

[0454] 125. A method for reducing the size of a tumor in a subject, comprising administering to the subject an amount of a pharmaceutical composition as described in embodiment 122 that effectively reduces the size of the tumor.

[0455] 126. A method for preventing cancer recurrence in a subject, comprising administering to the subject an amount of a pharmaceutical composition as described in embodiment 122 that is effective in preventing cancer recurrence.

[0456] 127. The method of any one of embodiments 123-126, wherein the pharmaceutical composition induces apoptosis of tumor cells.

[0457] 128. The method of any one of embodiments 123-126, wherein the pharmaceutical composition induces apoptosis in cells expressing P2RY8.

[0458] 129. A method for detecting a P2Y receptor family member 8 (P2RY8) protein or a fragment thereof in a sample, comprising contacting the sample with an antigen-binding protein as described in any one of embodiments 1-41, a conjugate as described in any one of embodiments 42-64, a bispecific antigen-binding protein as described in any one of embodiments 65-107, or a fusion protein as described in embodiment 108, and determining an immune complex comprising the antigen-binding protein, conjugate, or fusion protein binding the P2RY8 protein or a fragment thereof.

[0459] 130. A method for diagnosing P2Y receptor family member 8 (P2RY8) protein-positive cancer in a subject, comprising contacting a sample from the subject (e.g., a biological sample containing cells or tissue) with an antigen-binding protein as described in any one of embodiments 1-41, a conjugate as described in any one of embodiments 42-64, a bispecific antigen-binding protein as described in any one of embodiments 65-107, or a fusion protein as described in embodiment 108, and measuring an immune complex comprising the antigen-binding protein, conjugate, or fusion protein binding to the P2RY8 protein or a fragment thereof.

[0460] The following embodiments are provided for illustrative purposes only and are not intended to limit the scope of this disclosure in any way.

[0461] Example

[0462] Example 1: P2RY8 RNA expression in normal human tissues and cells, human tumors, and human cancer cell lines

[0463] This example demonstrates the analysis of P2RY8 RNA levels in normal human tissues, primary human cancers, and human cancer cell lines from the Translational Oncology Research Laboratory (TORL) of human cancer patients.

[0464] For example, the P2RY8 RNA expression in normal human tissues provided in the GTEx database GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_tpm was downloaded from the GTEx portal (gtexportal.org / home / datasets) and presented as a box plot. Figure 1 The Cancer Genome Atlas (TCGA) database, managed by the National Cancer Institute (NCI), was used to download P2RY8 RNA expression data from a total of 10,071 human cancer tissue samples from cBioPortal (cbioportal.org / ), and plotted using the ggplot2 package in the R programming language. Figure 2 This is a box plot from TCGA showing P2RY8 expression in human cancers, revealing high levels of P2RY8 RNA expression in various human cancers, particularly mature B-cell tumors and leukemia.

[0465] P2RY8 expression levels were measured using an Agilent 44K microarray (4x44K array chip, Agilent Technologies, Santa Clara, CA) and RNA sequencing (RNA-Seq) in a cohort of human cancer cell lines compiled by TORL. RNA sequencing was performed by BGI Americas (Cambridge, MA) using their "RNA Sequencing for Quantification" service. P2RY8 RNA expression in a total of 587 human cancer cell lines was analyzed. Figure 3 As shown, human cancer cell lines derived from patients with acute myeloid leukemia (AML), lymphoma, and myeloma (e.g., multiple myeloma) expressed the highest levels of P2RY8; while cancer cell lines derived from sarcomas and connective tissue cancers, prostate cancer, lung cancer, melanoma, colon cancer, endometrial cancer, pancreatic cancer, ovarian cancer, kidney cancer, liver cancer, breast cancer, upper gastrointestinal cancer, head and neck cancer, and bladder cancer typically showed low P2RY8 RNA expression.

[0466] Example 2: Generation of engineered cells to overexpress P2RY8

[0467] This example demonstrates the generation of cells engineered to overexpress P2RY8.

[0468] A model engineered to overexpress P2RY8 was generated. In short, the nucleotide sequence encoding the P2RY8 protein was introduced into a lentiviral vector containing a CMV promoter and an internal ribosome entry site (IRES) for encephalomyocarditis virus (EMCV). The IRES is located between the target gene (GOI) cDNA (P2RY8) and the puromycin cDNA. The prairie dog posttranscriptional regulatory element (WPRE) is located downstream of the puromycin cDNA. The vector also optionally expresses a GFP marker sequence or a MycDDK epitope tag as a fusion protein containing the P2RY8 protein and the GFP marker or MycDDK epitope tag.

[0469] The expression vector was transduced into HEK293T cells (for selection) and NIH3T3 cells (for immunization) via viral transduction. Positive transduced cells were selected based on their survival in culture medium containing puromycin (1 µg / ml). Positive cells were subcloned to obtain a stable and homogeneous clonal population of P2RY8 overexpressing cells.

[0470] The overexpression of P2RY8 protein was confirmed by using GFP tags and fluorescence microscopy to identify subclones of P2RY8-overexpressing cells.

[0471] In addition, using the P2RY8 monoclonal antibody (mAb) (Abcam, Waltham, MA; catalog number: ab169392), flow cytometry was performed on a BD Biosciences Accuri™ flow cytometer (San Jose, CA) using Alexa Fluor. ® The expression of P2RY8 protein on the cell surface was confirmed using the AF647 goat anti-mouse IgG secondary antibody (Biolegend, San Diego, CA; catalog number: 405322). In short, cells expressing P2RY8 were incubated with the anti-P2RY8 antibody on ice for 30 min, washed, and then incubated with the AF647 goat anti-mouse IgG antibody on ice for 30 min. Fluorescence was read using a BDBiosciences Accuri™ flow cytometer (San Jose, CA). In addition to the monoclonal antibody, a polyclonal P2RY8 antibody (Abnova, Taipei, Taiwan, CN; catalog number: PAB 16354) was also used to characterize cells expressing P2RY8.

[0472] In addition to producing human P2RY8 protein (UniProt accession number: Q86VZ1; Table 9, SEQ ID NO: 369; see also HEK293T cells and NIH3T3 cells) Figure 4 In addition to stable cell lines expressing the cell surface expression (as shown in Table 38), the expression was also found in cynomolgus macaques (cynomolgus macaques; NCBI RefSeq accession number: XP_005595888; Table 9, SEQ ID NO: 370; see also...). Figure 4 (and Table 38) and dog (NCBI RefSeq accession number: XP_013967130; Table 9, SEQ ID NO: 371; see also) Figure 4 P2RY8 (and Table 38) was expressed as a fusion protein with the mGFP marker by transient transfection in HEK293T cells (see Table 39) to characterize species selectivity for binding to anti-P2RY8 antibodies. Figure 4 This is a sequence alignment of the P2RY8 protein from humans, cynomolgus monkeys, and dogs.

[0473] Example 3: Generation of P2RY8-specific antibodies

[0474] P2RY8-specific antibodies were generated by immunizing Balb / c mice, CD1 mice, and F1 hybrids of Balb / c and C57BL / 6 mice with NIH3T3 stable cells overexpressing the full-length human P2RY8 protein (UniProt accession number: Q86VZ1; SEQ ID NO: 369). NIH3T3 stable cells expressing human P2RY8 were obtained by transducing a mammalian expression vector virus encoding the human P2RY8-myc-DDK fusion protein into NIH3T3 cells.

[0475] Spleen cells were harvested from immunized mice and fused with myeloma lines via BTX electrofusion (BTX, Holliston, MA) to generate hybridomas. 7000 primary hybridoma cultures were generated and cultured in 384-well plates. The ability of antibodies to bind to peptides and / or human cancer cells expressing the P2RY8 protein was assessed by flow cytometry. Potentially positive antibodies were rearranged into 96-well plates and further screened by flow cytometry against endogenous and artificial cell lines with sequence similarity to the human P2RY8 protein (e.g., artificial cell lines expressing monkey or dog P2RY8 protein).

[0476] Positive hybridomas producing antibodies that bind to human P2RY8 protein, but preferably not to canine P2RY8 protein, were identified, and nucleic acid sequences encoding the light and heavy chains of the immunoglobulin against human P2RY8 protein were isolated. Using the ExpiCHO™ expression system (ThermoFisher Scientific, Waltham, MA; catalog number: A29133), these nucleic acid sequences were used to generate P2RY8 antibodies formatted as full-length IgG antibodies (e.g., chimeric or humanized IgG1). The heavy and light chain variable regions of the antibodies were cloned into an antibody expression vector, which was developed in the laboratory based on pcDNA™ 3.4-TOPO. ® The vector (ThermoFisher Scientific, Waltham, MA; catalog number: A14697) was engineered. Following the protocol provided with the ExpiCHO™ Expression System Kit, the antibody expression vector was transfected into CHO cells, resulting in the production of bicistronic mRNA from which both the heavy and light chains of immunoglobulins were derived, and IRES drove the expression of the second immunoglobulin chain. The resulting antibody was purified using Protein A resin and / or size exclusion.

[0477] The cell surface binding of the antibody to the P2RY8 protein and the antibody IC50 were determined by FACS, in which the P2RY8 antibody was directly bound to Alexa Fluor according to the manufacturer's protocol. ®647 NHS ester (succinimide ester) (ThermoFisherScientific, Waltham, MA; catalog number: A20106) was used for conjugation. Alternatively, purified P2RY8 antibody was used with Alexa Fluor. ® In the indirect immunofluorescence assay of 647 goat anti-mouse IgG (minimum cross-reactivity) secondary antibody (Biolegend, San Diego, CA; catalog number: 405322).

