Anti-CD200r1 antibodies

Abstract

Disclosed herein are antibodies and antigen-binding fragments thereof that bind to CD200R1, as well as their uses, nucleic acids encoding these antibodies and antigen-binding fragments, vectors comprising these nucleic acids, and host cells comprising these nucleic acids or these vectors. Also disclosed are pharmaceutical compositions and conjugates comprising these antibodies, as well as methods of treatment using these antibodies.

Description

[0001] This application claims priority to PCT patent application PCT / CN2022 / 093965 filed on May 19, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to antibodies and antigen-binding fragments thereof that bind to CD200R1. Background Art

[0003] In normal tissues, CD200 is expressed on B cells, activated T cells, epithelial cells, neurons, and many other normal cell types. In addition, CD200 has been shown to be highly expressed in different cancers, including non-small cell lung cancer, renal cell carcinoma, ovarian cancer, leukemia, and many other cancers (Cancer Res 2020; 80 (Supplement 16): Abstract No. 937; Cancer Immunol Immunother. 2008; 57 (7): 987-996; Proc Natl Acad Sci US A. 2006; 103 (4): 1041-1046). Samalizumab, an anti-human antagonist CD200 antibody, has been used in phase I clinical trials to treat chronic lymphocytic leukemia and multiple myeloma (J Immunother Cancer. 2019 August 23; 7 (1): 227). Anti-CD200 antibodies also showed anti-tumor efficacy in a pancreatic ductal adenocarcinoma model (J Immunother Cancer. 2020 Jun;8(1):e000189).

[0004] CD200R1 is an Ig superfamily transmembrane glycoprotein expressed on the surface of myeloid cells and T cell subsets (Immunity. August 2000; 13 (2): 233-42; J Immunol 2003; 171: 3034-3046). Human CD200R1 interacts with CD200 and viral CD200 homologs. CD200-CD200R1 mediates inhibitory signaling pathways and plays a key role in inhibiting T cell, myeloid cell and NK cell function (Int. J. Mol. Sci. 2021, 22 (4), 1602). CD200R1 blockade has therapeutic potential for cancer. For example, in mouse studies, blocking the interaction of CD200 with CD200R1 through adenoviral delivery of soluble CD200R-Ig inhibited tumor growth (Mol Ther Oncolytics. 2021 Sep 14; 23: 138-150). When combined with thermal ablation therapy, anti-CD200R1 antibodies greatly inhibited tumor growth (Med Sci Monit. 2019 Mar 6; 25: 1718-1728).

[0005] Compared to anti-CD200 blocking antibodies, CD200R1 is highly expressed on T cells and myeloid cells in the tumor microenvironment. In addition, anti-CD200R1 antagonistic antibodies will block CD200-mediated as well as viral CD200-mediated inhibitory signaling pathways. Anti-CD200R1 antagonistic antibodies will target T cells and myeloid cells that overexpress CD200R1 and restore the function of these cells in tumors. Summary of the invention

[0006] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CD200R1.

[0007] The present invention also provides a bispecific antibody, which comprises the antibody of the present invention or an antigen-binding fragment thereof and a second antigen-binding region that specifically binds to a tumor-associated antigen or an immune checkpoint molecule.

[0008] The present invention also provides a nucleic acid encoding the antibody or bispecific antibody of the present invention.

[0009] The present invention also provides a vector comprising the nucleic acid of the present invention.

[0010] The present invention also provides a host cell comprising the vector or nucleic acid of the present invention.

[0011] The present invention also provides an antibody-drug conjugate (ADC), which comprises the antibody or antigen-binding fragment thereof of the present invention or the bispecific antibody of the present invention.

[0012] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention or the bispecific antibody of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0013] The present invention also provides an antibody of the present invention for use in therapy.

[0014] The present invention also provides an antibody of the present invention for use in treating cancer.

[0015] The present invention also provides an antibody of the present invention for activating T cells and / or myeloid cells in a cancer microenvironment.

[0016] The present invention also provides a method for determining that a subject has cancer or has a risk of developing cancer, the method comprising:

[0017] (a) obtaining a biological sample from the subject,

[0018] (b) contacting the sample with the antibody or antigen-binding fragment thereof of the present invention; and

[0019] (c) detecting binding of the antibody to the sample,

[0020] Wherein increased binding of the antibody to the sample compared to binding of the antibody to a control sample indicates that the subject has or is at risk for developing cancer. In some embodiments, the cancer is a cancer that is responsive to reduction, inhibition and / or blocking of CD200R1-mediated immunomodulatory function or activity.

[0021] The present invention also provides a method for imaging cancer in a subject, wherein the microenvironment of the cancer comprises T cells and / or myeloid cells that highly express CD200R1 on their surface, wherein the method comprises:

[0022] (a) administering to the subject an antibody or antigen-binding fragment thereof of the present invention, wherein the antibody is conjugated to a detectable marker; and

[0023] (b) detecting the presence of the marker.

[0024] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CD200R1, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), and wherein

[0025] (1) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 117, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 136; or

[0026] (2) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 111, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 124; or

[0027] (3) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 117, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 133; or

[0028] (4) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 118, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 134; or

[0029] (5) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 119, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 135; or

[0030] (6) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 120, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 137; or

[0031] (7) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 108, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 123; or

[0032] (8) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 109, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 124; or

[0033] (9) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 110, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 125; or

[0034] (10) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 114, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 128; or

[0035] (11) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 115, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 129; or

[0036] (12) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 116, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 130; or

[0037] (13) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 115, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 131; or

[0038] (14) The VH comprises HCDR 1-3 of the VH having the amino acid sequence shown in SEQ ID NO: 116, and the VL comprises LCDR 1-3 of the VL having the amino acid sequence shown in SEQ ID NO: 132.

[0039] In a second aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CD200R1, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein

[0040] (1) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 67, 80, 99, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 67, 80, 99, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 32, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 32, 46, respectively, by one or two amino acid residues; or

[0041] (2) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 26, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 26, 46, respectively, by one or two amino acid residues; or

[0042] (3) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 66, 80, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 66, 80, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 32, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 32, 46, respectively, by one or two amino acid residues; or

[0043] (4) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 66, 81, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 66, 81, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 33, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 33, 46, respectively, by one or two amino acid residues; or

[0044] (5) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 67, 76, 99, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 67, 76, 99, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 14, 32, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 14, 32, 46, respectively, by one or two amino acid residues; or

[0045] (6) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 66, 80, 100, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 66, 80, 100, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 14, 33, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 14, 33, 46, respectively; or

[0046] (7) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 91, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 91, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 10, 25, 44, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 10, 25, 44, respectively, by one or two amino acid residues; or

[0047] (8) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 26, 45, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 26, 45, respectively, by one or two amino acid residues; or

[0048] (9) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 93, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 93, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 25, 45, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 25, 45, respectively, by one or two amino acid residues; or

[0049] (10) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 96, respectively, or LCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 65, 79, 96, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 29, 49, respectively, or HCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 11, 29, 49, respectively, by one or two amino acid residues; or

[0050] (11) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 97, respectively, or LCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 65, 79, 97, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 30, 49, respectively, or HCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 11, 30, 49, respectively; or

[0051] (12) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 98, respectively, or LCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 65, 79, 98, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 31, 50, respectively, or HCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 11, 31, 50, respectively; or

[0052] (13) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 97, respectively, or LCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 65, 79, 97, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 30, 49, respectively, or HCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 11, 30, 49, respectively; or

[0053] (14) The VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, and 98, respectively, or LCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 65, 79, and 98 by one or two amino acid residues, respectively, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 31, and 50, respectively, or HCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 11, 31, and 50, respectively.

[0054] In some embodiments of the first aspect or the second aspect of the present invention, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD200R1, wherein

[0055] (1) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 136, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 117; or

[0056] (2) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 124, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 111; or

[0057] (3) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 133, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 117; or

[0058] (4) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 134, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 118; or

[0059] (5) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 135, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 119;

[0060] or

[0061] (6) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No 137, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No 120; or

[0062] (7) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 123, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 108; or

[0063] (8) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 124, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 109; or

[0064] (9) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 125, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 110; or

[0065] (10) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 128, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 114; or

[0066] (11) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 129, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 115; or

[0067] (12) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 130, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 116; or

[0068] (13) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 131, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 115;

[0069] or

[0070] (14) The VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 132, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 116.

[0071] In some embodiments of the first aspect or the second aspect of the present invention, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CD200R1, wherein the antibody or antigen-binding fragment comprises a heavy chain (HC) and a light chain (LC), and wherein

[0072] (1) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 170, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 151; or

[0073] (2) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 158, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 143; or

[0074] (3) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 158, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 147; or

[0075] (4) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 167, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 151; or

[0076] (5) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 168, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 152; or

[0077] (6) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 169, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 153; or

[0078] (7) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 171, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 154; or

[0079] (8) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 157, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 140; or

[0080] (9) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 158, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 141; or

[0081] (10) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 159, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 142; or

[0082] (11) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 162, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 146; or

[0083] (12) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 162, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 148; or

[0084] (13) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 163, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 149; or

[0085] (14) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 164, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 150; or

[0086] (15) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 165, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 149; or

[0087] (16) The LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID No: 166, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID No: 150.

[0088] In some preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention have binding activity to human CD200R1 and cynomolgus monkey CD200R1. In some preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention block the interaction between CD200R1 and CD200. In some preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention inhibit CD200 / CD200R1 signal transduction. In some preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention do not activate CD200 / CD200R1 signal transduction.

[0089] In some preferred embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0090] In some preferred embodiments, the antibody is an isotype selected from IgG, IgA, IgM, IgE and IgD. In some more preferred embodiments, the antibody is a subtype selected from IgG1, IgG2, IgG3 and IgG4.

[0091] In some preferred embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, ds-scFv and dAb.

[0092] In some preferred embodiments, the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody, which further comprises a second antigen binding region that specifically binds to a tumor-associated antigen or an immune checkpoint molecule.

[0093] In some preferred embodiments, the antibody or antigen-binding fragment is linked to a fluorescent label, a radioactive label, or a cytotoxic agent.

[0094] In a third aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the first aspect or the second aspect of the present invention.

[0095] In a fourth aspect, the present invention provides a vector comprising the nucleic acid of the third aspect of the present invention.

[0096] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid of the third aspect or the vector of the fourth aspect of the present invention.

[0097] In a sixth aspect, the present invention provides an antibody drug conjugate (ADC), which comprises the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention.

[0098] In a seventh aspect, the present invention provides a pharmaceutical composition comprising (i) the antibody or antigen-binding fragment thereof of the first or second aspect of the present invention, or the nucleic acid of the third aspect of the present invention, or the vector of the fourth aspect of the present invention, or the host cell of the fifth aspect of the present invention, or the ADC of the sixth aspect of the present invention; and optionally (ii) a pharmaceutically acceptable carrier or excipient.

[0099] In some preferred embodiments of the seventh aspect, the composition further comprises a second therapeutic agent selected from an antibody, a chemotherapeutic agent, a siRNA, an antisense oligonucleotide, a polypeptide and a small molecule drug.

[0100] In an eighth aspect, the present invention provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, ADC or pharmaceutical composition of the present invention. In some embodiments, the microenvironment of the cancer comprises T cells and / or myeloid cells that highly express CD200R1 on the surface. In some embodiments, the cancer is a cancer that responds to a reduction, inhibition and / or blocking of CD200R1-mediated immunomodulatory function or activity.

[0101] In a ninth aspect, the present invention provides an effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, ADC or pharmaceutical composition of the present invention for use in a method for treating cancer in a subject. In some embodiments, the microenvironment of the cancer includes T cells and / or myeloid cells that highly express CD200R1 on their surfaces. In some embodiments, the cancer is a cancer that responds to reduction, inhibition and / or blocking of CD200R1-mediated immunomodulatory function or activity.

[0102] In a tenth aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, an ADC or a pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer in a subject. In some embodiments, the microenvironment of the cancer includes T cells and / or myeloid cells that highly express CD200R1 on their surfaces. In some embodiments, the cancer is a cancer that responds to a reduction, inhibition and / or blocking of CD200R1-mediated immunomodulatory function or activity.

[0103] In some preferred embodiments of the eighth, ninth or tenth aspects, the cancer is selected from renal cancer, pancreatic duct adenocarcinoma, prostate cancer, melanoma, liver cancer, breast cancer, lung cancer (e.g., squamous cell carcinoma of the lung), mesothelioma, squamous cell carcinoma, ovarian cancer, papillary thyroid cancer, uterine cancer, brain cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, head and neck cancer, liposarcoma, leukemia and myeloma.

[0104] In some preferred embodiments of the eighth, ninth or tenth aspects, the method further comprises administering to the subject a second therapeutic agent. In some more preferred embodiments of the eighth, ninth or tenth aspects, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

[0105] In an eleventh aspect, the present invention provides a method for determining that a subject has cancer or is at risk of having cancer, wherein the microenvironment of the cancer comprises T cells and / or myeloid cells that highly express CD200R1 on their surface, wherein the method comprises:

[0106] (a) obtaining a biological sample from the subject,

[0107] (b) contacting the sample with the antibody or antigen-binding fragment thereof of the first aspect of the present invention; and

[0108] (c) detecting binding of the antibody to the sample,

[0109] Wherein increased binding of the antibody or antigen-binding fragment thereof to the samples, compared to binding of the antibody or antigen-binding fragment thereof to control samples, indicates that the subject has cancer or is at risk of developing cancer.

[0110] In some embodiments, the cancer is one that is responsive to reduction, inhibition and / or blocking of CD200R1 -mediated immunomodulatory function or activity.

[0111] In a twelfth aspect, the present invention provides a method for imaging cancer in a subject, wherein the microenvironment of the cancer comprises T cells and / or myeloid cells that highly express CD200R1 on their surface, wherein the method comprises:

[0112] (a) administering to the subject the antibody or antigen-binding fragment thereof of the first aspect of the invention, wherein the antibody is conjugated to a detectable marker; and

[0113] (b) detecting the presence of the marker.

[0114] In some embodiments, the cancer is one that is responsive to reduction, inhibition and / or blocking of CD200R1 -mediated immunomodulatory function or activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 Shown is the expression of human CD200R1 on human PBMCs from healthy donors and tumor infiltrating lymphocytes from renal cell carcinoma patients.

[0116] Figure 2 Shown is the expression profile of mouse CD200R1 after PD1 treatment of the mouse HKP tumor model.

[0117] Figure 3 Binding of anti-CD200R1 antibodies to CHOK1 cells overexpressing human CD200R1 isoform a (CHOK1 / hCD200R1a) is shown.

[0118] Figure 4 Binding of anti-CD200R1 antibodies to CHOK1 cells overexpressing cynomolgus CD200R1a (CHOK1 / cynoCD200R1a) is shown.

[0119] Figure 5 The efficacy of anti-CD200R1 antibodies in blocking the binding of CD200 protein to CHOK1 / hCD200R1a is shown.

[0120] Figure 6 The efficacy of anti-CD200R1 antibodies in blocking CD200-induced PathHunter CD200R1a signaling is shown.

[0121] Figure 7 Binding of variants of anti-CD200R1 antibodies to CHOK1 / hCD200R1a is shown.

[0122] Figure 8 Binding of variants of anti-CD200R1 antibodies to CHOK1 / cynoCD200R1a is shown.

[0123] Fig. 9 The efficacy of variants of anti-CD200R1 antibodies in blocking the binding of CD200 protein to CHOK1 / hCD200R1a is shown.

[0124] Fig.10The efficacy of variants of anti-CD200R1 antibodies in blocking CD200-induced PathHunter CD200R1a signaling is shown.

[0125] Fig.11 The ability of variants of anti-CD200R1 antibodies to induce PathHunter CD200R1a signaling is shown.

[0126] Fig.12 Binding of anti-CD200R1 antibodies to recombinant human CD200R1L protein is shown.

[0127] Fig.13 Binding of anti-CD200R1 antibodies to CHOK1 expressing different CD200R1 isoforms (CHOK1 / hCD200R1a and CHOK1 / hCD200R1d) is shown.

[0128] Fig.14 The efficacy of anti-CD200R1 antibodies in blocking the binding of CD200 protein to CHOK1 / hCD200R1a and CHOK1 / hCD200R1d is shown.

[0129] Fig.15 Binding of human anti-CD200R1 antibodies on human PBMCs from healthy donors and tumor infiltrating lymphocytes from renal cell carcinoma patients is shown.

[0130] Fig.16 Anti-CD200R1 antibody is shown in the antigen recall assay ( Fig.16 A) Induction of IL12 production and in MLR assay ( Fig.16 B) Enhances the function of anti-PD1-induced IFNγ production.

[0131] Fig.17 The anti-tumor effect of anti-CD200R1 antibody in the NCI-H226 CDX model of humanized NSG mice is shown.

[0132] Fig.18 The anti-tumor effect of anti-CD200R1 antibody in the OVCAR3 CDX model of humanized NSG mice is shown.

