CD200 Receptor 1 agonists

Field of the Invention

The present invention relates to agonistic ant.i-CD200 receptor 1 antibodies, polynucleotides encoding the antibodies or fragments, and methods of making and using the foregoing.

Background of the Invention

The immune system functions to protect individuals from infective agents, e.g., bacteria, multi-cellular organisms, and viruses, as well as from cancers, and includes several types of lymphoid and myeloid cells such as monocytes, macrophages, dendritic cells (DCs) , eosinophils, T cells, B cells, and neut ophils.

CD200 receptor 1 (CD200R1) is a member of the immunoglobulin superfamily having an inhibitory role in immunity. CD200R1 is expressed on myeloid cells such as macrophages, dendritic cells (DC), neutrophils, basophils and mast cells; and T cells (Wright et al (2000) Immunity 12:233-242; Wright et al (2003) J. Immunol. 171:3034- 3046) . Myeloid cells play important roles in maintaining chronic inflammation (Kinne et al (2000) Arthritis Res. 2:189-202; Kiefer et al (2001) Progr . Neurobiol . 64:109- 127; O'Shea et al (2002) Nat. Rev. Immunol. 2:37-45; Haraid et al C2003) J. Allergy Clin. Immunol. Ill: S5-S12; Liu et al (2004) Rheum Dis. Clin. North Am. 30:19-39) . They can be regulated through cell-cell interactions that trigger matched sets of act i ating and i h ib11ory receptors, in addition to being regulated by secreted factors (Barclay et al (2002) Trends in Immunol. 23: 285-290) . The regulation of myeloid cell activity by direct cell-cell contact allows a more localized control than that mediated by cytok ines .

CD200 is the only known CD200R1 ligand and is more broadly expressed on a variety of cells, such as thymocytes, 3 cells, some peripheral T cells, follicular DCs, endothelium, epithelium, smooth muscle, neurons of the central nervous system, and cells in reproductive organs. CD200 binding to CD200R1 initiates an inhibitory signal to myeloid cells and T cells and this signal may function as an additional or potentiating inhibitory control on damaging proinflammatory activity. CD200R1 is phosphorylated. on tyrosine residues upon ligand b nding . Phosphorylation, results in recruitment of adapter proteins Dok-1 a d Dok-2 , hic are i n tur phosphor1yated a d associate with RasGAP and SHIP, leading to inhibition of MAPK pathway {Zhang st al (2004), J. Immunol. 173:6786- 6793) .

A viral horaolog of CD200 exists and has been shown to mimic human CD200 in down-regulating macrophage activation through CD200R1. In humans, CD200 has been reported to be down regulated in multiple sclerosis (Koning et al (2007) Ann Neurol. 62:504-14), asthma exacerbation (Aoki et al

(2009) Clin. Exp. Allergy 39 : 213-21 ) , Alzheimer's disease

(Walker et al (2009) Exp. Neurol. 215:5-19) , Lichen planopilaris (Harries et al (2013) J Pathol. Epub ahead of print; Jun 20. doi : 10.1002 /path , 4233 ) , primary

hypertrophic osteoarthropathy (Ren et al (2013) Rheumatol Int. Epub a.hed fo print; Apr 18), and failed pregnancy

(Clark (2009) Am J Reprod Immunol. 2009 61:75-84) . CD200R1 agonists reduce pathology in various mouse models such as arthritis (Gorczynski et al (2001) Clin. Immunol. 101:328- 34; Gorczynski et al (2002) Clin. Immunol. 104:256-64), graft rejection. (Gorczynski et al (2002) Transplantation 73:1948-53.), abortion (Gorczynski et al (2002) Am. J. Reprod. Immunol. 48:18-26,), contact hypersensitivity

(Rosenblum et al (2004) Blood 103:2691-8), influenza- induced lung inflammation (Snelgrove et aJ (2008) Nat. Immunol. 9:1074-83), and KSV- induced immunoin flarnmatory lesion (Sarangi et al (2009) Clin. Immunol. 131:31-40) . Agonistic CD200R1 antibodies have been described; see e.g. Int. Pat. Publ . No. WO08/079352.

The need exists for agonists of CD2Q0R1, such as an i-CD200Rl monoclonal antibodies, for use in treatment of human disorders, such as inflammatory or autoimm ne disorders .

Brief Description of the Drawings

Figure 1A shows inhibition of 10 ng/ml

lipopolysaccharide (LPS) --induced IL-6 release in

differentiated U937 cells over-expressing human CD200R1 by antibodies H2RM190 and H2RM211. Data represents mean of quadruplicates +/- SEM.

Figure IB shows inhibition of 10 ng/ml LPS-induced IL-6 release in differentiated U937 cells ove --expressing human CD200R1 by antibody H2RM147 or CD200-FC fusion protein. Data represents mean of quadruplicates +/- SEM.

Figure 1C shows inhibition of 10 ng/ml LPS-induced IL-6 release in di ferentiated U937 ceils over-expressing human CD200R1 by antibodies H2RM226 and H2RM266. Data represents mean of quadruplicates +/- SEM.