[0478] Cells expressing P2RY8 were used in FACS assays to determine the ability of P2RY8 antibodies to bind to the cell surface P2RY8 protein. HEK293T cells engineered to express human P2RY8 fused with GFP, cynomolgus monkey P2RY8 fused with GFP, or canine P2RY8 fused with GFP were used as artificial models of P2RY8 expression (Tables 13 and 39). SU-DHL-5 (ATCC CRL-2958), SU-DHL-10 (ATCC CRL-2963), and Karpas-1106P (RRID: CVCL_1821) cells were used as endogenous models of P2RY8 expression.

[0479] For each cell type and each mAb used in each test, suspension cells are used directly without Versene. ® Treatment®; while adherent cells are treated with Versene®. ® (Instead of trypsin) it dissociates from the culture flask surface to protect cell surface proteins. Cells are collected, washed, resuspended, and then treated with Alexa Fluor. ® The labeled P2RY8 mAb was incubated at a predetermined concentration on ice in the dark for 30 min. P2RY8 mAb was then directly administered via Alexa Fluor. ® 647 NHS ester (succinimide ester) was used for labeling. Alternatively, unlabeled P2RY8 antibody could be detected using a fluorescently labeled secondary antibody. After washing, cells were read using a BD Accuri™ flow cytometer C6 to detect antibody-antigen protein binding in the FL4-H channel.

[0480] Example 4: Characterization of chimeric mouse IgG mAB

[0481] The ability of chimeric P2RY8 antibodies containing variable regions of heavy and light chains, as well as human constant IgG1-κ or λ constant regions that replace the mouse constant regions, to inhibit tumor growth or reduce tumor size in human tumor xenograft models was analyzed.

[0482] In vivo binding studies were conducted in xenograft mice injected with human cancer cell lines. In short, a xenograft model of human cancer cell lines was established in six-week-old SCID mice (Charles River Laboratories). Each cell line was subcutaneously injected with SU-DHL-5 (ATCC CRL-2958) 1×10⁻⁶ cells / mL under the following conditions. 7 10 cells, SU-DHL-10 (ATCC CRL-2963) 1×10 7 1.0 × 10⁻⁶ cells or SCC-3 (RRID: CVCL_1683) 7 Cells were injected using 50% matrix gel (BD Biosciences). A sufficient number of mice were injected to achieve 8 mice per treatment arm. Injection was performed when the tumor reached 150 to 300 mm. 3 When the average size was small, mice were randomly assigned to the treatment group. For treatment, each therapeutic antibody (P2RY8 mAb-690 (also referred to herein as 10-0690-m), P2RY8 mAb-716 (also referred to herein as 10-0716-m or P2RY8 mAb-716), P2RY8 mAb-720 (also referred to herein as 10-0720-m), P2RY8 mAb-724 (also referred to herein as 10-0724-m), or P2RY8 mAb-729) was administered. (Also referred to in this article as 10-0729-m), rituximab (an anti-CD20 chimeric antibody against B-cell lymphoma) and a non-targeted IgG1 control (Hu-IgG) were diluted in sterile saline to a working concentration of 1 mg / ml for intravenous (IV) injection via the tail vein. Mice were treated weekly with either anti-P2RY8 antibody or control IgG1 antibody at a dose of 10 mg / kg, or with rituximab at a dose of 10 mg / kg every 4 days until the end of the experiment or for the specified duration of administration. Tumor xenografts were measured three times weekly using calipers, and measurements were determined in mm by multiplying height × width × length. 3 Tumor volume is represented as a unit. Mice were treated for 2–5 weeks. At the end of the study, animals were euthanized, tumor tissue was excised and divided into several fractions, and stored as frozen or formalin-fixed paraffin-embedded (FFPE) tissue for biomarker analysis. All animal experiments were conducted according to protocols approved by the IACUC and UCLA Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA). Results are presented as mean volumes for each group. Error bars represent the standard error (SE) of the mean.

[0483] The results of xenotransplantation assays are shown in Figures 5-7. Figure 5A and Figure 5B As shown, in mice carrying P2RY8-positive human B-cell lymphoma cells from the SU-DHL-5 (ATCC CRL-2958) cell line, each of the anti-P2RY8 proteins—P2RY8 mAb-690, P2RY8 mAb-716, P2RY8 mAb-720, P2RY8 mAb-724, and P2RY8 mAb-729—as well as the anti-CD20 chimeric antibody rituximab, resulted in a significant mean change in tumor volume on day 24, relative to the control IgG1 antibody. All anti-P2RY8 antibodies were found to inhibit tumor growth, with P2RY8 mAb-690, P2RY8 mAb-716, and P2RY8 mAb-724 reducing tumor volume to below the initial volume at the start of the experiment. Figure 6A and Figure 6B As shown, in mice carrying another P2RY8-positive human B-cell lymphoma cell line from SU-DHL-10 (ATCC CRL-2963), anti-P2RY8 antibody and rituximab inhibited tumor growth and reduced tumor volume within 2 weeks compared to the control IgG1 antibody, but to different degrees than in SU-DHL-5 cells. In xenograft assays, the inhibition of tumor cell growth by anti-P2RY8 antibody depends on the presence of P2RY8 protein expression, as the antibody did not inhibit the growth of tumor cells from the human P2RY8-negative B-cell non-Hodgkin lymphoma cell line (SCC-3; RRID: CVCL_1683) in SCID mice.

[0484] As shown in Table 10 below, the anti-P2RY8 antibody of this disclosure exhibits a higher inhibitory effect on the growth of SU-DHL-5 tumor cells compared to SU-DHL-10 cells. This may be because the anti-P2RY8 antibody binds more to SU-DHL-5 cells than to SU-DHL-10 cells, as the former cells are stained with P2RY8 antibody to approximately 2-3 times more than the latter. This higher staining level suggests that SU-DHL-5 cells may have more P2RY8 protein on their cell surface, possibly 2-3 times more than SU-DHL-10 cells.

[0485] Tables 1 and 25-27 provide the names of expression vectors used to express chimeric anti-P2RY8 antibodies containing mouse heavy and light chain variable regions and human IgG1, as well as human κ or λ light chain constant regions replacing the mouse heavy and light chain constant regions. Tables 2, 14-18, and 25-27 provide the amino acid sequences of the heavy and light chain variable regions, as well as the CDR and frame regions defined by IMGT, for various anti-P2RY8 antibodies, including those of P2RY8 mAb-690, P2RY8 mAb-716, P2RY8 mAb-720, P2RY8 mAb-724, and P2RY8 mAb-729. The corresponding nucleic acid sequences encoding the heavy and light chain variable regions are provided in Tables 5 and 33, and the CDRs are provided in Table 36. Tables 4 and 32 provide amino acid sequences, while Tables 7 and 35 provide the encoding nucleic acids for the human IgG1 heavy chain constant region and the human Igκ or λ light chain constant region used to generate the full-length chimeric IgG1 anti-P2RY8 antibody in its complete form.

[0486] Example 5: Humanization of Antibodies

[0487] This embodiment demonstrates the humanization of the antibody of this disclosure.

[0488] Antibodies were selected for humanization analysis. The heavy chain variable (VH) and light chain variable (VL) sequences of the mouse monoclonal anti-P2RY8 antibody were compared with those from human V... H Genes and Human V L Known human germline sequence library of the κ gene (IMGT®, International Immunogenetic Information System) ®The website is imgt.org; Founders and Directors: Marie-Paule Lefranc, Montpellier, France; Giudicelli V., Duroux P., Rollin M., Aouinti S., Folch G., Jabado-Michaloud J., Lefranc M.-P., Kossida S, IMGT® Immunoinformatics Tools for Standardized V-DOMAIN Analysis. Methods Mol Biol. 2022; 2453:477-531. doi: 10.1007 / 978-1-0716-2115-8_24.PMID: 35622340; Lefranc MP., IMGT® immunoglobulin repertoire analysis and antibody humanization. Published in: Alt, FW, Honjo, T, Radbruch A. and Reth, M. (eds.), Molecular Biology of B Cells, 2nd Edition, Academic Press, Elsevier Ltd, London, UK, Chapter 26, 2014, pp. 481-514 (dx.doi.org, ISBN: 978-0-12-397933-9) was compared; the database used was IMGT. H Genes (F+ORF, 273 germline sequences) and IMGT human V L κ gene (F+ORF, 74 germline sequences). Recipient human germline is selected from the germline most closely related to the parental antibody sequence.

[0489] Human germline framework (i.e., non-CDR residues in VH and VL; abbreviated as FR) positions may need to be reverted to the corresponding parental mouse sequences to optimize binding of humanized antibodies. Sequences of humanized antibody versions are provided in the tables and figures. Tables 1 and 28-31 provide the names of expression vectors used to express humanized anti-P2RY8 IgG1-κ or -λ antibodies containing mouse CDRs and / or variants thereof. Tables 3, 19-24, and 28-31 provide the amino acid sequences of the heavy and light chain variable regions, as well as the CDR and framework regions defined by IMGT, for various humanized anti-P2RY8 antibodies; the corresponding nucleic acid sequences encoding the heavy and light chain variable regions are provided in Tables 6 and 34, and the CDR and framework regions are provided in Table 37. Tables 4 and 32 provide the amino acid sequences, while Tables 7 and 35 provide the corresponding nucleic acid sequences for the human IgG1 heavy chain constant region and the human Igκ or λ light chain constant region for producing the full-length humanized IgG1 anti-P2RY8 antibody in its complete form.