[0133] Fig.19 The anti-tumor effect of anti-CD200R1 antibody in the NCI-H292 CDX model of humanized NSG mice is shown.

[0134] Fig. 20 The anti-tumor effect of anti-CD200R1 antibody in the MDA-MB-231CDX model of humanized NSG mice is shown.

[0135] Sequence Listing

[0136] The sequences of the light chain, heavy chain, light chain variable region, heavy chain variable region, light chain and heavy chain CDRs are shown in Tables 1 to 3. The CDR sequences are defined according to the Chothia numbering system.

[0137] Table 1. Sequences of the heavy and light chains of the antibodies of the present invention

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] Table 2. Sequences of the heavy chain variable region and light chain variable region of the antibodies of the present invention

[0150]

[0151]

[0152]

[0153]

[0154] Table 3. Sequences of HCDR1-3 and LCDR1-3 of the antibodies of the present invention (Chothia system)

[0155]

[0156]

[0157] DETAILED DESCRIPTION

[0158] The above features and advantages of the present invention and additional features and advantages thereof will be more clearly understood from the following detailed description of the embodiments in conjunction with the accompanying drawings.

[0159] The embodiments described herein with reference to the accompanying drawings are illustrative, exemplary, and are used to generally understand the present invention. These embodiments should not be construed as limiting the scope of the present invention. Throughout the specification, identical or similar elements and elements with identical or similar functions are represented by the same reference numerals.

[0160] Unless otherwise stated or defined, all terms used have their ordinary meanings in the art, which are clear to those skilled in the art. For example, reference is made to standard manuals such as Leuenberger, HGW, Nagel, B. and KlbI, H., eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Vol. 1 to Vol. 3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., "Current protocols inmolecular biology", Green Publishing and Wiley InterScience, New York (1987); Roitt et al., "Immunology" (6th edition), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology" (6th edition), Garland Science Publishing / Churchill Livingstone, New York (2005), and the general background art cited above.

[0161] As used herein, the singular forms "a", "an", "and", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and in some embodiments reference to "an antibody" includes a plurality of antibodies, and so forth.

[0162] Unless otherwise stated or defined, the term "comprise" and variations thereof such as "include" and "comprising" should be understood to imply the inclusion of the stated elements or steps or groups of elements or steps, but not the exclusion of any other elements or steps or groups of elements or steps. The term "comprising" encompasses "including" as well as "consisting of", for example, a composition "comprising" X may consist of X alone, or may include some additional substances, such as X+Y.

[0163] The term "about" in relation to a value x is optional and means, for example, x±10% or x±5%.

[0164] As used herein, the term "antibody" refers to an immunoglobulin molecule with the ability to specifically bind to a specific antigen. Antibodies typically include a variable region and a constant region in each of a heavy chain and a light chain. The variable regions of the heavy and light chains of an antibody include a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to a host tissue or factor, which includes various cells of the immune system (such as effector cells) and components of the complement system, such as the first component C1q in the classical pathway of complement activation. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antigen-binding portion of an antibody.

[0165] A "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of a "framework" region interrupted by three "complementarity determining regions" or "CDRs". The framework region is used to align the CDRs to specifically bind to the epitope of the antigen. The CDRs contain the amino acid residues of the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL domain and the VH domain contain the following framework region (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively, and the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0166] The amino acid assignments for each VL and VH domain are consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a combination of the Chothia Kabat CDRs, where CDR-H1 is a combination of the Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the contact definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. Although the present disclosure can use CDRs defined according to any of these numbering systems, a preferred embodiment uses the Chothia defined CDRs.

[0167] As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two different antigen-binding regions. The antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0168] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, C200R1). Studies have shown that the antigen-binding function of an antibody can be achieved by a fragment of a full-length antibody.

[0169] Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, i.e., a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a portion of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul, ed., Supplementary Version 3, 1993); (iv) a Fd fragment consisting of a VH domain and a CH1 domain; (v) a Fd' fragment having a VH domain and a CH1 domain and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL domain and the VH domain of a single arm of an antibody; (vii) a dAb fragment (Ward et al., (1989) Nature 557(5):115-123. 341:544-546), which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); and (ix) a nanobody, i.e., a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, recombinant methods can be used to connect these domains by synthetic linkers, allowing these domains to be made into a single protein chain in which the VL region and the VH region pair to form a monovalent molecule (called single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) and a modified form of any of the aforementioned fragments, which together with the complementary light chain polypeptide form an antigen-binding region, wherein the modified forms of these fragments retain antigen-binding activity.

[0170] These antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments screened for utility in the same manner as intact antibodies.

[0171] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as a non-random binding reaction between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity, represented by the equilibrium constant (KD) of antigen-antibody dissociation, is a measure of the strength of binding between an antigenic determinant (epitope) and an antigen binding site on an antibody: the smaller the KD value, the stronger the binding strength between the antigenic determinant (epitope) and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD.

[0172] Avidity is a measure of the strength of binding between an antibody and its associated antigen. Avidity is related to both the affinity between an antigenic determinant (epitope) and its antigen binding site on an antibody and the number of associated binding sites present on the antibody. -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10 -8 M to 10 -12 M, and / or a dissociation constant (KD) of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 (such as at least 10 12 M -1 ) binding affinity. Any K D Value greater than 10 -4 M is generally considered to indicate non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), biomembrane interferometry (BLI) assays and sandwich competition assays, as well as different variations of these methods known per se in the art.

[0173] The term "epitope" refers to a site on an antigen that binds to an antibody. An epitope can be formed by continuous amino acids, or by non-continuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by continuous amino acids (also referred to as linear epitopes) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding (also referred to as conformational epitopes) are usually lost when treated with denaturing solvents. An epitope typically includes at least 3, more typically at least 5 or 8 to 10 amino acids in a unique spatial conformation. An epitope defines the minimum binding site of an antibody and is therefore a specific target of an antibody or its antigen-binding fragment.

[0174] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acids) have identical residues at the same position in an alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the % identity of the amino acid sequence to SEQ ID NO: Y, and is detailed as X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0175] In addition, when determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is substituted with another amino acid residue of similar chemical structure and has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example according to WO 04 / 037999, GB-A-2 357 768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and (preferred) types and / or combinations of such substitutions may be selected based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0176] Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0177] Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0178] Any amino acid substitutions applied to the polypeptides described herein may also be based on analyses of the frequency of amino acid variations between homologous proteins from different species, as developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978; analyses of structure-forming potentials, as developed by Chou and Fasman, Biochemistry 13:211, 1974, and Adv. Enzymol., 47:45-149, 1978; and analyses of protein hydrophobicity patterns, as developed by Eisenberg et al., Proc. Nat. Acad Sci. USA 81:140-144, 1984; Kyte & Doolittle, J Mol. Biol. 157:105-132, 198 1; and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353, 1986, the entireties of which are incorporated herein by reference.

[0179] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. That is, the antibodies that make up the population are identical except for a small amount of possible naturally occurring mutations. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology in this article, and should not be construed as requiring the production of antibodies by any particular method.

[0180] The term "bispecific antibody" in the context of the present invention is understood to mean an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as different target binding, but also includes binding to different epitopes in one target.

[0181] As used herein, the term "tumor-associated antigen" refers to an antigen that is differentially expressed in cancer cells compared to normal cells and, therefore, can be used to target cancer cells.

[0182] As used herein, the term "bispecific T cell engager" or "BiTE" refers to a single polypeptide chain molecule with two antigen binding domains, one of which binds to a T cell antigen and the second binds to an antigen present on the surface of a target (see PCT Publication WO 05 / 061547; Baeuerle et al., 2008, Drugs of the Future 33:137-147; Bargou et al., 2008, Science 321:974-977, which are incorporated herein by reference in their entirety). Thus, the BiTE of the present disclosure has an antigen binding region that binds to GPC3 and a second antigen binding region for a T cell antigen.

[0183] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0184] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0185] The term "pharmaceutically acceptable" means that the carrier or adjuvant is compatible with the other ingredients of the composition and is substantially not deleterious to the recipient thereof, and / or such carrier or adjuvant is approved or approvable for inclusion in a pharmaceutical composition for parenteral administration to humans.

[0186] As used herein, the terms "treatment" and "treating" refer to the administration of an agent or the performance of a procedure in order to obtain an effect. The effect may be a preventive effect that completely or partially prevents a disease or its symptoms, and / or may be a therapeutic effect that partially or completely cures a disease and / or its symptoms. As used herein, "treatment" may include treating a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease or disease symptom in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease (e.g., including a disease that may be associated with or caused by a primary disease); (b) inhibiting the disease, i.e., preventing the disease from developing; and (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any marker of success in the treatment or improvement or prevention of cancer, including any objective or subjective parameter, such as mitigation; relief; reducing symptoms or making the patient more tolerant to the disease condition; slowing the rate of degeneration or decline; or making the endpoint of degeneration less debilitating. The treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes administering an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate, stop or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to reducing, eliminating or preventing a disease, disease symptoms, or side effects of a disease in a subject.

[0187] As used herein, the term "effective amount" refers to an amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for the disease.

[0188] As used herein, the term "subject" refers to any mammalian subject in need of diagnosis, treatment or therapy. For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, livestock and farm animals, as well as laboratory animals, zoo animals, sports animals or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0189] The terms "hCD200R1" and "human CD200R1" are used interchangeably herein and refer to human CD200 receptor 1. The term includes any CD200R1 variants, isoforms and species homologs naturally expressed by human cells (including human T cells), or CD200R1 expressed on cells transfected with genes or cDNA encoding human CD200R1, which are naturally expressed on human cells. Preferably, the term refers to wild-type human CD200R1 having the amino acid sequence shown in SEQ ID NO: 172.

[0190] The terms "cyno / cynomolgus / Cynomolgus macaques" are used interchangeably herein and refer to the cynomolgus CD200 receptor 1. The term includes any CD200R1 variants, isoforms and species homologs naturally expressed by cynomolgus cells (including cynomolgus T cells), or CD200R1 expressed on cells transfected with genes or cDNAs encoding cynomolgus CD200R1, which are naturally expressed on cynomolgus cells. Preferably, the term refers to wild-type cynomolgus CD200R1 having the amino acid sequence shown in SEQ ID NO: 175.

[0191] As used herein, "human CD200R1 antagonist antibody" or "anti-human CD200R antagonist antibody" refers to an antibody that binds to human CD200R1 and, when administered in vitro or in vivo, inhibits CD200-CD200R1 signaling in T cells or other types of cells in the microenvironment.

[0192] The term "hCD200R1L" refers to human CD200 receptor 2. The term includes any CD200R1L variants, isoforms and species homologs naturally expressed by human cells (including human immune cells), or CD200R1L expressed on cells transfected with genes or cDNAs encoding human CD200R1L, which are naturally expressed on human cells. Preferably, the term refers to wild-type human CD200R1L having the amino acid sequence shown in SEQ ID NO: 174.

[0193] Anti-CD200R1 Antibody

[0194] The present invention provides an anti-CD200R1 antibody and an antigen-binding fragment thereof. In some embodiments, the anti-CD200R1 antibody specifically binds to human CD200R1 and cynomolgus monkey CD200R1, but does not bind to mouse CD200R1. In some embodiments, the anti-CD200R1 antibody blocks the binding of CD200 and CD200R1, or blocks the interaction between CD200 and CD200R1. In some embodiments, the anti-CD200R1 antibody has a stronger binding affinity for CD200R1 than the binding of CD200 to CD200R1. In some embodiments, the anti-CD200R1 antibody binds to CD200R1 expressed on the surface of a cell. In some embodiments, the anti-CD200R1 antibody blocks the binding of CD200R1 expressed on the surface of a cell and CD200. In some embodiments, the anti-CD200R1 antibody blocks the interaction between CD200R1 expressed on the surface of a cell and CD200. In some embodiments, the anti-CD200R1 antibody blocks CD200-induced CD200R1 signaling in a cell. In some embodiments, the anti-CD200R1 antibody does not bind to CD200R1L. In some embodiments, the anti-CD200R1 antibody has antagonist activity but does not exhibit agonist activity.

[0195] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CD200R1, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), and wherein

[0196] (1) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 117, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 136; or

[0197] (2) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 111, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 124; or

[0198] (3) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 117, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 133; or

[0199] (4) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 118, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 134; or

[0200] (5) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 119, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 135; or

[0201] (6) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 120, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 137; or

[0202] (7) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 108, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 123; or

[0203] (8) the VH comprises HCDRs 1-3 of the VH having the amino acid sequence set forth in SEQ ID NO: 109, and the VL comprises LCDRs 1-3 of the VL having the amino acid sequence set forth in SEQ ID NO: 124; or

[0204] (9) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 110, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 125; or

[0205] (10) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 114, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 128; or

[0206] (11) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 115, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 129; or

[0207] (12) the VH comprises HCDRs 1-3 of the VH having the amino acid sequence set forth in SEQ ID NO: 116, and the VL comprises LCDRs 1-3 of the VL having the amino acid sequence set forth in SEQ ID NO: 130; or

[0208] (13) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 115, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 131; or

[0209] (14) The VH comprises HCDR 1-3 of the VH having the amino acid sequence shown in SEQ ID NO: 116, and the VL comprises LCDR 1-3 of the VL having the amino acid sequence shown in SEQ ID NO: 132.

[0210] In a second aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to CD200R1, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), and wherein

[0211] (1) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 67, 80, 99, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 67, 80, 99, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 32, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 32, 46, respectively, by one or two amino acid residues; or

[0212] (2) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 66, 80, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 66, 80, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 32, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 32, 46, respectively, by one or two amino acid residues; or

[0213] (3) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 66, 81, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 66, 81, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 33, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 33, 46, respectively, by one or two amino acid residues; or

[0214] (4) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 67, 76, 99, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 67, 76, 99, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 14, 32, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 14, 32, 46, respectively, by one or two amino acid residues; or

[0215] (5) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 66, 80, 100, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 66, 80, 100, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 14, 33, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 14, 33, 46, respectively; or

[0216] (6) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 91, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 91, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 10, 25, 44, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 10, 25, 44, respectively, by one or two amino acid residues; or

[0217] (7) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 26, 45, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 26, 45, respectively, by one or two amino acid residues; or

[0218] (8) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 93, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 93, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 25, 45, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 25, 45, respectively, by one or two amino acid residues; or

[0219] (9) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 26, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 26, 46, respectively, by one or two amino acid residues; or

[0220] (10) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 96, respectively, or LCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 65, 79, 96, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 29, 49, respectively, or HCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 11, 29, 49, respectively, by one or two amino acid residues; or

[0221] (11) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 97, respectively, or LCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 65, 79, 97, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 30, 49, respectively, or HCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 11, 30, 49, respectively; or

[0222] (12) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 98, respectively, or LCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 65, 79, 98, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 31, 50, respectively, or HCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 11, 31, 50, respectively; or

[0223] (13) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 97, respectively, or LCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 65, 79, 97, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 30, 49, respectively, or HCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 11, 30, 49, respectively; or

[0224] (14) The VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, and 98, respectively, or LCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 65, 79, and 98 by one or two amino acid residues, respectively, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 31, and 50, respectively, or HCDR 1-3 having amino acid sequences that differ from SEQ ID NOs: 11, 31, and 50, respectively.

[0225] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD200R1, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), and wherein

[0226] (1) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 136, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 117; or

[0227] (2) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 133, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 117; or

[0228] (3) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 134, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 118; or

[0229] (4) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 135, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 119; or

[0230] (5) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No 137, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No 120; or

[0231] (6) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 123, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 108; or

[0232] (7) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 124, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 109; or

[0233] (8) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 125, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 110; or

[0234] (9) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 124, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 111; or

[0235] (10) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 128, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 114; or

[0236] (11) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 129, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 115; or

[0237] (12) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 130, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 116; or

[0238] (13) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 131, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 115; or

[0239] (14) The VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 132, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 116.

[0240] In some embodiments, VL comprises a functional variant of an amino acid sequence as shown in any one of SEQ ID NOs: 123 to 125, 128 to 137, which is formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to CD200R1. In some embodiments, VH comprises a functional variant of an amino acid sequence as shown in any one of SEQ ID NOs: 108 to 111, 114 to 120, which is formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to CD200R1.

[0241] The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with the amino acid sequence of the parent polypeptide. For example, a functional variant of any one of SEQ ID NOs: 123 to 125, 128 to 137 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to any one of SEQ ID NOs: 123 to 125, 128 to 137, respectively. For example, a functional variant of any one of SEQ ID NOs: 108 to 111, 114 to 120 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to any one of SEQ ID NOs: 108 to 111, 114 to 120.

[0242] In some embodiments, a functional variant of any one of SEQ ID NOs: 123 to 125, 128 to 137 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to any one of SEQ ID NOs: 123 to 125, 128 to 137 and is formed by insertion, deletion and / or substitution of one or more amino acids in any one of SEQ ID NOs: 123 to 125, 128 to 137. In some embodiments, a functional variant of any one of SEQ ID NOs: 108 to 111, 114 to 120 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to any one of SEQ ID NOs: 108 to 111, 114 to 120 and is formed by insertion, deletion and / or substitution of one or more amino acids in any one of SEQ ID NOs: 108 to 111, 114 to 120.