Figure 2A shows the ribbon representa ion of the K2RM147 Fab:CD200Rl complex as observed in the crystal structure (H2RM147 is named M147 in the Figure. CD200R = CD200R1.

Figure 2B shows the paratope and the epitope of

H2RM1 7 having the VH of SEQ ID NO: 10 and the vL of SEQ ID NO: 33. Epitope residue numbering is according to mature human CD200R1 (SEQ ID NO: 4) . HCDR1, HCDR2 and HCDR3 residue numbering is according to SEQ ID NO: 10, and LCDR1, LCDR2 and LCDR3 residue numbering is according to SEQ ID NO: 33. CD200R1 residues are shown in ellipses, and H2RM147 residues are boxed and grouped by the CDRs . Residues Nil and N20 highlighted in grey are glycosylated .

Figure 3 shows inhibition of 10 ng/ml LPS-induced IL- 6 elease i differentiated U937 cells over-expressing human CD200R1 by antibodies H2RM185 (M185) , H2RM187

(M187) , H2RM190 (M190) , H2RM200 (M200) and K2RM204 (M204) . CNT08937 : isotype control; H2RG14 is CD200 extracellular domain coupled to Fc (CD200-Fc) .

Figure 4 shows inhibition of 10 ng/ml LPS- induced IL-

6 release in differentiated U937 cells over-expressing human CD200R1 by indicated antibodies. KRM211-K2RM211. CNT08937: isotype control; H2RG14 is CD200-FC.

Figure 5 shows inhibition of 10 ng/ral LPS-induced IL-- 6 release in differentiated U937 cells expressing cyno CD200R1 by antibodies H2RM226 and H2RM266 at indicated doses in parenthesis at μg/πll.

Figure 6A shows the VL sequences of the parental antibody H2RM211 and antibodies generated through aff nity maturation of H2RM211. Parental VL chain: H2RL191. Rabat CDRs are underlined. SEQ ID MOs : for each VL is shown in parenthesis after the VL name.

Figure 6B shows the consensus VL and LCDR sequences derived, from affinity-matured H2RM211 light chains shown in Figure 6A. Rabat CDRs are underlined.

Figure 7A shows the VH sequences of the parental antibody H2RM147 and antibodies generated through affinity maturation of H2RM147. Parental VH chain: H2RH81. SEQ ID NOs : for each VH is shown, in parenthesis after the VH name. Kabat CDRs are underlined.

Figure 7B shows the VL sequences of the parental antibody H2RM147 and antibodies generated through affinity maturation of H2RM147. Parental VL chain: PH9L1. SEQ ID NOs: for each VL is shown in parent esis after the VL name. Rabat CDRs are underlined.

Figure 7C shows the consensus VH and HCDR sequences derived from affinity-matured H2RM147 heavy chains shown in Figure 7A. Kabat CDRs are underlined.

Figure 7D shows the consensus VL and LCDR sequences derived from affinity-matured H2RM147 light chains shown in Figure 7B. Kabat CDRs are underlined. Figure 8A shows inhibition of nontypeable Haemophilu influenzae (NTHi) -induced G-CSF release in differentiated and cigarette smoke extract (CSE) -pretreated U937 cells over-expressing human CD200R1 by CD200R agonists CD200-FC and K2RM147 (M147 in the Figure), whereas Fluticasone propionate (FP) increased the G-CSF release, ERK inhibitor (PD98059) inhibited G-CSF release at similar degree as H2R.M147, whereas p38 inhibitor (SB203680) had n.c effect. M: medium only.

Figure 8B shows inhibition of NTHi-induced IL-Ιβ release in differentiated and cigarette smoke extract (CSE) -pretreated U937 cells over- express ing human CD200R1 by CD200R agonists CD200-FC and H2RM147 (Ml 7 in the Figure) , whereas Fluticasone propionate (FP) had no effect. ERK inhibitor (PD98059) inhibited IL-lB release at similar degree as H2RM147, whereas p38 inhibitor (SB203680) had no effect. M: medium only.

Figure 8C shows inhibition of nNTHi-induced IL-6 release in dif erentiated and cigarette smoke extract (CSE) -pretreated U937 cells over-expressing human CD200R1 by CD200R agonists CD200-Fc and H2RM147 (Ml47 in the Figure) and Fluticasone propionate (FP) . FP potentiated the inhibitory effect of CD200 and H2RM147, Both ERK inhibitor (PD98059) and p38 inhibitor (S3203680) were effective in suppressing IL-6 release. M: medium only.

Figure 8D shows inhibition of nNTHi- induced TNF-a release in differentiated and cigarette smoke extract (CSE) -pretreated U937 cells over-expressing human CD200R1 by CD200R agonists CD200-Fc and H2RM1 7 (M147 in the Figure) and Fluticasone propionate (FP) , FP potentia ed the inhibitory effect of CD200 and H2RM147 and ERK inhibitor (PD98059) . p38 inhibitor (SB203680) alone was effective in suppressing IL-6 release. M: medium only. Summary of the invention

One embodiment of the inventio is an isolated agonistic antibody that speci ically binds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4) , compris ng certain heavy chain CDR and certain light chain CDR sequences as described herein.