[0490] The humanized antibody was constructed and expressed essentially as described in Example 3. The ability of the humanized antibody to bind to cells expressing the P2RY8 protein was characterized by indirect immunofluorescence microscopy and flow cytometry (FACS). Binding of the antibody to cells expressing P2RY8 was assessed using endogenously P2RY8-expressing cell models (e.g., KARPAS-1106P) and the HEK293T cell line overexpressing P2RY8. Cells not expressing the P2RY8 protein (e.g., HEK293T, SCC-3, or M202 (RRID: CVCL_D746)) and the unhumanized parental chimeric antibody served as controls.

[0491] Figure 8 and Figure 9 An example of the P2RY8-positive human large B-cell non-Hodgkin lymphoma cell line Karpas-1106P (RRID: CVCL_1821) is shown, stained with chimeric anti-P2RY8 antibody P2RY8 mAb-716 (also referred to herein as 716-m) and seven different candidate-derived versions of P2RY8 mAb-716. Briefly, Karpas-1106P cells grown in suspension were collected, resuspended, and incubated at 4°C for 30 min with 20 µg / ml of candidate-derived anti-P2RY8 antibody or control parental antibody. The cell pellet was then washed with 2% FBS-PBS to remove unbound excess antibody. The final cell pellet was resuspended in 2% FBS-PBS, and 49 µl of the cell suspension was mixed with 1 µl of 0.3 mg / ml Alexa Fluor. ®647 mouse anti-human IgG-Fc secondary antibody (Biolegend, San Diego, CA; catalog number: 409320) stock solution was mixed. Cells were incubated with the secondary antibody at 4°C for 30 min, then washed with 2% FBS-PBS, and observed under a Keyence fluorescence microscope (BZ-X710) at 40x magnification.

[0492] Fluorescence microscopy analysis revealed that among the seven candidate anti-P2RY8 antibodies derived from P2RY8 mAb-716, five of the seven candidate antibodies showed good surface staining against native P2RY8-positive cell lines, such as... Figure 8 and Figure 9 As shown, these five antibodies are indeed humanized anti-P2RY8 antibodies, namely, P2RY8 mAb-716-h3, P2RY8 mAb-716-h4, P2RY8 mAb-716-h5, P2RY8 mAb-716-h6, and P2RY8 mAb-716-h8. Candidate antibodies 716-h7 and 716-h9 do not stain P2RY8-expressing Karpas-1106P cells, therefore lacking P2RY8 binding activity and are not P2RY8 antibodies.

[0493] To determine the relative antigen-binding strength of humanized antibodies to the P2RY8 protein, indirect flow cytometry was performed using the P2RY8 mouse monoclonal antibody P2RY8mAb-716 and its humanized P2RY8 antibodies (P2RY8mAb-716-h3 to 716-h6 and P2RY8mAb-716-h1 and 716-h2). In short, three naturally occurring P2RY8-positive human lymphoma cell lines (KARPAS-1106P, SU-DHL-5, and SU-DHL-10), a P2RY8-negative human lymphoma cell line (SCC-3), and a P2RY8-negative human melanoma cell line (M202) were examined. The lymphoma cell lines were grown in suspensions, and single-cell suspensions were readily available. For the melanoma cell line M202, these adherent cells were first treated with Versene... ® Treatment was performed to obtain a single-cell suspension. After washing repeatedly with 2% FBS-PBS, the cells were resuspended in 2% FBS-PBS and incubated at 4°C with 20 µg / ml or 6 µg / ml P2RY8 antibody for 30 min. The cell pellet was then washed with 2% FBS-PBS to remove unbound excess antibody. The final cell pellet was resuspended in 2% FBS-PBS, and 49 µl of the cell suspension was mixed with 1 µl of 0.3 mg / ml Alexa Fluor. ®647 mouse anti-human IgG-Fc secondary antibody (Biolegend, San Diego, CA; catalog number: 409320) stock solution was mixed. Cells were incubated with the secondary antibody at 4°C for 30 min, followed by washing with 2% FBS-PBS. After washing with 2% FBS-PBS, cells were analyzed by flow cytometry on an iQue flow cytometer (Sartorius, Goettingen, Germany) according to the manufacturer's protocol. Alexa Fluor ® The 647 fluorescence signal was collected in the FL4-H channel.

[0494] As shown in Table 10, all six humanized P2RY8 antibodies (P2RY8mAb-716-h1 to 716-h6) exhibited good binding activity against native P2RY8-positive cell lines at antibody concentrations of 20 µg / ml and 6 µg / ml, while minimal or no specific binding was observed against P2RY8-negative cell lines, similar to the background secondary antibody control. The general rank of cell binding for P2RY8 antibodies was: Karpas-1106P > SU-DHL-5 > SU-DHL-10 >>> M202, SCC-3. Compared with the P2RY8mAb-716 chimeric monoclonal antibody, the humanized P2RY8 antibody showed similar or better antibody binding activity to the natural P2RY8 positive cell lines.

[0495] Similar indirect flow cytometry analyses were performed on an artificial cell model expressing P2RY8, a stable HEK293T cell line overexpressing human P2RY8-mGFP fusion protein (HEK293T P2RY8mGFP clone 5), and the P2RY8-negative parental HEK293T cell line. Since HEK293T cells are adherent, Versene was used... ® The cultured cells were dissociated to obtain a single-cell suspension. Furthermore, when the human P2RY8 protein was expressed as a fusion protein with mGFP, the relative amount of human P2RY8 expression in HEK293T cells could be monitored based on mGFP fluorescence, which was recorded in the FL1-H channel of an iQue flow cytometer.

[0496] Table 11 shows that all six humanized P2RY8 antibodies exhibited robust binding to the stable HEK293T P2RY8mGFP clone 5 cell line overexpressing P2RY8 at antibody concentrations of 20 µg / ml and 6 µg / ml. Minimal or no binding was observed with the parental HEK293T cell line lacking P2RY8 expression. Compared to the P2RY8mAb-716 chimeric monoclonal antibody upon which the humanized P2RY8 antibodies are based, the humanized P2RY8 antibodies showed similar antibody-binding activity to the stable HEK293T P2RY8mGFP clone 5 cells. Monitoring of GFP fluorescence showed consistent human P2RY8-mGFP expression in HEK293T P2RY8mGFP clone 5 cells used for comparing P2RY8 antibody binding activity.

[0497] Based on in vitro antigen binding data, four humanized antibodies were selected: 10-0716-h3 (also known as P2RY8mAb-716-h3, P2RY8mAb-716-h3, P2RY8hAb 0716-h3, or 716-h3), 10-0716-h4 (also known as P2RY8mAb-716-h4, P2RY8mAb-716-h4, P2RY8hAb 0716-h4, or 716-h4), 10-0716-h5 (also known as P2RY8mAb-716-h5, P2RY8mAb-716-h5, P2RY8hAb 0716-h5, or 716-h5), and 10-0716-h6. (Also known as P2RY8mAb-716-h6, P2RY8mAb-716-h6, P2RY8hAb 0716-h6, or 716-h6) for further testing and development. The antibody is derived from 10-0716-m (also known as P2RY8 mAb-716, P2RY8mAb-716, 716-m, 716-m (hIgG1), or 0716m).

[0498] Example 6: Measurement of antibody affinity using KinExA 4000

[0499] This example demonstrates the nanomolar dissociation constant (K2) of the selected humanized P2RY8 antibody against the P2RY8 protein expressed on the cell surface. D ).

[0500] The antibody equilibrium dissociation constant (Ki) of whole-cell surface proteins was measured using a KinExA 4000 (Sapidyne Instruments, Boise, ID). D(Value). In short, PMMA beads (Sapidyne Instruments, Boise, ID; catalog number: 440176) were pre-coated with goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA; catalog number: 109-005-003) overnight at 4°C to a final concentration of 30 µg / ml. The beads were then blocked by adding bovine serum albumin (BSA; Research Products International, Mount Prospect, IL; catalog number: A30075-100.0) to 10 mg / ml and incubating at room temperature for 1 hour. P2RY8-engineered overexpressing cell line HEK293T P2RY8-mGFP clone 5 cells were used with Versene... ® The solution (Thermo Fisher Scientific, Waltham, MA; catalog number: 15040066) was dissociated and equilibrated overnight at 4°C in 2% FBS-DMEM with 500 pM or 50 nM P2RY8 mAb (four humanized P2RY8 mAbs, including 716-h3, 716-h4, 716-h5, 716-h6, and 716 mouse parental mAbs). Cell concentrations were set at 1 × 10⁻⁶. 7 Starting with cells / ml, perform serial 2-fold dilutions up to 10 spots. The next day, centrifuge the cells at 1500 rpm for 10 min and save the supernatant. Use Alexa Fluor diluted 0.5 µg / ml in 1% BSA-PBS. ® 647 AffiniPure goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA; Catalog No.: 109-605-003) was used to detect free antibodies in the supernatant. Antibody-only solution (100% signal) and non-specific binding (NSB, buffer only) were also included in the measurement. The two antibody curves were analyzed using n-curve analysis with KinExA Pro version 4.4.26 software from the manufacturer, and K was calculated. D .

[0501] Table 12 shows that the humanized P2RY8 antibodies 716-h3 (P2RY8mAb-716-h3), 716-h4, h3 (P2RY8mAb-716-h4), 716-h5 (P2RY8mAb-716-h5), and 716-h6 (P2RY8mAb-716-h6) exhibit dissociation constants of approximately 4 to 6 nM for the P2RY8 protein displayed on the surface of HEK293T cells (HEK293T P2RY8mGFP clone 5 stable cell line) overexpressing human P2RY8 protein. This single nanomolar K D The KD measured against the parental 716 monoclonal antibody (P2RY8mAb-716) is nearly an order of magnitude lower, indicating that the binding affinity of the humanized anti-P2RY8 antibody is significantly improved compared to the original parental antibody on which the CDR of the humanized antibody is based. The dissociation constants of the four humanized antibodies are similar to each other.

