Antigen binding proteins

Abstract

Provided are methods for treating a tumor using an anti-CD200R1 therapy, and methods for selecting a subject for treatment of tumor with an anti-CD200R1 therapy based on CD200 expression of cells of the tumor.

Description

ANTIGEN BINDING PROTEINSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 653,437, filed May 30, 2024, which is hereby incorporated by reference in its entirety for all purposes.FIELD

[0002] The present disclosure relates to methods for treating a tumor using an anti- CD200R1 therapy, and methods for selecting a subject for treatment of tumor with an a nti- CD200R1 therapy based on CD200 expression of cells of the tumor.REFERENCE TO SEQUENCE LISTING

[0003] The official copy of the Sequence Listing is submitted concurrently with the specification as an WIPO Standard ST.26 formatted XML file with file name “09402- 015PV1.xml”, a creation date of May 29, 2024, and a size of 2,802,823 bytes.BACKGROUND TO THE INVENTION

[0004] Cell surface transmembrane glycoprotein CD200 receptor 1 (referred to herein as “CD200R1” and also known in the art as CD200 receptor 1 , CD200R, HCRTR2, OX2R, and MOX2R) is a human protein expressed on the surface of various immune cells, including myeloid cells and CD4+ T cells. CD200R1 is a cell surface glycoprotein containing two immunoglobulin-like domains. CD200R1 and its binding partner, CD200, are both highly conserved type I paired membrane glycoproteins, consisting of two immunoglobulin (Ig)-like domains (V and C) that belong to the Ig protein superfamily. CD200R1 is expressed mainly on subsets of T cells and myeloid lineage cells. CD200, however, is expressed more widely on a variety of human cells including neurons, epithelial cells, endothelial cells, fibroblasts, and lymphoid cells. CD200R1 acts to regulate expression of pro-inflammatory molecules such as tumor necrosis factor (TNF-a), and interferons. The binding of CD200R to its ligand CD200 has been found to signal an immunosuppressive activity including inhibiting T-cell immune response and natural killer (NK) cell cytotoxic activity, promoting macrophage secretion of indoleamine-2,3 dioxygenase (IDO), and triggering regulatory T cell (Treg) expansion (see e.g., Gorczynski, “CD200:CD200R-mediated regulation of immunity.” ISRN Immunol. 2012; 2012). CD200 is understood to have an immune checkpoint function on dendritic cells and lymphoid effector cells, modulating activation inflammatory immune responses and contributing to the maintenance of self-tolerance (see e.g., Rygiel T P, Meyaard L. “CD200R signaling in tumor tolerance and inflammation: a tricky balance." Curr Opin Immunol. 2012; 24(2):233-8). CD200 is overexpressed in a wide variety of solid andhematological tumor cell types, including chronic lymphocytic leukemia (CLL) multiple myeloma (MM), acute myeloid leukemia (AML) and others (see e.g., McWhirter et al. “Antibodies selected from combinatorial libraries block a tumor antigen that plays a key role in immunomodulation.’’ Proc Natl Acad Sci USA. 2006; 103(4): 1041-6). A finding of decreased anti-tumor cytotoxic T cell (CTL) response correlated with aggressive tumor progression, and reduced patient survival has been associated with the overexpression of CD200 on tumor cells and correlated. Accordingly, CD200 has been targeted for cancer immunotherapy including the development of a humanized antibody, Samalizumab, that specifically binds to CD200 and blocks its ligation to the CD200R1 , and which was in clinical trials for reduction of tumor burden in patients with advanced chronic lymphocytic leukemia (CLL) (see e.g., Mahadevan et al., “Phase I study of samalizumab in chronic lymphocytic leukemia and multiple myeloma: blockade of the immune checkpoint CD200,” J. Immunotherapy Cancer 7, 227 (2019)). CD200R1 has also been targeted for cancer immunotherapy and currently tested in a clinical trial (NCT05199272).SUMMARY OF THE INVENTION

[0005] The present disclosure relates generally to methods for treating a tumor using an anti-CD200R1 therapy, and methods for selecting a subject for treatment of tumor with an anti-CD200R1 therapy based on CD200 expression of cells of the tumor. This summary is intended to introduce the subject matter of the present disclosure, but does not cover each and every embodiment, combination, or variation that is contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0006] In a first aspect, the present disclosure provides a method of treating a tumor, which method comprises administering a therapeutically effective amount of an anti-CD200R1 antibody, wherein cells in the tumor exhibit membrane CD200 expression. In one embodiment, the anti-CD200R1 antibody is 23ME-00610. In one embodiment, the tumor is selected from the group consisting of adrenal gland cancer, bladder cancer, sarcomas, microsatellite instability-high (MSI-H) cancer (including solid MSI-cancer), TMB (tumor mutational burden)-high tumor, mismatch repair deficient (dMMR) cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, EGJ adenocarcinoma, esophageal cancer, gallbladder cancer, gastric cancer (e.g., gastrointestinal carcinoid (Gl carcinoid)), head and neck cancer, heart cancer, hepatocellular carcinoma, kidney cancer, liver cancer, melanoma, mesothelioma (e.g., pleural mesothelioma), non-small cell lung cancer, ovarian cancer, epithelial ovarian cancer, endometrial cancer, pediatric solid cancers, pancreatic cancer, prostate cancer, spleen cancer, small cell lung cancer, testicular cancer, thyroidcancer (e.g., medullary thyroid cancer or follicular thyroid cancer), blood cancers (e.g., diffuse large B cell lymphoma (DLBCL), leukemias, lymphomas, myelomas), renal cell carcinoma, clear cell renal carcinoma, neuroendocrine tumors (e.g., malignant pheochromocytoma and paraganglioma), and uterine cancer. In some embodiments, the cancer is selected from lung cancer (e.g., small cell lung cancer), skin cancer (e.g., melanoma), pancreatic cancer, endometrial cancer, prostate cancer, colorectal cancer, ovarian cancer, mesothelioma, and bladder cancer. In one embodiment, the treatment is in a subject in need thereof. In one embodiment, the CD200 expression is determined by IHC. In one embodiment, the measure of the membrane of CD200 expression is provided by an H score. In one embodiment, a control of disease is observed with patients with detectable CD200 expression, or control of disease is greater when compared to patients with no detectable CD200 expression. In one embodiment, a clinical benefit of treatment to patients is observed with patients with detectable CD200 expression, or clinical benefit is greater when compared to patients with no detectable CD200 expression. In one embodiment, a reduction in growth or size of tumor is observed with patients with detectable CD200 expression, or reduction in growth or size of tumor is greater when compared to patients with no detectable CD200 expression. In another embodiment, a control of disease, clinical benefit or reduction in growth or size of tumor, is observed with patients with moderate to high CD200 expression. In one embodiment, the progression free survival rate is higher, or the overall survival rate is improved in patients with tumors with high CD200 expression relative to patients with no detectable CD200 tumor expression with anti-CD200R1 antibody treatment.

[0007] In a first aspect, the present disclosure provides a method of treating a tumor, which method comprises administering a therapeutically effective amount of an anti-CD200R1 antibody, wherein cells in the tumor exhibit membrane CD200R1 expression. In one embodiment, the anti-CD200R1 antibody is 23ME-00610. In one embodiment, the tumor is selected from the group consisting of adrenal gland cancer, bladder cancer, sarcomas, microsatellite instability-high (MSI-H) cancer (including solid MSI-cancer), TMB (tumor mutational burden)-high tumor, mismatch repair deficient (dMMR) cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, EGJ adenocarcinoma, esophageal cancer, gallbladder cancer, gastric cancer (e.g., gastrointestinal carcinoid (Gl carcinoid)), head and neck cancer, heart cancer, hepatocellular carcinoma, kidney cancer, liver cancer, melanoma, mesothelioma (e.g., pleural mesothelioma), non-small cell lung cancer, ovarian cancer, epithelial ovarian cancer, endometrial cancer, pediatric solid cancers, pancreatic cancer, prostate cancer, spleen cancer, small cell lung cancer, testicular cancer, thyroid cancer (e.g., medullary thyroid cancer or follicular thyroid cancer), blood cancers (e.g., diffuse large B cell lymphoma (DLBCL), leukemias, lymphomas, myelomas), renal cellcarcinoma, clear cell renal carcinoma, neuroendocrine tumors (e.g., malignant pheochromocytoma and paraganglioma), and uterine cancer. In some embodiments, the cancer is selected from lung cancer (e.g., small cell lung cancer), skin cancer (e.g., melanoma), pancreatic cancer, endometrial cancer, prostate cancer, colorectal cancer, ovarian cancer, mesothelioma, and bladder cancer. In one embodiment, the treatment is in a subject in need thereof. In one embodiment, the CD200R1 expression is determined by IHC. In one embodiment, the measure of the membrane of CD200R1 expression is provided by an H score. In one embodiment, a control of disease is observed with patients with detectable CD200R1 expression, or control of disease is greater when compared to patients with no detectable CD200R1 expression. In one embodiment, a clinical benefit of treatment to patients is observed with patients with detectable CD200R1 expression, or clinical benefit is greater when compared to patients with no detectable CD200R1 expression. In one embodiment, a reduction in growth or size of tumor is observed with patients with detectable CD200R1 expression, or reduction in growth or size of tumor is greater when compared to patients with no detectable CD200R1 expression. In another embodiment, a control of disease, clinical benefit or reduction in growth or size of tumor, is observed with patients with moderate to high CD200R1 expression. In one embodiment, the progression free survival rate is higher or the overall survival rate is improved in patients with tumors with high CD200R1 expression relative to patients with no detectable CD200R1 tumor expression with anti-CD200R1 antibody treatment.

[0008] In another aspect, the present disclosure provides a method for selecting a subject for treatment of a tumor using an anti-CD200R1 antibody or an inhibitor of the CD200 / CD200R1 pathway, which method comprises determining the expression of CD200 on cells of the tumor, and treating the subject with the antibody wherein the cells of the tumor express CD200. In one embodiment, the anti-CD200R1 antibody is 23ME-00610.

[0009] In another aspect, the present disclosure provides a method for selecting a subject for treatment of a tumor using an anti-CD200R1 antibody or an inhibitor of the CD200 / CD200R1 pathway, which method comprises determining the expression of CD200R1 on cells of the tumor, and treating the subject with the antibody wherein the cells of the tumor express CD200R1 . In one embodiment, the anti-CD200R1 antibody is 23ME- 00610.

[0010] In another aspect, the present disclosure provides a method of selecting a subject for treatment of a tumor using an anti-CD200R1 antibody, the method comprising: (a) determining if the level of expression of CD200 of cells of the tumor is within a clinically established range indicating susceptibility to treatment with an anti-CD200R1 antibody; (b) selecting the subject for treatment with the antibody if the cells of the tumor express CD200 within the clinically established range; and (c) treating the subject with the antibody if thecells of the tumor express CD200 within the clinically established range. In one embodiment, the anti-CD200R1 antibody is 23ME-00610.

[0011] In another aspect, this disclosure also provides a method of treating a tumor, comprising administering a therapeutically effective amount of an anti-CD200R1 antibody, wherein cells in the tumor (tumor cells or stromal cells) exhibit membrane CD200 expression, and wherein the administering of an anti-CD200R1 antibody results in a reduction in the growth or size of the tumor. In one embodiment, the administering of an anti-CD200R1 antibody results in a control of disease. In one embodiment, the administering of an anti-CD200R1 antibody results in a clinical benefit of treatment to patients. In one embodiment, the administering of an anti-CD200R1 antibody results in a higher progression free survival rate. In one embodiment, the administering of an anti- CD200R1 antibody results in an improved overall survival rate. In one embodiment, the anti- CD200R1 antibody is 23ME-00610.

[0012] In another aspect, this disclosure also provides a method of treating a tumor, comprising administering a therapeutically effective amount of an anti-CD200R1 antibody, wherein cells in the tumor (tumor cells or stromal cells) exhibit membrane CD200R1 expression, and wherein the administering of an anti-CD200R1 antibody results in a reduction in the growth or size of the tumor. In one embodiment, the administering of an anti-CD200R1 antibody results in a control of disease. In one embodiment, the administering of an anti-CD200R1 antibody results in a clinical benefit of treatment to patients. In one embodiment, the administering of an anti-CD200R1 antibody results in a higher progression free survival rate. In one embodiment, the administering of an anti- CD200R1 antibody results in an improved overall survival rate. In one embodiment, the anti- CD200R1 antibody is 23ME-00610.BRIEF DESCRIPTION OF THE FIGURES

[0013] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0014] FIG. 1A and FIG. 1B depict 23ME-00610 enhancement of PBMC-mediated tumorcell killing in a tumor cell line (COV644-GFP) that endogenously expresses CD200. FIG. 1A depicts the tumor cell growth curve over time with either isotype control or 23ME-00610 treatment at various concentrations. The change in GFP signal overtime relative to isotype, in the killing phase, was used to calculate the EC50. FIG. 1B shows a representative 23ME-00610 dose curve of tumor cell killing relative to isotype control. Mean and standard error (N=4 replicates for each concentration) using PBMC from one donor are shown.