Another embodiment of the invention is an isoleited agonistic antibody that specifically binds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4) , comprising certain heavy chain variable regions (VH) and certain light chain variable regions (VL) sequences as described herein.

Another embodiment of the invention is an isolated agonistic antibody that specifically binds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4), wherein the antibody competes for binding to human CD200R1 (SEQ ID NO: 4) with an isolated antibody comprising the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 33.

Another embodiment of the invention is an isolated agonistic antibody that specifically binds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4) , wherein the antibody competes for binding to human CD200R1 (SEQ ID NO: 4) with an isolated antibody comprising the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 35.

Another embodiment of the invention is an isolated agonistic antibody that specifically binds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4) comprising the VH and the VL of SEQ ID NOs : 12 and 35 or 34 and 33, produced by a method comprising:

incorporating into an expression, vector

a first synthetic polynucleotide encoding the VH of SEQ ID NOs: 12 and a second synthetic polynucleotide encoding the VL of SEQ ID NOs: 35; or

a first synthetic polynucleotide encoding the VH of SEQ ID NOs: 34 and a second synthetic polynucleotide encoding the VL of SEQ ID NOs: 33; transforming a host cell with the expression vector; culturing the host cell in culture medium under condi ions wherein the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 35 or the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 33 are expressed; and

recovering the antibody comprising

the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 35; or the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 33 rom the host cell or culture medium.

Another embodiment of the invention is an isolated synthetic polynucleotide encoding the VH of SEQ ID NOs :

10, 12, 34, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51 or 55; or the VL of SEQ ID NOs: 11, 13, 33, 35, 56, 57, 58, 59,

60, 61, 62, 63, 64, 65, 66, 67 or 68.

Another embodiment of the invention is a vector comprising an isolated synthetic polynucleotide of the invention

Another embodiment of the inventio is a host cell comprising the vector of the invention.

Another embodiment of the invention is a method of producing an antibody of the invention, comprising culturing the host cell of the invention and recovering the antibody from the cell.

Another embodiment of the invention is a

pharmaceutical composition comprising an isolated antibody of the invention and a pharmaceutically accepted carrier.

Another embodiment of the invention is a method of treating or preventing a human CD200Rl-mediated condition comprising administering a therapeutically effective amount of an isolated antibody of the invention to a patient in need thereof for a time sufficient to treat or prevent the CD200R1 -mediated condition.

Detailed. Descxiption of the Invention

All publications, includi g but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments onl and is not i tended to be limiti g.

Unless defined otherwise, all technical and scientific terms used herein heive the same meaning as commonly understood by one of o dina y skill in the art to which the invention perta ns .

Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention., exemplary materials and methods are described herein. In describing and claiming the present invention, the following

termino1ogy wi11 be used .

"Specific binding" or "specifically binds" or "binds" as used herein refers to antibody binding to a

predetermined antigen with greater affinity than for other antigens . Typically, the antibody binds to a

predetermined antigen with a dissociation constant (K _E ) of about 1x10 ^" ' M or less, for example about 1x10 ^"' M or less, about IxlO ^"9 M or less, about IxlO ^"10 M or less, about 1x10 ^" - ^ll M or less, or about IxlO ^"1 ' M or less, typically with a K _D that is at least ten fold less than its K _c for binding to a non-specific antigen or epitope (e.g., 3S.A, casei ) . The dissociation constant can be measured using standard procedures. Antibodies that specifically bind to a predetermined antigen may, however, have cross-reactivity to other related antigens, for example to the same predetermined antigen from, other species (homologs) , such as human or monkey, for example Macaca fascicularis

(cynomolgus, cyno) or Pan troglodytes (chimpanzee, chimp) .

"Monoclonal antibody that specifically binds human CD200R1" refers to antibodies that specifically bind human CD200R1 isoform d having the sequence shown in. SEQ ID NO: "Agonist" or "agonistic" or "antibody agonists" or "agonistic antibody" as used herein refers to a CD200R1 antibody or antibody fragment that activates the CD200R1- medilated inhibitory activity to the cell. Agonistic CD200R1 antibodies can be identified using assays for CD200R1 -mediated inhibitory activity such as measuring reduction by the agonistic antibody of lipopolysaccharide (LPS) -induced. IL-6 secretion, in differentiated human lymphoma cell line U937 overexpressing human CD200R1 using the "U937 IL-6 secretion assay" as described herein in Example 2. The agonistic CD200R1 antibody may inhibit the LPS -induced IL-6 secretion by 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% ,

"CD200" or "huCD200" as used herein refers to the human CD200 protein having the amino acid sequence shown in SEQ ID NO: 26 and in GenBank Accession Number

NP__005935.