[0502] Example 7: Growth inhibition and tumor size reduction of anti-P2RY8 humanized antibody in an in vivo xenograft model

[0503] This embodiment demonstrates that the humanized anti-P2RY8 antibody of this disclosure inhibits the growth of P2RY8-positive tumor cells and reduces tumor size in an in vivo xenograft model.

[0504] In vivo binding studies of the four selected anti-P2RY8 humanized antibodies from Example 5 were conducted in xenograft mice injected with human cancer cell lines. In short, a xenograft model of human cancer cell lines was established in six-week-old SCID mice (Charles River Laboratories). Each cell line was subcutaneously injected with SU-DHL-5 (ATCC CRL-2958) 1×10⁻⁶ cells / mL. 7 1.0 × 10⁶ cells, SCC-3 (RRID: CVCL_1683) 7 Cells were injected using 50% matrix gel (BD Biosciences). A sufficient number of mice were injected to achieve 8 mice per treatment arm. Injection was performed when the tumor reached 150 to 300 mm. 3Mice were randomly assigned to treatment groups when their mean size was reached. For treatment, each therapeutic antibody (humanized P2RY8 mAb-716-h3 to -h6 or chimeric P2RY8 mAb-716), rituximab (an anti-CD20 chimeric antibody against B-cell lymphoma), and non-targeted IgG1 control (Hu-IgG) were diluted to a working concentration of 1 mg / ml in sterile saline and administered via intravenous (IV) injection into the tail vein. Mice were treated weekly with either the anti-P2RY8 antibody or the control IgG1 antibody at a dose of 10 mg / kg, or with rituximab at a dose of 10 mg / kg every 4 days until the end of the experiment or for the specified duration of administration. Tumor xenografts were measured three times weekly using calipers, and measurements were determined in mm by multiplying height × width × length. 3 Tumor volume is represented as a unit. Mice were treated for 2–5 weeks. At the end of the study, animals were euthanized, tumor tissue was excised and divided into several fractions, and stored as frozen or formalin-fixed paraffin-embedded (FFPE) tissue for biomarker analysis. All animal experiments were conducted according to protocols approved by the IACUC and UCLA Animal Research Committee. Data were analyzed using StudyLog software from StudyDirector (San Francisco, CA). Results are presented as mean volumes for each group. Error bars represent the standard error (SE) of the mean.

[0505] The results of xenotransplantation assays are shown in Figures 10 and 11. Figure 10A and Figure 10B As shown, in mice carrying P2RY8-positive human B-cell lymphoma cells from the SU-DHL-5 cell line, each of the four selected humanized anti-P2RY8 antibodies (P2RY8 mAb-716-h3, P2RY8 mAb-716-h4, P2RY8 mAb-716-h5, and P2RY8 mAb-716-h6), the chimeric parental P2RY8 mAb-716 antibody against the human P2RY8 protein, and the anti-CD20 chimeric antibody rituximab resulted in a significant mean change in tumor volume observed on day 20, relative to the control human IgG1 antibody (Hu-IgG), and this change persisted long after the last administration at week 5. Tumors in mice treated with the control human IgG1 antibody reached approximately 3000 mm². 3 The tumor volume was such that euthanasia of the mice was required around day 18.

[0506] During the experimental period of approximately 54–62 days, compared to the control IgG1 antibody, the anti-P2RY8 antibody and rituximab inhibited tumor growth and reduced tumor volume. Figure 10ANotably, the humanized P2RY8 mAb-716-h6 and chimeric P2RY8 mAb-716 antibodies inhibited tumor growth, resulting in tumor volume remaining the same as the initial tumor volume at the end of the study period (day 54 or day 62). Most notably, the humanized P2RY8 antibody P2RY8 mAb-716-h3 reduced tumor volume to below its original size, resulting in almost undetectable tumor masses. Figure 10A This makes the humanized P2RY8mAb-716-h3 antibody the lead antibody among the four selected humanized anti-P2RY8 antibodies.

[0507] The amino acid sequences of the heavy and light chain variable regions of P2RY8mAb-716-h3 (also referred to herein as P2RY8mAb-716-h3, P2RY8hAb 0716-h3, 10-0716-h3, or 716-h3) are provided as SEQ ID NO: 177 and 185, respectively. The IMGT-defined CDR1 (SEQ ID NO: 186), CDR2 (SEQ ID NO: 187), and CDR3 (SEQ ID NO: 188) of the light chain variable region of 10-0716-h3 are identical to the IMGT-defined CDRs (SEQ ID NO: 58-60) of the light chain of the chimeric P2RY8 mAb-716 (also referred to herein as 10-0716-m, 0716m, or 716-m) antibody upon which the CDRs of the light chain variable region of 10-0716-h3 are based. The IMGT-defined CDR3 (SEQ ID NO: 180) of the 10-0716-h3 heavy chain variable region is identical to the IMGT-defined CDR3 (SEQ ID NO: 58-60) of the heavy chain of the chimeric P2RY8 mAb-716 antibody. For the IMGT-defined CDR1 (SEQ ID NO: 178) and CDR2 (SEQ ID NO: 179) of the 10-0716-h3 heavy chain variable region, these two heavy chain CDRs are variants of the IMGT-defined CDR1 (SEQ ID NO: 50) and CDR2 (SEQ ID NO: 51) of the heavy chain of the chimeric P2RY8 mAb-716 antibody. A single amino acid difference exists at the first amino acid of each CDR, where alanine is replaced by glycine to obtain heavy chain CDR1, and valine is replaced by isoleucine to obtain heavy chain CDR2 of the 10-0716-h3 humanized anti-P2RY8 antibody.

[0508] The expression vector p10-0716-h3 for expressing the humanized anti-P2RY8 antibody p2RY8 mAb-716-h3 was deposited on November 14, 2024, at the American Center for Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia 20110-2209, with accession number PTA-127827. This deposit will be maintained in accordance with the terms of the Budapest Treaty on International Recognition of Microbial Deposits for Patent Proceedings.

[0509] The inhibition of tumor cell growth and / or reduction of tumor volume by anti-P2RY8 antibody in xenograft assays depended on the presence of P2RY8 protein expression, as the antibody had no significant effect on the growth of tumor cells derived from the human P2RY8-negative B-cell non-Hodgkin lymphoma cell line (SCC-3; RRID: CVCL_1683) in SCID mice (Figure 11).

[0510] Example 8: Internalization of chimeric and humanized anti-P2RY8 IgG1 antibody

[0511] This embodiment demonstrates the internalization of the anti-P2RY8 antibody of this disclosure.

[0512] Primary antibody using Alexa Fluor ® Antibody labeling was performed using the 647 antibody labeling kit (ThermoFisher Scientific, Waltham, MA; catalog number: A20006). For antibody internalization studies, P2RY8-positive Karpas-1106P and SU-DHL-5 cells were cultured in Ibidi USA μ-SLIDE 4-well chambers (Fisher Scientific, Pittsburgh, PA; catalog number: NC0515977); HEK293T cells were cultured in Ibidi chamber slides and transfected with the P2RY8mGFP fusion protein expression vector. Hoechst 33342 DNA dye was used for nuclear staining (Abcam, Waltham, MA; catalog number: ab228551, final concentration 1µM). The time progression of cell staining with 25µg / ml antibody and imaging using a Keyence fluorescence microscope (BZ-X710) continued until internalization was complete.

[0513] Figures 12-15 This demonstrates the successful transfection of P2RY8-positive Karpas-1106P cells with the P2RY8mGFP fusion protein expression vector. Figure 12 and Figure 13 ), P2RY8 positive SU-DHL-5 cells ( Figure 14) and P2RY8-positive HEK293T cells ( Figure 15 Rapid internalization of the chimeric P2RY8mAb-716 (also referred to as 0716m in this paper) was observed. Note: The P2RY8 antibody initially stained the cell surface peripherally (red for AF647 fluorescence), and in many cases this staining was uniform, followed by loss or heterogeneity of peripheral staining and an increase in punctate staining within the cells. The nucleus is located in... Figure 12 and Figure 15 It appears blue in the middle, while GFP fluorescence is in... Figure 15 The last two time points are shown in green. The phase contrast overlay on the fluorescence image is illustrated in... Figure 12 (Two images below) Figure 13 (see image below) and Figure 14 (See figure below). Therefore, the internalization of chimeric P2RY8 mAb-716 observed in P2RY8-positive Karpas-1106P cells is consistent with the ability of P2RY8 antibody to induce rapid internalization of the P2RY8 protein / chimeric P2RY8 mAb-716 antibody complex in P2RY8-positive cancer cells.

[0514] The internalization of the lead humanized anti-P2RY8 antibody was also investigated in P2RY8-positive Karpas-1106P lymphoma cells. Figure 16 As shown, the lead humanized anti-P2RY8 antibody P2RY8 mAb-716-h3 undergoes rapid internalization after binding to the P2RY8 protein exposed on the cell surface. Binding of the P2RY8 antibody disclosed herein induces rapid internalization of the P2RY8 protein / humanized P2RY8 antibody complex in P2RY8-positive cancer cells, as observed in P2RY8-positive Karpas-1106P lymphoma cells. A superimposed image of AF647 fluorescence corresponding to the P2RY8 mAb-716-h3 antibody site on phase-contrast images of the Karpas-1106P cell field at the last four time points (last four subplots) is shown.