[0015] FIG. 2 shows representative images of various syngeneic tumor models stained for CD200 to highlight the lack of tumor cell expression in CT26, EMT6, MC38, E0771 and MTB2 tumors and positive cytoplasmic and membranous expression of CD200 by IHC in the Cloudman S91 syngeneic tumor cells (bottom right panel, the black arrow highlights the cytoplasmic and membranous CD200 expression while the white arrow highlights melanin pigment in this syngeneic melanoma tumor).

[0016] FIG. 3 depicts the ability of the blocking antibody, anti-CD200 (0X90), to reduce tumor growth compared to treatment with an isotype control (murine I gG 1 silent Fc antibody), in an in vivo S91 melanoma mouse model.

[0017] FIG. 4 depicts RNA expression of CD200 in tumor tissues from The Cancer Genome Atlas (TCGA), with the highest expression in mesothelioma (MESO), pheochromocytoma and paranganglioma (PCPG), and renal cancer (KIRC). Tumor samples included MESO (mesothelioma), PCPG (pheochromocytoma and paraganglioma), KIRC (renal cancer), THCA (thyroid carcinoma), LGG (brain lower grade glioma), KIRP (kidney renal papillary cell carcinoma), OV (ovarian serous cystadenocarcinoma), GBM (glioblastoma multiforme), THYM (thymoma), TGCT (testicular germ cell tumors), PAAD (pancreatic adenocarcinoma), SARC (sarcoma), UCEC (uterine corpus endometrial carcinoma), BRCA (breast invasive carcinoma), UVM (uveal melanoma), DCS (uterine carcinosarcoma), STAD (stomach adenocarcinoma), LUSC (lung squamous cell carcinoma), LUAD (lung adenocarcinoma), KICH (kidney chromophobe), COAD (colon adenocarcinoma), READ (rectum adenocarcinoma), BLCA (bladder urothelial carcinoma), PRAD (prostate adenocarcinoma), CHOL (cholangiosarcoma), HNSC (head and neck squamous cell carcinoma), ESCA (esophageal carcinoma), ACC (adrenocortical carcinoma), LIHC (liver hepatocellular carcinoma), and CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma).

[0018] FIG. 5A and FIG. 5B show the quantification of CD200 protein expression in IHC stained tumor tissue samples. In FIG. 5A, CD200 expression was quantified as the % of tumor area (inclusive of tumor, stromal, and endothelial cells with positive staining using Visiopharm image analysis software (H0rsholm, Denmark)). Mesotheliomas and PNETs (pancreatic neuroendocrine tumors) had the highest CD200 tumor cell expression by tumor area. Tumor samples included PNET (pancreatic neuroendocrine tumor), mesothelioma, OVCC (ovarian clear cell carcinoma), OVCA (ovarian carcinoma), RCCC (clear cell renal carcinoma), DLBCL (diffuse large B cell lymphoma), Kaposi sarcoma, carcinoid tumor, SCLC (small cell lung cancer), MSI-H (microsatellite instability - high), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), CRC (colorectalcarcinoma), sarcoma tumor and TCC (transitional cell carcinoma). In FIG. 5B, the CD200 expression was semi-quantitatively assessed in the tumor cell compartment specifically. CD200 expression analysis was performed by a board-certified veterinary pathologist by assessing staining intensity and percentages of positive cells at each intensity. The graph shows the percentage of samples per tumor type that have high-, moderate-, low- or no CD200 expression. Mesothelioma and carcinoids ranked as the top two tumor types with the highest tumor cell CD200 expression.

[0019] FIG. 6 shows quantification of CD200R1 expression in intratumoral immune cells in IHC stained biobanked human solid tumor tissue. Tumor samples included RCCC (clear cell renal carcinoma), OVCA (ovarian carcinoma), MSI-H (microsatellite instability - high) tumor, mesothelioma, OVCC (ovarian clear cell carcinoma), carcinoid tumor, SCLC (small cell lung cancer), PNET (pancreatic neuroendocrine tumor), and Kaposi sarcoma tumor. Each tumor was scored for CD200R1 by counting the percentage of positively staining immune cells per mm2of tumor area. 0 = number of unique biobanked tumor biopsies. Mean and standard deviation of CD200R1 expression of each tumor type are shown.

[0020] FIG. 7A and FIG. 7B show the potential role of CD200 expression on efficacy of anti- CD200 (0X90 clone) treatment (twice weekly, 20 mg / kg, n=10 / group) for decreasing in vivo tumor growth of S91 tumors in S91 WT (wild-type) (FIG. 7A) or CD200KO (FIG. 7B) tumor bearing-mice. Efficacy of MOPC 21 (isotype control antibodies) was also shown for comparison in both FIG. 7A and FIG. 7B. Plots shown are mean tumor volume + / - SEM. Adjusted area under the curve (aAUC) was used to establish statistical significance.Treatment with 0X90 resulted in significant tumor growth inhibition (FDR adjusted p-value = 0.018) compared to treatment with the isotype control (MOPC 21). A similar study performed in CD200 KO S91 tumors showed no difference in tumor growth between 0X90 and MOPC 21 (isotype control) treatment groups (FDR adjusted p-value = 0.696) indicating that having CD200 tumor cell expression is essential for tumor growth inhibition with anti- CD200 treatment in this model.

[0021] FIG. 8 shows an association between higher tumor expression of CD200 and potential clinical benefit in some patients treated with 23ME-00610.

[0022] FIG. 9 shows tumor response and CD200 tumor cell expression in neuroendocrine patients from the Phase 1 and Phase 2a portions of the Phase 1 / 2a clinical trial(NCT05199272). Tumor response (as measured by % change in SoD of target lesions at baseline) was associated with moderate to high membranous tumor CD200 expression. (SoD = sum of target lesion diameters; NA = not available as 3 responders did not have archival tissue for IHC and did not have CD200 expression data).

[0023] FIG. 10 shows progression-free survival (PFS) for patients with cancerthat were enrolled on the Phase 1 / 2a clinical trial (NCT05199272) and treated with 23ME-00610,stratified by their tumor CD200 expression, with either low tumor CD200 expression (H-score ^10, solid line) or moderate to high tumor CD200 expression (H-score > 10, dashed line).DETAILED DESCRIPTION OF THE INVENTION

[0024] The present disclosure provides methods of treating a cancer, such as a tumor, comprising administering an antibody, such as a humanized antibody that is an anti- CD200R1 antibody, thereby inhibiting, decreasing, and / or fully blocking the function of CD200R1 as a cell surface receptor involved in immune regulation, particularly the function of CD200R1 as an inhibitor of immune cell (e.g., T cell and NK cell) activation. Accordingly, it is contemplated that a method of treatment with anti-CD200R1 antibody herein can be effective for treatment of diseases mediated by the function of CD200R1 or its target ligand, CD200, such as cancers and viral infections. Further, it is contemplated that a method of treatment with anti-CD200R1 antibody herein can further comprise administering a second therapeutic, such as antibodies that target immune checkpoint molecules and other hallmark mechanisms of oncology including, but not limited to, PD1 , TIGIT, LAG3, PVRIG, CD96, KIR, TIM-3, CRT AM, EGF, FGF and VEGF family members and their receptors, and various tumor antigens. In some embodiments, a method of the present disclosure can comprise administering a bispecific antibody comprising anti-CD200R1 binding specificity and another binding specificity of an antibody, for example but not limited to an immune checkpoint molecule such as PD1 , TIGIT, LAG3, PVRIG, KIR, TIM-3, and CRTAM, orthose targeting hallmark mechanisms of cancer, including but not limited to EGF, FGF and VEGF family members and their receptors, or tumor surface antigens.

[0025] Overview of Terminology and Techniques

[0026] For the descriptions herein and the appended claims, the singular forms “a,” and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.”

[0027] Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50,” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc.

[0028] Generally, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common techniques and methodologies described in Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (hereinafter “Sambrook”); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 2011) (hereinafter “Ausubel”); Antibody Engineering, Vols. 1 and 2, R. Kontermann and S. Dubel, eds., Springer-Verlag, Berlin and Heidelberg (2010); Monoclonal Antibodies: Methods and Protocols, V. Ossipow and N. Fischer, eds., 2nd Ed., Humana Press (2014); Therapeutic Antibodies: From Bench to Clinic. Z. An, ed., J. Wiley & Sons, Hoboken, N.J. (2009); and Phage Display. Tim Clackson and Henry B. Lowman, eds., Oxford University Press, United Kingdom (2004).

[0029] All publications, patents, patent applications, and other documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. For purposes of interpreting this disclosure, the following description of terms will apply and, where appropriate, a term used in the singular form will also include the plural form and vice versa.

[0031] “CD200R1” as used herein, refers to the cell surface transmembrane glycoprotein CD200 receptor 1 , and encompasses the CD200R1 proteins of human, cynomolgus monkey (herein referred to in some cases as “cyno”), rhesus monkey, and their various isoforms.Amino acid sequences of various exemplary CD200R1 proteins and isoforms are known in the art and are provided in Table 1 below.

[0032] “CD200R1 mediated condition” or“CD200R1 mediated disease,” as used herein, encompasses any medical condition associated with the specific binding of CD200R1 to a ligand (e.g., CD200). For example, specific binding of CD200 expressed on cell surfaces to other cells expressing the CD200R1 receptor can affect the binding of CD200 expressing cells to other immune regulatory molecules, which can alter activation of lymphocytes (e.g., T cells). Accordingly, CD200R1 mediated diseases can include, but are not limited to, any disease or condition mediated by and / or responsive to antagonists or inhibitors of binding between CD200R1 or CD200 expressing cells, and / or any disease or condition responsive to inhibition of immune checkpoint inhibitors, including, but not limited to, cancers. Specific exemplary cancers are provided elsewhere herein.

[0033] “Immune checkpoint molecule,” as used herein, refers to a molecule that functions to regulate an immune system pathway and thereby prevent it from attacking cells unnecessarily. Many immune checkpoint molecules, both inhibitory and co-stimulatory, are targets for immunotherapy (e.g., with blocking antibodies to block immune inhibition or with agonists to promote immune stimulation) in the treatment of cancer and viral infections. Exemplary immune checkpoint molecules targeted for cancer immunotherapy include, but are not limited to, PD1, TIGIT, LAG3, PVRIG, KIR, TIM-3, CRTAM, CTLA-4, BTLA, CD244, CD160, LIGHT, GITR, 4-1 BB, 0X40, CD27, TMIGD2, ICOS, CD40, CD47, SIRPa, NKG2D, NKG2A, TNFRSF25, CD33, CEA, Epcam, GPC3, CD73, CD83, CD39, TRAIL, CD226, and VISTA.

[0034] “Antibody,” as used herein, refers to a molecule comprising one or more polypeptide chains that specifically binds to, or is immunologically reactive with, a particular antigen. Exemplary antibodies of the present disclosure include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific (or hetero conjugate) antibodies (e.g., bispecific antibodies), monovalent antibodies (e.g., singlearm antibodies), multivalent antibodies, antigen-binding fragments (e.g., Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimetics).

[0035] “Anti-CD200R1 antibody” or “antibody that binds CD200R1 ” refers to an antibody that binds CD200R1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD200R1 . In some embodiments, the extent of binding of an anti-CD200R1 specific antibody to an unrelated, non-CD200R1 antigen is less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the binding of the antibody to CD200R1 as measured, e.g., by a radioimmunoassay (RIA) or surface plasmon resonance (SPR). In some embodiments, an antibody that binds to CD200R1 has adissociation constant (KD) of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <1 pM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M).

[0036] “Full-length antibody,” “intact antibody,” or “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0037] “Antibody fragment” refers to a portion of a full-length antibody which is capable of binding the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; monovalent, or single-armed antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0038] “Class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), e.g., IgG 1 , lgG2, lgG3, lgG4 , lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, E, y, and p, respectively.

[0039] “Humanized antibody” refers to a chimeric antibody comprising amino acid sequences from non-human HVRs and amino acid sequences from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0040] “Human antibody” refers to an antibody which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibodyencoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0041] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). “Binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

[0042] “Binds specifically” or “specific binding” refers to binding of an antibody to an antigen with an affinity value of no more than about 1 x10-7M. In some embodiments, an antibody may have a secondary affinity for an antigen other than the antigen to which it binds specifically, where “secondary affinity” will generally refer to binding of an antibody to a secondary antigen with an affinity value of more than about 10 nM as described elsewhere herein. Where an antibody may have a secondary affinity for a secondary antigen, such an antibody will nevertheless bind specifically to the primary antigen.

[0043] “Functional antigen binding site” of an antibody is one which is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen.

[0044] “Isolated antibody” refers to an antibody which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS- PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87.

[0045] “Substantially similar” or “substantially the same,” as used herein, refers to a sufficiently high degree of similarity between two numeric values (for example, one associated with a test antibody and the other associated with a reference antibody), such that one of skill in the art would consider the difference between the two values to be of little or no biological and / or statistical significance within the context of the biological characteristic measured by said values (e.g., KD values).