"Antibodies" as used herein, is meant in a. broad, sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, human- adapted, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies formed rom at least two Intact antibodies or antibody fragments, dime ic, tetrameric or multimeric antibodies, and single chain antibodies.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain, constant domain amino acid, sequence. IgA and IgG are further sub-classified as the isot pes IgAi, IgA ₂ , IgGi, lgG ₂ , IgG ₃ and IgG ₄ . Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa ( ) and lambda (λ) , based on the amino acid sequences of their constant domains .

3 "Antibody fragments" refers to a portion of an immunoglobulin molecule that retains the heavy chain and/or the light chain antigen binding site, such as a heavy chain, complementarity determining regions (HCDR) 1, 2 and 3, a light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH) , or a light chain variable region (VL) . Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments l nked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an an ibody; a dAb fragment (Ward et al (1989) Nature

341:544-- 546), which consists of a VH domain. VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains pair

intramolecula 1 y, or i termol ecula l y in those cases when, the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Pat. Publ . No. WO1998/44001, Int. Pat. Publ. No. WO1988/01649; Int. Pat. Publ. No.

WO1994/13804 ; Int. Pat. Publ. No. WO1992/01047 , These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

"Isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody agonists that specifically binds human CD200R1 is substantially free of antibodies that speci ically bind antigens other than human CD200R1) . An isolated antibody that specifically binds human CD200R1 may, however, have cross- reactivity to other antigens, such as orthologs of human CD200R1, such as Macaca fascicularis (cynomolgus) CD200R1. Moreover, an isolated antibody may be

substantially free of other cellular material and/or chemicals .

An antibody variable region consists of a "framework" region interrupted by three "antigen binding sites". The antigen binding sites are defined using various erms: (i) Complementarity Determining Regions (CDRs) , three in the VH (HCDR1 , HCDR2 , HCDR3 ) and three in the VL ( LCDR1 , LCDR2, LCDR3) are based on sequence variability { u and Rabat (1970) J. Exp. Med, 132:211-50; Rabat et al

Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) . (ii) "Hyperva iable regions", ^W HVR", or ^W HV", three in the VH (HI, H2 , H3) and three in the VL (LI, L2, L3) refer to the regions of an antibody variable domains which are hypervariafole in structure as defined, by Chothia and Lesk. (Chothia and Lesk (1987) Mol . Biol. 196:901-17) . Other terms include "IMGT-CDRs"

(Lefranc et al (2003) Dev Comparat Immunol 27:55-77) and "Specificity Determining Residue Usage" (SDRU) (Almagro

(2004) Mol. Recognit. 17:132-43) . The International ImMunoGeneTics ( IMOT) database (http://www imgt org) provides a standardized numberi g and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al

(2003) Dev Comparat Immunol 27:55-77.

"Chothia residues" as used herein are the antibody VL and VH residues numbered according to Al-Lazikani (A1-- Lazikani et al (1997) J. Mol. Biol. 273:927-48) .

"Framework" or "framework sequences" are the remaining sequences of a variable region other than those defined to be an igen bi ding site. Because e antigen binding site can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigen-binding site was defined.

"Humanized antibody" refers to an antibody in which the antigen binding site is derived rom non-human species and the variable region frameworks are derived from, human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions so that the framework may not be an exact copy of expressed human i mmunoglobui in or germline gene sequences.

"Human-adapted" antibodies or "human framework adapted (HFA) " antibodies refers to humanized antibodies adapted eiccording to methods described in U.S. Pat. Publ . No. US2009/0118127. Human-adapted antibodies are humanized by selecting the acceptor human frameworks based on the maximum CDR and FR similarities, length

compatibilities and sequence similarities of CDR1 and CDR2 loops and a portion of light chain CDR3 loops.

"Humein antibody" refers to an antibody having heavy and l ght chain, variable regions in which both the framework and the antigen binding site reg ons are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin.

Human an ibody comprises heavy or light chain variable regions that are "derived from" sequences of human origin if the variable regions of the antibody are obtained from a system that uses human germline

immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin, gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci as described herein. "Human antibody" may contain amino acid

differences when compared to the human germline or rearranged, immunoglobulin sequences due to for example nat rally occurring somatic mutations or i tentional introduction of substitutions. Typically, "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, "human antibody" may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knapplk et al (2000) J. Mol . Biol. 296:57-86), or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al (2010) J. Mol. Biol. 397:385-96, and Int. Pat. Publ . No. WO2009/085462.

Antibodies in which antigen binding sites are derived from a non-human species are not included in the definition of "human antibody" .

Isolated humanized antibodies are synthetic. Human antibodies, while derived from human immunoglobulin sequences, can be generated using systems such as phage display inco rporating synthetic CDRs and/or synthetic frameworks, or can be subjected to n vitro mutagenesis for example during affinity maturation to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vi vo .

"Recombinant antibody" as used herein includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to a heterologous DNA sequence . "Monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition, A monoclonal antibody composition displays a single bindi g specifici y and affini y for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes.