[0515] Example 9: Species Specificity of Lead Antibodies

[0516] This embodiment demonstrates the binding specificity of the lead humanized anti-P2RY8 antibody P2RY8 mAb-716-h3 to the P2RY8 protein in humans and cynomolgus monkeys.

[0517] Primary antibody using Alexa Fluor ® Antibody labeling was performed using the 647 antibody labeling kit (ThermoFisher Scientific, Waltham, MA; catalog number: A20006). For antibody internalization, Alexa Fluor was used. ®Immunofluorescence assays were performed using 647 goat anti-mouse IgG (minimum cross-reactivity) secondary antibody (Biolegend, San Diego, CA; catalog number: 405322). The lead humanized anti-P2RY8 antibody P2RY8 mAb-716-h3 was used for indirect flow cytometry assays to quantify the binding of the lead antibody to human, cynomolgus macaque, and canine P2RY8 proteins expressed on the surface of HEK293T cells. HEK293T cells engineered to stably express human P2RY8 fused with GFP, cynomolgus macaque P2RY8 fused with GFP, or canine P2RY8 fused with GFP were used as artificial models of P2RY8 expression (Table 39). Cells were subjected to Versene... ® (Instead of trypsin) detached from the culture flask surface to protect cell surface proteins and obtain a single-cell suspension. Cells were collected, washed, resuspended in 2% FBS-PBS, and incubated at 4°C with 4 µg / ml P2RY8 mAb-716-h3 antibody for 30 min. The cell pellet was then washed with 2% FBS-PBS to remove unbound excess antibody. The final cell pellet was resuspended in 2% FBS-PBS, and 49 µl of the cell suspension was mixed with 1 µl of 0.3 mg / ml Alexa Fluor. ® 647 mouse anti-human IgG-Fc secondary antibody (Biolegend, San Diego, CA; catalog number: 409320) stock solution was mixed. Cells were incubated with the secondary antibody at 4°C for 30 min, followed by washing with 2% FBS-PBS. After washing with 2% FBS-PBS, cells were analyzed by flow cytometry on a BD Accuri™ C6 flow cytometer. The FL4-H channel was used to collect AF647 fluorescence signals, while the FL1-H channel was used to collect GFP fluorescence signals. The negative control consisted of P2RY8-negative HEK293T cells (HEK203T parent), which were co-incubated only with secondary AF647-labeled anti-human IgG-Fc secondary antibody (anti-human 2° only) and without antibody (antibody-free).

[0518] Table 13 shows that the lead anti-P2RY8 antibody P2RY8 mAb-716-h3 bound to HEK293T cells expressing human P2RY8 (HEK293T human P2RY8 mGFP clone 5) as expected. Furthermore, the antibody also bound to HEK293T cells expressing cynomolgus macaque P2RY8 protein (610-HEK293T macP2RY8 mGFP hybrid population), but not to HEK293T cells expressing dog P2RY8 protein (618-HEK293T dogP2RY8 mGFP hybrid population) or showed very low affinity. After subtracting background binding (anti-human 2° only) and normalizing the expression level of the P2RY8-mGFP fusion protein (GFP signal in the 716-h3 column), the binding affinity of the lead antibody to human P2RY8 protein can be estimated to be approximately 330 times that to dog P2RY8 protein. Therefore, the P2RY8 mAb-716-h3 lead antibody binds with good affinity, previously characterized as being in the nanomolar range (see Table 12), but does not bind to canine P2RY8 protein or has very low affinity.

[0519] Table 2: Exemplary mouse models of chimeric P2RY8 antibody V H Mouse V L and CDR sequence

[0520]

[0521]

[0522]

[0523]

[0524]

[0525] Table 3: Exemplary humanized V antibodies for humanized P2RY8 H Humanized V L and CDR sequence

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532] Table 4: Exemplary amino acid sequences of the human immunoglobulin light chain constant regions κ (IGKC) and λ2 (IGLC2) and the human immunoglobulin heavy chain constant region γ1 (IGHG1) of the humanized P2RY8 antibody.

[0533]

[0534] Table 5: Mouse V encoding chimeric P2RY8 antibody H and mouse V L Exemplary nucleic acid sequences

[0535]

[0536]

[0537]

[0538] Table 6: Humanized V encoding humanized P2RY8 antibody H and humanized V L Exemplary nucleic acid sequences

[0539]

[0540]

[0541]

[0542]

[0543] Table 7: Exemplary nucleic acid sequences encoding the human immunoglobulin light chain constant regions κ (IGKC) and λ2 (IGLC2) and the human immunoglobulin heavy chain constant region γ1 (IGHG1) of the humanized P2RY8 antibody.

[0544]

[0545] Table 8: Exemplary amino acid sequences of mGFP markers or mycDDK epitope tags

[0546]

[0547] Table 9: Exemplary amino acid sequence of P2RY8 protein

[0548]

[0549] Table 10: Flow cytometry analysis of six humanized P2RY8 antibodies using native positive cell lines via iQUE

[0550]

[0551] Table 10 shows six humanized anti-P2RY8 antibodies: 716-h1 (also referred to herein as 10-0716-h1, P2RY8mAb-716-h1, P2RY8mAb-716-h1, or P2RY8hAb 0716-h1), 716-h2 (also referred to herein as 10-0716-h2, P2RY8 mAb-716-h2, P2RY8mAb-716-h2, or P2RY8hAb 0716-h2), 716-h3 (also referred to herein as 10-0716-h3, P2RY8 mAb-716-h3, P2RY8mAb-716-h3, or P2RY8hAb 0716-h3), and 716-h4 (also referred to herein as 10-0716-h4, P2RY8...). mAb-716-h4, P2RY8mAb-716-h4 or P2RY8hAb 0716-h4, 716-h5 (also referred to herein as 10-0716-h5, P2RY8 mAb-716-h5, P2RY8mAb-716-h5 or P2RY8hAb 0716-h5), or 716-h6 (also referred herein as 10-0716-h6, P2RY8 mAb-716-h6, P2RY8mAb-716-h6 or P2RY8hAb 0716-h6) or chimeric anti-P2RY8 antibody 716-m (also referred herein as P2RY8 mAb-716 and 716-m) (hIgG1), in which the mouse heavy and light chain constant regions are replaced by the human IgG1-κ antibody constant region, was used to demonstrate binding activity with a group of human P2RY8 receptor-positive natural human cancer cell lines: human large B-cell lymphoma cell line Karpas-1106P (RRID: CVCL_1821), human large B-cell lymphoma cell line SU-DHL-5 (ATCC CRL-2958), and human B-cell lymphoma cell line SU-DHL-10 (ATCC CRL-2963); or P2RY8 receptor-negative cell lines: human B-cell non-Hodgkin lymphoma cell line SCC-3 (RRID: CVCL_1683) and human melanoma cell line M202 (RRID: CVCL_D746). The secondary antibody was used in conjunction with Alexa Fluor. ® Mouse anti-human IgG-Fc conjugated with AF647 fluorescent dye (Biolegend, San Diego; catalog number: 409320, clone HP6017) was used to detect antibody-bound cells, and fluorescence was measured on an Intellicyt iQue® advanced flow cytometer platform. Median fluorescence values ​​for two different primary antibody concentrations are reported.

[0552] Table 11: Flow cytometry analysis of six humanized P2RY8 antibodies using overexpression (OE) positive cell lines via iQUE

[0553]

[0554] Table 11 shows the binding activity of six humanized anti-P2RY8 antibodies and chimeric mouse-human anti-P2RY8 antibodies, as described in Table 10, measured on the human embryonic kidney cell line HEK293T or HEK293T P2RY8mGFP clone 5 cell line (a stable HEK293T cell line overexpressing the human P2RY8 receptor-mGFP fusion protein (as provided in Table 39)). The upper part of the table shows AF647 fluorescence measured at two different primary antibody concentrations; the lower part shows GFP fluorescence.

[0555] Table 12: KD-HEK293T P2RY8 mGFP clone 5 cells of P2RY8 hAb as determined by KinExA 4000

[0556]

[0557] Table 12 summarizes four humanized anti-P2RY8 antibodies (10-0716-h3 (also referred to herein as P2RY8 mAb-716-h3), 10-0716-h4 (also referred herein as P2RY8 mAb-716-h4), 10-0716-h5 (also referred herein as P2RY8 mAb-716-h5), or 10-0716-h6 (also referred herein as P2RY8 mAb-716-h6)) or chimeric anti-P2RY8 antibodies (10-0716-m (also referred herein as P2RY8 mAb-716-h6)) that bind to HEK293T cells stably overexpressing human P2RY8-mGFP fusion protein (HEK293TP2RY8mGFP clone 5) or HEK293T cells transiently transfected with human P2RY8-mGFP fusion protein (HEK293TP2RY8mGFP hybrid population) that bind to human P2RY8-mGFP fusion protein. The dissociation constant K determined by mAb-716,0716m) D .

[0558] Table 13: Toxicity Model Flow Cytometry Tests: Read by BD Accuri C6

[0559]

[0560] Table 13 shows the flow cytometry assessment of the binding activity of the humanized anti-P2RY8 antibody P2RY8 mAb-716-h3 to HEK293 cells overexpressing mGFP fluorescent fusion proteins of human P2RY8 (UniProt accession number: Q86VZ1; SEQ ID NO: 369), monkey P2RY8 (NCBI RefSeq accession number: XP_005595888.1; SEQ ID NO: 370), or dog P2RY8 (NCBI RefSeq accession number: XP_013967130.1; SEQ ID NO: 371) (Table 8; mGFP protein sequence is SEQ ID NO: 367; fusion protein is also shown in Table 39). The upper part of the table shows the P2RY8 signal detected by fluorescently labeled secondary antibody against the cell-bound anti-P2RY8 antibody; the lower part of the table shows the GFP signal.