[0243] In the case of functional variants, the number of inserted, deleted and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, more preferably 15% to 20%. For example, the number of inserted, deleted and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and most preferably 1 to 2. In a preferred embodiment, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0244] In some embodiments, insertions, deletions and / or substitutions may be made at the framework (FR) regions, such as at FR1, FR2, FR3 and / or FR4.

[0245] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions in which one amino acid in the following groups (a) to (e) is substituted by another amino acid residue in the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0246] Particularly preferred conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0247] In a preferred embodiment, VL comprises the amino acid sequence as shown in SEQ ID NO: 136, and VH comprises the amino acid sequence as shown in SEQ ID NO: 117; or VL comprises the amino acid sequence as shown in SEQ ID NO: 124, and VH comprises the amino acid sequence as shown in SEQ ID NO: 111; or VL comprises the amino acid sequence as shown in SEQ ID NO: 133, and VH comprises the amino acid sequence as shown in SEQ ID NO: 117; or VL comprises the amino acid sequence as shown in SEQ ID NO: 134, and VH comprises the amino acid sequence as shown in SEQ ID NO: 118; or VL comprises the amino acid sequence as shown in SEQ ID NO: 135, and VH comprises the amino acid sequence as shown in SEQ ID NO: 119; or VL comprises the amino acid sequence as shown in SEQ ID NO: 137, and VH comprises the amino acid sequence as shown in SEQ ID NO: 120; or VL comprises the amino acid sequence as shown in SEQ ID NO: 123, and VH comprises the amino acid sequence as shown in SEQ ID NO: 108; or VL comprises the amino acid sequence as shown in SEQ ID NO: 137, and VH comprises the amino acid sequence as shown in SEQ ID NO: 120; or VL comprises the amino acid sequence as shown in SEQ ID NO: 131 and VH comprises the amino acid sequence as shown in SEQ ID NO: 132 and VH comprises the amino acid sequence as shown in SEQ ID NO: 136.

[0248] Based on the amino acid sequence of the constant region of the heavy chain of the antibody, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of the antibody can be classified as lambda (λ) chain or kappa (κ) chain. The antibodies disclosed herein can be any of the above types or subtypes.

[0249] In some embodiments, the antibody can be an isotype selected from IgG, IgA, IgM, IgE and IgD. In some embodiments, the antibody can be a subtype selected from IgG1, IgG2, IgG3 and IgG4. In a preferred embodiment, the antibody is an IgG1 antibody.

[0250] The antibodies disclosed herein may be complete antibodies or antigen-binding fragments thereof. The antigen-binding fragment may be any fragment of an antibody that retains the ability to specifically bind to CD200R1. Examples of antigen-binding fragments include, but are not limited to, Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity determining regions (CDRs); nanobodies; linear antibodies comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) and a modified form of any of the aforementioned fragments, wherein the modified forms of these fragments retain antigen-binding activity.

[0251] In some embodiments, the antigen binding fragment may be selected from Fab, Fab', F(ab')2, Fv, scFv and ds-scFv. In a preferred embodiment, the antigen binding fragment is Fab or scFv.

[0252] In some embodiments, the antibody can be a monoclonal antibody. In some embodiments, the antibody comprises a heavy chain (HC) and a light chain (LC), and wherein

[0253] (1) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 170, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 151; or

[0254] (2) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 158, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 143; or

[0255] (3) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 158, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 147; or

[0256] (4) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 167, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 151; or

[0257] (5) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 168, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 152; or

[0258] (6) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 169, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 153; or

[0259] (7) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 171, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 154; or

[0260] (8) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 157, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 140; or

[0261] (9) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 158, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 141; or

[0262] (10) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 159, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 142; or

[0263] (11) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 162, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 146; or

[0264] (12) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 162, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 148; or

[0265] (13) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 163, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 149; or

[0266] (14) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 164, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 150; or

[0267] (15) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 165, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 149; or

[0268] (16) The LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID No: 166, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID No: 150.

[0269] In some embodiments, the light chain includes a functional variant of any one of the amino acid sequences formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to any one of SEQ ID NOs: 157 to 159 and 162 to 171. In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence shown in any one of SEQ ID NOs: 140 to 143 and 146 to 154, which is formed by insertion, deletion and / or substitution of one or more amino acids, provided that the functional variant retains the ability to bind to CD200R1.

[0270] For example, a functional variant of any one of SEQ ID NOs: 157 to 159 and 162 to 171 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to any one of SEQ ID NOs: 157 to 159 and 162 to 171, respectively. For example, a functional variant of any one of SEQ ID NOs: 140 to 143 and 146 to 154 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to any one of SEQ ID NOs: 140 to 143 and 146 to 154, respectively.

[0271] In some embodiments, the number of inserted, deleted and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, more preferably 15% to 20%. For example, the number of inserted, deleted and / or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. In a preferred embodiment, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0272] In some embodiments, insertions, deletions and / or substitutions may be made at the framework (FR) regions, such as FR1, FR2, FR3 and / or FR4; and / or at the constant regions, such as CL, CH1, CH2 and / or CH3.

[0273] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0274] In a preferred embodiment, the light chain comprises the amino acid sequence as shown in SEQ ID NO: 170, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 151; or the light chain comprises the amino acid sequence as shown in SEQ ID NO: 158, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 143; or the light chain comprises the amino acid sequence as shown in SEQ ID NO: 158, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 147; or the light chain comprises the amino acid sequence as shown in SEQ ID NO: 167, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 151; or the light chain comprises the amino acid sequence as shown in SEQ ID NO: 168, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 152; or the light chain comprises the amino acid sequence as shown in SEQ ID NO: 169, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 153; or the light chain comprises the amino acid sequence as shown in SEQ ID NO: 171, and the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 154; or the light chain comprises the amino acid sequence as shown in SEQ ID NO: 172. NO: 157, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 140; or the light chain comprises the amino acid sequence shown in SEQ ID NO: 158, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 141; or the light chain comprises the amino acid sequence shown in SEQ ID NO: 159, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 142; or the light chain comprises the amino acid sequence shown in SEQ ID NO: 162, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 146; or the light chain comprises the amino acid sequence shown in SEQ ID NO: 162, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 148; or the light chain comprises the amino acid sequence shown in SEQ ID NO: 163, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 149; or the light chain comprises the amino acid sequence shown in SEQ ID NO: 164, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 150; or the light chain comprises the amino acid sequence shown in SEQ ID NO: 165, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: The light chain comprises the amino acid sequence shown in SEQ ID NO:149; or the light chain comprises the amino acid sequence shown in SEQ ID NO:166, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:150.

[0275] In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region can be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or is derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0276] In one embodiment, the Fc region is an effector function defective type. For example, the Fc region can be an IgG1 isotype, or a non-IgG1 type, such as IgG2, IgG3 or IgG4, which has been mutated so that the ability to mediate effector functions such as ADCC has been reduced or even eliminated. Such mutations have been described in, for example, Dall'Acqua WF et al., J Immunol. 177 (2): 1129-1138 (2006) and Hezareh M, J Virol.; 75 (24): 12161-12168 (2001). In some embodiments, the Fc region of the antibody comprises a wild-type IgG1 Fc with L234A, L235A and G237A mutations.

[0277] In some embodiments, the antibody is mutated at one or more post-translational modification sites. In one embodiment, the Fc region comprises a mutation that removes an acceptor site for Asn-linked glycosylation, or otherwise manipulates the Fc region to eliminate the effector function of the antibody.

[0278] In some embodiments, the antibody is a bispecific or multispecific antibody. In some embodiments, the antibody is a bispecific antibody, which further comprises a second antigen binding region that binds a second antigen. In some embodiments, the second antigen can be a tumor-associated antigen, an immune checkpoint molecule, or an immune cell antigen.

[0279] The anti-CD200R1 antibody of the present invention is a naturally occurring antibody that has binding activity to both human CD200R1 and cynomolgus monkey CD200R1. The Fc engineered antibody of the present invention has no ADCC effect and effectively blocks CD200-CD200R1 interaction, inhibits CD200-induced CD200R1 signal transduction pathway, and activates immune response; the antibody also does not show agonistic activity, and is therefore a potential antibody molecule suitable for the development of antagonistic antibodies.

[0280] Bispecific Antibodies

[0281] In a second aspect, the present application provides a bispecific or multispecific antibody. In some embodiments, the antibody is a bispecific antibody, which further comprises a second antigen binding region that binds a second antigen. In some embodiments, the second antigen can be a tumor-associated antigen, an immune checkpoint molecule, or an immune cell antigen.

[0282] Many tumor-associated antigens associated with specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens that can potentially stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as those that are usually silent (i.e., not expressed) in normal cells, those that are only expressed at certain stages of differentiation, and those that are expressed over time, such as embryonic antigens and fetal antigens. Other tumor antigens are encoded by mutant cell genes, such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., mutant p53), and fusion proteins produced by internal deletions or chromosomal translocations. Other tumor antigens can be encoded by viral genes, such as those carried by RNA tumor viruses and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be produced by those skilled in the art.

[0283] Examples of tumor-associated antigens include, but are not limited to, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, CD200R1, MUC-1, mutant p53, mutant Ras, ROR1, VEGFR2, and combinations thereof.

[0284] In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen can be selected from T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD44, CD62L, CD69, ICOS, 41-BB (CD137) and NKG2D, or any combination thereof.

[0285] In some embodiments, the second antigen is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule can be selected from PD-1, PD-L1, CTLA-4, etc.

[0286] In some embodiments, the bispecific antibody comprises a single polypeptide chain comprising a first antigen binding region and a second antigen binding region, and optionally an Fc region.

[0287] The Fc region can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can contain one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or is derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0288] In one embodiment, the Fc region is an effector function defective type. For example, the Fc region can be an IgG1 isotype, or a non-IgG1 type, such as IgG2, IgG3 or IgG4, which has been mutated so that the ability to mediate effector functions such as ADCC has been reduced or even eliminated. Such mutations have been described, for example, in Dall'Acqua WF et al., J Immunol. 177 (2): 1129-1138 (2006) and Hezareh M, J Virol.; 75 (24): 12161-12168 (2001).

[0289] In one embodiment, the Fc region comprises a mutation that removes an Asn-linked glycosylated acceptor site, or the Fc region is otherwise manipulated to change the glycosylation properties. For example, in an IgG1 Fc region, an Asn-linked glycosylation site can be removed using an N297Q mutation. Thus, in a specific embodiment, the Fc region comprises an IgG1 wild-type sequence having an N297Q mutation. For example, in an IgG1 Fc region, an Asn-linked glycosylation site can be removed using an N297Q mutation. Thus, in a specific embodiment, the Fc region comprises an IgG1 wild-type sequence having an N297Q mutation.

[0290] In another embodiment, the Fc region is glycoengineered to reduce fucose, thereby enhancing ADCC, for example by adding compounds to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al., (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, for example, as described in Yamane-Ohnuki et al., (2004) Biotechnol. Bioeng 87:614. ADCC may alternatively be used et al., (1999) Nature Biotech 17: 176. In another embodiment, the Fc region has been engineered to enhance complement activation, for example, as described in Natsume et al., (2009) Cancer Sci. 100: 2411.

[0291] In some embodiments, the Fc region comprises a modification or mutation that can inhibit Fc homodimerization. In some embodiments, the Fc region comprises a variant of the human IgG1 Fc wild-type sequence. The variant may comprise amino acid substitutions at positions T366 and Y407 of human IgG1 (Kabat numbering). Preferably, T366 is substituted by L (leucine). Preferably, Y407 is substituted by I (isoleucine), F (phenylalanine), L (leucine), M (methionine), H (histidine), K (lysine), S (serine), Q (glutamine), T (threonine), W (tryptophan), A (alanine), G (glycine) or N (asparagine). More preferably, Y407 is substituted by H. In one embodiment, T366 is substituted by L and Y407 is substituted by H.

[0292] In some embodiments, the Fc region can be a monomeric human IgG1 Fc, as described in PCT Application No. PCT / US2018 / 016524, which is incorporated herein by reference in its entirety.

[0293] In some embodiments, the bispecific antibody comprises a first polypeptide chain comprising a VL of a first antigen-binding region and a VL of a second antigen-binding region, and optionally an Fc region; and a second polypeptide chain comprising a VH of a first antigen-binding region and a VH of a second antigen-binding region, and optionally an Fc region. The Fc region may be those as described above.

[0294] In some embodiments, the first polypeptide chain further comprises a light chain constant region (CL). In some embodiments, the first polypeptide chain comprises a monomeric human IgG1 Fc as described above. In some embodiments, the first polypeptide chain comprises, from N-terminus to C-terminus: a VL of a first antigen binding region, a VL of a second antigen binding region, a CL and an Fc.

[0295] In some embodiments, the second polypeptide chain further comprises a heavy chain constant region (CH), such as CH1. In some embodiments, the first polypeptide chain comprises a monomeric human IgG1 Fc as described above. In some embodiments, the second polypeptide chain comprises, from N-terminus to C-terminus: a VH of the first antigen binding region, a VH of the second antigen binding region, CH1 and Fc.

[0296] Nucleic Acids

[0297] In a third aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0298] The term "polynucleotide" or "nucleic acid" includes both single-stranded nucleotide polymers and double-stranded nucleotide polymers. The nucleotides comprising nucleic acids can be modified forms of ribonucleotides or deoxyribonucleotides or any type of nucleotides. The modifications include base modifications (such as bromouridine and inosine derivatives), ribose modifications (such as 2', 3'-dideoxyribose) and internucleotide linkage modifications (such as thiophosphates, dithiophosphates, selenophosphates, diselenphosphates, anilinothiophosphates (phosphoroanilothioate), anilinophosphates (phoshoraniladate) and phosphoramidates.

[0299] For example, the present invention provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of the heavy chain variable region sequences disclosed herein.

[0300] For example, the present invention provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of the light chain variable region sequences disclosed herein.

[0301] For example, the present invention provides nucleic acid molecules encoding the following items: (i) any of the heavy chain variable region sequences disclosed herein; and (ii) any of the light chain variable region sequences disclosed herein. The present invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98% or at least 99% identical to nucleic acids encoding the following items: (i) any of the heavy chain variable region sequences disclosed herein; and (ii) any of the light chain variable region sequences disclosed herein.

[0302] For example, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence of any one of the heavy chain variable region sequences disclosed herein.

[0303] In some embodiments, the invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising any one of the groups of three CDR sequences disclosed herein.

[0304] The present invention also provides a nucleic acid molecule encoding a heavy chain variable region sequence, which comprises a CDR sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein.

[0305] In some embodiments, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence, which heavy chain variable region sequence comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR1, CDR2, and CDR3 of any group of three CDR sequences disclosed herein, respectively.

[0306] For example, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence, which comprises the CDR sequence of any one of the light chain variable region sequences disclosed herein.

[0307] In some embodiments, the invention provides a nucleic acid molecule encoding a light chain variable region sequence comprising any one of the groups of three CDR sequences disclosed herein.

[0308] The present invention also provides a nucleic acid molecule encoding a light chain variable region sequence, which comprises a CDR sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein.

[0309] In some embodiments, the present invention provides a nucleic acid molecule encoding a light chain variable region sequence, which light chain variable region sequence comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the CDR1, CDR2, and CDR3 of any group of three CDR sequences disclosed herein, respectively.

[0310] For example, the present invention provides nucleic acid molecules encoding the following items: (i) a heavy chain variable region sequence comprising a CDR sequence of any of the heavy chain variable region sequences disclosed herein; and (ii) a light chain variable region sequence comprising a CDR sequence of any of the light chain variable region sequences disclosed herein. In some embodiments, the present invention provides nucleic acid molecules encoding the following items: (i) a heavy chain variable region sequence comprising any of the groups of three CDR sequences disclosed herein; and (ii) a light chain variable region sequence comprising any of the groups of three CDR sequences disclosed herein. The present invention also provides nucleic acid molecules encoding the following items: (i) a heavy chain variable region sequence, which comprises a CDR sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR sequence of any of the heavy chain variable region sequences disclosed herein; and (ii) a light chain variable region sequence, which comprises a CDR sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR sequence of any of the light chain variable region sequences disclosed herein. In some embodiments, the present invention provides nucleic acid molecules encoding: (i) a heavy chain variable region sequence, which comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence that are at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR1, CDR2, and CDR3 of any group of three CDR sequences disclosed herein, respectively; and (ii) a light chain variable region sequence, which comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence that are at least 90%, at least 95%, at least 98% or at least 99% identical to the CDR1, CDR2, and CDR3 of any group of three CDR sequences disclosed herein, respectively.

[0311] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding an anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the present invention provides a deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0312] Thus, a deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein is used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from pancreatic ductal adenocarcinoma, prostate cancer, melanoma, liver cancer, breast cancer, lung cancer (e.g., squamous cell carcinoma of the lung), squamous cell carcinoma, ovarian cancer, leukemia, and myeloma.