"Substantially identical" as used herein means that the two antibody variable region amino acid sequences being compared are identical or have ^insubstantial di ffe re ces " . Insubstantia 1 di f fe e ces are substitut ions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in an antibody variable region sequence that do not adversely affect antibody properties. Amino acid sequences substantially identical to the variable region sequences disclosed herei are within he scope of the invention. In some embodiments, the sequence identity can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.

In some embodiments, the sequence identity can be about 95%, 96%, 97%, 98%, 99% or higher. Percent identity can be determined for example by pairwise alignment using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA) . The protein sequences of the present invention can be used as a query sequence to perform a search against public or patent databases to, for example, identify related sequences. Exemplary programs used to perform such searches are the XBLAST or BLASTP programs (http //www ncbi nlm/nih gov), or the GenomeQuest™ (GenomeQuest, Westborough, MA.) suite using the defau11 se11irigs.

Epitope" as used herein means a portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surfi3.ce groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be corr.posed of contiguous and/or discontiguous amino acids that form a. conformational spa i l unit. For a

d iscontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule.

"Bispecific" as used herein refers to an antibody or molecule that binds two distinct antigens or two distinct epitopes withi an antigen.

"Monospecific'' as used herein refers to an antibody that binds one antigen or one epitope .

"In combination with" as used herein means that the described a.gents can be administered to an animal together in a mixture, concurre tly as si gle agents or

sequentially as single agents in any order,

"Vector" means a non-natural polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain a cDNA encoding a protein of interest and additional elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these pol nucleotides in. a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DMA or RNA molec les or a hyb id, of these.

"Expression vector" means a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a

polypeptide encoded by a polynucleotide sequence present in the expression vector.

"Polynucleotide" means a. molecule comprising a chain of nucleotides covalently linked by a s gar--phosphate backbone or other equivalent covalent chemistry. Double and single-stranded DNAs and RNAs are typical examples of polynucleotides .

"Complementary DNA" or "cDNA" refers to a well known synthetic polynucleotide that shares the arrangement of sequence elements found in native mature mRNA species with contiguous exons, with the intervening introns present in genomic DNA are removed. The codons encoding the initiator methionine may or may not be present in cDNA . cDNA may be synthesized for example by reverse

transcription or synthetic gene assembly.

"Synthetic" or "non-natural" as used herein refers to a polynucleotide or a polypeptide molecule not present in na t are .

Table 1.

Ami o acid Three- letter One- letter

code code

A la. i e Ala A

Arginine Arg R

Asparagine Asn N

Aspartate Asp D

Cyste ine Cys C

Glutamate Qln Q

Glutami e Gl Ξ

Glyci e Gly G

Histidine His H

I soleucine He I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser c;

Threonine Thr T Tryptop an Trp W

Tyrosine Tyr Y

Valine Val

"Polypeptide" or "protein" means a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids ma be referred to as "peptides".

Conventional one and three-letter amino acid codes are used herein as shown in Table 1.

CD200 Receptor 1 (CD200R1) and CD200 Receptor 1 -like proteins (CD20GR1L)

CD200R1 been ide ti fied from arious species including human, mouse, rat, and non-human primates based on overall sequence homology. In humans, CD200R1 is present in four alternatively spliced forms, a-d (SEQ ID NOs : 1-4) . Human CD200R1 is an inhibitory receptor transmitting an inhibitory signal to myeloid cells upon ligand human CD200 { SEQ ID NO: 26) binding, or upon binding of an agonistic antibody,

SEQ ID NO: 26:

AWLCTAQVQWTQDEREQLYTPASLKCSLQNAQEALIVTWQK KAVSPENMVTFSEN HGWIQPAYKDKINI QLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTV YVQPIVSLHYKFSEDHLNITCSATARPAPMVTWKVPRSGIENSTVTLSHPNGTTSVTS ILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISI LLY KRHRMQDREP

CD200R1 lacks the i m no ecepto t asi e-based inhibitory motif (ITIM) doma n commonly found in immune inhibiting receptors that cause recruitment of

phosphatases and downregulation of inflammatory pathways. Instead, the CD200R1 cytoplasmic tail contains 3 tyrosine residues as potential phosphorylation sites. One of these tyrosine residues is located within a NPXY motif, which is phosphorylated upon ligation of the ecepto (Zhang et al (2004) J. Immunol. 173:6786-6793), and subsequently binds adapter proteins Dokl and Dok2. Phosphorlyation of the adapter proteins recruits RasGAP and SHIP, and inhibition of the Ras/MAPK activation pathways (Zhang et al (2004) J. Immunol. 173:6786-6793) . CD200R1 orthologs in. mouse (mouse CD200R1, SEQ ID NO: 28 and cyno {Macaca

fascicularis ; SEQ ID NO: 5) share 59 and 90 % identity to the human CD200R1 isoform a in their extracellular domains a.nd also transmit an inhibitory signal to myeloid, cells upon CD200 or agonis c antibody binding.