[0046] “Substantially different,” as used herein, refers to a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values.

[0047] “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0048] “Immunoconjugate” refers to an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0049] “T reatment,” “treat” or “treating” refers to clinical intervention in an attempt to alter the natural course of a disorder in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desired results of treatment can include, but are not limited to, preventing occurrence or recurrence of the disorder, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disorder, preventing metastasis, decreasing the rate of progression, amelioration or palliation of a disease state, and remission or improved prognosis. For example, treatment can include administration of a therapeutically effective amount of pharmaceutical formulation comprising an anti-CD200R1 antibody to a subject to delay development or slow progression of a disease or condition mediated by CD200R1 or disease or condition in which CD200R1 may play a role in the pathogenesis and / or progression.

[0050] “Pharmaceutical formulation” refers to a preparation in a form that allows the biological activity of the active ingredient(s) to be effective, and which contain no additional components which are toxic to the subjects to which the formulation is administered. A pharmaceutical formulation may include one or more active agents. For example, a pharmaceutical formulation may include an anti-CD200R1 antibody as the sole active agent of the formulation or may include an anti-CD200R1 antibody and one or more additional active agents, such as e g., an immune checkpoint inhibitor.

[0051] By “sole active agent”, as used herein, is meant that the agent referred to is the only agent present in the formulation, or used in the therapy, that provides, or would be expected to provide, the relevant pharmacological effect to treat the subject for the condition, consistent with the description of “treatment” as provided herein. A pharmaceutical formulation comprising a sole active agent does not exclude the presence of one or more non-active agents, such as e.g., a pharmaceutically acceptable carrier, in the formulation. A “non-active agent” is an agent that would not be expected to provide, or otherwise significantly contribute to, the relevant pharmacological effect intended to treat the subject for the condition.

[0052] “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to the subject to whom it is administered. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0053] “Therapeutically effective amount” refers to the amount of an active ingredient or agent (e.g., a pharmaceutical formulation) to achieve a desired therapeutic or prophylactic result, e.g., to treat or prevent a disease, disorder, or condition in a subject. In the case of a CD200R1 mediated disease or condition, the therapeutically effective amount of the therapeutic agent is an amount that reduces, prevents, inhibits, and / or relieves to some extent one or more of the symptoms associated with the disease, disorder, or condition. Forcancer therapy, efficacy in vivo can, for example, be measured by assessing the growth of a primary tumor, occurrence and / or growth of secondary tumor(s), occurrence and / or number of metastases, duration, severity, and / or recurrence of symptoms, the response rate (RR), duration of response, and / or quality of life.

[0054] “Concurrently,” as used herein, refers to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).

[0055] “Individual” or “subject” refers to a mammal, including but not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0056] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.

[0057] Human CD200R1 (“hu-CD200R1”) is a transmembrane glycoprotein that is expressed on the surface of cells, notably, myeloid cells and T cells. The hu-CD200R1 UniProt sequence Q8TD46-1 encodes a 325 amino acid isoform referred to as isoform 1. The hu-CD200R1 UniProt sequence Q8TD46-4 encodes a 348 amino acid isoform referred to in as isoform 1. The predominantly observed hu-CD200R1 isoform in human populations, however, is a 348 amino acid sequence variant of UniProt sequence Q8TD46-4 isoform 4 with a E335Q substitution. This variant of isoform 4 (Q8TD46-4+E335Q) represents one of two major haplotypes that account for >99 / 6 of all individuals observed in the 1000 Genomes project (https: / / www.intenationalgenome.org / ). This haplotype (referred to herein as “Alt”) is the most frequently observed in individuals of European ancestry with a frequency of 0.54. The other major haplotype (referred to herein as “Ref’) is observed in individuals of European ancestry with a frequency of 0.46 and corresponds with the human genome reference sequence. The Alt haplotype has the extracellular domain (ECD, amino acids 1- 266) of the Q8TD46-4 sequence, and the intracellular domain (ICD, amino acids 291-348) of the Q8TD46-4 sequence with the E335Q substitution. The Ref haplotype has the extracellular domain (ECD, amino acids 1-266) of Q8TD46-4 sequence with three substitutions, R112K, P144T and Q200H, and the intracellular domain (ICD, amino acids 291-348) of the Q8TD46-4 sequence.

[0058] A 240 amino acid segment of the hu-CD200R1-iso4 Alt and Ref haplotype ECD sequences (positions 27-266) are set forth herein as SEQ ID NO: 1 and 2, respectively, inTable 1 below. A 215 amino acid segment of hu-CD200R1-iso1 Alt and Ref haplotype ECD sequences (positions 29-243) are set forth herein as SEQ ID NO: 3 and 4, respectively, in Table 1 below.

[0059] Recombinantly prepared segments of cyno-CD200R1 and rhesus CD200R1 , analogous to hu-CD200R1 isoform 4 are set forth as SEQ ID NO: 5 and 6, respectively, in Table 1 below.

[0060] The hu-CD200R1 receptor target ligand, hu-CD200 protein can be found at UniProt P41217 and is set forth herein as SEQ ID NO: 7. The corresponding cyno-CD200R1 CD200 proteins are also provided herein as SEQ ID NO: 8. All CD200 polypeptides are a C-terminal fusion with effectorless human IgG Fc as SEQ ID NO: 9.

[0061] Table 1 below provides a summary description of the sequences of the various CD200R1 and CD200 polypeptides of the present disclosure, and their sequence identifiers.The sequences also are included in the accompanying Sequence Listing.

[0062] TABLE 1: CD200R1 , CD200, and associated sequences

[0063] Methods of treating subjects

[0064] Provided herein are methods of treating cancer in a subject, comprising administering a therapeutically effective amount of an anti-CD200R1 antibody to the subject, wherein cells of the cancer exhibit membrane CD200 expression.

[0065] The cancer being treated can comprise a solid tumor, a carcinoma, a metastasis, other configuration of cancer cells within the subject, or a combination thereof. The cancer can be of a specific type. In some embodiments, the cancer can be selected from the cancer is selected from adrenal gland cancer, bladder cancer, sarcomas, microsatellite instability- high (MSI-H) cancer (including solid MSI-cancer), TMB (tumor mutational burden)-high tumor, mismatch repair deficient (dMMR) cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, EGJ adenocarcinoma, esophageal cancer, gallbladder cancer, gastric cancer (e.g., gastrointestinal carcinoid (Gl carcinoid)), head and neck cancer, heart cancer, hepatocellular carcinoma, kidney cancer, liver cancer, melanoma, mesothelioma (e.g., pleural mesothelioma), non-small cell lung cancer, ovarian cancer, epithelial ovarian cancer, endometrial cancer, pediatric solid cancers, pancreatic cancer, prostate cancer, spleen cancer, small cell lung cancer, testicular cancer, thyroid cancer (e.g., medullary thyroid cancer or follicular thyroid cancer), blood cancers (e.g., diffuse large B cell lymphoma (DLBCL), leukemias, lymphomas, myelomas), renal cell carcinoma, clear cell renal carcinoma, neuroendocrine tumors (e.g., malignant pheochromocytoma and paraganglioma), and uterine cancer. In some embodiments, the cancer is selected from lung cancer (e.g., small cell lung cancer), skin cancer (e.g., melanoma), pancreatic cancer, endometrial cancer, prostate cancer, colorectal cancer, ovarian cancer, mesothelioma, and bladder cancer.

[0066] Subjects can be mammal subjects suspected of having cancer or confirmed to have cancer. For example, a subject can be a mouse, rat, dog, monkey, or a human.

[0067] Treating can comprise administering to the subject (e.g., a human subject in need thereof) afflicted with disease, such as disease mediated by CD200 and / or CD200R1 , such as a cancer, a therapeutic antibody, such as those disclosed in U.S. Pat. No. 11 ,787,861 (which is incorporated by reference herein), which exemplifies, anti-CD200R1 antibodies, including h10F6 (also referred to herein as 23ME-00610), but can include other appropriate therapeutic antibodies. Treatment can comprise administering an IV infusion of the antibody to the subject at a safe and effective dose. For example, the dose can be 600 mg, 1400 mg, or another dose determined to be safe and effective.

[0068] Before, during, or after treatment, the cancer of the subject can be described as having clinical benefit (CB) or no response (NR). If determined during or after treatment, determination of stable disease (SD), partial response (PR), complete response (CR) or progressive disease (PD) for a subject can be a clinical outcome of the treatment.

[0069] In some embodiments, treatment of a subject having cancer cells that exhibit CD200 expression can result in the subject having clinical benefit. “Clinical Benefit” (CB) includes but is not limited to stable disease for 6 months or longer (SD), any tumor shrinkage, a partial response (PR) or complete response (CR), a higher progression free survival rate, an improved overall survival rate, as defined by RECIST criteria. In some embodiments, a subject having cancer cells that exhibit CD200 expression can be more likely to have clinical benefit (CB) (vs. no response (NR)) during the treatment phase than a subject having cancer cells that do not exhibit CD200 expression.

[0070] In some embodiments, a subject having cancer cells that exhibit CD200 expression such that an H score determined from an assay (e.g., IHC) from a sample of said cancer cells is greater than a threshold has an increased likelihood of having clinical benefit (vs. no response) for the duration of the treatment than a subject having cancer cells that either do not exhibit CD200 expression, or that exhibit CD200 expression such that an H score determined from an assay (e.g., IHC) from a sample of said cancer cells is less than the threshold. The threshold can be, for example, an H value of 5, 10, 15, or 20. In some embodiments, the H value of the threshold is 10. In some embodiments, the H value of the threshold can be 100. Additionally, the duration of the treatment can be at least 1 day, at least 1 week, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or a range between any two foregoing values.

[0071] In some embodiments, a subject having cancer cells that exhibit CD200 expression such that an H score determined from an assay (e.g., IHC) from a sample of said cancercells is greater than a threshold has an increased likelihood of having tumor shrinkage (vs. progressive disease) for the duration of the treatment than a subject having cancer cells that either do not exhibit CD200 expression, or that exhibit CD200 expression such that an H score determined from an assay (e.g., IHC) from a sample of said cancer cells is less than the threshold. The threshold can be, for example, an H value of 5, 10, 15, or 20. In some embodiments, the H value of the threshold is 10. In some embodiments, the H value of the threshold can be 100. Additionally, the duration of the treatment can be at least 1 day, at least 1 week, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or a range between any two foregoing values. In some embodiments, tumor shrinkage can comprise reduction in tumor mass of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 99%, or a range between any two foregoing values.

[0072] Membrane CD200 expression

[0073] Membrane CD200 expression can be determined by an acceptable method, such as by an assay, which can be performed on a sample of a tumor or cancer cells from a subject (e.g., a human subject that has or is suspected of having a cancerthat can comprise a tumor), such as from a biopsy, from a blood sample, or from another sample type.

[0074] In some embodiments, membrane CD200 expression can be determined using an immunohistochemistry technique. Given an appropriate detection molecule, the details of performing immunohistochemistry on a sample will be clear to a person skilled in the art. Other acceptable techniques can include (but are not limited to) immunofluorescence, in situ hybridization (ISH), and fluorescence in situ hybridization (FISH), or a combination of appropriate techniques.

[0075] Other acceptable assays for measuring membrane CD200 are envisioned, for example quantum dot luminescent labels, oxyblot immunochemical detection, polymer immunocomplexes, ‘coupled’ probing approach, in situ renaturation of proteins for detecting enzyme activities in crude or purified preparations, immunochromatographic assay, western- phosphatase assay and the use of Congo red dye, a cosmetic color named Alta, Pro-Q Emerald 488 dye or amine-reactive dye in combination with alkaline phosphatase-and horseradish peroxidase-antibody conjugates for the simultaneous trichromatic fluorescence detection of proteins.

[0076] For an assay (e.g., IHC) to effectively measure membrane CD200 expression (e.g., surface CD200 expression), a detection molecule (e.g., an IHC antibody) having adequate specificity and selectivity for CD200 as well as sufficient binding kinetics may be employed. In some embodiments, the detection molecule can be specific to membrane CD200.However, wherein the detection molecule measures CD200 without specificity to membrane CD200, a pathologist or other person skilled in the art can assess the assay (e.g., stained slides, etc.) and determine whether the expression is of surface CD200.

[0077] An assay (such as one employing an IHC antibody or other detection molecule) to determine CD200 expression (e.g., surface CD200 expression) can be precise. This can be due to the precision of the antibody or other detection molecule used in the assay. For example, such an assay can have inter-run, inter-day, inter-operator, and / or inter-instrument precision, resulting in at least 95%, 96%, 97%, 98%, 99%, or 100% concordance for the results of such inter-condition assays. In some embodiments, an assay (such as one employing an IHC antibody) to determine CD200 expression (e.g., surface CD200 expression) can have 100% concordance for inter-run, inter-day, inter-operator, and / or interinstrument results.

[0078] An assay (such as one employing an IHC antibody or other detection molecule) to determine CD200 expression (e.g., surface CD200 expression) can have specificity for CD200. This can be due to the specificity of the antibody or other detection molecule used in the assay.