[0313] In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of the cell.

[0314] Therefore, ribonucleic acid (RNA) comprising a deoxynucleotide sequence encoding an anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein can be used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from pancreatic ductal adenocarcinoma, prostate cancer, melanoma, liver cancer, breast cancer, lung cancer (e.g., squamous cell carcinoma of the lung), squamous cell carcinoma, ovarian cancer, leukemia and myeloma.

[0315] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a vector or delivery agent.

[0316] Carrier

[0317] In a fourth aspect, the present invention further provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding the anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein or the bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0318] In some embodiments, the vector is a recombinant expression vector capable of expressing a polypeptide comprising a heavy chain variable region or a light chain variable region of an anti-CD200R1 antibody. For example, the present invention provides a recombinant expression vector comprising any of the above nucleic acid molecules.

[0319] Any vector may be suitable for use in the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papillomatosis virus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP and pLXIN-Luc.

[0320] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses. For example, vectors can be selected from pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors useful in the context of the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0321] Recombinant expression vectors can be prepared using standard recombinant DNA techniques, which are described, for example, in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing Associates and John Wiley & Sons, NY, 1994. 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 papilloma virus, etc.

[0322] Thus, the vector can be used to treat a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from pancreatic ductal adenocarcinoma, prostate cancer, melanoma, liver cancer, breast cancer, lung cancer (e.g., lung squamous cell carcinoma), squamous cell carcinoma, ovarian cancer, leukemia, and myeloma.

[0323] The vectors of the present invention can be introduced into cells. In some embodiments, the vectors of the present invention can be introduced into cells in vitro or ex vivo. Optionally, the cells introduced with the vectors can then be administered into a subject. In some embodiments, the vectors of the present invention can be introduced into cells in vivo.

[0324] For example, the vector can be an adenovirus vector, which comprises a nucleotide sequence encoding an anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein. The vector can be administered to a subject and then enter the subject's cells in vivo, thereby integrating the nucleotide sequence encoding the anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein or a bispecific antibody or antigen-binding fragment thereof disclosed herein into the genome of the cell, which then expresses the anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein, so as to treat the diseases disclosed herein.

[0325] Host cells

[0326] In a fifth aspect, the present invention also provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.

[0327] Any cell can be used as a host cell for nucleic acids or vectors disclosed herein. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, true bacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example Salmonella typhimurium; Serratia, for example Serratia marcescens. marcescans and Shigella; Bacilli, such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHOK1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0328] The host cells of the present invention are prepared by introducing the vector disclosed herein or the nucleic acid disclosed herein in vitro or ex vivo. The host cells of the present invention can be administered to a subject, and the host cells express the anti-CD200R1 antibody or antigen-binding fragment thereof disclosed herein in vivo to treat the diseases disclosed herein.

[0329] Antibody Drug Conjugates

[0330] In a sixth aspect, the present invention provides an antibody drug conjugate (ADC), which comprises the antibody or antigen-binding fragment thereof according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention.

[0331] In the context of the present disclosure, "conjugate" refers to an antibody or antibody fragment (such as an antigen binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, a toxin, a therapeutic agent, a detectable marker, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates". When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody drug conjugate" or "ADC". Other antibody conjugates include, for example, multispecific (such as bispecific or trispecific) antibodies.

[0332] In some embodiments, the effector molecule can be a detectable marker or an immunotoxin. Specific non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38 and PE40), diphtheria toxin (DT), botulinum toxin or its modified toxins, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds, usually causing death by hepatotoxicity. However, PE and DT can be modified to be used as an immunotoxin by removing the natural targeting components of the toxin (such as the domain 1a of PE and the B chain of DT) and replacing them with different targeting parts (such as antibodies). The term "conjugated" or "connected" can refer to making two polypeptides into a continuous polypeptide molecule. In one embodiment, an antibody is connected to an effector molecule. In another embodiment, the antibody connected to the effector molecule is further connected to a lipid or other molecule, to a protein or peptide, to increase its half-life in vivo. The connection can be carried out by chemical or recombinant means. In one embodiment, the linkage is chemical, wherein the reaction between the antibody portion and the effector molecule has resulted in a covalent bond formed between the two molecules, thereby forming one molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the effector molecule.

[0333] The present invention provides immunoconjugates comprising a monoclonal antibody or antigen binding fragment disclosed herein and an effector molecule. In some embodiments, the effector molecule is a toxin, such as but not limited to Pseudomonas exotoxin or a variant thereof. In other embodiments, the effector molecule is a detectable label, such as but not limited to a fluorophore, an enzyme, or a radioisotope.

[0334] The disclosed monoclonal antibodies may be conjugated to therapeutic agents or effector molecules. Immunoconjugates include, but are not limited to, molecules in which a therapeutic agent is covalently linked to an antibody. A therapeutic agent is an agent having a specific biological activity against a specific target molecule or a cell carrying a target molecule. Those skilled in the art will appreciate that therapeutic agents may include various drugs such as vinblastine, daunomycin, etc.; cytotoxins such as natural or modified Pseudomonas exotoxin or diphtheria toxin; encapsulating agents (such as liposomes) containing pharmacological compositions; radioactive agents such as 125 I. 32 P. 14 C. 3 H and 35 S and other labels; target moieties; and ligands.

[0335] The choice of a particular therapeutic agent depends on the particular target molecule or cell and the desired biological effect. Thus, for example, the therapeutic agent may be a cytotoxin for causing death of a particular target cell, such as a tumor cell. Conversely, when it is desired to cause a non-lethal biological response, the therapeutic agent may be conjugated to a non-lethal pharmaceutical agent or a liposome containing a non-lethal pharmaceutical agent.

[0336] Using the therapeutic agents and antibodies described herein, a skilled artisan can easily construct multiple clones containing functionally equivalent nucleic acids, such as nucleic acids that differ in sequence but encode the same effector portion or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.

[0337] The effector molecule can be connected to the antibody of interest using any number of methods known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. The method of attaching the effector molecule to the antibody varies according to the chemical structure of the effector. Polypeptides generally contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody to cause the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or connect additional reactive functional groups. Derivatization may include connecting any of a number of known linker molecules. The linker can be any molecule used to connect the antibody to the effector molecule. The linker can form a covalent bond with both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art, including but not limited to straight or branched carbon linkers, heterocyclic carbon linkers or peptide linkers. When the antibody and the effector molecule are polypeptides, the linker can be connected to these constituent amino acids through the side groups of the constituent amino acids (such as through a disulfide bond with cysteine), or to the α-carbon amino group and carboxyl group of the terminal amino acid.

[0338] In some cases, when the immunoconjugate has reached its target site, it is desirable to release the effector molecule from the antibody. Therefore, in these cases, the immunoconjugate will contain a bond that is cleavable near the target site.

[0339] Cleavage of the linker can be facilitated by enzymatic activity or by conditions to which the immunoconjugate is subjected within the target cell or near the target site to release the effector molecule from the antibody.

[0340] In view of the numerous methods reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one skilled in the art will be able to determine the appropriate method for attaching a given agent to an antibody or other polypeptide.

[0341] The antibodies disclosed herein may be derivatized or linked to another molecule (such as another peptide or protein). Typically, the antibody or portion thereof is derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, the antibody functionality may be linked (by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0342] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to produce bispecific antibodies). Suitable cross-linking agents include those with heterobifunctional groups having two distinct reactive groups separated by a suitable spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester), or those with homobifunctional groups (such as disuccinimidyl suberate). Such linkers are commercially available.

[0343] The antibody can be conjugated to a detectable marker; for example, the detectable marker can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic inspection, and laparoscopy. Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). The antibody or antigen binding fragment can also be conjugated to an enzyme for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When the antibody or antigen binding fragment is conjugated to a detectable enzyme, detection can be performed by adding additional reagents that use the additional reagents to produce a distinguishable reaction product. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. The antibody or antigen binding fragment can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin protein binding. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent label.

[0344] The antibody can be fused to a self-labeled protein tag (e.g., HaloTag). For example, a protein tag can be cloned at the end of a constant region. HaloTag is a self-labeled protein tag derived from a bacterial enzyme (haloalkane dehalogenase) that is designed to be covalently bound to a synthetic ligand. In some cases, the synthetic ligand comprises a chloroalkane linker (Los et al., (2008) ACS Chem Biol. 3 (6): 373-82) connected to a fluorophore such as a near-infrared fluorophore.

[0345] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0346] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter gene (such as a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag). In some embodiments, the labels are connected by spacer arms of various lengths to reduce potential steric hindrance.

[0347] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect expression of the target antigen by X-ray, emission spectroscopy or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I.

[0348] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life or increasing tissue binding.

[0349] Toxins can be used with monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxins and their subunits, and botulinum toxins A to F. These toxins can be easily obtained from commercial sources (Sigma Chemical Company, St. Louis, MO). The toxins considered also include variants of toxins described herein (see, for example, see U.S. Patent Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, "Pseudomonas exotoxin" refers to full-length natural (naturally occurring) PE or modified PE. Such modifications may include, but are not limited to: elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions, and addition of one or more sequences at the carboxyl terminus (see, for example, Siegall et al., Biol. Chem. 264: 14256-14261, 1989).

[0350] The PE used in the monoclonal antibodies described herein may include native sequences, cytotoxic fragments of native sequences, and conservatively modified variants of native PE and its cytotoxic fragments. The cytotoxic fragments of PE include cytotoxic fragments with or without subsequent proteolysis or other treatments in target cells. The cytotoxic fragments of PE include PE40, PE38, and PE35. For additional descriptions of PE and its variants, see, e.g., U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; U.S. Patent Application Publication No. 2015 / 0099707; PCT Publication Nos. WO 99 / 51643 and WO 2014 / 052064; Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol. Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta 1333:C1-C6, 1997.

[0351] Also contemplated herein are protease-resistant PE variants and PE variants with reduced immunogenicity, such as, but not limited to, PE-LR, PE-6X, PE-8X, PE-LR / 6X, and PE-LR / 8X (see, e.g., Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA 105(32):11311-11316, 2008; and PCT Publication Nos. WO 2007 / 016150, WO 2009 / 032954, and WO 2011 / 032022, which are incorporated herein by reference).

[0352] In some examples, PE is a variant resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113 (16): 3792-3800, 2009; PCT Publication No. WO 2009 / 032954). In other examples, PE is a variant named PE-LR / 6X (PCT Publication No. WO 2011 / 032022). In other examples, PE variants are PE with reduced immunogenicity. In other examples, PE is a variant named PE-LR / 8M (PCT Publication No. WO 2011 / 032022).

[0353] Modification of PE may occur in any of the aforementioned variants, including cytotoxic fragments of PE (e.g., PE38, PE-LR, and PE-LR / 8M). The modified PE may include any substitution, such as substitution of one or more amino acid residues within one or more T cell epitopes and / or B cell epitopes of PE, or deletion of one or more T cell epitopes and / or B cell epitopes (see, e.g., U.S. Patent Application Publication No. 2015 / 0099707).

[0354] Contemplated PE forms also include deimmunized forms of PE, such as forms with domain II deletion (e.g., PE24). Deimmunized forms of PE are described in, for example, PCT publications WO 2005 / 052006, WO 2007 / 016150, WO 2007 / 014743, WO 2007 / 031741, WO 2009 / 32954, WO 2011 / 32022, WO 2012 / 154530, and WO 2012 / 170617.

[0355] The antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing tumor or viral antigens on the surface. Therefore, the antibodies disclosed herein can be directly or via a joint connected to a drug that will be delivered directly to cells expressing cell surface antigens. This can be used for treatment, diagnosis or research purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates or recombinant viruses. Nucleic acid therapy and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent crosslinking with single-stranded or double-stranded DNA, and oligonucleotides that form triplexes.

[0356] Alternatively, the molecule attached to the antibody can be an encapsulation system, such as a nanoparticle, a liposome or a micelle, which contains a therapeutic composition, such as a drug, a nucleic acid (e.g., an antisense nucleic acid), or another therapeutic moiety that is preferably protected from direct exposure to the circulatory system. Methods for preparing liposomes attached to antibodies are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).

[0357] The antibodies described herein may also be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such uses include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive labels (e.g., 3 H. 125 I. 35 S.14 C or 32 P), enzymes (such as horseradish peroxidase, alkaline phosphatase and other enzymes commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, etc.) beads.

[0358] The means for detecting such labels are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or scintillation counters, and fluorescent markers can be detected using photodetectors to detect emitted illumination. Enzyme labels are usually detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, while colorimetric labels are detected by simply visualizing the colored label.

[0359] ADC is a compound consisting of a tumor antigen-specific antibody (or its antigen-binding fragment) and a drug (usually a cytotoxic agent, such as an anti-microtubule agent or a cross-linking agent). Because ADC can specifically target cancer cells, the drug may be more effective than the agents used for standard chemotherapy. The most common cytotoxic drugs currently used with ADC have an IC50 that is 100 to 1000 times more effective than conventional chemotherapeutic agents. Common cytotoxic drugs include anti-microtubule agents, such as maytansinoids and auristatins (e.g., auristatin E and auristatin F). Other cytotoxins used with ADC include pyrrolobenzodiazepines (PDB), which covalently bind to the minor groove of DNA to form interchain crosslinks. In many cases, ADC includes antibodies and drugs in a ratio of 1:2 to 1:4 (Bander, Clinical Advances in Hematology & Oncology 10 (8; suppl 10): 3-7, 2012).

[0360] Antibodies and drugs can be connected by cleavable or non-cleavable joints. However, in some cases, it is desirable to have a joint that is stable in the circulation to prevent the systemic release of cytotoxic drugs, which may cause significant off-target toxicity. Non-cleavable joints prevent the release of cytotoxic agents before ADC is internalized by target cells. Once entering the lysosome, the digestion of antibodies by lysosomal proteases will lead to the release of cytotoxic agents (Bander, Clinical Advances in Hematology & Oncology 10 (8; suppl 10): 3-7, 2012).

[0361] A method for site-specific and stable conjugation of drugs to monoclonal antibodies is via glycan engineering. Monoclonal antibodies have a conserved N-linked oligosaccharide chain at the Asn297 residue in the CH2 domain of each heavy chain (Qasba et al., Biotechnol Prog 24:520-526, 2008). Using a mutant 1,4-galactosyltransferase (Y289L-Gal-Tl; U.S. Patent Application Publication Nos. 2007 / 0258986 and 2006 / 0084162, which are incorporated herein by reference), 2-keto-galactose is transferred to the free GlcNAc residues on the antibody heavy chain to provide a chemical treatment for conjugation.

[0362] Based on the terminal galactose residues-complete galactosylation (two galactose residues; IgG-G2), one galactose residue (IgG-G1) or complete degalactosylation (IgG-GO), the oligosaccharide chains attached to the monoclonal antibody can be divided into three groups. The monoclonal antibody is treated with 1,4-galactosidase to convert the antibody into the IgG-GO glycoform. The mutated 1,4-galactosyltransferase is able to transfer 2-keto-galactose or 2-azido-galactose from their respective UDP derivatives to the GlcNAc residues on the IgG-G1 and IgG-GO glycoforms. Chemical treatment of the transferred sugar enables a variety of molecules to be conjugated to the monoclonal antibody via the glycan residues (Qasba et al., Biotechnol Prog 24: 520-526, 2008).

[0363] Provided herein are ADCs comprising a drug (such as a cytotoxic agent) conjugated to a monoclonal antibody that binds (such as specifically binds) to CD200R1. In some embodiments, the drug is a small molecule. In some examples, the drug is a cross-linking agent, an anti-microtubule agent, and / or an anti-mitotic agent, or any cytotoxic agent suitable for mediating killing of tumor cells.

[0364] Exemplary cytotoxic agents include, but are not limited to, PDB, auristatin, maytansinoid, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU-159682, anthracycline, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, combretastain, dolastatin, duocarmycin, enediyne, geldanamycin, indoline-benzodiazepine dimer, puromycin, tubulysin, hemiasterlin, spliceostatin or pladienolide, as well as stereoisomers, isosteres, analogs, and derivatives thereof having cytotoxic activity.

[0365] In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). The natural product anthramycin (PBD) was first reported in 1965 (Leimgruber et al., J Am Chem Soc, 87:5793-5795, 1965; Leimgruber et al., J Am Chem Soc, 87:5791-5793, 1965). Since then, many PBDs have been reported, including both naturally occurring analogs and synthetic analogs (Gerratana, Med Res Rev 32 (2): 254-293, 2012; and U.S. Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099). As an example, PBD dimers recognize and bind specific DNA sequences and have been shown to be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies, and the resulting ADCs have been shown to have anticancer properties (see, e.g., US2010 / 0203007). Exemplary attachment sites on PBD dimers include five-membered pyrrole rings, tethers between PBD units, and N10-C11 imine groups (see WO 2009 / 016516; US2009 / 304710; US2010 / 047257; US 2009 / 036431; US2011 / 0256157; and WO 2011 / 130598).