SEQ ID NO: 28:

TDKNQTTONNSSSPLTQYNTTYSYQIGTKALLCCFSI PLTKAYLI WI IKLRGLPSCT

lAYKYDTKTNETSCLGRNITWASTPDHSPELQISAYTLQKEGTYTCETVTPEGNFEK N YDLQVLVPPEYTYFPEKNRSAVCEAMAGKPAAQIS SPDGDCVTTSESHSNGTYTVRS TCH EQN VSDVSCIVSHLTGNQSLSIELSRGGNQSLRPYIPYI IPSIIILIIIGCIC LLKISGFRKCKLPKLEATSAIEEDEMQPYASYTEKSNPLYDTY KVEAFPVSQGEYNG TDCLTLSAIGI

CD200R1 related proteins, CD200 Receptor 1-like (CD200R1L) proteins include the human CD200R1L (SEQ ID NO: 27), the cyno CD200R1I, (SEQ ID NO: 6), and several mouse CD200R1L proteins (Wright et al (2003) J. Immunol.

171:3034-46, 2003) . Some of the mouse CD200R1L proteins have been shown to associate with the activating adaptor protein, DNAX activating protein DAP12 (Hatherley et al (2005) J Immunol 175: 2469-74), and therefore activate myeloid cells .

The human CD200R1L protein (SEQ ID NO: 27) lacks two cysteine residues that are conserved throughout the CD200 receptor family members, and overexpression of the protein has been unsuccess l unless the protein is enginee ed to include the cysteines, in which case surface expression is achieved with Dapl2, indicating potential activation function for CD200R1L ( U.S. Pat, No. 8,212,008) . The cyno CD200R1L has been shown to be activating receptor on myeloid cells (U.S. Pat. No. 8,212,008) .

Despite their name based on homology to the

extracellular domain of CD200R1, the CD200R1L proteins do not appear to bind CD200 and at present the ligand(s) for these additional receptors remains unknown (Hatherly et al (2005) J, Immunol . 1 5:2469-74) .

"Human CD200 receptor 1" OR "hu.CD200Rl" or

wbumanCD200Rl as used herein refers to the human CD200R1. The human CD200R1 is a type 1 membrane protein with two im unoglobulin-like domains in the extracellular portion of the molecule. Several splice variants of human CD200R1 exist and inclu.de isoforms a and d having amino acid sequences shown in SEQ ID MOs : 1 and 4, respectively and in GenBank Accession Numbers NP_620161 and NPJ740750, respectively. Truncated human CD200R1 variants lacking one immunoglobulin domain are for example isoform b and i soform c, shown in SEQ ID NOs : 2 and 3, respectively, and in GenBank Accession Numbers NP_620385 and NP_620386 , respectively .

SEQ ID NO: 1 :

LCMDEKQI Q YSKVLAEVNTSWPVKMATNAVLCCPPIALRNL11 ITWEI ILRGQPSC

TKAYKKETNETKETNCTDERITWVSRPDQNSDLQIRTVAITHDGYYRCIMVTPDGNF K RGYHLQVLVTPEVTLFQ RNRTAVCKAVAGKPAAHJSWIPEGDCATKQEYWSNGTVTV KSTCHWEVH VSTVTCHVSHLTGN SLYIELLPVPGAKKSAKLYIPYIILTIIILTIV GFI LLKVNGCRKYKLNKTESTP EEDEMQPYASYTEKNNPLYDTTNKVKASEALQS EVDTDLHTL

SEQ ID NO: 2:

CMDEKQI QNYSKVLAEVNTSWPVKMATNAVLCCPPIALRNL11 IT EI ILRGQPSCT KAYKKETNETKETNCTDERITWVSRPDQNSDLQIRTVAITHDGYYRCIMVTPDGNFHR GYHLQVLGKEHKI LP.YFTS PDL

SEQ ID NO: 3:

CMDEKQI TQNYSKVLAEVNTSWPVKMATNAVLCCPPIALRNLI I ITWEI ILRGQPSCT KAYKKETNETKETNCTDERITWVSRPDQNSDLQIRTVAITHDGYYRCIMYTPDGNFHR GYHLQVLGKEHHI LRYFTS PDL

SEQ ID NO: 4:

LCMDEKQITQNYSKVLAEVNTSWPVKMATNAVLCCPPIALRNL11 ITWEI ILRGQPSC TKAYKKETNETKETNCTDERITWVSRPDQNSDLQIRTVAITHDGYYRCIMVTPDGNFH RGYHLQVLVTPEVTLFQNRNRTAVCKAVAGKPAAHISWI PEGDCATKQEYWSNGTVTV KS CKWEV.HNYSTVTCHYSHLTGNKSLYIELJJPVPGA.KKS AKLYIPYI ILTI I ILTIV GFIWLLKVNGCRKYKLNKTESTPWEEDEMQPYASYTEKNNPLYDTTNKVKASEALQS EVDTDLHTL ^w Cyno CD200 receptor 1" or cynoCD200Rl" refers to the Macaca fascicularis { cynomolgus ) CD200R1 having the sequence shown in SEQ ID NO: 5.