[0079] An assay (such as one employing an IHC antibody) to determine CD200 expression (e.g., surface CD200 expression) can be adequately sensitive, for example such that at least a threshold amount of CD200 in a sample is detected by said assay. This can be due to the sensitivity of the antibody or other detection molecule used in the assay.

[0080] An assay (such as one employing an IHC antibody) to determine CD200 expression (e.g., surface CD200 expression) can be accurate. This can be due to the accuracy of the antibody or other detection molecule used in the assay.

[0081] An assay to determine CD200 expression (e.g., surface CD200 expression) can be qualitative or quantitative. A qualitative assay can, for example, distinguish between cells having CD200 expression (e.g., surface CD200 expression) and cells not having CD200 expression. In some embodiments, a pathologist reviewing a qualitative assay can distinguish between cells having no CD200 expression, low CD200 expression, moderate CD200 expression, or high CD200 expression. The CD200 expression determined by said pathologist can be surface CD200 expression or total CD200 expression.

[0082] An assay to determine CD200 expression (e.g., surface CD200 expression) can be semi-quantitative or quantitative. Such an assay can provide a measure of CD200 expression (e.g., surface CD200 expression), for example as a numerical value. A semi- quantitative or quantitative assay can use a computer, camera, detector and / or detection system to provide a numerical value descriptive of the level of CD200 expression on a cell, cell membrane, tissue, tumor, pixel, area, or other region of interest. This can be done automatically (e.g., by a computer) or manually (e.g., by a scientist, pathologist, physician, orother individual), or a combination thereof. In some embodiments, an algorithm can be used to determine the numerical value. The numerical value can be an absolute value of CD200 expression, or can be a relative value (e.g., none / low / moderate / high, or a relative value compared with a standard or control sample).

[0083] In some embodiments, an H score or another quantitative, semi-quantitative, or statistical method can be used to provide a measure of CD200 expression or CD200R1 expression. Such a method (e.g., an H score) can comprise evaluation of CD200 expression or CD200R1 expression (e.g., surface expression) in a sample subjected to immunohistochemistry, immunofluorescence, ISH, FISH, or other method of determination or measurement of CD200 expression or CD200R1 expression (e.g., a spatial method of determination or measurement). An H-score can be used to summarize CD200 expression or CD200R1 expression, while accounting for variable staining intensity from cell to cell (or pixel to pixel or region of interest to region of interest) while also accounting for the percentage of cells that have positive staining. An H-score assessment can comprise information regarding signal intensity (e.g., determined as 0 (no evidence of staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining)), and the percentage of stained cells at each intensity. Other assessments comprising similar or overlapping information can also be employed. In some embodiments, the H score can be calculated as a weighted sum of the percentage of stained cells at each intensity level. For example, the H-score can be calculated as:H-score = (OxPo) + (1xPi) + (2xP2) + (3xP3)

[0084] wherein Pois the percentage of cells having expression in a first bin (e.g., low expression), Pi is the percentage of cells having expression in a second bin (e.g., low expression), P2is the percentage of cells having expression in a third bin (e.g., medium expression), and P3is the percentage of cells having expression in a fourth bin (e.g., high expression).

[0085] An H-score can be an integer ranging from 0 to 300. In some embodiments, an H score can be at least 1 , at least 5, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, or at least 100.

[0086] Other quantitative, semi-quantitative, or statistical methods are also envisioned which can be used to provide a measure of CD200 expression or CD200R1 expression. For example, a combined positive score (CPS) can be employed, which can be determined as the number of all CD200 or CD200R1 stained cells independent of cell type, divided by the total number of viable tumor cells, multiplied by 100.

[0087] In some embodiments, the level of CD200 expression or CD200R1 expression can be converted into a numerical H-score of: no CD200 expression or CD200R1 expression which includes an H-score of 0, low CD200 expression or CD200R1 expression, whichincludes an H-score of about 1 to 10; moderate CD200 expression or CD200R1 expression, which includes an H-score of about 11 to 99; or high CD200 expression or CD200R1 expression, which includes an H-score of about 100 to 300.

[0088] Treating the patient with a potential therapeutic can be based upon these H scores. For example, if the treatment methods are carried out on a patient with a low-H-score, that patient would have CD200 expression or CD200R1 expression corresponding to an H-score of about 1 or above. If the treatment methods are carried out on a patient with a moderate H-score, that patient would have CD200 expression or CD200R1 expression corresponding to an H-score of about 11 or above. If the treatment methods are carried out on a patient with a high H-score, that patient would have CD200 expression or CD200R1 expression corresponding to an H-score of about 100 or above. In another example, if the treatment methods are carried out on a patient with a “positive CD200 score” or “expresses CD200” which denotes that the level of CD200 is at or over the level of expression needed for a tumor that is indicated for treatment with a CD200R1 antagonist in which patients are selected for treatment based on CD200 expression levels. In some embodiments, a “positive level of CD200" is one in which more than 1 % of the cells in the tumor have (membrane) staining. In some embodiments, a “positive level” in regard to CD200 is more than 1% staining, for example, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50% or 75% or above, or iterations between those values, of the cells of the tumor are stained at any staining intensity. In some embodiments, a “positive” refers to the intensity of the stain on cells in the tumor at any percentage of cells “positive,” where a “positive” refers to intensity levels “IHC intensity 1 ”, “IHC intensity 2” or “IHC intensity 3”. In some embodiments, “positive” refers to the intensity of the stain on cells on tumor cells on a minimum percentage of tumor cells positive such as 5%, 10%, 20% or higher, where a “positive” refers to intensity levels “IHC intensity 1”, “IHC intensity 2” or “IHC intensity 3.” In another example, if the treatment methods are carried out on a patient with a “positive CD200R1 score” or “expresses CD200R1 ” which denotes that the level of CD200R1 is at or over the level of expression needed for a tumor that is indicated for treatment with a CD200R1 antagonist in which patients are selected for treatment based on CD200R1 expression levels. In some embodiments, a “positive level of CD200R1” is one in which more than 1% of the cells in the tumor have (membrane) staining. In some embodiments, a “positive level” in regard to CD200R1 is more than 1% staining, for example, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50% or 75% or above, or iterations between those values, of the cells of the tumor are stained at any staining intensity. In some embodiments, a “positive” refers to the intensity of the stain on cells in the tumor at any percentage of cells positive, where a “positive” refers to intensity levels “IHC intensity 1”, “IHC intensity 2” or “IHC intensity 3”. In some embodiments, a “positive” refers to the intensity of the stain on tumor cells on a minimumpercentage of tumor cells positive such as 5%, 10%, 20% or higher, where a “positive” refers to intensity levels “IHC intensity 1 ,” “IHC intensity 2,” or “IHC intensity 3”.

[0089] Treating a subject in need thereof

[0090] This disclosure provides a method for immunotherapy of a subject afflicted with cancer, which method comprises: (a) determining expression of CD200R1 of immune cells of test tissue sample obtained from a subject afflicted with cancer of the tissue; (b) based on the determination that a proportion of tumor cells in the test tissue sample express CD200 above a predetermined threshold level on the cell surface; and (c) based on the determination administering a composition comprising a therapeutically effective amount of an anti-CD200R1 antibody to the subject.

[0091] Therapeutic antibodies

[0092] Therapeutic antibodies herein generally comprise high affinity to CD200R1. These antibodies may be used, for example, to treat subjects (e.g., a human subject in need thereof) afflicted with disease, such as disease mediated by CD200 and / or CD200R1 . Generally, said therapeutic antibodies can be administered to a subject (e.g., a human subject in need thereof). Therapeutic antibodies useful in the methods of the present disclosure include, but are not limited to, the anti-CD200R1 antibodies disclosed in U.S. Pat. No. 11 ,787,861 , which is hereby incorporated by reference herein for all purposes. The anti- CD200R1 antibodies, disclosed in U.S. Pat. No. 11 ,787,861 , include h10F6 (also referred to herein as 23ME-00610), but it is contemplated that other appropriate therapeutic antibodies can be used in the methods of the present disclosure.

[0093] The present disclosure provides structures of anti-CD200R1 antibodies in terms of the amino acid and encoding nucleotide sequences of the various well-known immunoglobulin features (e.g., HVRs, FRs, VH, VL domains, and full-length heavy and light chains). Table 2 below provides a summary description of anti-CD200R1 antibody sequences of the present disclosure, and their sequence identifiers. The sequences are included in the accompanying Sequence Listing.

[0094] TABLE 2: Anti-CD200R1 antibody sequences

[0095] 1. Binding Affinity, Blocking, and Cell-Signaling Inhibition of Anti-CD200R1 Antibodies

[0096] In some embodiments, the anti-CD200R1 antibodies provided herein have an equilibrium dissociation constant (KD) for binding to CD200R1 of<100 nM <10 nM,<1 nM <0.1 nM,<0.01 nM, or<0.001 nM (e.g., 10-8M or less, from 10~8M to 10~13M, e.g., from 10-9M to 10“13M).

[0097] In some embodiments, the binding affinity is measured by equilibrium dissociation constant (KD) to a hu-CD200R1 isoform / haplotype polypeptide of SEQ ID NO: 1, 2, 3, and / or4. In some embodiments, the anti-CD200R1 is capable of binding with an affinity of an equilibrium dissociation constant (KD) for binding to CD200R1 of<100 nM <10 nM,<1 nM <0.1 nM,<0.01 nM, or<0.001 nM (e.g., 10-8M or less, from 10-8M to 10-13M, e.g., from 10-9M to 10“13M) to all four hu-CD200R1 isoform / haplotype polypeptides of SEQ ID NO: 1 , 2, 3, and 4. In at least one embodiment, the anti-CD200R1 is capable of binding with comparable (e.g., within 20%) or equivalent affinity in the range of 10-8M or less to the hu- CD200R1 polypeptides of SEQ ID NO: 1 and 2.

[0098] In at least one embodiment, the anti-CD200R1 antibody is characterized by binding to hu-CD200R1-iso4 and hu-CD200R1-iso1 with a binding affinity of 1 x103M or less, 1 xio-9M or less, 1 xi o-10M or less, or 1 xio-11M or less. In some embodiments, wherein the binding affinity is measured by equilibrium dissociation constant (KD) to a hu-CD200R1- iso4 polypeptide of SEQ ID NO: 1 and / or 2, and a hu-CD200R1-iso1 polypeptide of SEQ ID NO: 3 and / or 4.

[0099] In at least one embodiment, the anti-CD200R1 antibody is characterized by binding to hu-CD200R1-iso4-Alt and hu-CD200R1-iso4-Ref with a binding affinity of 1 x10“8M or less, l xio-9M or less, 1xio-10M or less, or 1xW11M or less. In some embodiments, wherein the binding affinity is measured by equilibrium dissociation constant (KD) to a hu- CD200R1-iso4-Alt polypeptide of SEQ ID NO: 1 , and a hu-CD200R1-iso4-Ref polypeptide of SEQ ID NO: 2.

[0100] It is contemplated that the various anti-CD200R1 antibodies generated as disclosed herein include antibodies capable of high-affinity binding to cyno-CD200R1 , and / or to both hu-CD200R1 and cyno-CD200R1 . More specifically, in some embodiments, the anti- CD200R1 antibodies of the present disclosure bind to cyno-CD200R1 with a binding affinity of 1xio-sM or less, 1 xio-9M or less, 1 xW10M or less, or 1 xio-11M or less. In some embodiments, the binding affinity is measured as the equilibrium dissociation constant (KD) for binding to the hu-CD200R1 polypeptide of SEQ ID NO: 5. In some embodiments, the anti-CD200R1 antibodies of the present disclosure bind to cyno-CD200R1 with a binding affinity of 1 xio-8M or less, 1 xio-9M or less, 1 xW10M or less, or 1 xio-11M or less. In some embodiments, the binding affinity is measured as the equilibrium dissociation constant (KD) for binding to the cyno-CD200R1 polypeptide of SEQ ID NO: 5. In some embodiments, the anti-CD200R1 antibodies of the present disclosure bind to both hu-CD200R1 and cy- CD200R1 with a binding affinity of 1 xi o-8M or less, 1 xio-9M or less, 1 xio-10M or less, or 1 xW11M or less. In some embodiments, the binding affinity is measured as the equilibrium dissociation constant (KD) for binding to a hu-CD200R1 polypeptide of SEQ ID NO: 1 , 2, 3, or 4, and a cyno-CD200R1 polypeptide of SEQ ID NO: 5.

[0101] Generally, binding affinity of a ligand to its receptor can be determined using any of a variety of assays and expressed in terms of a variety of quantitative values. SpecificCD200R1 binding assays useful in determining affinity of the antibodies are disclosed in the Examples herein. Additionally, antigen binding assays are known in the art and can be used herein including without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, enzyme-linked immunoabsorbent assay (ELISA), “sandwich” immunoassays, surface plasmon resonance based assay (such as the BIAcore assay as described in W02005 / 012359), immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, flow cytometric and fluorescence activated cell sorting (FACS) assays, and the like.