[0366] In some embodiments, ADC comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine, and are mitotic inhibitors that work by inhibiting tubulin polymerization. Maytansine was first separated from East African shrub Maytenus serrata (U.S. Patent number 3,896,111). Subsequently, it was found that some microorganisms also produced maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent number 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0367] In some embodiments, the ADC comprises an antibody conjugated to dolastatin or auristatin or an analog or derivative thereof (see U.S. Pat. Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatin is a derivative of the marine mollusk compound dolastatin-10. Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., Antimicrob Agents and Chemother 45(12):3580-3584, 2001), and have anticancer activity (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob Agents Chemother 42:2961-2965, 1998). Examples of dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB), auristatin EFP (AEFP), MM AD (monomethyl auristatin D or monomethyl dolastatin 10), MMAF (monomethyl auristatin F or N-methyl valine-valine-dolaisoleucine-dolaproline-phenylalanine), MMAE (monomethyl auristatin E or N-methyl valine-valine-dolaisoleucine-dolaproline-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, e.g., U.S. Patent Publication No. 2013 / 0129753).

[0368] In some embodiments, the ADC comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin antibiotic family and its analogs are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Res 53:3336-3342, 1993; Lode et al., Cancer Res 58:2925-2928, 1998). Exemplary methods for preparing ADCs with calicheamicin drug moieties are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0369] In some embodiments, ADC comprises anthracycline. Anthracycline is an antibiotic compound that exhibits cytotoxic activity. It is believed that anthracycline can kill cells by many different mechanisms, including inserting drug molecules into the DNA of cells, thereby inhibiting DNA-dependent nucleic acid synthesis; inducing the production of free radicals, and then reacting these free radicals with cell macromolecules, causing cell damage; and / or causing the interaction of drug molecules with cell membranes. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, valrubicin and mitoxantrone and their derivatives. For example, PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clin Cancer Res 11 (4): 1608-1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin that has a 2-methoxymorpholinyl group on the glycosylamino group of doxorubicin (Grandi et al., Cancer Treat Rev 17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).

[0370] In some embodiments, the ADC may further comprise a linker. In some examples, the linker is a bifunctional or multifunctional moiety that can be used to connect one or more drug moieties to an antibody to form an ADC. In some embodiments, a linker having a reactive functional group for covalently linking a drug and an antibody is used to prepare an ADC. For example, a cysteine ​​thiol of an antibody can form a bond with a reactive functional group of a linker or a drug-linker intermediate to prepare an ADC.

[0371] In some examples, the linker has a functional group that can react with the free cysteine ​​present on the antibody to form a covalent bond. Exemplary linkers with such reactive functional groups include maleimide, haloacetamide, oc-haloacetyl, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0372] In some examples, the linker has a functional group that can react with the electrophilic group present on the antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some cases, the heteroatom of the reactive functional group of the linker can react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting examples include hydrazides, oximes, amino groups, hydrazine, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0373] In some examples, the linker is a cleavable linker that facilitates the release of the drug. Examples of cleavable linkers include acid-labile linkers (e.g., including hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive), photolabile linkers, and disulfide-containing linkers (Chari et al., Cancer Res 52:127-131, 1992; U.S. Pat. No. 5,208,020).

[0374] The ADCs disclosed herein can be used alone or in combination with another therapeutic agent and / or in combination with any standard therapy for treating cancer (such as surgical resection of the tumor, chemotherapy or radiotherapy) to treat cancer, wherein the microenvironment of the cancer includes T cells and / or myeloid cells that highly express CD200R1 on their surface, or the cancer is responsive to reduction, inhibition and / or blocking of CD200R1-mediated immunomodulatory function or activity.

[0375] Pharmaceutical composition

[0376] In a seventh aspect, the present invention provides a pharmaceutical composition comprising (i) the antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the bispecific antibody of the second aspect of the present invention, or the nucleic acid of the third aspect of the present invention, or the vector of the fourth aspect of the present invention, or the host cell of the fifth aspect of the present invention, or the ADC of the sixth aspect of the present invention; and optionally (ii) a pharmaceutically acceptable carrier or excipient.

[0377] The present invention provides pharmaceutical compositions comprising antibodies of the present invention. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). For example, in some embodiments, the composition for intravenous administration is typically a solution in a sterile isotonic aqueous buffer.

[0378] The antibodies or agents of the present invention (also referred to herein as "active compounds") and their derivatives, fragments, analogs and homologues can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise an antibody or agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. that are compatible with drug administration. Suitable carriers are described in the latest edition of "Remington's Pharmaceutical Sciences", which is a standard reference text in this field, and is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous solvents such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, these media or agents are considered to be used in the composition. Supplementary active compounds may also be incorporated into the composition.

[0379] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or glucose. The pH value can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multidose vials made of glass or plastic.

[0380] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of preparation and storage; and it must be preserved to resist the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing the following substances: for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. For example, by using a coating (such as lecithin), by maintaining the required particle size in the case of a dispersion, and by using a surfactant to maintain appropriate fluidity. The preventive effect on microorganisms can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it will be preferred to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. The sustained absorption of the injectable composition can be achieved by including an agent that delays absorption (for example, aluminum monostearate and gelatin) in the composition.

[0381] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into an appropriate solvent with one or more of the components listed above, as required, followed by sterilization filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from the components listed above. For sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produce powders of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.

[0382] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be mixed with an excipient and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a fluid carrier used as a mouthwash, wherein the compound in the fluid carrier is orally administered and spit out or swallowed after rinsing the mouth. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants such as colloidal dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as mint, methyl salicylate or orange flavoring.

[0383] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0384] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream well known in the art.

[0385] The compounds may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0386] In one embodiment, the active compound is prepared with carriers that protect the compound from rapid discharge from the body, such as controlled release formulations including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such preparations are obvious to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These materials can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0387] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect when associated with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the art of compounding such active compounds for the treatment of individuals, and are directly dependent on these factors.

[0388] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0389] The present invention provides therapeutic compositions comprising an anti-CD200R1 antibody or antigen-binding fragment thereof of the present invention. The therapeutic compositions according to the present invention will be administered with suitable carriers, excipients and other agents, which are incorporated into formulations to improve transfer, delivery, tolerability, etc. A large number of suitable formulations can be found in the formulary known to all pharmaceutical chemists: "Remington's Pharmaceutical Sciences" (Mack Publishing Company, Easton, Pennsylvania). These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

[0390] Treatment

[0391] In an eighth aspect, the present invention provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, ADC or pharmaceutical composition of the present invention.

[0392] In a ninth aspect, the present invention provides an effective amount of an antibody or antigen-binding fragment thereof, a bispecific antibody, a nucleic acid, a vector, a host cell, an ADC or a pharmaceutical composition of the present invention for use in a method of treating cancer in a subject.

[0393] In a tenth aspect, the present invention provides use of an antibody or antigen-binding fragment thereof, a bispecific antibody, a nucleic acid, a vector, a host cell, an ADC or a pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer in a subject.

[0394] The antibodies provided herein can be administered to slow or inhibit the progression of cancer, or to inhibit the metastasis of cancer. In these applications, a therapeutically effective amount of the composition is administered to a subject in an amount sufficient to inhibit the growth, replication or metastasis of cancer cells or to inhibit the signs or symptoms of cancer. Suitable subjects may include those diagnosed with cancer, wherein the microenvironment of the cancer includes T cells and / or myeloid cells that highly express CD200R1 on the surface, such as mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, triple-negative breast cancer, or ovarian cancer.

[0395] Provided herein is a method for treating a subject's cancer by administering to the subject a therapeutically effective amount of an antibody as described herein. Also provided herein is a method for inhibiting the subject's cancer metastasis by administering to the subject a therapeutically effective amount of an antibody as described herein. In some embodiments, the cancer is pancreatic ductal adenocarcinoma, prostate cancer, melanoma, liver cancer, breast cancer, lung cancer (e.g., squamous cell carcinoma of the lung), squamous cell carcinoma, ovarian cancer, leukemia, or myeloma.

[0396] The administration of the antibodies disclosed herein may also be accompanied by the administration of other anticancer agents or therapeutic treatments (such as surgical resection of tumors). Any suitable anticancer agent may be administered in combination with the antibodies disclosed herein. Exemplary anticancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvival agents, biological response modifiers, antihormones (e.g., antiandrogens), and antiangiogenic agents. Other anticancer treatments include radiotherapy and other antibodies that specifically target cancer cells.

[0397] Another common treatment for some types of cancer is surgery, such as surgical removal of metastatic tumors. Another example of treatment is radiation therapy, such as administering radioactive material or energy (such as external beam radiation therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical removal.

[0398] Diagnosis and testing methods

[0399] In an eleventh aspect, the present invention provides a method for determining that a subject has cancer or is at risk of having cancer, wherein the microenvironment of the cancer comprises T cells and / or myeloid cells that highly express CD200R1 on their surface, wherein the method comprises:

[0400] (a) obtaining a biological sample from the subject,

[0401] (b) contacting the sample with the antibody or antigen-binding fragment thereof of the first aspect of the present invention; and

[0402] (c) detecting binding of the antibody to the sample,

[0403] Wherein increased binding of the antibody or antigen-binding fragment thereof to the samples, compared to binding of the antibody or antigen-binding fragment thereof to control samples, indicates that the subject has cancer or is at risk of developing cancer.

[0404] In a twelfth aspect, the present invention provides a method for imaging cancer in a subject, wherein the microenvironment of the cancer comprises T cells and / or myeloid cells that highly express CD200R1 on their surface, wherein the method comprises:

[0405] (a) administering to the subject the antibody or antigen-binding fragment thereof of the first aspect of the invention, wherein the antibody is conjugated to a detectable marker; and

[0406] (b) detecting the presence of the marker.

[0407] Provided herein are methods for detecting CD200R1 protein in vitro or in vivo. In some cases, CD200R1 expression is detected in a biological sample. The sample can be any sample, including but not limited to a blood sample, tissue from a biopsy, autopsy, and pathological specimens. Biological samples also include tissue sections, for example, frozen sections for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

[0408] Provided herein is a method for determining whether a subject has cancer by contacting a sample from the subject with a CD200R1-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample, wherein the microenvironment of the cancer includes T cells and / or myeloid cells that highly express CD200R1 on their surface. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample indicates that the subject has cancer, wherein the microenvironment of the cancer includes T cells and / or myeloid cells that highly express CD200R1 on their surface.

[0409] In another embodiment, a method for confirming a cancer diagnosis in a subject is provided by contacting a sample from a subject diagnosed with cancer with a CD200R1-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample, wherein the microenvironment of the cancer includes T cells and / or myeloid cells that highly express CD200R1 on their surface. An increase in binding of the antibody to the sample compared to binding of the antibody to a control sample confirms a cancer diagnosis in the subject, wherein the microenvironment of the cancer includes T cells and / or myeloid cells that highly express CD200R1 on their surface.

[0410] In some examples of the disclosed methods, the monoclonal antibody is directly labeled.

[0411] In other examples, the method further comprises contacting the sample with a second antibody that specifically binds to the monoclonal antibody; and detecting binding of the second antibody. Increased binding of the second antibody to the sample as compared to binding of the second antibody to a control sample can detect cancer in the subject or verify a diagnosis of cancer in the subject, wherein the microenvironment of the cancer includes T cells and / or myeloid cells that highly express CD200R1 on their surface.

[0412] In some instances, the cancer is mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, triple-negative breast cancer, or ovarian cancer.

[0413] In some examples, the control sample is a sample from a subject without cancer. In certain examples, the sample is a blood or tissue sample.

[0414] In some embodiments of the diagnostic and detection methods, the anti-CD200R1 antibody is directly labeled with a detectable label. In another embodiment, the anti-CD200R1 antibody (first antibody) is unlabeled, and the second antibody or other molecules that can bind to the first antibody are labeled. As is well known to those skilled in the art, a secondary antibody is selected that is capable of specifically binding to a particular type and class of the first antibody. For example, if the first antibody is human IgG, the secondary antibody can be anti-human IgG. Other molecules that can bind to antibodies include, but are not limited to, protein A and protein G, both of which are commercially available.

[0415] Suitable labels for antibodies or secondary antibodies include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I. 131 I. 35 S or 3 H.

[0416] In another embodiment, CD200R1 in a biological sample can be measured by a competitive immunoassay using a CD200R1 protein standard labeled with a detectable substance and an unlabeled anti-CD200R1 antibody. In this assay, the biological sample, the labeled CD200R1 protein standard, and the anti-CD200R1 antibody are combined, and the amount of the labeled CD200R1 protein standard bound to the unlabeled antibody is measured. The amount of CD200R1 in the biological sample is inversely proportional to the amount of the labeled CD200R1 protein standard bound to the anti-CD200R1 antibody.

[0417] The immunoassays and methods disclosed herein can be used for many purposes. In one embodiment, an anti-CD200R1 antibody can be used to detect the production of CD200R1 in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of CD200R1 in a biological sample (such as a tissue sample or a blood or serum sample). In some examples, CD200R1 is cell surface CD200R1. In other examples, the CD200R1 protein is soluble (e.g., in a cell culture supernatant, or in a body fluid sample such as a blood or serum sample).

[0418] In one embodiment, a kit for detecting CD200R1 in a biological sample (such as a blood sample or a tissue sample) is provided. For example, in order to verify a cancer diagnosis in a subject, a biopsy may be performed to obtain a tissue sample for histological examination. The kit for detecting the polypeptide will generally include a monoclonal anti-CD200R1 antibody, such as any of the monoclonal antibodies disclosed herein. In another embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzyme label).

[0419] In one embodiment, the kit includes instructional materials that disclose methods of using the anti-CD200R1 antibody. The instructional materials may be written in electronic form (such as a computer disk or CD), or may be visual (such as a video file). The kit may also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit may additionally contain a device for detecting the label (such as an enzyme substrate for the enzyme label, a filter set for detecting the fluorescent label, an appropriate secondary label (such as a secondary antibody), etc.). The kit may additionally include buffers and other reagents routinely used to implement the specific method. These kits and appropriate contents are well known to those skilled in the art.

[0420] In one embodiment, the diagnostic kit includes an immunoassay. Although the details of the immunoassay may vary depending on the particular format used, the method of detecting CD200R1 in a biological sample generally includes the steps of contacting the biological sample with an anti-CD200R1 antibody. The antibody is allowed to specifically bind under immunoreactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0421] The antibodies disclosed herein can also be used in immunoassays, such as but not limited to radioimmunoassay (RIA), ELISA or immunohistochemical assays. The antibodies can also be used in fluorescence activated cell sorting (FACS). FACS uses multiple color channels, low angle and obtuse angle light scattering detection channels and impedance channels, as well as other more complex detection levels, to separate or sort cells (see U.S. Patent No. 5,061,620). As disclosed herein, any monoclonal antibody that binds to CD200R1 can be used in these assays. Therefore, the antibodies can be used in conventional immunoassays, including but not limited to ELISA, RIA, FACS, tissue immunohistochemistry, Western blotting or immunoprecipitation.

[0422] Example

[0423] The present invention is further illustrated by the following examples, which are not intended to limit the present invention. The experimental steps without specified conditions in the following examples were carried out according to conventional steps and conditions, or according to the instructions.

[0424] Example 1. Evaluation of CD200 and CD200R1 expression on tumor infiltrating lymphocytes (TILs)

[0425] CD200 and CD200R1 have been shown to be upregulated in the tumor microenvironment of various cancers, which may contribute to immunosuppressive functions (Pathobiology 2021;88:218–227; Cancers (Basel). 2021 Mar;13(5):1024). The expression of CD200R1 and PD1 on tumor-infiltrating lymphocytes (TILs) from patients with renal cell carcinoma (RCC) and PBMCs from healthy donors was analyzed. Briefly, human blood samples from healthy donors were purchased from Research Blood Components (Watertown, MA). PBMCs were isolated by using Lymphoprep (STEMCELL). Fresh tumor tissues from cancer patients were provided by MTGroup biospecimens (Los Angeles, CA). Tumor tissues were dissociated and digested in 500 μg / ml DNase (Roche), 1 mg / ml Dispase (Gibco Life Tech), and 1 mg / ml Collagenase P (Roche). Single cell suspensions were collected, washed, and used for analysis. The expression of CD200, CD200R1, and PD1 was analyzed by FACS staining. PBMC or tumor single cell suspensions were stained with CD200 antibodies (BioLegend), CD200R1 antibodies (BioLegend), and PD1 antibodies (BioLegend) and analyzed. The CD45 positive cell population in the tumor single cell suspension was identified as TIL.

[0426] For PBMCs from healthy donors, CD200R1 expression levels were low on T cells and non-T cells from healthy PBMCs. For tumor-infiltrating lymphocytes (TILs) from patients with renal cell carcinoma, CD200R1 expression was highly upregulated on T cells from the tumor microenvironment, especially on PD1+ T cells ( Figure 1 A); CD200R1 is also upregulated in non-T cell populations ( Figure 1 A); In addition, CD200 expression is also upregulated on T cells (especially on PD1+ T cells). ( Figure 1 B).