SEQ ID NO: 5:

LCMDEKQITQNKSKVLAEVNIS PVQMARNAVLCCPPIEFRNLIVIT EIILRGQPSC TKTYRKDTNETKETNCTDERITWVSTPDQNSDLQIHPVAITHDGYYRCIMATPDGNFH RGYHLQVLV PEy LFESRNRTAVCKAVAGKPAAQISW PAGDCAPTEQEY GNGTVT YKSTCHWEGHNVS YTCHVSHLTGNKSLYIELLPVPGAKKSAKLYMPYVIL I I ILTI YGFIWLLKISGCRKYNLNKTESTSVVEEDEMQPYASYTEKNNPLYDTTNKYKASQALQ SEVGTDLHTL

"Human CD200 receptor 1-like" or "humanCD200RlL" as used herein refers to the human CD200R1L protein having the sequence shown in SEQ ID NO: 27.

SEQ ID NO: 27:

GKQMTQNYSTIFAEGNISQPVLMDINAVLCCPPIALRNLIIIT EIILRGQPSCTKAY KKETNETKET CTVERITWVSP.PDQNSDLQIRPVDTTHDGYYP.GIVVTPDGNFI-IRGYH LQVLVTPEV LFQ5P.NITAVCKAVTGKPAAQI SWI PEGS ILATKQEYWGNGTVTVKST CPWEGHKSTVTCHVSHLTGNKSLSVKL SGLRTSGSPALSLLI ILYVKLSLFVVILVT TGFVFFQRINHVRKYL

"Cyno CD200 receptor 1-iike" or ^w cynoCD200RlL" as used herein refers to the Macaca fascicuiaris (cynomolgus) CD200R1L protein having the sequence shown in SEQ ID NO: 6.

SEQ ID NO: 6

SCMDGKQMTQNYSKMSAEGNISQPVLMDTNANLCCPPIEFRNLIVIVWEIIIRGQPSC TKAYRKETNETKETNCTDKRITWVSTPDQNSDLQIHPVAITHDGYYRCIMATPDGNFH RGYHLQVLVTPEVTLFQSR RTAVCKAVAGKPAAQIS I PAGDCAPTEHEY G GTVT VES C: GDH ASTVTCI-IVSfILTG KSLYIKLNSGLRTSGSPALDLLIILYVKLSLFV YILVTTGFVFFQRINYVRKSL

Compositions of matter

The present invention, provides agonistic monoclonal antibodies tha bind human CD200R1, and therefore activa e the human CD200R1 -mediated inhibitory activity in a cell. The antibodies of the invention cross -react with cyno CD200R1, and optionally do not cross-react with the cyno activating receptor CD200R1L. The antibodies of the invention bind CD200R1 with higher affinity and may be more potent in activating the human CD200R1-mediated inhibitory activity in the cell than the natural ligand CD200. The present invention provides synthetic

polynucleotides encoding the antibodies and fragments thereof, vectors and hos ceils, and methods of making and using the antibodies of the invention.

One embodiment of the invention is an isolated monoclonal antibody agonist or fragment thereof that specifically binds human CD200 receptor 1 (CD200R1) having the sequence of SEQ ID NO: 4, wherein the monoclonal antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining regions (HCDR) 1 (HCDRl ) , 2 (HCDR2 ) and 3 (HCDR3 ) , and a light chain variable region (VL) comprising a light chain complementarity determining reg ons (LCDR) 1 (LCDRl), 2 (LCDR2) and 3 (LCDR3), wherein the antibody cross-reacts with Macaca fascicularis (cynomolgus) CD200R1 of SEQ ID NO: 5.

Another embodiment of the invention is an isoleited monoclonal an ibody agonist or fragment thereof that specifically binds human CD200 receptor 1 (CD200R1) having the sequence of SEQ ID NO: 4, wherein:

the HCDRl comprises the sequence of SEQ ID NOs : 14,

20 or 38;

the HCDR2 comprises the sequence of SEQ ID NOs: 15, 21 or 39;

the HCDR3 comprises the sequence of SEQ ID Os: 16 or 22;

the LCDRl comprises the sequence of SEQ ID Os: 17, 23 or 36;

the LCDR2 comprises the sequence of SEQ ID NOs: 18 or 24; and

the LCDR3 comprises the sequence of SEQ ID NOs: 19, 25, 37 or 40.

Another embodiment of the invention is an isolated monoclonal antibody agonist or fragment thereof that specifically binds human. CD200 receptor 1 (CD200R1) having

2 the sequence of SEQ ID NO: 4, wherein the HCDR1, the HCDR2, the HCDR3 , the LCDR1, the LCDR2 and the LCDR3 comprise the sequence of

SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively;

SEQ ID NOs: 20, 21, 22, 23, 24 and 25, respectively;

SEQ ID NOs: 14, 15, 16, 36, 18 and 37, respectively;

SEQ ID NOs: 38, 39, 16, 36, 18 and 37, respectively; or

SEQ ID NOs: 20, 21, 22, 23, 24 and. 40, respectively.