[0102] Accordingly, in some embodiments, the binding affinity is expressed as KD values and reflects intrinsic binding affinity (e.g., with minimized avidity effects). The anti-CD200R1 antibodies of the present disclosure exhibit strong binding affinities for the extracellular domains of the four distinct CD200R1 isoform / haplotype combinations that are predominant in humans: hu-CD200R1-iso4-Alt (SEQ ID NO: 1); hu-CD200R1-iso4-Ref (SEQ ID NO: 2); hu-CD200R1-iso1-Alt (SEQ ID NO: 3); and hu-CD200R1-iso1-Ref (SEQ ID NO: 4). The extracellular domains of these four isoforms four hu-CD200R1 polypeptides of SEQ ID NOs: 1-4, exhibit KD values of between 10 nM and 1 pM. Accordingly, anti-CD200R1 antibodies of the present disclosure may compete with antibodies having lower affinity for the same or overlapping epitopes of CD200R1.

[0103] In some embodiments, the anti-CD200R1 antibodies provided herein decrease, inhibit, and / or fu I ly- block binding of CD200 to CD200R1 , and thereby block immune regulation and / or immune signaling mediated by CD200R1 , including the activation of T cells. The ability of the antibodies to inhibit these immune regulatory and / or immune signaling pathways mediated by CD200R1 -expressing cells binding to CD200-expressing cells can be assayed in vitro using known cell-based assays including the various cell-based assays described in the Examples of the present disclosure. Accordingly, in some embodiments, the CD200R1 antibodies of the present disclosure are characterized by one or more of following functional properties based on the ability to decrease, inhibit, and / or fully-block cellular signaling pathways mediated by CD200R1 binding to CD200.

[0104] In at least one embodiment, the CD200R1 antibody of the present disclosure blocks hu-CD200-Fc binding to hu-CD200R1-iso4-Alt (SEQ ID NO: 1), hu-CD200R1-iso4-Ref (SEQ ID NO: 2), hu-CD200R1-iso1-Alt (SEQ ID NO: 3), and hu-CD200R1-iso1-Ref (SEQ ID NO: 4) measured by ELISA with an IC50 of 10 nM or less, 7 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less.

[0105] In at least one embodiment, the CD200R1 antibody of the present disclosure blocks hu-CD200-Fc binding to hu-CD200R1 expressed on a cell with an IC50 of 2.5 nM or less, 1 nM or less, or 0.5 nM or less; optionally, wherein the cell is a U937 cell stably expressing hu- CD200R1.

[0106] In at least one embodiment, the CD200R1 antibody of the present disclosure blocks CD200 binding to CD200R1 expressed on a human T cell with an antibody IC50 concentration of 10 nM or less, 5 nM or less, 1 nM or less, or 0.1 nM or less; optionally, wherein the cell is a CD8+T-cell or a CD4+T-cell.

[0107] In at least one embodiment, the CD200R1 antibody of the present disclosure binds to human T-cells with an EC50 of 2.5 nM or less, 1 nM or less, or 0.5 nM or less; optionally, wherein the human T-cells are CD4+T-cells or CD8+T-cells.

[0108] In at least one embodiment, the CD200R1 antibody of the present disclosure increases IFNy production from human tumor cells by at least 1 .2-fold, 1.5-fold, 2-fold, or more, with an antibody concentration of 100 nM or less, 50 nM or less, or 10 nM or less; optionally, wherein the tumor cell type is selected from colorectal, endometrial, lung, melanoma, ovarian, pancreatic, or prostate.

[0109] In at least one embodiment, the CD200R1 antibody increases IFNy and / or IL-2 production from CD200-Fc coated human T cells relative to IgG control by at least 1.2-fold, 1.5-fold, 2-fold, or more.

[0110] In at least one embodiment, the CD200R1 antibody increases activation of CD4+T- cells and / or Cd8+T-cells by at least 1 .5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold or more.

[0111] In at least one embodiment, the CD200R1 antibody blocks NFkp transcription induced by binding between CD200 and CD200R1 expressing cell-lines; optionally, wherein the cell lines are a CD200R1 -expressing K562 reporter cells and CD200-expressing 293T cells.

[0112] In at least one embodiment, the CD200R1 antibodies of the present disclosure are capable of blocking CD200R1 -mediated cell-signaling and not act as an agonist of CD200R1 activity, or otherwise inadvertently agonize CD200R1 signaling. One experimental measure of induction of CD200R1 agonist activity is the induction of pDok2 activity in cells treated with CD200. Accordingly, in at least one embodiment, the anti-CD200R1 antibody of the present disclosure blocks induction of pDok2 activity in U937 monocytic cell lines treated with soluble CD200-Fc.

[0113] 2. Antibody Fragments

[0114] In some embodiments, the anti-CD200R1 antibody of the present disclosure can be an antibody fragment. Antibody fragments useful with the binding determinants the present disclosure include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2-, Fv, scFv fragments, monovalent, single domain antibody, one-armed or single-arm antibody, and other fragments described herein and known in the art. Accordingly, in some embodiments of the anti- CD200R1 antibodies of the present disclosure, the antibody is an antibody fragmentselected from the group consisting of F(ab')2-, Fab', Fab, Fv, single domain antibody (VHH), single-arm antibody, and scFv.

[0115] For a review of various antibody fragments, see e.g., Hudson et al. Nat. Med. 9:129- 134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO93 / 16185; and U.S. Pat. Nos. 5,571 ,894 and 5,587,458.For a description of Fab and F(ab')2- fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046. Other monovalent antibody forms are described in, e.g., W02007 / 048037, WO2008 / 145137, WO2008 / 145138, and W02007 / 059782. Monovalent, single-armed antibodies are described, e.g., in W02005 / 063816. Diabodies are antibody fragments with two antigenbinding sites that may be bivalent or bispecific (see e.g., EP0404097, WO93 / 01161 ; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444- 6448 (1993)).

[0116] In some embodiments, the antibody fragments are single-domain antibodies which comprise all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516).

[0117] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0118] 3. Chimeric and Humanized Antibodies

[0119] In some embodiments, the anti-CD200R1 antibody of the present disclosure can be a chimeric antibody. (See e.g., chimeric antibodies as described in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). In one embodiment, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, a chimeric antibody is a “class switched: antibody in which the class or subclass has been changed from that of the parent antibody. It is contemplated that chimeric antibodies can include antigen-binding fragments thereof.

[0120] In some embodiments, the anti-CD200R1 antibody of the present disclosure is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non- human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, CDRs, (or portions thereof) are derived from a non-human antibody, andFRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve antibody specificity or affinity.

[0121] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821 ,337, 7,527,791 , 6,982,321, and 7,087,409; Kashmiri et al, Methods 36:25-34 (2005) (describing SDR (a-HVR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

[0122] Human framework regions that may be used for humanization include but are not limited to framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol, 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271 :2261 1-22618 (1996)).

[0123] 4. Human Antibodies

[0124] In some embodiments, the anti-CD200R1 antibody of the present disclosure can be a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117- 1125 (2005). See also, e.g., XENOMOUSE™technology in U.S. Pat. Nos.6,075,181 and 6,150,584; HUMAB® technology in U.S. Pat.No.5, 770, 429; K-M MOUSE® technology in U.S. Pat. No.7, 041, 870; and VELOCIMOUSE® technology in U.S. Pat. Appl. Pub. No. US 2007 / 0061900). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

[0125] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse- human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. See, e.g., Kozbor J. Immunol, 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51 -63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991). Human antibodies generated via human B- cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103 :3557- 3562 (2006). Additional methods include those described, for example, in U.S. Pat. No.7, 189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

[0148] Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0126] 5. Library-Derived Antibodies

[0127] In some embodiments, the anti-CD200R1 antibody of the present disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. The use of phage display for preparation of affinity matured variants of the humanized version of the anti-CD200R1 antibody of the present disclosure are described in the Examples disclosed herein. Other methods for producing such library- derived antibodies can be found in e.g., , Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001); McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119- 132(2004).

[0128] 6. Multispecific Antibodies

[0129] In some embodiments, the anti-CD200R1 antibody of the present disclosure is a multispecific antibody, e.g., a bispecific antibody. In some embodiments, the multispecific antibody is a monoclonal antibody having at least two different binding sites, each with a binding specificity for a different antigen, at least one of which specifically binds CD200R1.

[0130] In some embodiments, the multispecific antibody is a bispecific antibody comprising a specificity for CD200R1 and a specificity for another antigen that mediates immune regulation, immune signaling, and / or is expressed on a cancer or tumor cell. In some embodiments of the bispecific antibody, the other specificity is for an antigen that is an immune checkpoint molecule selected from PD1 , TIGIT, LAG3, PVRIG, KIR, TIM-3, CRTAM, CTLA-4, BTLA, CD244, CD160, LIGHT, GITR, 4-1 BB, 0X40, CD27, TMIGD2, ICOS, CD40, CD47, SIRPa, NKG2D, NKG2A, TNFRSF25, CD33, CEA, Epcam, GPC3, CD73, CD83, CD39, TRAIL, CD226, and VISTA. In some embodiments, the anti-CD200R1 bispecific antibody, the other antigen for which the antibody has specificity is selected from PD1 , TIGIT, LAG3, PVRIG, KIR, TIM-3, and CRTAM.

[0131] In some embodiments, at least one binding site specifically binds a cytotoxic agent. In exemplary embodiments, an anti-CD200R1 antibody of the present disclosure is a bispecific antibody and can be used to localize a cytotoxic agent to cells which express CD200R1.

[0132] Techniques for making multispecific antibodies include, but are not limited to, recombinant co- expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see e.g., Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBOJ.10: 3655 (1991)). “Knob-in-hole" engineering can also be used to generate bispecific antibodies useful with the anti-CD200R1 antibodies of the present disclosure. Techniques for knob-in-hole engineering are known in the art and described in e.g., U.S. Patent No.5, 731 ,168.

[0133] Multispecific antibodies can also be made by engineering “electrostatic steering” effects that favor formation of Fc-heterodimeric antibody molecules rather than homodimers (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Pat. No.4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol, 148(5): 1547-1553 (1992)); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); using single- chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol, 152:5368 (1994)); or tri-specific antibodies (see e.g., Tutt et al., J. Immunol.147: 60 (1991).

[0134] 7. Antibody Variants

[0135] In some embodiments, variants of the anti-CD200R1 antibody of the present disclosure are also contemplated. For example, antibodies with improved binding affinityand / or other biological properties of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristic of CD200R1 antigen binding.

[0136] A. Substitution, Insertion, and Deletion Variants

[0137] In some embodiments, anti-CD200R1 antibody variants having one or more amino acid substitutions in addition to those described herein are provided. Sites for mutagenesis can include the HVRs and FRs. Typical “conservative” amino acid substitutions and / or substitutions based on common side-chain class or properties are well-known in the art and can be used in the embodiments of the present disclosure. The present disclosure also contemplates variants based on non-conservative amino acid substitutions in which a member of one amino acid side chain class is exchanged for an amino acid from another class.

[0138] Amino acid side chains are typically grouped according to the following classes or common properties: (1) hydrophobic: Met, Ala, Vai, Leu, lie, Norleucine; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) chain orientation influencing: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Techniques are well-known in the art for amino acid substitution into an antibody and subsequent screening for desired function, e.g., retained / improved antigen binding, decreased immunogen.

[0139] Amino acid substitution variants can include substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described in the Examples herein. Briefly, one or more HVR residues are mutated, and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0140] A method for identifying residues or regions of an antibody that may be targeted for mutagenesis is “alanine scanning mutagenesis” (see e.g., Cunningham and Wells (1989) Science, 244: 1081-1085). In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine whether theinteraction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen can be determined. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0141] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0142] Substitutions can be made in HVRs to improve antibody affinity. Such alterations may be made in “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol.Biol.207: 179-196 (2008)) with the resulting variant VH or VL being tested for binding affinity. In some embodiments, affinity maturation can be carried out by constructing and reselecting from secondary libraries (see e.g., in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. HVR-H3 and HVR-L3 in particular are often targeted.

[0143] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots.” In some embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0144] B. Glycosylation Variants

[0145] In some embodiments, the anti-CD200R1 antibody of the present disclosure is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be carried out by altering the amino acid sequence such that one or more glycosylation sites can be created or removed.

[0146] In embodiments where the antibody comprises an Fc region, the carbohydrate attached to the Fc region can be altered. Typically, native antibodies produced bymammalian cells comprise a branched, biantennary oligosaccharide attached by an N- linkage to the asparagine at about position 297 (“N297”) of the CH2 domain of the Fc region (see, e.g., Wright et al. TIBTECH 15:26-32 (1997)). The oligosaccharide may include various carbohydrates, such as mannose, N-acetyl glucosamine (GIcNAc), galactose, and sialic acid, as well as a fucose attached to a GIcNAc in the “stem” of the bi-antennary oligosaccharide structure. In some embodiments, the modifications of the oligosaccharide of an Fc region of an antibody can create a variant with certain improved properties.