[0427] Example 2. Analysis of CD200R1 expression in PD1-treated mice

[0428] The HKP1 (KrasG12Dp53- / -) orthotopic, immunocompetent, syngeneic preclinical model of non-small cell lung cancer (NSCLC) was used to explore the therapeutic efficacy of PD-1 / PD-L1 inhibition. CD4 and CD8 lung tumor infiltrating lymphocytes were obtained from C57Bl / 6 mice treated with IgG antibody (IgG2a, as control) or anti-PD1 antibody on day 14, day 17, or day 24 and sorted into RLT lysis buffer for mRNA sequencing (as shown in JCI Insight. 2018 Jul 12; 3(13): e96836.). mRNA sequencing data were downloaded from NCBI (GSE114300).

[0429] To investigate the association between CD200R1 expression and the therapeutic effect of anti-PD1 therapy, the expression of gene cd200r1 was analyzed in the IgG2a treatment group with tumor growth progression, the anti-PD1 treatment group with tumor regression, the anti-PD1 treatment group with partial tumor regression, and the anti-PD1 treatment group with tumor growth progression. The results showed that the expression of cd200r1 gene (mRNA) on CD4+ and CD8+ T cells in the PD1-progression group was upregulated ( Figure 2 ).

[0430] Example 3. Generation of anti-CD200R1 antibody molecules

[0431] 3.1 Immunization of mice

[0432] The Harbour H2L2 mice of the present invention (obtained from Harbour Antibodies BV) are transgenic mice carrying a human immunoglobulin immune spectrum, and the antibodies produced by the transgenic mice have fully human sequences. The Harbour H2L2 mice were immunized multiple times using a soluble recombinant human CD200R1 isoform a (CD200R1a)-Fc fusion protein (SEQ ID NO: 178) as an antigen. The antigen protein is mixed with an immune adjuvant to form an immunogenic agent, which is then injected subcutaneously or intraperitoneally via the groin.

[0433] In each round of immunization, each mouse received a total injection dose of 100 μL. In the first round of immunization, each mouse received an immunization of an immunogenic reagent, which was prepared by mixing 50 μg of antigenic protein with complete Freund's adjuvant (Sigma, #F5881) in a volume ratio of 1:1. In each subsequent round of booster immunization, each mouse received an immunization of an immunogenic reagent, which was prepared by mixing 25 μg of antigenic protein with Sigma Adjuvant System adjuvant (Sigma, #S6322). Typically, there are 6 to 7 rounds of booster immunization. Immunization was performed on day 0, day 14, day 28, day 42, day 56, day 70, day 84, and day 98; and antibody titers in mouse serum were measured on day 49 and day 77. Five days after the last round of booster immunization, spleen B cells were isolated from Harbour H2L2 mice.

[0434] 3.2 Serum titer determination

[0435] At the above-mentioned specific time points (day 49 and day 77), the mouse sera were collected, and the titer of antibodies binding to CD200R1 in the sera was determined by the FACS method.

[0436] cDNA encoding human CD200R1 isoform a (CD200R1a) (NP_620161.1, SEQ ID NO: 172) or isoform d (NP_740750.1, SEQ ID NO: 173) was obtained by gene synthesis and cloned into the lentiviral vector pGWLV11 (AZENTA Life sciences). Lentiviral particles were produced and then transfected into CHOK1 cells to generate CHOK1 / hCD200R1a cells and CHOK1 / hCD200R1d cells, which highly express human CD200R1 isoform a (CD200R1a) or isoform d (AZENTA Life sciences), respectively.

[0437] Serially diluted mouse serum was incubated with CHOK1-hCD200R1a cells at 4°C for 1 hour; after washing the cells twice, secondary anti-mouse IgG (H+L) (Life technologies, A11006) was added and incubated at 4°C for 1 hour, and then the resulting cells were washed twice, resuspended, and detected by flow cytometry (BD, CantoII). CHOK1 cells were used as background controls. Immune mice with high serum titers against CD200R1 were selected.

[0438] 3.3 Screening of anti-CD200R1 antibodies using hybridoma technology

[0439] cDNA encoding cynomolgus monkey CD200R1a (NP_001305106.1, SEQ ID NO: 175) was obtained by gene synthesis and cloned into the lentiviral vector pGWLV11 (AZENTA Life sciences). Lentiviral particles were produced and then transfected into CHOK1 cells to generate CHOK1 / cynoCD200R1a cells, which highly express cynomolgus monkey CD200R1 isoform a (AZENTA Life sciences).

[0440] The selected immunized mice with high serum titers of anti-CD200R1 were used for the final round of immunization and then sacrificed. Splenocytes and SP2 / 0 myeloma cells (ATCC, CRL-1581) were electrofused at a cell ratio of 4:1, and the electrofusion parameters were as follows: V1: 50 V, t1: 15 s, V2: 600 V, t2: 20 μs, t3: 0.5 s, n: 1, t4: 7 s, V+ / -: +, and attenuation: on. The cells were resuspended in DMEM medium containing 20% ​​FBS and HT and cultured at 1×10 5 Cells / 100 μL / well were inoculated. After 24 hours, DMEM containing 20% ​​FBS and 2×HT was added at 100 μL / well for further culture. The supernatant was then collected and the antibody titer was detected. Usually, 9 to 15 days after fusion, hybridoma supernatants derived from mouse spleen cells immunized with recombinant human CD200R1-his protein were collected and preliminarily screened by ELISA to detect binding to recombinant human CD200R1-his protein (SinoBiological, Cat: 11218-H08H). Positive clones were then further verified by FACS, and the binding ability to CHOK1 / hCD200R1a and CHOK1 / cynoCD200R1a was detected.

[0441] In addition, the blocking effect of positive clones on the binding of human CD200 to CHOK1 / hCD200R1a was tested by FACS method. Positive wells with blocking effect were further subcloned by limiting dilution method and further screened by ELISA and FACS. Clones with significant binding to human and cynomolgus CD200R1a were selected for sequencing.

[0442] In this example, the variable domain sequences of the anti-CD200R1 monoclonal antibody molecules obtained from immunized Harbour H2L2 mice are human antibody sequences.

[0443] 3.4 Preparation and purification of fully human recombinant IgG1 antibodies

[0444] After obtaining the sequences encoding the light chain variable region and the heavy chain variable region of each screened antibody molecule, the nucleic acid sequences encoding the light chain variable region and the heavy chain variable region of each antibody molecule are fused with the nucleic acid sequences encoding the light chain constant domain and the heavy chain constant domain of a human antibody, and expressed by recombinant DNA technology to obtain recombinant antibody molecules.

[0445] Specifically, the nucleic acid sequence encoding the heavy chain variable region (VH) of the antibody is gene synthesized and cloned into a mammalian cell expression plasmid vector (pTT5 mammalian expression vector), which contains a nucleic acid sequence encoding the heavy chain constant domain of the human IgG1 antibody to encode the full-length heavy chain. The nucleic acid sequence encoding the light chain variable domain (VL) of the antibody is gene synthesized and cloned into a mammalian cell expression plasmid vector (pTT5 mammalian expression vector), which contains a nucleic acid sequence encoding the light chain constant region of the human Igκ antibody to encode the full-length light chain.

[0446] The plasmid encoding the antibody heavy chain and the plasmid encoding the antibody light chain are simultaneously transfected into human embryonic kidney cells HEK293, and the purified recombinant anti-CD200R1 antibody with the light chain and heavy chain correctly assembled in pairs can be obtained through conventional recombinant protein expression and purification techniques.

[0447] Specifically, in FreeStyle TM HEK293 cells were propagated in F17 expression medium (Thermo, #A1383504). Before transient transfection, cells were adjusted to a concentration of 6 × 10 5 cells / mL to 8×10 5 cells / mL and cultured in a shaker at 37°C with 8% CO2 for 24 hours until the concentration reached approximately 1.2 × 10 6 Cells / mL. Take 30mL of cultured cells. The above-mentioned plasmid containing the nucleic acid sequence encoding the heavy chain of the antibody and the plasmid containing the nucleic acid sequence encoding the light chain of the antibody are mixed in a ratio of 2:3, and a total of 30μg of plasmid is dissolved in 1.5mL Opti-MEM reduced serum medium (Thermo, 31985088), and the medium is filtered through a 0.22μm filter for sterilization. Then, 1.5mL Opti-MEM is mixed with 120μL 1mg / mL PEI (Polysciences, Inc., #23966-2) and allowed to stand for 5 minutes. PEI is slowly added to the plasmid and incubated at room temperature for 10 minutes. The mixed solution of plasmid and PEI is slowly dripped into the culture bottle while shaking, and cultured in a shaker at 37°C with 8% CO2 for 5 days. Cell viability is measured after 5 days. The culture is collected and centrifuged at 3300g for 10 minutes, and then the supernatant is collected and centrifuged at high speed to remove impurities. The cells containing MabSelect TMThe column was loaded with a gravity column (Bio-Rad, #7311550) (GE Healthcare Life Science, #71-5020-91AE) and equilibrated with PBS (pH 7.4) and rinsed with 2 to 5 column volumes of PBS. The supernatant sample was loaded into the column and rinsed with 5 to 10 column volumes of PBS, followed by elution of the target protein with 0.1 M glycine at pH 3.5. The eluate was adjusted to neutral with Tris-HCl at pH 8.0, and the concentrate and buffer were exchanged into PBS buffer using an ultrafiltration tube (Millipore, UFC901024) to obtain a purified solution of the anti-CD200R1 antibody. Finally, the purified solution was purified by using a NanoDrop (Thermo Scientific TM NanoDrop TM One) to determine the concentration of the purified solution, and then the solution was aliquoted and stored for future use.

[0448] 3.5 Sequence of fully human recombinant anti-CD200R1 antibody

[0449] After screening and sequencing, the amino acid sequences of the light chain variable region and heavy chain variable region, light chain, heavy chain and CDR of the CD200R1 antibody in the present application defined according to the Chothia scheme are listed in Table 4.

[0450] The reference antibody PR002906 used in each example of the present application is the corresponding recombinant IgG1 antibody of huDX182. The sequence of huDX182 is derived from patent application number US8212008B2. The heavy chain sequence and light chain sequence of PR002906 are shown in SEQ ID NO: 139 and SEQ ID NO: 156, respectively.

[0451] The reference antibody PR002903 used in each example of the present application is the corresponding recombinant antibody of hB7V3V2-hG2G4. The sequence of hB7V3V2-hG2G4 is derived from patent application number US8075884B2. The heavy chain sequence and light chain sequence of PR002903 are shown in SEQ ID NO: 138 and SEQ ID NO: 155, respectively.

[0452] The reference antibody PR006207 used in each embodiment of the present application is the corresponding recombinant antibody of I-4P. The sequence of I-4P is derived from patent application number WO2020055943A1. The heavy chain sequence and light chain sequence of PR006207 are shown in SEQ ID NO: 144 and SEQ ID NO: 160, respectively.

[0453] The reference antibody PR200691 used in various embodiments of the present application is derived from patent application number WO2021096753A1. The heavy chain sequence and light chain sequence of PR200691 are shown in SEQ ID NO: 181 and SEQ ID NO: 182, respectively.

[0454] The reference antibody PR200526 (ie, IgG1) was used as a negative control, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 179, and the amino acid sequence of the light chain is shown in SEQ ID NO: 180.

[0455] Table 4. Sequence numbering of anti-CD200R1 antibodies

[0456] Antibody ID Light chain Heavy chain V L VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 PR002903 155 138 121 106 60 74 89 8 23 42 PR002906 156 139 122 107 61 75 90 9 24 43 PR006207 160 144 126 112 63 77 94 12 27 47 PR005474 157 140 123 108 62 76 91 10 25 44 PR005509 158 141 124 109 62 76 92 11 26 45 PR005512 159 142 125 110 62 76 93 11 25 45 PR005514 158 143 124 111 62 76 92 11 26 46 PR006475 161 145 127 113 64 78 95 13 28 48 PR006480 162 146 128 114 65 79 96 11 29 49

[0457] Example 4. Measurement of the binding affinity of anti-CD200R1 antibodies to CD200R1a

[0458] To determine the binding affinity of anti-CD200R1 antibodies to CD200R1a proteins of various species (mouse, human, cynomolgus monkey), we used The RED96e instrument was used for biomembrane interferometry (BLI) assay. Antibodies were prepared as described in Example 3.4 and diluted to 5 μg / mL using freshly prepared 1× kinetic buffer (10× kinetic buffer (Catalog No. 18-1105, ForteBio) with PBS (Catalog No. E607016-0500, BBI Life Sciences)) and captured on the surface of anti-human Fc (AHC) Octet biosensor (Catalog No. 18-5060, ForteBio) to achieve a capture level between 0.9 nm and 1.2 nm. The biosensor with captured anti-CD200R1 antibody was then immersed in a well containing a 2-fold serial dilution of CD200R1a protein to detect the association signal, followed by a dissociation step in a well containing 1× kinetic buffer. The association phase and dissociation phase are shown in Table 5. The sensorgram was recorded and the reference signal was subtracted before curve fitting using ForteBio Data Analysis 11.0 software. The association rates (k) were calculated using a simple one-to-one Langmuir binding model. on ) and dissociation rate (k off ). The equilibrium dissociation constant (K D ) is calculated as k off / k on The binding affinity of human CD200R1a to human CD200 was determined using the same protocol using Fc-tagged human CD200 protein instead of anti-CD200R1 antibody and using 2-fold serial dilutions of human CD200R1a protein.

[0459] All screened antibodies (PR005474, PR005509, PR005512, PR005514, PR006475 and PR006480) do not bind to mouse CD200R1 (data not shown). The data of the binding affinity of anti-CD200R1 antibodies to human CD200R1a and cynomolgus CD200R1a are summarized in Table 5 below. As shown in Table 5, antibody clones PR005474, PR005509, PR005512, PR005514, PR006475 and PR006480 have higher binding affinity to human CD200R1a than hCD200 or reference antibody PR002906. Antibodies PR005474, PR005509, PR005512 and PR006480 had higher binding affinity to cynomolgus monkey CD200R1a than hCD200 or the reference antibody PR002906.

[0460] Table 5. Binding affinity of anti-CD200R1 antibodies to human or cynomolgus monkey CD200R1a

[0461]

[0462] Example 5. Detection of the binding of anti-CD200R1 antibodies to CHOK1 / hCD200R1a and CHOK1 / cynoCD200R1a by FACS

[0463] This example aims to study the in vitro binding activity of human anti-CD200R1 monoclonal antibodies to human / cynomolgus CD200R1a expressed on the cell surface. Anti-CD200R1 antibodies were prepared as described in Example 3.4, and cell-level binding experiments were performed using CHOK1 / hCD200R1a and CHOK1-cynoCD200R1a.

[0464] Briefly, CHOK1 / hCD200R1a cells and CHOK1-cynoCD200R1a cells were digested and collected, and then resuspended in PBS containing 2% BSA. The cell density was adjusted to 1 × 10 6Cells / mL. The cells were inoculated in 96-well V-bottom plates (Corning, #3894) at 100 μL / well, followed by addition of test antibodies and reference antibodies at serial dilution concentrations, each at 100 μL / well. The cells were incubated in the dark for 1 hour at 4 °C. Afterwards, the cells in each well were rinsed twice with 100 μL of precooled PBS containing 2% BSA, and centrifuged at 500 g for 5 minutes at 4 °C, and the supernatant was discarded. 100 μL of fluorescent secondary antibody (AffiniPure goat anti-human IgG conjugated to Alexa Fluor 488, Fcγ fragment specific, Jackson, #109-545-098, diluted at a ratio of 1:1000) was added to each well, and the plate was incubated in the dark for 1 hour at 4 °C. The cells in each well were rinsed twice with 100 μL of precooled PBS containing 2% BSA, and centrifuged at 500 g for 5 minutes at 4 °C, and the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL of pre-chilled PBS containing 2% BSA and the fluorescence signal values ​​were read using a BD CantoII flow cytometer.

[0465] The results of antibody binding to CHOK1 / hCD200R1a are shown in Figure 3 ( Figure 3 A to Figure 3 C). Compared with the reference antibody PR002906, the anti-CD200R1 antibodies PR005474, PR005509, PR005512, PR005514 and PR006475 have stronger binding activity to human CD200R1a expressed on the surface of CHOK1 cell line in vitro. The results of antibody binding to CHOK1 / cynoCD200R1a are shown in Figure 4 ( Figure 4 A to Figure 4 C). Compared with the reference antibody PR002906, the anti-CD200R1 antibodies PR005474, PR005509, PR005512, PR005514, PR006475 and PR006480 showed stronger cross-binding activity to cynomolgus monkey CD200R1a expressed on the surface of CHOK1 cell line in vitro.