[0561] The following section describes Tables 14-39.

[0562] Table 14 shows the mouse heavy chain variable region (V) of two chimeric anti-P2RY8 antibodies, 10-0690-m and 10-0695-m. H ) and light chain variable region (V L The amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by immunogenetic methods (IMGT).

[0563] Table 15 shows the mouse heavy chain variable region (V) of two chimeric anti-P2RY8 antibodies, 10-0701-m and 10-0716-m. H ) and light chain variable region (V L The amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0564] Table 16 shows the mouse heavy chain variable region (V) of two chimeric anti-P2RY8 antibodies, 10-0720-m and 10-0724-m. H ) and light chain variable region (V L The amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0565] Table 17 shows the mouse heavy chain variable region (V) of two chimeric anti-P2RY8 antibodies, 10-0725-m and 10-0728-m. H ) and light chain variable region (V LThe amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0566] Table 18 shows the variable region (V) of the mouse heavy chain containing chimeric anti-P2RY8 antibody 10-0729-m. H ) and light chain variable region (V L The amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0567] Table 19 shows the humanized heavy chain variable region (V) of two humanized chimeric anti-P2RY8 antibodies, 10-0716-h1 and 10-0716-h2. H ) and light chain variable region (V L The amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0568] Table 20 shows the humanized heavy chain variable region (V) of two humanized chimeric anti-P2RY8 antibodies, 10-0716-h3 and 10-0716-h4. H ) and light chain variable region (V L The amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0569] Table 21 shows the humanized heavy chain variable region (V) of two humanized chimeric anti-P2RY8 antibodies, 10-0716-h5 and 10-0716-h6. H ) and light chain variable region (V L The amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0570] Table 22 shows the humanized heavy chain variable region (V) of two humanized chimeric anti-P2RY8 antibodies, 10-0716-h8 and 10-0724-h3. H ) and light chain variable region (V L The amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0571] Table 23 shows the humanized heavy chain variable region (V) of two humanized chimeric anti-P2RY8 antibodies, 10-0724-h4 and 10-0724-h5. H ) and light chain variable region (V LThe amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0572] Table 24 shows the humanized heavy chain variable region (V) of the humanized chimeric anti-P2RY8 antibody 10-0724-h6. H ) and light chain variable region (V L The amino acid sequence of the region, as well as the length, position, and amino acid sequence of the complementarity-determining region (CDR) and framework region (FR) as defined by the IMGT method.

[0573] Table 25 shows the mouse heavy chain variable region (V) of three chimeric anti-P2RY8 antibodies 10-0690-m, 10-0695-m, and 10-0701-m. H ) and mouse light chain variable region (V L The amino acid sequence of the antibody, as well as the amino acid sequences of the complementarity-determining region (CDR) and frame region (FR) defined by immunogenetic methods (IMGT), and the name of the expression vector that produces the antibody.

[0574] Table 26 shows the mouse heavy chain variable region (V) of three chimeric anti-P2RY8 antibodies 10-0716-m, 10-0720-m, and 10-0724-m. H ) and mouse light chain variable region (V L The amino acid sequence of the antibody, as well as the amino acid sequences of the complementarity-determining region (CDR) and frame region (FR) defined by the IMGT method, and the name of the expression vector that produces the antibody.

[0575] Table 27 shows the mouse heavy chain variable region (V) of three chimeric anti-P2RY8 antibodies 10-0725-m, 10-0728-m, and 10-0729-m. H ) and mouse light chain variable region (V L The amino acid sequence of the antibody, as well as the amino acid sequences of the complementarity-determining region (CDR) and frame region (FR) defined by the IMGT method, and the name of the expression vector that produces the antibody.

[0576] Table 28 shows the heavy chain variable region (V) of three humanized anti-P2RY8 antibodies, 10-0716-h1, 10-0716-h2, and 10-0716-h3. H ) and light chain variable region (V L The amino acid sequence of the antibody, as well as the amino acid sequences of the complementarity-determining region (CDR) and frame region (FR) defined by the IMGT method, and the name of the expression vector that produces the antibody.

[0577] Table 29 shows the heavy chain variable region (V) of three humanized anti-P2RY8 antibodies, 10-0716-h4, 10-0716-h5, and 10-0716-h6. H ) and light chain variable region (V L The amino acid sequence of the antibody, as well as the amino acid sequences of the complementarity-determining region (CDR) and frame region (FR) defined by the IMGT method, and the name of the expression vector that produces the antibody.

[0578] Table 30 shows the heavy chain variable region (V) of three humanized anti-P2RY8 antibodies, 10-0716-h8, 10-0724-h3, and 10-0724-h4. H ) and light chain variable region (V L The amino acid sequence of the antibody, as well as the amino acid sequences of the complementarity-determining region (CDR) and frame region (FR) defined by the IMGT method, and the name of the expression vector that produces the antibody.

[0579] Table 31 shows the heavy chain variable regions (V1) of two humanized anti-P2RY8 antibodies, 10-0724-h5 and 10-0724-h6. H ) and light chain variable region (V L The amino acid sequence of the antibody, as well as the amino acid sequences of the complementarity-determining region (CDR) and frame region (FR) defined by the IMGT method, and the name of the expression vector that produces the antibody.

[0580] Table 32 shows the amino acid sequences of the human immunoglobulin light chain κ constant region (IGKC; UniProt ID: P01834), the human immunoglobulin light chain λ constant 2 region (IGLC2; UniProt ID: P0DOY2), and the human immunoglobulin heavy chain constant region γ1 (IGHG1; UniProt ID: P01857) in some embodiments of the humanized anti-P2RY8 antibody of the present invention.

[0581] Table 33 shows the mouse heavy chain variable regions (V) encoding nine chimeric anti-P2RY8 antibodies: 10-0690-m, 10-0695-m, 10-0701-m, 10-0716-m, 10-0720-m, 10-724-m, 10-0725-m, 10-0728-m, and 10-0729-m. H ) and mouse light chain variable region (V L The nucleic acid sequence of ).

[0582] Table 34 shows the humanized heavy chain variable regions (V1, V2, V3, V4, V5, V6, V8, V3, V4, V5, V6, V8, V3, V4, V5, V6, V8, V8, V9, V10 ... H ) and humanized light chain variable region (V L The nucleic acid sequence of ).

[0583] Table 35 shows the nucleic acid sequences encoding the human immunoglobulin light chain κ constant region (IGKC), human immunoglobulin light chain λ constant region 2 (IGLC2), and human immunoglobulin heavy chain constant region γ1 (IGHG1) for generating full-length humanized anti-P2RY8 antibodies in some embodiments of the present invention.

[0584] Table 36 shows the nucleic acid sequences of the CDRs encoding the heavy and light chain variable regions of chimeric anti-P2RY8 antibodies 10-0690-m, 10-0695-m, 10-0701-m, 10-0716-m, 10-0720-m, 10-0724-m, 10-0725-m, 10-0728-m, and 10-0729-m.

[0585] Table 37 shows the heavy chain variable regions (V1, V2, V3, V4, V5, V6, and V8) of humanized anti-P2RY8 antibodies encoding 10-0716-m (10-0716-h1, 10-0716-h2, 10-0716-h3, 10-0716-h4, 10-0716-h5, 10-0716-h6, and 10-0716-h8) and humanized antibodies encoding 10-0724-m (10-0724-h3, 10-0724-h4, 10-0724-h5, and 10-0724-h6), as defined by the IMGT method. H ), light chain variable region (V L Nucleic acid sequences of the complementarity-determining region (CDR) and framework region (FR).

[0586] Table 38 shows the amino acid sequences of human P2RY8 protein (UniProt ID: Q86VZ1; SEQ ID NO: 369), cynomolgus macaque P2RY8 protein (NCBI Ref Seq: XP_005595888; SEQ ID NO: 370), and dog P2RY8 protei...

Claims

1. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 177) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 185) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

2. The antigen-binding protein of claim 1, comprising: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 178) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 179) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 180) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 186) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 187) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 188) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

3. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 145) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 153) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

4. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 161) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 169) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

5. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 193) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 201) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

6. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 209) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 217) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

7. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 225) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 233) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

8. An antigen-binding protein comprising: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 241) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 249) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

9. An antigen-binding protein comprising: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 146) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 147) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 148) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, which contains the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 154) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 155) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 156) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

10. An antigen-binding protein comprising: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 162) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 163) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 164) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, which contains the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 170) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 171) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 172) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

11. An antigen-binding protein comprising: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 194) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 195) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 196) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 202) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 203) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 204) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

12. An antigen-binding protein comprising: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 210) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 211) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 212) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 218) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 219) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 220) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

13. An antigen-binding protein comprising: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 226) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 227) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 228) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, which contains the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 234) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) The light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 235) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 236) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

14. An antigen-binding protein comprising: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 242) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 243) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 244) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, which contains the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 250) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 251) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 252) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

15. The antigen-binding protein of claim 9, further comprising: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 149) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWVSY (SEQ ID NO: 150) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYC (SEQ ID NO: 151) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 152) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 157) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 158) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 159) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 160) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

16. The antigen-binding protein of claim 10, further comprising: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 165) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWVSY (SEQ ID NO: 166) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYC (SEQ ID NO: 167) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 168) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 173) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 174) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYC (SEQ ID NO: 175) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 176) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

17. The antigen-binding protein of claim 2, further comprising: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 181) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 182) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 183) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 184) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 189) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 190) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 191) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 192) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

18. The antigen-binding protein of claim 11, further comprising: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 197) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 198) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 199) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 200) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 205) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 206) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYC (SEQ ID NO: 207) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 208) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

19. The antigen-binding protein of claim 12, further comprising: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 213) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 214) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 215) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 216) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 221) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 222) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYC (SEQ ID NO: 223) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 224) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

20. The antigen-binding protein of claim 13, further comprising: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 229) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 230) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 231) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 232) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 237) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 238) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYC (SEQ ID NO: 239) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 240) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

21. The antigen-binding protein of claim 14, further comprising: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 245) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 246) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 247) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 248) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 253) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 254) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 255) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 256) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

22. An antigen-binding protein comprising: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 145) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 153) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

23. An antigen-binding protein comprising: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 161) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 169) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

24. An antigen-binding protein comprising: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 177) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 185) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

25. An antigen-binding protein comprising: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 193) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 201) or a variant sequence differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

26. An antigen-binding protein comprising: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 209) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 217) or variant sequences differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

27. An antigen-binding protein comprising: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 225) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 233) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

28. An antigen-binding protein comprising: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 241) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 249) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

29. The antigen-binding protein as claimed in any of the preceding claims, wherein the variant sequence has at least about 80% or at least or about 85% sequence identity.