[0147] In some embodiments, the anti-CD200R1 antibody of the present disclosure can be a variant of a parent antibody, wherein the variant comprises a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such an antibody may be from about 1% to about 80%, from about 1% to about 65%, from about 5% to about 65%, or from about 20% to about 40%. The amount of fucose can be determined by calculating the average amount of fucose within the sugar chain at N297, relative to the sum of all glyco-structures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry (see e.g., WO 2008 / 077546). N297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, N297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies.

[0148] In some embodiments, the fucosylation variants can have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108, or US 2004 / 0093621 . Examples of “defucosylated” or “fucose-deficient” antibodies and associated methods for preparing them are disclosed in e.g., US2003 / 0157108; US2003 / 0115614; US20Q2 / QI64328; US2004 / 0093621 ; US2004 / 0132140; US2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; W02000 / 61739; WO2001 / 29246; W02003 / 085119; W02003 / 084570; W02005 / 035586; W02005 / 035778; W02005 / 053742; W02002 / 031140; Okazaki et al. J, Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech, Bioeng. 87: 614 (2004).

[0149] Cell lines useful for producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (see e.g., Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108, and WO2Q04 / 056312), and knockout cell lines, such as alpha-1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al, Biotechnol. Bioeng., 94(4):680-688 (2006); and W02003 / 085107).

[0150] C. Fc Region Variants

[0151] In some embodiments, an anti-CD200R1 antibody of the present disclosure can comprise one or more amino acid modifications in the Fc region (i.e., an Fc region variant). The Fe region variant may comprise a human Fc region sequence (e.g., a human IgG 1 ,lgG2, lgG3, or lgG4 Fc region) comprising an amino acid substitution at one or more amino acid residue positions. A wide range of Fc region variants known in the art that are useful with the anti-CD200R1 antibodies of the present disclosure are described below.

[0152] In some embodiments, the anti-CD200R1 antibody can be an Fc region variant which has altered effector function. In some embodiments, the antibody with altered effector function can possess some (but not all of) the effector functions, decreased effector function, or none of the effector functions (e.g., effectorless) of the parent antibody. Effectorless Fc region variants can be more desirable for certain applications where effector function (such as ADCC) is unnecessary or deleterious, and / or in vivo half- life of the antibody is important.

[0153] Fc region variant antibodies with reduced effector function, or which are effectorless, can include an amino acid substitution at one or more of the following Fc region positions: 238, 265, 269, 270, 297, 327 and 329. (see, e.g., U.S. Patent No. 6,737,056). Such Fc region variants can include amino acid substitutions at two or more of positions 265, 269, 270, 297 and 327. Such Fc region variants can also include substitutions of both residues 265 and 297 to alanine (see e.g., US Pat, No. 7,332,581). In some embodiments, the anti- CD200RI antibodies of the present disclosure are effectorless Fc region variants. In some embodiments, the effectorless Fc region variants of the anti-CD200R1 antibodies comprise one or more amino acid substitutions selected from N297G (see e.g., Shields, R. et al, “High Resolution Mapping of the Binding Site on Human IgGI for FcyRI, FcyRII, FcyRIII and FcRn and Design of IgGI Variants with Improved Binding to the FcyR*”, Journal of Biological Chemistry, 276(9): 6591-6604 (2001)); N297A (see e.g.. Friend, P.J. et al, “Phase I Study of an Engineered Aglycosylated Humanized CD3 Antibody in Renal Transplant Rejection”, Transplantation, 68(11): 1632-7 (1999)); P331S / K322A (see e.g., Tawara, T. et al., “Complement Activation Plays a Key Role in Antibody-Induced Infusion Toxicity in Monkeys and Rats”, J Immunol, 180(4): 2.294-8 (2008)); S228P / L235E (see e.g., Newman, R. et al., “Modification of the Fc Region of a Primatized IgG Antibody to Human CD4 Retains Its Ability to Modulate CD4 Receptors but Does Not Deplete CD4(+) T Cells in Chimpanzees”, Clin Immunol, 98(2): 164-74 (2001); or L234A / L235A / P329G (also referred to as “LALAPG”) (see e.g., Schlotliauer, T. et al., “Novel human IgG 1 and lgG4 Fc-engineered antibodies with completely abolished immune effector functions”, Protein Eng. Des. Sei., 29(10): 457-- 466 (2.016); Lo, M. et al, "Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice", Journal of Biological Chemistry, 292(9): 3900-3908 (2017). In other embodiments, the effectorless Fc region variants of the anti-CD200R1 antibodies comprise the amino acid substitutions L234A / L235A (“LALA”) (Woodie, E. Steve et al, Transplantation, 68(5): 608-616 (1999)).

[0154] Accordingly, in some embodiments, the effectorless Fc region variants of the anti- CD200R1 antibodies comprise one or more amino acid substitutions selected from N297A orN297G. In some embodiments, the effectorless Fc region variants of the anti-CD200R1 antibodies comprise the pair of amino acid substitutions P331S / K322A. In other embodiments, the effectorless Fc region variants of the anti-CD200R1 antibodies comprise the amino acid substitutions L234A / L235A (LALA) or L234A / L235A / P329G (LALAPG). In some embodiments, wherein the anfi-CD20GRI is of isotype lgG2 or lgG4, the anti- CD200R1 antibody comprises the amino acid substitutions S228P and / or L235E.

[0155] Fc region variants having improved or diminished binding to FcRs are disclosed in e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al, j. Biol. Chern. 9(2): 6591- 6604 (2001). Fc region variants having improved ADCC can comprise one or more amino acid substitutions at e.g., positions 298, 333, and / or 334 of the Fc region (based on EU numbering). Fc region variants having altered (i.e., either improved or diminished) Clq binding and / or Complement Dependent Cytotoxicity (CDC), as described in e.g., US Pat. No. 6, 194,551 , W099 / 51642, and Idusogie et al, j. Immunol. 164: 4178- 4184 (2000). Fc region variants with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) are disclosed in e.g., US2G05 / 0014934 A 1 (Hinton et al.). Such Fc region variants comprise amino acid substitutions at one or more of positions: 238, 256, 265, 272, 286, 303, 305, 307, 311 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, and 434. Other Fc region variants with increased half-lives include the set of YTE mutations at positions 252, 254, and 256 (i.e., M252Y / S254T / T256E) described in e.g., US 7658921 B2 (Dali’Acqua et al). Other examples of Fc region variants can be found in e.g., U.S. Pat. Nos. 5,648,260 and 5,624,821 ; and W094 / 29351.

[0156] Generally, in vitro and / or in vivo cytotoxicity assays can be carried out to confirm the reduction / depletion of CDC and / or ADCC activities in an Fc region variant. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRTII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, et al., Proc. Nat 'I Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, et ah, Proc. Nat’l Acad. Sci. USA 82: 1499-1502 (1985); 5,821 ,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox96* non-radioactive cytotoxicity assay (Promega, Madison, WT).Useful effector ceils for such assays include peripheral blood mononuclear cells (PJBMC) and Natural Killer (NK) ceils. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652- 656 (1998). Clq binding assays may also be carriedout to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in W02006 / 029879 and W02005 / 100402.

[0157] To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and MJ. Glennie, SW 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can be performed using methods known in the art (see, e.g., Petkova, et al., Inti. Immunol. 18(12): 1759-1769 (2006)).

[0158] D. Cysteine Engineered Antibody Variants

[0159] In some embodiments, it is contemplated that the anti-CD200RI antibody described herein can be substituted at specific non-HVR positions with cysteine residues so as to create reactive thiol groups.

[0160] Such engineered “thioMAbs” can be used to conjugate the antibody to e.g., drug moieties or linker-drug moieties and thereby create immunoconjugates, as described elsewhere herein. Cysteine engineered antibodies can be generated as described in e.g., U.S. Pat. No. 7,521 ,541. In some embodiments, any one or more of the following antibody residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.

[0161] E. Antibody Derivatives

[0162] In some embodiments, the anti-CD200R1 antibody of the present disclosure may be further modified (i.e., derivatized) with non-proteinaceous moieties. Non-proteinaceous moieties suitable for derivatization of the antibody include, but are not limited to, water soluble polymers, such as: polyethylene glycol (PEG), copolymers of ethylene glycol and propylene glycol, carboxy- methylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1 , 3-dioxolane, poly-1, 3, 6- trioxane, ethylene / maleic anhydride copolymer, poly-amino acid homo-polymers or random co-polymers, and dextran or poly(n-vinyl pyrrolidone- polyethylene glycol, polypropylene glycol homo-polymers, polypropylene oxide / ethylene oxide co-polymers, polyoxy-ethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. In some embodiments, modification of the antibody can be carried out using methoxy-polyethylene glycol propionaldehyde. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody, e.g., whether the antibody derivative will be used in a therapy under defined conditions.

[0163] 8. Immunoconjugates

[0164] In some embodiments, the anti-CD200R1 antibody of the present disclosure can also be an immunoconjugate, wherein the immunoconjugate comprises an anti-CD200R1 antibody conjugated to one or more cytotoxic agents. Suitable cytotoxic agents contemplated by the present disclosure include chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0165] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) in which an anti-CD200RI antibody, as described herein, is conjugated to one or more drugs.

[0166] In some embodiments, an immunoconjugate of the present disclosure comprises an anti-CD200R1 antibody as described herein conjugated to a drug or therapeutic agent for the treatment of a CD200R1 -mediated disease or condition.

[0167] In some embodiments, an anti-CD200R1 antibody as described herein can be conjugated to an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarein, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0168] In some embodiments, an immunoconjugate of the present disclosure comprises an anti- CD200R1 antibody as described herein conjugated to a radioactive isotope (i.e.., a radioconjugate). A variety of radioactive isotopes are available for the production of such radioconjugates. Examples include, but are not limited to,64Cu,89Zr,211At,131l,125l,90Y,186Re,188Re,153Sm,212Bi,32P,212Pb, and radioactive isotopes of Lu. In some embodiments, a radioactive isotope can be or comprise18F, FOG. In some embodiments, the immunoconjugate may comprise a radioisotope for scintigraphic detection, or a spin label for NMR detection or MRI. Suitable radioisotopes or spin labels can include, as123T,131l,111ln,13C,19F,15N,170, various isotopes of Gd, Mo, and Fe.

[0169] Immunoconjugates of an anti-CD200R1 antibody and a cytotoxic agent, can be made using a variety of well-known bifunctional reagents and chemistries suitable for conjugating to proteins. Such reagents include but are not limited to: N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC), iminothioiane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HQ), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis-(p-azidobenzoyl)- liexancdiamine), bis-diazonium derivatives (e.g., bis- (p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2, 6-diisocyanate), and bis-active fluorine compounds (e.g., l,5-difluoro-2,4- dinitrobenzene).

[0170] Reagents for preparing immunoconjugates of the present disclosure can also include commercially available “cross-linking” reagents such as: BMPS, EMCS, GMBS, HBVS, LC- SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4- vinylsulfone)benzoate) (see e.g., , Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

[0171] 9. Synthetic Antibodies

[0172] In some embodiments, the anti-CD200R1 antibody of the present disclosure can be a synthetic antibody comprising a set of CDRs or HVRs from an anti-CD200R1 immunoglobulin (e.g., HVR-L1 , etc.) grafted onto a scaffold or framework other than an immunoglobulin scaffold or framework, such as an alternative protein scaffold, or an artificial polymer scaffold.

[0173] Exemplary alternative protein scaffolds contemplated for preparation of synthetic antibodies of the present disclosure can include, but are not limited to: fibronectin, neocarzinostatin CBM4-2, lipocalins, T-cell receptor, protein-A domain (protein Z), Im9, TPR proteins, zinc finger domains, pVIII, avian pancreatic polypeptide, GCN4, WW domain Src homology domain 3, PDZ domains, TEM-1 beta- lactamase, thioredoxin, staphylococcal nuclease, PHD-finger domains, CL-2, BPTI, APPI, HPSTI, ecotin, LACI-D1, LDTI, MTI-II, scorpion toxins, insect defensin-A peptide, EETI-II, Min-23, CBD, PBP, cytochrome b-562, Ldl receptor domains, gamma-crystallin, ubiquitin, transferrin, and / or C-type lectin- like domains.

[0174] Exemplary artificial polymer (non-protein) scaffolds useful for synthetic antibodies are described in e.g., Fiedler et al., (2014) “Non-Antibody Scaffolds as Alternative Therapeutic Agents,” in Handbook of Therapeutic Antibodies (eds S. Dlibel and J. M. Reichert), Wiley- VCH Verlag GmbH & Co.; Gebauer et al., Curr. Opin. Chem. Biol, 13:245-255 (2009); Binz et al, Nat. Biotech., 23(10): 1257-1268 (2005).EXAMPLES

[0175] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.Example 1: Incucyte tumor cell killing assay

[0176] To determine whether anti-CD200R1 antibodies enhance in vitro PBMC-mediated tumor cell killing, a tumor cell line (COV644-GFP) that endogenously expresses CD200 was evaluated upon treatment with 23ME-00610 or isotype control.