[0466] Example 6. Evaluation of the blocking effect of anti-CD200R1 antibodies on the binding of human CD200 protein to CHOK1 / hCD200R1a

[0467] To investigate the activity of human anti-CD200R1 antibodies in blocking the binding of human CD200 to its receptor CD200R1a in vitro, human CD200 / human CD200R1a binding and blocking experiments at the cellular level were performed using CHOK1 / hCD200R1a.

[0468] Briefly, CHOK1 / hCD200R1a was digested and resuspended in PBS containing 2% BSA. The cell density was adjusted to 1 × 10 6 Cells / mL, cells were seeded in 96-well V-bottom plates (Corning, catalog number: 3894) at 100 μL cells / well, followed by addition of serial dilutions of test antibody and reference antibody, each at 100 μL / well, with hIgG1 used as a control. The cells were placed at 4°C and incubated in the dark for 1 hour. Afterwards, centrifuged at 4°C for 5 minutes, the supernatant was discarded, and then 1 μg / mL biotin-labeled human CD200 protein (Acro Biosystems, B77-H82F5) was added at 50 μL / well, and incubated in the dark at 4°C for 30 minutes. 100 μL / well of pre-cooled PBS containing 2% BSA was added, the cells were rinsed twice, centrifuged at 500g for 5 minutes at 4°C, and the supernatant was discarded. Fluorescent secondary antibody (PE streptavidin, BD, catalog number: 554061, 1:200) was added at 100 μL / well and incubated in the dark at 4°C for 30 minutes. The cells were washed twice with 200 μL / well of pre-cooled PBS and centrifuged at 500g for 5 minutes at 4°C, and the supernatant was discarded. Finally, the cells were resuspended with 200 μL / well of pre-cooled PBS and the fluorescence signal value was read with an ACEA Novocyte 3000 flow cytometer. The IC50 value was calculated.

[0469] The results are shown in Figure 5 Compared with PR006475 and the reference antibody PR002906, the antibodies PR005474, PR005509, PR005512, PR005514 and PR006480 of the present invention showed excellent blocking efficiency.

[0470] Example 7. Evaluation of Anti-CD200R1 Antibodies for Inhibition of CD200-Mediated CD200R1a Signaling

[0471] Cells expressing CD200R1a Jurkat CD200R1a signaling cell line was purchased from Eurofins DiscoverX Products LLC (Catalog No. 93-1136C19) and used to detect the interaction between human CD200 and its receptor CD200R1. Binding of CD200R1a expressed on Jurkat CD200R1 signaling cells) results in the assembly of β-galactosidase and subsequent luminescent readout. Blocking of the CD200-CD200R1a interaction by anti-CD200R1 antibodies will reduce or eliminate the luminescent signal.

[0472] The cDNA encoding human CD200 (NP_005935.4, SEQ ID NO: 185) was obtained by gene synthesis and cloned into the lentiviral vector pGWLV11 (AZENTA Life sciences). Lentiviral particles were produced and transfected into CHOK1 cells to construct CHOK1 / huCD200 cells, which highly express human CD200 (AZENTA Life sciences).

[0473] Specifically, collect Jurkat CD200R1 signaling cells were used as 8×10 5 The CHOK1 / hCD200 cells were digested and plated at 2×10 6 100 μl / well of anti-CD200R1 antibody (antibodies PR005474, PR005509, PR005512, PR005514, PR006475 and PR006480 of the present invention, and reference antibody PR002903, and control antibody IgG1) were serially diluted and added to the plate at 100 μl / well. The plate was then incubated at room temperature for 2 hours. After incubation, the plate was added Flash Detection Kit reagent was added and incubated for another 30 minutes. Luminescence was measured on an Envision microplate reader (PerkinElmer, model 2105).

[0474] The results are shown in Figure 6 ( Figure 6 A and Figure 6 In B). Antibodies PR005474, PR005509, PR005512, PR005514 and PR006480 showed superior blocking efficiency compared to the reference anti-CD200 antibody PR002903.

[0475] Example 8. Maturation of humanized anti-CD200R1 antibodies PR006480 and PR005514

[0476] 8.1 Ablation of ADCC effector function by Fc region mutation

[0477] Gene synthesis encoding antibody heavy chain variable region (VH) nucleic acid sequence, and cloned into pTT5 mammalian cell expression vector, the vector contains the nucleic acid sequence encoding the heavy chain constant domain of human IgG1 antibody, and L234A, L235A and G237A mutations (according to EU numbering, leucine is replaced by alanine at positions 234 and 235, and glycine is replaced by alanine at position 237) are introduced into the CH2 region of the IgG1 heavy chain constant region to eliminate ADCC effector function. Then, the plasmid encoding the antibody heavy chain and the plasmid encoding the antibody light chain are transfected into CHOK1 or HEK 293 mammalian host cell lines by the method described in Example 3.4, respectively, to obtain recombinant antibody protein.

[0478] The antibody sequences of PR006895 and PR006839 derived from PR006480 and PR005514, respectively, are listed in Table 6.

[0479] Table 6. Pass PR0 0648 Antibodies obtained by Fc mutation of PR005514

[0480]

[0481]

[0482] 8.2 Antibody Sequence Analysis and Optimization

[0483] In this example, amino acid mutations were introduced into potential post-translational modification (PTM) sites in the antibody PR006895 and PR006839 sequences to obtain new antibody molecules (referred to as PTM variants). According to the Chothia protocol definition, the amino acid sequences of the light chain variable domain and heavy chain variable domain, light chain, heavy chain (human IgG1) and CDR of the PTM variants in this example are listed in Table 7. All designed PTM variants were purified by the method described in Example 3.4 to obtain purified recombinant antibodies, and further verified in subsequent functional experiments.

[0484] Table 7. Antibodies obtained after PTM mutation

[0485]

[0486] Example 9. Measurement of the binding affinity of PTM variants derived from PR006480 and PR005514 to CD200R1a

[0487] The method for determining the binding affinity of PTM variants derived from PR006480 and PR005514 to human CD200R1a and cynomolgus CD200R1a recombinant proteins was similar to that in Example 4. The data of the binding affinity of anti-CD200R1 antibodies to human CD200R1a and cynomolgus CD200R1a are summarized in Table 8. The results showed that PR007223, PR007973 and PR007221 had high binding affinity to human CD200R1a and cynomolgus CD200R1a, and had no binding to mouse CD200R1.

[0488] Table 8. Binding affinity of variants derived from PR006480 and PR005514

[0489]

[0490]

[0491] Example 10. Evaluation of Binding Epitope Competition of PTM Variants Derived from PR006480 and PR005514

[0492] To determine whether anti-CD200R1 antibodies bind to human CD200R1a at different or similar binding epitopes, ForteBio The RED96e platform was used to perform epitope competition experiments with anti-CD200R1 antibodies. Human CD200R1a protein was diluted to 3 μg / ml with kinetic buffer (10× kinetic buffer (Catalog No. 18-1105, ForteBio)) and then loaded onto an anti-Penta HIS biosensor (Catalog No. 18-5120, ForteBio) to achieve a capture level of 0.3 nm. A tandem competition assay format was used, and it included two association steps. First, the biosensor loaded with the antigen was bound to each antibody (i.e., the first antibody, the 1st Ab) at a saturation concentration of 400 nM for 300 seconds to reach equilibrium, and then bound to 400 nM of a competitive antibody (i.e., the second antibody, the 2nd Ab) for 300 seconds. When the first antibody was replaced by the dynamic buffer, the second binding signal was recorded as 100% signal for each antibody. All binding data were analyzed using ForteBio Data Analysis 11.0 software. The inhibition rate was calculated by the following formula:

[0493] Inhibition rate (%) = (AB) / A*100,

[0494] "A" represents 100% signal for each antibody; and

[0495] "B" represents the signal from the secondary antibody binding step.

[0496] If the inhibition rate obtained is greater than 80(%), it means that the epitopes of the two antibodies completely overlap; if the inhibition rate is less than 40(%), it means that the epitopes of the two antibodies are different or far away from each other.

[0497] The results in Table 9 show that antibodies PR007223, PR007973 and PR007221 compete significantly with each other, indicating that they may share the same binding epitope of human CD200R1a. In addition, whether it is the first Ab or the second Ab, the reference antibody PR006207 does not block the antigen binding of other antibodies, indicating that PR006207 binds to an epitope of human CD200R1a that is different from the epitope bound by PR007223, PR007973 and PR007221.

[0498] Table 9. Inhibition rate of antibodies in epitope competition assay

[0499]

[0500]

[0501] Example 11. Detection of binding of anti-CD200R1 antibody variants to CHOK1 / hCD200R1a and CHOK1-cynoCD200R1a by FACS

[0502] This example aims to study the in vitro binding activity of human anti-CD200R1 monoclonal antibodies to human / cynomolgus CD200R1a, which have been engineered to eliminate ADCC effector function and remove PTM. Antibody binding experiments at the cell level are as described in Example 5.

[0503] The results of binding of anti-CD200R1 antibody to CHOK1 / hCD200R1a cells are shown in Figure 7 ( Figure 7 A and Figure 7 B). Variant antibodies PR007221, PR007223, PR007442 and PR007444 have stronger binding activity than reference antibody PR002906, and have comparable or stronger binding activity than their parent antibodies, i.e., PR006480 and PR006895. Variant antibodies PR007630, PR007631, PR007972, PR007973 and PR007974 have stronger binding activity than reference antibody PR002906, and have comparable or stronger binding activity than their parent antibodies, i.e., PR005514 and PR006839.

[0504] The results of binding of these antibodies to CHOK1 / cynoCD200R1a are shown in Figure 8 ( Figure 8 A and Figure 8 B). Compared with the reference antibody PR002906, variant antibodies PR007221, PR007223, PR007442, PR007444, PR007630, PR007631, PR007972, PR007973 and PR007974 showed better cross-binding activity to cynomolgus monkey CD200R1a. Variant antibodies PR007221, PR007223, PR007442 and PR007444 have stronger binding activity than their parent antibodies, i.e., PR006480 and PR006895. Variant antibodies PR007630, PR007631, PR007972, PR007973 and PR007974 have comparable binding activity compared to their parent antibodies, i.e., PR005514 and PR006839.

[0505] Example 12. Evaluation of the blocking effect of variants of CD200R1 antibodies on the binding of human CD200 protein to CHOK1 / hCD200R1a

[0506] To investigate the activity of human anti-CD200R1 antibodies in blocking the binding of human CD200 to its receptor CD200R1a in vitro, the antibody blocking assay described in Example 6 was performed at the cellular level using antibody variants engineered to abolish ADCC effector function and remove PTMs.

[0507] Fig. 9 ( Fig. 9 A and Fig. 9 The results in B) show that the variant antibodies PR007221, PR007223, PR007442, PR007444, PR007630, PR007631, PR007972, PR007973 and PR007974 showed better blocking activity compared to the reference antibody PR002906.

[0508] Example 13. Evaluation of variant anti-CD200R1 antibodies for inhibition of CD200-mediated CD200R1a signaling

[0509] As described in Example 7, The antagonistic activity of anti-CD200R1 antibodies was investigated using Jurkat CD200R1a reporter cells. The results are shown in Fig.10 ( Fig.10 A to Fig.10 D) in.

[0510] As shown in the results, the variant antibodies PR007221, PR007223, PR007442, PR007444, PR007630, PR007631, PR007972, PR007973 and PR007974 showed superior blocking activity compared to the reference antibody PR002903 and its parent antibodies PR005514 and PR006839, PR006480 and PR006895, respectively.

[0511] Example 14. Evaluation of variants of anti-CD200R1 antibodies for activation of CD200R1a signaling

[0512] This embodiment aims to use Jurkat CD200R1 reporter cells were used to investigate whether the variant anti-CD200R1 antibodies of the present invention possess any agonistic activity.

[0513] In short, collect Jurkat CD200R1 cells were collected at 8×10 5 The cells were resuspended at 2×10 cells / ml and then seeded into VieePlate 96-well plates (PerkinElmer, catalog number 6005181) at 25 μL cells / well. CHOK1-hCD32b (BPS Biosciences, catalog number 79511, FcGR2B-CHO K1) cells were digested and plated at 2×10 6 Resuspend at 100 cells / ml and seed at 25 μL cells / well. Serially dilute the anti-CD200R1 antibody and add 100 μl / well to the plate. PR200526 was used as a negative control. The plate was then incubated at room temperature for 2 hours. After incubation, add Flash Detection Kit reagent was added and incubated for another 30 minutes. Luminescence was measured on an Envision microplate reader (PerkinElmer, model 2105).

[0514] Fig.11 ( Fig.11 A and Fig.11 The results of B) showed that compared with the reference antibody PR002906, the parent antibodies PR6480, PR005514 and the variant antibodies PR006895, PR007221, PR007442, PR007223, PR007444, PR006839, PR007630, PR007631, PR007972 and PR007973 did not show any agonistic activity.

[0515] Example 15. Evaluation of cross-reactivity of anti-CD200R1 antibodies with CD200R1L

[0516] To test whether the antibodies had cross-reactivity with CD200R1L, the antibodies were tested for binding to CD200R1L.

[0517] Specifically, the cell surface glycoprotein CD200 receptor 2 isoform 1 precursor CD200R1L recombinant protein (SinoBiologicals, 11620-H08H, SEQ ID NO: 174) was diluted to 1 μg / mL with PBS, added to a 96-well plate (Corning, #9018) at 100 μl / well, and incubated overnight at 4°C. After discarding the liquid, the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20), 250 μl of 2% BSA blocking solution was added to the plate, and incubated at 37°C for 1 hour. The blocking solution was discarded, and the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% Tween-20). The anti-CD200R1 antibody was diluted and added at 100 μl / well, incubated at 37°C for 1 hour, and the isotype antibody was used as a control. After washing the plate with PBST buffer (pH 7.4, containing 0.05% Tween-20) for 3 times, goat anti-human HRP secondary antibody (Invitrogen, #A18805) was diluted 5000 times, added to the plate, and incubated in the dark at 37°C for 1 hour. After washing the plate with PBST buffer (pH 7.4, containing 0.05% tween-20) for 3 times, 100 μl / well of TMB (Biopanda, #TMB-S-003) was added, and the plate was placed in the dark at room temperature for about 30 minutes; stop solution (BBI life sciences, #E661006-0200) was added to each well at 50 μl / well to stop the reaction, and the absorbance (OD450) at 450 nm was measured in a microplate reader (Perkin Elemer, #Enspire).

[0518] Fig.12 The results in indicated that the anti-CD200R antibodies PR007223, PR007973, and PR007221 did not cross-react with CD200R1L.

[0519] Example 16. Detection of the binding of anti-CD200R1 antibodies to CHOK1 / hCD200R1a and CHOK1 / hCD200R1d by FACS

[0520] This example aims to study the in vitro binding activity of variant anti-CD200R1 monoclonal antibodies to CHOK1 / hCD200R1a and CHOK1 / hCD200R1d. Briefly, CD200R1 antibodies (PR007973 and PR007223) and reference antibodies (PR200526 and PR200691) were labeled with Alexa Fluor 647 using a protein labeling kit (Invitrogen, A20173). CHOK1 / hCD200R1a cells and CHOK1 / hCD200R1d cells were digested and collected, and then these cells were resuspended in PBS containing 2% BSA, respectively. The cell density was adjusted to 1×10 6 The binding of anti-CD200R1 antibodies was analyzed as described in Example 5.

[0521] The results are shown in Fig.13 ( Fig.13 A and Fig.13 In B). PR007973 showed high binding activity to both CD200R1 isoform a and isoform d compared to the PR200691 reference antibody. PR007223 showed high binding activity to CD200R1 isoform a, but weaker binding to CD200R1 isoform d.

[0522] Example 17. Evaluation of the blocking effect of anti-CD200R1 antibodies on the binding of human CD200 protein to CHOK1 / hCD200R1a and CHOK1 / hCD200R1d

[0523] To investigate the activity of human anti-CD200R1 antibodies in blocking the binding of human CD200 to its receptors CD200R1a and CD200R1d in vitro, the cellular human CD200 / human CD200R1 binding and blocking experiments described in Example 6 were performed using CHOK1 / CD200R1a and CHOK1 / CD200R1d.

[0524] The results are shown in Fig.14 Compared to the PR200691 control antibody, both PR007223 and PR007973 showed excellent blocking activity.

[0525] Example 18. Evaluation of the binding of anti-CD200R1 antibodies to immune cells in the tumor microenvironment

[0526] To investigate the binding of anti-CD200R1 antibodies to tumor infiltrating lymphocytes (TILs), human CD200R1 antibodies were labeled with Alexa Fluor 647 using a protein labeling kit (Invitrogen, A20173). The binding of CD200R1 antibodies to PBMCs and dissociated renal cell carcinoma single cell suspensions was analyzed as described in Example 1.

[0527] The results are shown in Fig.15 Compared with the PR200691 reference antibody, PR007973 showed high binding affinity to PD1-positive T cells ( Fig.15 A), and excellent binding to PBMCs and CD11b-positive myeloid cells from patients with renal cell carcinoma ( Fig.15 B). PR007223 showed high binding affinity to PBMCs from healthy donors and bound to PD1-positive T cells and CD11b-positive myeloid cells from PBMCs and kidney cells at high concentrations (20 μg / ml).