30. The antigen-binding protein as claimed in any of the preceding claims, wherein the variant sequence has at least about 90% sequence identity or at least or about 95% sequence identity.

31. The antigen-binding protein as described in any of the preceding claims, wherein: (a) The antigen-binding protein binds to human P2Y receptor family member 8 (P2RY8) protein (SEQ ID NO: 369); (b) The antigen-binding protein has a dissociation constant (K0) of approximately less than 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, or 0.25 nM. D () Binds to the extracellular domain of human P2RY8; (c) The antigen-binding protein preferentially binds to human P2RY8 compared to canine P2RY8 (SEQ ID NO: 371); (d) The antigen-binding protein does not bind to canine P2RY8; or (e) Any combination thereof.

32. The antigen-binding protein of claim 31, wherein the human P2RY8 (SEQ ID NO: 369) is expressed on the cell surface; the canine P2RY8 (SEQ ID NO: 371) is expressed on the cell surface; and the antigen-binding protein preferentially binds to the human P2RY8 compared to the canine P2RY8, with the former binding at least about 10, 20, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, or 325 times more strongly than the latter.

33. The antigen-binding protein as described in any of the preceding claims, wherein it is an antibody or an antibody fragment that binds to an antigen.

34. The antigen-binding protein of claim 33, wherein the antibody is a monoclonal antibody.

35. The antigen-binding protein of claim 33 or 34, wherein the antibody is a human antibody or a humanized antibody.

36. The antigen-binding protein according to any one of claims 33-35, wherein it is IgG.

37. The antigen-binding protein of claim 36, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

38. The antigen-binding protein of claim 36 or 37, wherein the IgG is IgG1.

39. The antigen-binding protein of claim 33, wherein the antibody fragment binding the antigen is selected from the group consisting of scFv, F(ab')2, Fab, Fab' and Fv.

40. The antigen-binding protein as described in any of the preceding claims, which inhibits tumor growth in xenograft mice injected with human cancer cells.

41. The antigen-binding protein of any of the preceding claims, wherein the antigen-binding protein comprises: (a) an Fc polypeptide, or (b) an Fc polypeptide comprising fucosylated glycans.

42. A conjugate comprising an antigen-binding protein as described in any of the preceding claims or such antigen-binding proteins or antigen-binding fragments thereof as described herein.

43. The conjugate of claim 42, wherein it comprises a cytotoxic agent or a chemotherapeutic agent.

44. The conjugate of claim 43, wherein the chemotherapeutic agent is an antimitotic agent that inhibits cell division by blocking microtubule polymerization.

45. The conjugate of claim 44, wherein the antimitotic agent is aurestatin.

46. ​​The conjugate of claim 45, wherein the auristatin is monomethyl auristatin E (MMAE).

47. The conjugate of any one of claims 43-46, wherein the agent is conjugated to the antigen-binding protein via a cleavable linker.

48. The conjugate of claim 47, wherein the cuttable connector is VC-PAB.

49. The conjugate according to any one of claims 42-48, wherein the antigen-binding protein is an antibody.

50. The conjugate of claim 49, wherein the antibody is a monoclonal antibody.

51. The conjugate of claim 49 or 50, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

52. The conjugate according to any one of claims 49-51, wherein the antibody is an IgG antibody, optionally wherein the IgG is IgG1.

53. The conjugate according to any one of claims 42-52, wherein the average number of pharmaceutical units conjugated to each antigen-binding protein is in the range of 1 to 8, preferably wherein the average number of pharmaceutical units conjugated to each antigen-binding protein is in the range of 3 to 8.

54. The conjugate according to any one of claims 42-53, wherein the conjugate is a heteroconjugate.

55. The conjugate according to any one of claims 42-53, wherein the conjugate is a homogeneous conjugate.

56. The conjugate according to any one of claims 42-55, wherein the pharmaceutical agent is conjugated at a specific site of the antigen-binding protein.

57. The conjugate of claim 56, wherein the specific site is an unpaired cysteine ​​residue.

58. The conjugate according to any one of claims 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 145 and SEQ ID NO:

153.

59. The conjugate according to any one of claims 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 161 and SEQ ID NO:

169.

60. The conjugate of any one of claims 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 177 and SEQ ID NO:

185.

61. The conjugate according to any one of claims 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 193 and SEQ ID NO:

201.

62. The conjugate according to any one of claims 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 209 and SEQ ID NO:

217.

63. The conjugate according to any one of claims 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 225 and SEQ ID NO:

233.

64. The conjugate according to any one of claims 42-57, wherein the conjugate comprises a polypeptide conjugated with VC-PAB-MMAE, the polypeptide comprising the amino acid sequences shown in SEQ ID NO: 241 and SEQ ID NO:

249.

65. A bispecific antigen-binding protein comprising: an antigen-binding protein as described in any one of claims 1-41, an antigen-binding protein of Table 3 or a fragment thereof, wherein the antigen-binding protein or the fragment thereof binds to human P2Y receptor family member 8 (P2RY8) protein (SEQ ID NO: 369); and a second antigen-binding protein that binds to a second antigen.

66. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 177) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 185) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

67. The bispecific antigen-binding protein of claim 66, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 178) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 179) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 180) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 186) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 187) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 188) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

68. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 145) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 153) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

69. The bispecific antigen-binding protein of claim 68, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 146) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 147) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 148) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, which contains the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 154) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 155) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 156) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

70. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 161) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 169) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

71. The bispecific antigen-binding protein of claim 70, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 162) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 163) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 164) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, which contains the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 170) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 171) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 172) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

72. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 193) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 201) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

73. The bispecific antigen-binding protein of claim 72, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 194) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 195) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 196) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 202) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 203) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 204) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

74. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 209) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 217) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

75. The bispecific antigen-binding protein of claim 74, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 210) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 211) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 212) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, comprising the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 218) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 219) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 220) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

76. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 225) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 233) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

77. The bispecific antigen-binding protein of claim 76, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 226) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 227) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 228) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, which contains the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 234) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) The light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 235) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 236) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

78. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) CDR1-3 derived from the heavy chain variable region, wherein the heavy chain variable region contains an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 241) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (b) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 249) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

79. The bispecific antigen-binding protein of claim 78, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain CDR1, comprising the amino acid sequence: GYSITSDYA (SEQ ID NO: 242) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) Heavy chain CDR2, comprising the amino acid sequence: ITYSGTT (SEQ ID NO: 243) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) Heavy chain CDR3, comprising the amino acid sequence: ARGYWYFDV (SEQ ID NO: 244) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (d) Light chain CDR1, which contains the amino acid sequence: ESVDSYGYTF (SEQ ID NO: 250) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (e) Light chain CDR2, comprising the amino acid sequence: RAS (SEQ ID NO: 251) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (f) The light chain CDR3, comprising the amino acid sequence: QQSNEDPFT (SEQ ID NO: 252) or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or Any combination of two or more of (g) (a)-(f).

80. The bispecific antigen-binding protein of claim 66, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 181) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 182) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 183) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 184) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 189) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 190) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 191) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 192) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

81. The bispecific antigen-binding protein of claim 68, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 149) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWVSY (SEQ ID NO: 150) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYC (SEQ ID NO: 151) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 152) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 157) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 158) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 159) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 160) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

82. The bispecific antigen-binding protein of claim 70, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 165) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWVSY (SEQ ID NO: 166) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYC (SEQ ID NO: 167) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 168) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 173) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 174) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYC (SEQ ID NO: 175) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 176) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

83. The bispecific antigen-binding protein of claim 72, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 197) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 198) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 199) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 200) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 205) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 206) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYC (SEQ ID NO: 207) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 208) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

84. The bispecific antigen-binding protein of claim 74, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 213) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 214) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 215) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 216) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 221) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 222) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYC (SEQ ID NO: 223) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 224) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

85. The bispecific antigen-binding protein of claim 76, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 229) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 230) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 231) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 232) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQLTQSPSSLSASVGDRATITCRAS (SEQ ID NO: 237) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 238) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYC (SEQ ID NO: 239) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 240) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

86. The bispecific antigen-binding protein of claim 78, wherein the antigen-binding protein binding the P2RY8 protein further comprises: (a) Heavy chain FR1, which contains the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVS (SEQ ID NO: 245) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (b) Heavy chain FR2, comprising the amino acid sequence: WNWIRQAPGKGLEWMGY (SEQ ID NO: 246) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (c) Heavy chain FR3, which contains the amino acid sequence: TYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYC (SEQ ID NO: 247) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (d) Heavy chain FR4, comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 248) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (e) Light chain FR1, comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 253) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (f) Light chain FR2, which contains the amino acid sequence: MHWYQQKPGKAPKLLIY (SEQ ID NO: 254) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (g) Light chain FR3, which contains the amino acid sequence: NVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 255) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; (h) Light chain FR4, comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 256) or a variant sequence differing by only one or two amino acids or having at least or about 85%, 90%, or 95% sequence identity; or Any combination of two or more of (i) (a)-(h).

87. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 177) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 185) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

88. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 145) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 153) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

89. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWVSYITYSGTTTYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 161) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 169) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

90. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 193) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 201) or a variant sequence differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

91. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 209) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 217) or variant sequences differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

92. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 225) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 233) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

93. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein comprises: (a) Heavy chain variable region, which has an amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYADSLKGRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGYWYFDVWGQGTTVTVSS (SEQ ID NO: 241) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (b) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDSYGYTFMHWYQQKPGKAPKLLIYRASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPFTFGQGTKLEIK (SEQ ID NO: 249) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

94. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein is selected from the group consisting of: (a) An antigen-binding protein, comprising: (i) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 257) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 265) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; (b) An antigen-binding protein comprising: (i) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 273) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 281) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; (c) Antigen-binding proteins, comprising: (i) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 289) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 297) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and (d) Antigen-binding proteins, which include: (i) CDR1-3 derived from the heavy chain variable region, the heavy chain variable region comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 305) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) CDR1-3 derived from the light chain variable region, wherein the light chain variable region contains an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 313) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

95. The bispecific antigen-binding protein of claim 65, wherein the antigen-binding protein binding the P2RY8 protein is selected from the group consisting of: (a) An antigen-binding protein, comprising: (i) Heavy chain variable region having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 257) or a variant sequence differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or (ii) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 265) or variant sequences differing from it by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; (b) An antigen-binding protein comprising: (i) Heavy chain variable region having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 273) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) Light chain variable region, which has an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 281) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; (c) Antigen-binding proteins, comprising: (i) Heavy chain variable region having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 289) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) Light chain variable region, which has an amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 297) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and (d) Antigen-binding proteins, which include: (i) Heavy chain variable region having the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNWIRQAPGKGLEWMGYITYSGTTTYNPSLKSRITISRDTSKNTFYLQLNSVRAEDTAVYYCARGFWYFDVWGQGTTVTVSS (SEQ ID NO: 305) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity; and / or (ii) Light chain variable region, which has an amino acid sequence: DIQLTQSPSSLSASVGDRATITCRASESVDTYGYNFLHWYQQKPGKAPKLLIYRASNVQSGIPSRFSGSGSRTDFTLTISSVQPEDFATYYCQQSNEDLLTFGQGTKLEIK (SEQ ID NO: 313) or a variant sequence differing by only one or two amino acids or having at least or about 85% sequence identity.

96. The bispecific antigen-binding protein of any one of claims 65-95, wherein the second antigen is a cell surface protein expressed by a T cell, optionally a component of the T cell receptor (TCR), such as CD3.

97. The bispecific antigen-binding protein of any one of claims 65-95, wherein the second antigen is a co-stimulatory molecule that assists in T cell activation, such as CD40 or 4-1BB (CD137).

98. The bispecific antigen-binding protein of any one of claims 65-95, wherein the second antigen is an Fc receptor, optionally an Fc γ receptor, an Fc-α receptor, or an Fc-ε receptor.

99. The bispecific antigen-binding protein of claim 98, wherein the Fc receptor is CD64 (Fc-γRI), CD32 (Fc-γRIIA), CD16A (Fc-γRIIIIA), CD16b (Fc-γRIIIIb), FcεRI, CD23 (Fc-εRII), CD89 (Fc-εRI), Fcα / µR, or FcRn.

100. The bispecific antigen-binding protein of claim 98 or 99, wherein the Fc receptor is CD16A.

101. The bispecific antigen-binding protein of any one of claims 65-95, wherein the second antigen is an immune checkpoint molecule, such as a protein involved in the immune checkpoint pathway, optionally A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7.

102. The bispecific antigen-binding protein of claim 101, wherein the immune checkpoint molecule is PD-1, LAG3, TIM3, or CTLA4.

103. The bispecific antigen-binding protein according to any one of claims 65-95, wherein the second antigen is CD3.

104. The bispecific antigen-binding protein according to any one of claims 65-95, wherein the second antigen is CD3E.

105. The bispecific antigen-binding protein according to any one of claims 65-95, wherein the second antigen is CD16A.

106. The bispecific antigen-binding protein according to any one of claims 65-105, comprising scFv, Fab, or F(ab')2 of any of the currently disclosed P2RY8 antibodies.

107. The bispecific antigen-binding protein according to any one of claims 65-106, comprising the structure of a nanobody, BiTE®, DART, TandAb, CrossMab, or HSAbody.

108. A fusion protein comprising an antigen-binding protein as described in any of the preceding claims or the antigen-binding proteins or antigen-binding fragments thereof described herein.

109. A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein as described in any one of claims 1-41, a bispecific antigen-binding protein as described in any one of claims 65-107, or a fusion protein as described in claim 108.

110. The nucleic acid of claim 109, wherein the nucleic acid is complementary DNA (cDNA).

111. A vector comprising the nucleic acid as described in claim 109 or 110.

112. The vector of claim 111, wherein the vector further comprises an internal ribosome entry site (IRES).

113. A host cell comprising the nucleic acid as claimed in claim 109, the cDNA as claimed in claim 110, or the vector as claimed in claim 111 or 112.

114. The host cell of claim 113, wherein the host cell is a bacterial cell.

115. The host cell of claim 113, wherein the host cell is a eukaryotic cell.

116. The host cell of claim 113 or 115, wherein the host cell is a mammalian cell.

117. The host cell of claim 116, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

118. A method for producing an antigen-binding protein that binds to P2Y receptor family member 8 (P2RY8) protein, comprising: (i) culturing the host cell as described in any one of claims 113-117 in a cell culture medium, and (ii) harvesting the antigen-binding protein from the cell culture medium.

119. A method for producing a bispecific antigen-binding protein, said bispecific antigen-binding protein comprising an antigen-binding protein that binds to P2Y receptor family member 8 (P2RY8) protein, said method comprising: (i) culturing the host cell as described in any one of claims 113-117 in a cell culture medium, and (ii) harvesting the bispecific antigen-binding protein from the cell culture medium.

120. A method for producing a fusion protein, said fusion protein comprising an antigen-binding protein that binds to P2Y receptor family member 8 (P2RY8) protein, said method comprising: (i) culturing the host cell as described in any one of claims 113-117 in a cell culture medium, and (ii) harvesting the fusion protein from the cell culture medium.

121. A method for preparing a pharmaceutical composition comprising combining an antigen-binding protein as described in any one of claims 1-41, a conjugate as described in any one of claims 42-64, a bispecific antigen-binding protein as described in any one of claims 65-107, a fusion protein as described in claim 108, a nucleic acid as described in claim 109 or 110, a carrier as described in claim 111 or 112, a host cell as described in any one of claims 113-117, or a combination thereof, with a pharmaceutically acceptable carrier, diluent, or excipient.

122. A pharmaceutical composition comprising an antigen-binding protein as described in any one of claims 1-41, a conjugate as described in any one of claims 42-64, a bispecific antigen-binding protein as described in any one of claims 65-107, a fusion protein as described in claim 108, a nucleic acid as described in claim 109 or 110, a carrier as described in claim 111 or 112, a host cell as described in any one of claims 113-117, or a combination thereof; and a pharmaceutically acceptable carrier, diluent, or excipient.

123. A method of treating a subject with cancer expressing P2RY8, comprising administering to the subject an amount of the pharmaceutical composition of claim 122 that is effective in treating the cancer.

124. A method of inhibiting tumor growth in a subject, comprising administering to the subject an amount of the pharmaceutical composition of claim 122 that effectively inhibits tumor growth.

125. A method for reducing the size of a tumor in a subject, comprising administering to the subject an amount of the pharmaceutical composition of claim 122 that effectively reduces the size of the tumor.

126. A method for preventing cancer recurrence in a subject, comprising administering to the subject an amount of the pharmaceutical composition of claim 122 that is effective in preventing cancer recurrence.

127. The method of any one of claims 123-126, wherein the pharmaceutical composition induces apoptosis in tumor cells.

128. The method of any one of claims 123-126, wherein the pharmaceutical composition induces apoptosis in cells expressing P2RY8.

129. A method for detecting a P2Y receptor family member 8 (P2RY8) protein or a fragment thereof in a sample, comprising contacting the sample with an antigen-binding protein as claimed in any one of claims 1-41, a conjugate as claimed in any one of claims 42-64, a bispecific antigen-binding protein as claimed in any one of claims 65-107, or a fusion protein as claimed in claim 108, and determining an immune complex comprising the antigen-binding protein, conjugate, or fusion protein bound to the P2RY8 protein or a fragment thereof.

130. A method for diagnosing P2Y receptor family member 8 (P2RY8) protein-positive cancer in a subject, comprising contacting a sample from the subject (e.g., a biological sample containing cells or tissue) with an antigen-binding protein as claimed in any one of claims 1-41, a conjugate as claimed in any one of claims 42-64, a bispecific antigen-binding protein as claimed in any one of claims 65-107, or a fusion protein as claimed in claim 108, and determining an immune complex comprising the antigen-binding protein, conjugate, or fusion protein binding to the P2RY8 protein or a fragment thereof.

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