[0177] Engineering GFP-Expressinq COV644 Tumor Cell Line

[0178] The human ovarian epithelial-mucinous cancer cell line, COV644, purchased from Sigma Aldrich (Catalog #07071908-1VL), was engineered to stably express the GFP using a lentiviral construct with a cytomegalovirus (CMV) promoter (COV644-GFP). Approximately 2 weeks after lentiviral transduction, GFP-positive cells were selected using the Sony SH800S cell sorter system as a pool population.

[0179] Flow Cytometry to Measure CD200 Expression on COV644-GFP Cells

[0180] The engineered, adherent COV644-GFP cell line was maintained in Dulbecco’s modified eagle media (DMEM)-high glucose (4.5 g / L) supplemented with 10% heat- inactivated fetal bovine serum (FBS), 1X GlutaMax, and 1X penicillin / streptomycin. Cells were sub-cultured as recommended by the vendor and passaged once cells were 80% confluent.

[0181] To test CD200 surface expression, COV644-GFP cells were harvested by brief exposure with 1X trypsin-0.25% EDTA solution and washed in complete media. Cells were resuspended in FACS buffer and stained with anti-CD200 clone OX104-allophycocyanin (APC) or APC-conjugated MOPC21 isotype control (Biolegend) for 20 minutes at 4 °C. Unbound staining antibody was washed away with 2 repeats of FACS buffer wash steps before analyzing on a Beckman Coulter Cytoflex LX.

[0182] IncuCyte Live Tumor Growth Monitoring Method

[0183] COV644-GFP tumor cells were seeded at 3,000 cells / well in a CellCarrier-96 black (Perkin Elmer), clear-bottom, tissue culture-treated plate and cultured at 37 °C. Each study utilized the inner 60 wells; outer wells were filled with media to provide an evaporation barrier. To ensure uniform and accurate cell seeding, COV644 suspensions were passed through a 30 micron cell strainer to remove cell aggregates. Cells were allowed to adhere and acclimate to the assay plate for 24 hours. On the day of the study, media was carefully aspirated away and 0.1 mL of antibody dilutions made in Roswell Park Memorial Institute (RPMI) complete media (supplemented with 10% heat-inactivated FBS, 1x penicillin / streptomycin, sodium pyruvate, GlutaMax, nonessential amino acids and - mercaptoethanol) were dispensed into the assay plate at a 2X concentration. Six replicates of the isotype control and 4 replicates of 23ME-00610 dilutions were tested in each study. Cryopreserved PBMCs isolated from fresh human leukopaks (StemCell Technologies) were quickly thawed in a 37 °C water bath and washed in pre-warmed RPMI complete media. Cells were strained and counted on a Beckman Coulter Vi-Cell and adjusted to 300,000 cells / mL. PBMCs were activated with a 2X concentration of SEB. Lastly, 0.1 mL of PBMCsequivalent to 30,000 cells per well were transferred to the assay plate. A PBMC effector to tumor cell ratio of 10:1 was used. The assay plate was placed on a steady surface at room temperature for 5 to 10 minutes to allow PBMCs to settle evenly to the well bottom. Finally, the plate was loaded into the IncuCyte S3 instrument chamber and 4 images per well were captured at 2 or 3 hour intervals for the duration of the study. The tumor cell growth was tracked as a defined metric of green area per well normalized to 2, 3, or 4 hour reading and plotted over time. For data analysis, the PBMC priming phase was defined as the period before the isotype control treatment groups reached maximum green fluorescence, and the killing phase was defined as the period after that time point. Tumor-cell cytotoxicity was evident in GFP signal vs. time plots as a decrease in GFP signal to levels lower than those observed at the initial seeding density. The change in GFP signal overtime relative to the isotype control, measured after the priming phase, was used to calculate the EC5o.

[0184] To quantify the tumor cell killing potency of 23ME-00610, the AUC of the killing phase was converted to COV644 tumor cell killing relative to isotype control using the following equation:

[0185] Six-point dose responses were analyzed using a 3-parameter, logio[23ME-00610] vs response, non-linear curve fit.

[0186] Results

[0187] Killing of a tumor cell line that endogenously expresses CD200, COV-644-GFP, by PBMCs was evaluated upon treatment with 23ME-00610 or isotype control. CD200R1+PBMCs were primed with SEB and treated with a titration of 23ME-00610 or isotype control. COV-644-GFP cells were co-cultured with PBMCs for 120 hours and GFP signal was monitored as a readout of tumor-cell number. Tumor-cell killing was observed as a decrease in total GFP signal at the end of the experiment relative to the GFP signal observed at the beginning of the experiment. The change in GFP signal over time relative to isotype, in the killing phase, was used to calculate the ECso (FIG. 1A). A representative dose response curve of tumor-cell killing relative to isotype control is provided in FIG. 1B. 23ME-00610 showed an increase in tumor-cell killing relative to isotype control, as measured by the decrease in total GFP at the end of the experiment relative to the GFP signal observed at the beginning of the experiment.Example 2: In vivo tumor growth

[0188] In vivo efficacy of anti-CD200 antibodies was determined in a melanoma mouse model.

[0189] S91 cells were confirmed to express endogenous CD200 by flow cytometry (data not shown). Cloudman S91 melanoma tumor cell line was the only syngeneic tumor cell line tested by IHC that expressed cytoplasmic / membranous CD200 (FIG. 2). Tumors in anti- CD200 (0x90) treated mice displayed a significant decrease in tumor volume compared with tumors in mice treated with isotype control. This difference in tumor volume between groups increased overtime (FIG. 2).

[0190] In vivo S91 Melanoma Model in Mice

[0191] Cloudman S91 murine melanoma cells were purchased from ATCC and cultured in F-K12 media containing 15% Non-Heat-inactivated horse serum, 2.5% fetal bovine serum and 1% Penicillin / Streptavidin.

[0192] CD200 expression in S91 tumors was assessed by flow cytometry. Tumors were collected in RPMI in C-tubes (Miltenyi) and mechanically dissociated by using GentleMACS dissociator (Miltenyi). Cell suspensions were then filtered through a 70 pm strainer and red blood cells were lysed by using ammonium-chloride-potassium (ACK) buffer. Cells were then incubated at room temperature with Live / Dead fixable and an additional 15 min with mouse Fc block. After washing the cells with PBS, cells were stained on ice for 30 min with fluorochrome- conjugated antibodies, washed, and fixed with 2% formaldehyde for flow cytometric analysis on Cytoflex flow cytometer (Beckman Coulter). Antibodies used to stain: CD200 (Clone: 0X90; Biolegend, PE).

[0193] CD200 expression in S91 , CT26, EMT6, MC38, E0771 and MTB2 tumors was assessed by IHC. MC38, CT26, E0771 , MTB2, EMT6, and S91 tumors were collected, processed, and embedded into FFPE blocks. Blocks were then sectioned to produce unstained slides. These slides then had CD200 IHC performed on them and were analyzed for CD200 expression / staining patterns. Prominent cytoplasmic / membranous staining of CD200 was observed in only S91 tumor cells (F G. 2). All other tumor cell lines were CD200 negative (FIG, 2). Cloudman S91 , a syngeneic melanoma tumor, was the only syngeneic tumor cell that expressed CD200 and was used for in vivo efficacy studies.

[0194] All in vivo experiments were conducted with the approval of the 23andMe IACUC prior to execution. Six to eight weeks old female DBA / 2 mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). Mice were injected subcutaneously with 0.5 million Cloudman S91 cells with 50% Matrigel. Once the tumors reached 80-100 mm3 volume, they were randomized into 2 groups of 15 mice and treated by IP injection twice a week with either MOPC1 isotype (murine IgG 1 silent Fc antibody) or 0X90 anti-CD200 Abs (20 mg / Kg). The scientist who performed the experiment was blinded on the treatment during the entire study. Tumor volume and body weight were measured twice a week. Mice inwhich tumors reached 2000 mm3 volume or became necrotic or started to interfere with their well-being were sacrificed. For the RNA-seq study, tumors from 10 mice in both groups were collected, cut in several small pieces, and transferred into RNA later after the 3rd and the 5th dose of each antibody.

[0195] Results

[0196] S91 cells were confirmed to express endogenous CD200 by flow cytometry (data not shown). Cloudman S91 melanoma tumor cell line was the only syngeneic tumor cell of those tested by IHC that expressed cytoplasmic / membranous CD200 (FIG. 2).

[0197] Tumors in anti-CD200 (0X90) treated mice displayed a significant decrease in tumor volume compared with tumors in mice treated with isotype control. This difference in tumor volume between groups increased overtime (FIG. 3).Example 3: Human prevalence in biobanked samples

[0198] The prevalence of CD200R1 or CD200 expression on tumors was determined.

[0199] The expression of CD200R1 or CD200 was evaluated on normal, healthy human tissue and selected tumors by analysis of public RNA expression databases (The Cancer Genome Atlas [TCGA]) and immunohistochemistry. For analysis of the public TCGA database, relative CD200R1 and CD200 expression on tumor samples was compared using matched normal tissue samples.

[0200] Results

[0201] To evaluate the RNA expression of CD200R1 or CD200 in cancers, publicly available data from the Tumor Cell Genome Atlas was queried. CD200 RNA expression was detectable in most tumor types (see Table 3), with highest expression in mesothelioma (MESO), pheochromocytoma and paranglioma (PCPG), and renal cancer (KIRC) and enriched expression of CD200 RNA over NAT was observed in KIRP (kidney renal papillary cell carcinoma), LUSC (lung squamous cell carcinoma), LUAD (lung adenocarcinoma) and STAD (stomach adenocarcinoma) (FIG. 4).

[0202] TABLE 3: Tumor Tissues Tested For Expression

[0203] Protein expression of CD200 was evaluated by IHC in 15 tumor types (PNET (pancreatic neuroendocrine tumor), mesothelioma, OVCC (ovarian clear cell carcinoma), OVCA (ovarian carcinoma), RCCC (clear cell renal carcinoma), DLBCL (diffuse large B cell lymphoma), Kaposi sarcoma, carcinoid tumor, SCLC (small cell lung cancer), MSI-H (microsatellite instability - high) tumor, NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), CRC (colorectal carcinoma), sarcoma tumor and TCC (transitional cell carcinoma), selected based on CD200R1 and CD200 RNA expression. All tumor types with overall high CD200 RNA expression showed cancer cell CD200 expression in a subset of samples tested. In ovarian adenocarcinomas, a significant contribution to overall CD200 by cancer-associated fibroblasts (CAFs) was observed. In addition, endothelial signal was present in all tumors and was an important contributor to the total CD200 signal in renal cell carcinoma. The tumors with highest CD200 expression were mesotheliomas and PNETs, where expression was primarily contributed by cancer cells. Additional tumors with high CD200 expression in a subset of IHC stained tumor samples tested were ovarian carcinoma (OVCA), carcinoid tumor, small cell lung (SCLC) and clear cell renal cancer (RCCC) (FIGS. 5A and 5B). CD200 expression was quantified as the % of tumor area with positive staining using Visiopharm image analysis software (H0rsholm, Denmark).

[0204] Protein expression of CD200R1 was evaluated by IHC in a series of 9 tumor types (RCCC (clear cell renal cancer), OVCA (ovarian carcinoma), MSI-H (microsatellite instability - high) tumor, mesothelioma, OVCC (ovarian clear cell carcinoma), carcinoid tumor, SCLC (small cell lung cancer), PNET (pancreatic neuroendocrine tumor), and Kaposi sarcoma tumor), selected based on CD200R1 and CD200 RNA expression. CD200R1 expression is detectable in intratumoral immune cells in all evaluated tumor types with the highest expression in RCCC, OVCA and MSI-H tumors. (FIG. 6).Example 4: In vivo CD200 knockout Tumor Growth

[0205] To evaluate the role of CD200 tumor cell expression on anti-CD200 efficacy, a CD200 KO S91 melanoma cell line was generated to analyze in vivo efficacy of anti-CD200 antibodies.

[0206] CD200 gene was knocked out in S91 tumor cells using CRISPR-Cas9 based gene editing protocol from the manufacturer (Synthego) protocol of CRISPRevolution sgRNA EZ Kit. Briefly, a sgRNA targeting exon 3 of CD200 gene and a non-targeting control sgRNA were designed using Synthego CRISPR design tool. sgRNAs were reconstituted to 100uM in nuclease-free duplex buffer (IDT Cat# 11 — 01-03-01). RNP complexes were formed for each sgRNA reaction in sterile Eppendorf tubes by adding 3uL (300pmol) of reconstituted sgRNA to 2uL of TrueCut Cas9 Protein v2 (ThermoFisher Cat# A36499) and incubating at room temperature for 10 min. S91 tumor cells were harvested and resuspended in SF buffer from SF Cell Line 4D-Nucleofector™ X Kit S (Cat# V4XC-2032) at 50,000 cells / 100uL. 10,000 cells (20uL) were added in each well of the nucleofection strip along with 5uL of RNP complex prepared as described above per sgRNA reaction. Nucleofection was performed using Lonza nucleofector unit’s A549 nucleofection program. Nucleofected cells were plated in complete RP I medium and passaged for 7 days before testing expression by flow cytometry using murine CD200-APC antibody (clone OX-90, Cat# 123810). More than 98% CRISPR editing was observed. Further flow sorting was performed on Sony SH800S Cell Sorter to obtain S91 cells with 100% CD200 CRISPR editing.