[0528] Example 19. Functional activity of anti-CD200R1 antibodies on human primary cells

[0529] The functional activity of anti-CD200R1 antibodies in inhibiting CD200-CD200R1 signaling was tested by using an antigen recall assay and a mixed lymphocyte reaction assay (MLR).

[0530] In the antigen recall assay, PBMCs from four different healthy HLA-A2+ donors were isolated and incubated with 100ng / ml IL4, 100ng / ml IL10 and 100ng / ml M-CSF for 72 hours. The cells were washed and resuspended in culture medium. Anti-CD200R1 antibodies PR007223, PR000150 (anti-PD1 antibodies, pembrolizumab analogs, HC / LC SEQ ID NOs: 183 and 184, produced as described in Example 4) and isotype control PR200526 were added at a final concentration of 50nM. CEF peptides (JPT, PM-CEF-E) were added at a final concentration of 1μg / ml. The cells were cultured for another 7 days. The supernatant was collected and IL12 / IL23p40 was detected by MSD (Meso Scale Discovery, MSD QuickPlex SQ120).

[0531] In the MLR assay, monocytes are isolated from human PBMCs using a monocyte isolation kit (STEMCELL, catalog number 19359), and these cells are cultured for 6 days with 50ng / ml IL4 and 50ng / ml M-CSF. Cells are collected as single DCs. T cells are isolated from PBMCs from different healthy donors using a human T cell isolation kit (STEMCELL, catalog number 17951). Single DCs and T cells are mixed together, and anti-CD200R1 antibodies PR007973 and PR000150 are added at a final concentration of 50nM. Cells are cultured for another 4 days. Supernatants are collected and IFNg is detected by MSD (Meso Scale Discovery, MSDQuickPlex SQ120).

[0532] The results are shown in Fig.16 ( Fig.16 A and Fig.16 In B). In an antigen recall assay, the anti-CD200R1 antibody PR007223 greatly enhanced the production of IL12p40 compared to the isotype control. PR007973 greatly increased the production of IFNg in the presence of the anti-PD1 antibody PR000150.

[0533] Example 20. Evaluation of the anti-tumor efficacy of anti-CD200R1 antibodies in CD200-positive tumors

[0534] To investigate the anti-tumor activity of anti-CD200R1 antibodies, CD200-positive tumor cell lines in humanized NCG mice (Gempharmatech Co), human lung squamous cell line NCI-H226 (ATCC, CRL-5826), and human ovarian cancer cell line OVCAR3 (ATCC, HTB-161) CDX models were used for in vivo studies.

[0535] On the day of tumor cell inoculation, NCI-H226 tumor cells were resuspended in a mixture of PBS and Matrigel (1:1). Each NCG mouse was subcutaneously inoculated with 5×10 6 NCI-H226 cells. When the average tumor volume of the mice reached about 90mm 3 At 4 pm, the mice were randomly divided into 6 mice / group. Then, each mouse was intravenously inoculated with 5×10 6 The next day, anti-CD200R1 antibodies (PR005514 and PR006480), negative control antibody IgG1, and positive anti-CD200 antibody PR002903 were administered via the tail vein at a dose of 20 mg / kg twice a week for 4 weeks. Tumor volume and body weight were measured twice a week. Tumor volume was calculated according to the following formula:

[0536] Tumor volume (mm 3 ) = 0.5 × long diameter of tumor × short diameter of tumor 2 .

[0537] Fig.17 The anti-tumor effect of anti-CD200R1 antibody in the NCI-H226 CDX model of humanized NCG mice is shown. Compared with the positive control anti-CD200 antibody PR002903, which showed a 35.3% tumor growth inhibition (TGI) rate on day 20 after treatment, the anti-CD200R antibody PR005514 showed 45.2% TGI, and PR006480 showed 60.2% TGI.

[0538] In the second experiment, OVCAR3 cells were resuspended in a mixture of PBS and Matrigel (1:1). 5 × 10 cells were inoculated subcutaneously in each NCG mouse. 6 OVCAR3 cells. When the average tumor volume of mice reached about 90mm 3 At 4 pm, the mice were randomly divided into 6 mice / group. Then, each mouse was intravenously inoculated with 5×10 6 The next day, anti-CD200R1 antibody (PR006480) and negative control hIgG1 were administered via the tail vein at a dose of 20 mg / kg twice a week for 3 weeks. Tumor volume and body weight were measured twice a week. Tumor volume was calculated according to the following formula:

[0539] Tumor volume (mm 3 ) = 0.5 × long diameter of tumor × short diameter of tumor 2 .

[0540] Fig.18 The anti-tumor effect of anti-CD200R1 antibody in the OVCAR3 CDX model of humanized NCG mice was shown. The results showed that PR006480 showed a 42.4% tumor inhibition rate TGI on day 13 after treatment.

[0541] Example 21. Evaluation of the anti-tumor efficacy of anti-CD200R1 antibodies in CD200-negative tumors

[0542] To investigate the antitumor activity of anti-CD200R1 antibodies in CD200-negative tumor cell lines, the human lung cancer cell line NCI-H292 (ATCC, CRL-1848) and human breast cancer cell MDA-MB-231 (ATCC, HTB-26) CDX model in humanized NCG mice (Gempharmatech Co) were used for in vivo studies.

[0543] On the day of tumor cell inoculation, NCI-H292 tumor cells were resuspended in a mixture of PBS and Matrigel (1:1). Each NCG mouse was subcutaneously inoculated with 5×10 6 NCI-H292 cells. When the average tumor volume of mice was about 90mm 3 At 4 pm, the mice were randomly divided into 6 mice / group. Then, each mouse was intravenously inoculated with 5×10 6 The next day, anti-CD200R1 antibodies (PR005514, PR005512, PR005509, PBS as negative control) were administered via the tail vein at a dose of 20 mg / kg twice a week for 4 weeks. Tumor volume and body weight were measured twice a week. Tumor volume was calculated according to the following formula:

[0544] Tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 .

[0545] Fig.19 The anti-tumor effect of anti-CD200R1 antibody in the NCI-H292 CDX model of humanized NCG mice is shown. The study showed that 14 days after anti-CD200R antibody treatment, PR005514 showed a 56.2% TGI value; PR005512 showed a 44.5% TGI; and PR005509 showed a 53.6% TGI.

[0546] In the MDA-MB-231CDX model, MDA-MB-231 tumor cells were resuspended in a mixture of PBS and Matrigel (1:1). Each NCG mouse was subcutaneously inoculated with 5×10 6 MDA-MB-231 cells. The average tumor volume of the mice was about 90 mm 3 At 4 pm, the mice were randomly divided into 6 mice / group. Then, each mouse was intravenously inoculated with 5×10 6 The next day, anti-CD200R1 antibodies (i.e., antibodies PR006895 and PR007223 of the present invention and negative control antibody hIgG1) were administered via the tail vein at a dose of 20 mg / kg twice a week for 4 weeks. Tumor volume and body weight were measured twice a week. Tumor volume was calculated according to the following formula:

[0547] Tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 .

[0548] Fig. 20The anti-tumor effect of anti-CD200R1 antibody in the MDA-MB-231CDX model of humanized NCG mice was shown. It was shown that PR006895 showed a 55.5% tumor inhibition rate TGI and PR007223 showed a 55.0% TGI 17 days after anti-CD200 antibody treatment.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CD200R1, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), and wherein (1) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 117, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 136; or (2) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 111, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 124; or (3) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 117, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 133; or (4) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 118, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 134; or (5) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as set forth in SEQ ID NO: 119, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as set forth in SEQ ID NO: 135; or (6) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 120, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 137; or (7) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 108, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 123; or (8) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 109, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 124; or (9) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 110, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 125; or (10) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 114, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 128; or (11) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 115, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 129; or (12) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 116, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 130; or (13) the VH comprises HCDRs 1-3 of a VH having an amino acid sequence as shown in SEQ ID NO: 115, and the VL comprises LCDRs 1-3 of a VL having an amino acid sequence as shown in SEQ ID NO: 131; or (14) The VH comprises HCDR 1-3 of VH having the amino acid sequence shown in SEQ ID NO: 116, and the VL comprises LCDR 1-3 of VL having the amino acid sequence shown in SEQ ID NO:

132.

2. An antibody or antigen-binding fragment thereof that specifically binds to CD200R1, wherein the antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein (1) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 67, 80, 99, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 67, 80, 99, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 32, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 32, 46, respectively, by one or two amino acid residues; or (2) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 26, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 26, 46, respectively, by one or two amino acid residues; or (3) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 66, 80, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 66, 80, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 32, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 32, 46, respectively, by one or two amino acid residues; or (4) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 66, 81, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 66, 81, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 33, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 33, 46, respectively, by one or two amino acid residues; or (5) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 67, 76, 99, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 67, 76, 99, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 14, 32, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 14, 32, 46, respectively, by one or two amino acid residues; or (6) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 66, 80, 100, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 66, 80, 100, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 14, 33, 46, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 14, 33, 46, respectively, by one or two amino acid residues; or (7) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 91, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 91, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 10, 25, 44, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 10, 25, 44, respectively, by one or two amino acid residues; or (8) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 92, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 92, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 26, 45, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 26, 45, respectively, by one or two amino acid residues; or (9) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 62, 76, 93, respectively, or LCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 62, 76, 93, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 25, 45, respectively, or HCDR 1-3 having amino acid sequences that differ from the sequences of SEQ ID NOs: 11, 25, 45, respectively, by one or two amino acid residues; or (10) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 96, respectively, or LCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 65, 79, 96, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 29, 49, respectively, or HCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 11, 29, 49, respectively; or (11) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 97, respectively, or LCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 65, 79, 97, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 30, 49, respectively, or HCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 11, 30, 49, respectively; or (12) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 98, respectively, or LCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 65, 79, 98, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 31, 50, respectively, or HCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 11, 31, 50, respectively; or (13) the VL comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 65, 79, 97, respectively, or LCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 65, 79, 97, respectively, by one or two amino acid residues, and the VH comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 11, 30, 49, respectively, or HCDR 1-3 having amino acid sequences that differ from those of SEQ ID NOs: 11, 30, 49, respectively; or (14) The VL comprises LCDR 1-3 having the amino acid sequences shown in SEQ ID NOs: 65, 79, and 98, respectively, or LCDR1-3 having the amino acid sequences that differ from the sequences shown in SEQ ID NOs: 65, 79, and 98 by one or two amino acid residues, respectively, and the VH comprises HCDR 1-3 having the amino acid sequences shown in SEQ ID NOs: 11, 31, and 50, respectively, or HCDR1-3 having the amino acid sequences that differ from the sequences shown in SEQ ID NOs: 11, 31, and 50, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein (1) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 136, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 117; or (2) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 124, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 111; or (3) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 133, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 117; or (4) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 134, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 118; or (5) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 135, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 119; or (6) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 137, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 120; or (7) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 123, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 108; or (8) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 124, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 109; or (9) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 125, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 110; or (10) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 128, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 114; or (11) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 129, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 115; or (12) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 130, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 116; or (13) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 131, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 115; or (14) The VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 132, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No:

116.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a heavy chain (HC) and a light chain (LC), and wherein (1) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 170, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 151; or (2) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 158, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 143; or (3) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 158, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 147; or (4) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 167, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 151; or (5) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 168, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 152; or (6) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 169, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 153; or (7) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 171, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 154; or (8) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 157, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 140; or (9) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 158, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 141; or (10) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 159, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 142; or (11) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 162, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 146; or (12) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 162, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 148; or (13) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 163, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 149; or (14) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 164, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 150; or (15) the LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 165, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No: 149; or (16) The LC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID No: 166, and the HC comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID No:

150.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is of an isotype selected from IgG, IgA, IgM, IgE and IgD.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is of a subtype selected from IgG1, IgG2, IgG3 and IgG4.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, ds-scFv, dAb and sdAb.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is linked to a fluorescent label, a radioactive label or a cytotoxic agent. 11 . A bispecific antibody, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and a second antigen-binding region that specifically binds to a tumor-associated antigen or an immune checkpoint molecule. 12 . A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to claim 1 or the bispecific antibody according to claim 11 . A vector comprising the nucleic acid according to claim 12 .

14. A host cell comprising the nucleic acid according to claim 12 or the vector according to claim 13. 15 . An antibody drug conjugate (ADC), comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the bispecific antibody according to claim 11 .

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the bispecific antibody according to claim 11, or the nucleic acid according to claim 12, or the vector according to claim 13, or the host cell according to claim 14, or the antibody-drug conjugate according to claim 15, and optionally a pharmaceutically acceptable carrier or excipient.

17. The pharmaceutical composition of claim 16, wherein the composition further comprises a second therapeutic agent selected from the group consisting of antibodies, chemotherapeutic agents, siRNA, antisense oligonucleotides, polypeptides and small molecule drugs.

18. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the bispecific antibody according to claim 11, or the nucleic acid according to claim 12, or the vector according to claim 13, or the host cell according to claim 14, the antibody-drug conjugate according to claim 15, or the pharmaceutical composition according to claim 16 or 17.

19. A method for activating T cells and / or myeloid cells in a cancer microenvironment in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the bispecific antibody according to claim 11, or the nucleic acid according to claim 12, or the vector according to claim 13, or the host cell according to claim 14, the antibody-drug conjugate according to claim 15, or the pharmaceutical composition according to claim 16 or 17.

20. The method of claim 18 or 19, wherein the cancer is selected from pancreatic ductal adenocarcinoma, prostate cancer, melanoma, liver cancer, breast cancer, lung cancer (e.g., lung squamous cell carcinoma), squamous cell carcinoma, ovarian cancer, leukemia, and myeloma.

21. The method of any one of claims 18 to 20, further comprising administering to the subject a second therapeutic agent, optionally the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, an siRNA, an antisense oligonucleotide, a polypeptide, and a small molecule drug.

22. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the bispecific antibody according to claim 11, or the nucleic acid according to claim 12, or the vector according to claim 13, or the host cell according to claim 14, the antibody-drug conjugate according to claim 15, or the pharmaceutical composition according to claim 16 or 17 in the preparation of a medicament for treating cancer in a subject.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the bispecific antibody according to claim 11, or the nucleic acid according to claim 12, or the vector according to claim 13, or the host cell according to claim 14, the antibody-drug conjugate according to claim 15, or the pharmaceutical composition according to claim 16 or 17 in the preparation of a medicament for activating T cells and / or myeloid cells in a cancer microenvironment in a subject.

24. The use according to claim 22 or 23, wherein the cancer is selected from pancreatic ductal adenocarcinoma, prostate cancer, melanoma, liver cancer, breast cancer, lung cancer (eg, lung squamous cell carcinoma), squamous cell carcinoma, ovarian cancer, leukemia and myeloma.

25. The use according to any one of claims 22 to 24, further comprising administering to the subject a second therapeutic agent, optionally the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, a siRNA, an antisense oligonucleotide, a polypeptide and a small molecule drug.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the bispecific antibody according to claim 11, the nucleic acid according to claim 12, the vector according to claim 13, the host cell according to claim 14, the antibody-drug conjugate according to claim 15, or the pharmaceutical composition according to claim 16 or 17, for treating cancer in a subject.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the bispecific antibody according to claim 11, or the nucleic acid according to claim 12, or the vector according to claim 13, or the host cell according to claim 14, the antibody-drug conjugate according to claim 15, or the pharmaceutical composition according to claim 16 or 17, for activating T cells and / or myeloid cells in a cancer microenvironment in a subject.

28. Use of an antibody or antigen-binding fragment thereof, a bispecific antibody, a nucleic acid, a vector, a host cell, an antibody drug conjugate or a pharmaceutical composition for use according to claim 26 or 27, wherein the cancer is selected from pancreatic ductal adenocarcinoma, prostate cancer, melanoma, liver cancer, breast cancer, lung cancer (e.g., squamous cell carcinoma of the lung), squamous cell carcinoma, ovarian cancer, leukemia and myeloma.

29. The antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid, vector, host cell, antibody-drug conjugate or pharmaceutical composition for use according to claim 26 or 27, further comprising administering to the subject a second therapeutic agent, optionally the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, siRNA, an antisense oligonucleotide, a polypeptide and a small molecule drug.

30. A method for determining whether a subject has cancer or is at risk of developing cancer, wherein the microenvironment of the cancer comprises T cells and / or myeloid cells that highly express CD200R1 on their surface, wherein the method comprises: (a) obtaining a biological sample from the subject, (b) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10; and (c) detecting binding of the antibody to the sample, Wherein increased binding of the antibody or antigen-binding fragment thereof to the sample, compared to binding of the antibody or antigen-binding fragment thereof to a control sample, indicates that the subject has the cancer.

31. A method for imaging a cancer in a subject, wherein the microenvironment of the cancer comprises T cells and / or myeloid cells that highly express CD200R1 on their surface, wherein the method comprises: (a) administering to the subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody is conjugated to a detectable marker; and (b) detecting the presence of the marker.

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