[0207] In vivo Materials and Methods

[0208] Strain: DBA / 2J

[0209] Source: Jackson Labs

[0210] Sex: Female

[0211] Age at study onset: 6-8 weeks

[0212] Diet: Normal chow

[0213] For inoculation, cells from CD200 S91 NT (non targeting) and CD200 KO S91 cell lines were maintained in vitro as monolayer culture in F-K12 media, 15% Non-HeatInactivated Horse Serum ,2.5%Non-Heat Inactivated FBS,1% Pen Strep. The cells in an exponential growth phase were harvested and counted for tumor inoculation.

[0214] Each mouse was inoculated subcutaneously in the right front flank region in Matrigel -PBS (1 :1) for tumor development. The date of tumor cells inoculation was denoted as day 0. The inoculation media was Matrigel-PBS (1 :1). 500K cells / mouse per 10Oul were inoculated on the right flank while mice were anesthetized by isoflurane. Tumor cells were kept on ice and all inoculations were completed within 2 hrs of cell harvest.

[0215] Antibodies used in the study were purchased from Absolute Antibody and included: isotype control MOPC-21 (4 mg / ml; Lot. #T2131A16), anti-OX90 (4 mg / ml; Lot. # T2039B21), and anti-PD1 (4180; (20.80 mg / ml).

[0216] All mice arrived at the facility and rested for three days prior to any manipulation. Mice were injected subcutaneously with 0.5 million Cloudman S91 Cells with 50% Matrigel. Once the tumor reached 80-100mm3 volume, mice were randomized in two groups of 10 mice and treated by s.c. injection over the shoulders into the loose skin over the neck twice a week with either of 20 mg / kg of MOPC-21 isotype control (AB00178-1.4-BT Absolute Antibody) or anti-CD200 Ab, 0X90 clone (AB00542-1 ,4-BT Absolute Antibody, 20mg / kg, mlgG1-Fc-silent) for a total of 6 doses. Tumor volume and body weight were measured twice a week. Measurements for tumor growth studies were conducted under blinded conditions.

[0217] An adjusted Area Under the Curve (aAUC) statistical test was used to quantitatively assess tumor growth over time for each mouse. The analysis was performed on data through the last dose.

[0218] Statistical analysis was performed using a one-sided test to determine if the ratio of mean adjusted Area Under the Curve (aAUC) between groups was significantly less than 1 , indicating lower tumor growth, with significance assessed at an FDR-adjusted alpha level of 0.05.

[0219] Results

[0220] To evaluate the role of S91 CD200 expression on anti-CD200 efficacy, a CD200 KO S91 melanoma cell line was generated.

[0221] Treatment with anti-CD200 (0X90 clone) did not affect tumor growth in S91 tumors that did not express CD200 (FIG. 7B), as there was no significant difference by aAUC between isotope control and 0X90 treatment groups. These results suggest that CD200 expression on tumor cells is important for 0X90 anti-CD200 Ab (0X90 clone) efficacy, in the S91 melanoma tumor model (FIG. 7A).Example 5: Human prevalence of CD200 in patients receiving anti-CD200R1 antibody therapy

[0222] To determine whether there is an association between higher tumor expression of CD200 and higher probability of clinical benefit, CD200 IHC was performed on tumor tissues from patients treated with 23ME-00610. Clinical benefit (CB) in this example (but not limited to) was defined as stable disease for 6 months or longer duration (SD), any tumor shrinkage, a partial response (PR) or complete response (CR) as defined by RECIST criteria.

[0223] 1. Collection of tissue samples from patients

[0224] Eighty patients having cancer were treated with the anti-CD200R1 antibody 23ME- 00610 according to NCT05199272. Formalin fixed paraffin embedded (FFPE) archival or tumor tissue biopsied prior dosing were evaluated for CD200 and CD200R1 expression by IHC. Best clinical response to anti-CD200R1 treatment was defined by RECIST (Response Evaluation Criteria in Solid Tumors) criteria (Eisenhauer et al., New Response Evaluation Criteria in Solid Tumors: revised RECIST guideline (version 1.1), Eur J Cancer (2009); 45(2): 228-47). Evaluable patients were classified as having clinical benefit (CB) or no response (NR).

[0225] 2. IHC analysis of tissue samples

[0226] A Ventana anti-CD200 IHC assay was used for CD200 staining and quantification. For this IHC assay, an anti-CD200 antibody having inter-day, inter-operator, and interinstrument precision that yields 100% concordance for the IHC assay was employed. In control analyses, the antibody (i) stained 13 out of 14 cell lines as expected based on CD200 expression, (ii) demonstrated immunoreactivity against CD200 in normal human tissue and human tumor samples, and (iii) demonstrated immunoreactivity in ovarian carcinoma, renal clear cell carcinoma, and small cell lung cancer FFPE specimens, wherein 69.8% of ovarian (30 out of 43 tumor samples), 79.5% of renal clear cell carcinoma (31 out of 39 tumor samples), and 28.9% of small cell lung cancer (13 out of 45 tumor samples) samples demonstrated positivity in the tumor cell membrane. The process of carrying out an IHC assay with an appropriate antibody will be clear to those skilled in the art.

[0227] Tumor samples collected from patients provided as above were subjected to the anti- CD200 IHC assay, and the resulting images were assessed by pathologists to determine an H score for the tumor sample from each patient. In this example, the H scores were then divided into bins of 0 (i.e., no CD200 expression), 1-10 (low CD200 expression), 11-99 (i.e., moderate CD200 expression) or 100+ (high CD200 expression) and compared with the clinical benefit to the patient (may include but not limited to clinical benefit such as defined by prolonged stable disease, partial (PR) or complete response (CR), as defined by RECIST criteria, response rate, tumor control or shrinkage, progression free or overall survival. In this example, clinical benefit to the patient was defined as having stable disease (SD) for 6 months or longer and / or tumor shrinkage versus no response defined as progressive disease (PD) during the anti-CD200R1 antibody treatment.

[0228] Results

[0229] Of 66 patients that progressed while being treated with an anti-CD200R1 antibody and had evaluable archival or baseline tumor tissue: (i) 25 patients had tumors with no CD200 expression with H-scores of 0 (38%); (ii) 17 patients had H-scores between 1 and 10 (26%); (iii) 14 patients had H-scores between 11-99 (21%); and (iv) 10 patients had H- scores between 100-300 (15%). Of 13 patients with clinical benefit (CB) (with either (a) stable disease (SD) > 6 months or (b) partial response or (c) any tumor shrinkage): (i) 5 patients had high CD200 expression with H-score (between 100-300) (39%); (ii) 3 patients had moderate CD200 expression with H-score (between 11-99) (23%); (iii) 2 patients had low CD200 expression with H-score (between 1-10) (15%); and (iv) 3 patients had no CD200 expression with H-score of 0 (23%). Median H-score for patients with clinical benefit (CB) (n=13) (defined as stable disease (SD) > 6 months and / or any tumor shrinkage, or partial response (PR)) was 45 (IQR 145.5), compared to 2.5 (IQR 52.2) for patients with no response (NR) (defined as progressive disease (PD)) (n=66 patients with evaluable tumors) (FIG. 8). These results show an association between higher tumor expression of CD200 and higher probability of clinical benefit, which in this example is defined as stable disease (SD) for 6 months or longer duration and / or tumor shrinkage, and / or partial response (PR), in some patients treated with 23ME-00610.

[0230] In NCT05199272 trial participants with neuroendocrine neoplasms, results showed an association between moderate to high membranous CD200 expression and tumor response, which suggests these patients are more likely to have clinical benefit with 23ME- 00610 treatment which is consistent with the mechanism of action of CD200R1 pathway inhibition (FIG. 9).Example 6: Progression-Free Survival (PFS) Probability

[0231] To determine whether patients with high CD200 expression were more likely to benefit from 23ME-00610 treatment, progression-free survival (PFS) probabilities were determined for patients treated with 23ME-00610 that have tumors with moderate to high tumor CD200 expression relative to patients with low to undetectable tumor CD200 expression.

[0232] Results

[0233] Progression-free-survival probabilities were plotted separately for patients with tumor CD200 H-score > 10 and patients with tumor CD200 H-score < 10, using Kaplan-Meier estimates (FIG. 10). Patients with moderate to high CD200 (i.e., >10) appeared to be more likely to benefit from 23ME-00610 treatment with longer duration free of disease progression or death, compared with those who had lower CD200 (i.e., <10).Example 7: Human prevalence of CD200R1 in patients receiving anti-CD200R1 antibody therapy

[0234] To determine whether there is a potential association between CD200R1 expression within tumors and clinical benefit from 23ME-00610 treatment, IHC may be performed on tumor tissues from patients treated with 23ME-00610, similar to Example 4. In vitro studies showed that CD200-mediated inhibition was alleviated by CD200R1 blockage with an anti- CD200R1 antibody (FIG. 3) and suggests that CD200R1 is required for in vitro inhibitory activity of anti-CD200R1.

[0235] These results may show a potential association between CD200R1 expression within the tumor microenvironment and clinical benefit in patients treated with 23ME-00610.

[0236] While the foregoing disclosure of the present invention has been described in some detail by way of example and illustration for purposes of clarity and understanding, this disclosure including the examples, descriptions, and embodiments described herein are for illustrative purposes, are intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to one skilled in the art that various modifications or changes to the examples, descriptions, and embodiments described herein can be made and are to be included within the spirit and purview of this disclosure and the appended claims. Further, one of skill in the art will recognize a number of equivalent methods and procedure to those described herein. All such equivalents are to be understood to be within the scope of the present disclosure and are covered by the appended claims.

[0237] Additional embodiments of the invention are set forth in the following claims.

[0238] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application or other document were individually specifically indicated to be incorporated by reference herein in its entirety for all purposes and were set forth in its entirety herein. In case of conflict, the present specification, including specified terms, will control.

Claims

CLAIMSWhat is claimed is:1 . A method of treating a tumor, comprising administering a therapeutically effective amount of an anti-CD200R1 antibody, wherein cells in the tumor exhibit membrane CD200 expression.

2. A method of selecting a subject for treatment of a tumor using an anti-CD200R1 antibody, the method comprising:(a) determining if the level of expression of CD200 of cells of the tumor is within a clinically established range indicating susceptibility to treatment with an anti-CD200R1 antibody;(b) selecting the subject for treatment with the antibody if the cells of the tumor express CD200 within the clinically established range; and(c) treating the subject with the antibody if the cells of the tumor express CD200 within the clinically established range.

3. A method of treating a tumor, comprising administering a therapeutically effective amount of an anti-CD200R1 antibody, wherein cells in the tumor exhibit membrane CD200 expression, and wherein the administering of an anti-CD200R1 antibody results in a reduction in the growth or size of the tumor.

4. The method of any one of claims 1-3, wherein the anti-CD200R1 antibody is 23ME- 00610.

5. The method of any one of claims 1-4, wherein the tumor is selected from the group consisting of adrenal gland cancer, bladder cancer, sarcomas, microsatellite instability- high (MSI-H) cancer (including solid MSI-cancer), TMB (tumor mutational burden)-high tumor, mismatch repair deficient (dMMR) cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, EGJ adenocarcinoma, esophageal cancer, gallbladder cancer, gastric cancer (e.g., gastrointestinal carcinoid (Gl carcinoid)), head and neck cancer, heart cancer, hepatocellular carcinoma, kidney cancer, liver cancer, melanoma, mesothelioma (e.g., pleural mesothelioma), non-small cell lung cancer, ovarian cancer, epithelial ovarian cancer, endometrial cancer, pediatric solid cancers, pancreatic cancer, prostate cancer, spleen cancer, small cell lung cancer, testicular cancer, thyroid cancer (e.g., medullary thyroid cancer or follicular thyroid cancer), blood cancers (e.g., diffuse large B cell lymphoma (DLBCL), leukemias, lymphomas, myelomas), renal cell carcinoma, clear cell renal carcinoma, neuroendocrine tumors (e.g., malignant pheochromocytoma and paraganglioma), and uterine cancer. In some embodiments, the cancer is selected from lung cancer (e.g., small cell lung cancer), skin cancer (e.g.,melanoma), pancreatic cancer, endometrial cancer, prostate cancer, colorectal cancer, ovarian cancer, mesothelioma, and bladder cancer.

6. The method of claim 1 or claim 3, wherein the treatment is in a subject in need thereof.

7. The method of any one of claims 1-6, wherein the subject is human.

8. The method of claim 1 , wherein membrane CD200 expression is determined by IHC.

9. The method of claim 8, wherein a measure of the membrane CD200 expression is provided by an H score.

10. The method of claim 2, wherein the expression is on the surface of the cells of the tumor.11 . The method of claim 3, wherein the administering results in a reduction in the growth or size of the tumor.

12. The method of claim 3, wherein the progression free survival rate is higher than without.

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