In another embodiment, the isolated monoclonal antibody agonist or fragment thereof that specifically binds human CD200 receptor 1 (CD200R1) having the sequence of SEQ ID NO: 4 comprises the VH and the VL, wherein the VH and the VL comprises the sequence of

SEQ ID NOs: 10 and 11, respecti ely;

SEQ ID NOs: 12 and 13, respectively;

SEQ ID NOs: 10 and 33, respectively;

SEQ ID NOs: 34 and 33, respectively; or

SEQ ID NOs: 12 and 35, respectively.

Another embodiment of the invention is an isolated agonistic antibody that specifically binds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4), comprising a heavy- chain complementarity determining region (HCDR) 1 (HCDR1), 2 (H.CDR2) and 3 (H.CDR3) of SEQ ID NOs : 143, 144 and 145, respectively .

Another embodiment of the invention is an isolated agonistic antibody that specifically binds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4), comprising a heavy chain complementarity determining region (HCDR) 1 (HCDR.l), 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 143, 144 and 145, respectively, and a light chain complementarity

determining region (LCDR) 1 (LCDR1), 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs: 146, 147 and 148, respectively,

Agonistic antibodies of the invention comprising the HCDR1 , HCDR2 , HCDR3 , LCDR1 , LCDR2 and LCDR3 sequences of SEQ ID NOs: 143, 144, 145, 146, 147 and 148 bind human CD200R1 with higher affinity and may agonize CD200R1 inhibitory singaling with higher potency than human CD200. Human CD200 binds human CD200R1 with a KD of about 1.7xl0 ^~8 M.

In some embodiments, the antibodies of the invention bind human CD200R1 with an affinity constant (K _D ) of

about 1.7xl0 ^~8 M or less;

about IxlO ^"9 M or less; or

between about 6x10 ^"9 M and about 7xl0 ^~]0 M, when the K is measured using FroteOn XPR36 using a protocol described in Example 2.

In some embodiments, the antibodies of the invention bind cyno olgus CD200R1 of SEQ ID NO: 5 with the K _D of between about 2.5x10 ^""7 M to about 4xl0 ^~10 M,

In some emboidments, the antibody of the invention comprises

the HCDR1 of SEQ ID NOs: 14, 38, 69 or 70;

the HCDR2 of SEQ ID NOs: 15, 39, 71, 72, 73, 74, 75,

76 or 142;

the HCDR3 of SEQ ID NOs: 16, 77 or 79;

the LCDR1 of SEQ ID NOs: 17, 36, 84 or 85;

the LCDR2 of SEQ ID NOs: 18, 89 or 90; and

the LCDR3 of SEQ ID NOs: 19, 37, 91, 92, 93, 94 or

95 ,

In some embodiments, the antibody of the invention comprises

the VH of SEQ ID NOs: 10, 34, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51 or 55; and

the VL of SEQ ID NOs: 11, 33, 56 , 57, 58 , 59 , 60 , 61 , 62 , 63 , 64 or 65 .

Exemplary antibodies that bind human CD200R1 with higher affinity than human CD200, and which are provided in the invention and described herein are antibodies comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of

SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively; SEQ ID NOs : 14, 15, 16, 36, 18 and 19, respectively;

SEQ ID NOs : 14, 15, 16, 36, 18 and 37, respective1y;

SEQ ID NOs : 70, 15, 16, 36, 18 a d 37, respect,ively;

SEQ ID NOs : 38, 39, 16, 36, 18 a d 37, respect,ively;

SEQ ID NOs : 69, 71, 16, 36, 18 and 37, respectively;

SEQ ID NOs : 14, 73, 16, 36, 18 and 37, respectively;

SEQ ID NOs : 38, 74, 16, 36, 18 and 37, respectively;

SEQ ID NOs : 38, 75, 16, 36, 18 and 37, espect ively;

SEQ ID NOs : 38, 76, 16, 36, 18 and. 37, espect ively;

SEQ ID NOs : 38, 142, 16, 36 , 18 and 37,

respectively;

SEQ ID NOs : 38, 39, 1 ' n' ! 36, 18 and 37, respective1y;

SEQ ID NOs : 38, 74, 1 ' n' ! 36, 18 and 37, respective1y;

SEQ ID NOs : 38, 39, 79, 36, 18 and 37, respect,ively;

SEQ ID NOs : 69, 72, 16, 36, 89 and 37, respectively;

SEQ ID NOs : 14, 15, 16, 36, 89 and 91, respectively;

SEQ ID NOs : 14, 15, 16, 36, 90 and 91, respectively;

SEQ ID NOs : 14, 15, 16, 84, 90 and 91, respectively;

SEQ ID NOs : 14, 15, 16, 17, 18 and 92, respective 1

SEQ ID NOs : 14, 15, 16, 84, 18 a d 92, respecti el ;

SEQ ID NOs : 14, 15, 16, 36, 89 and 93, respectiveIy;

SEQ ID NOs : 14, 15, 16, 85, 90 and 94, respectiveIy;

O 2

SEQ ID NOs : 14, 15, 16, 85, 18 a n.d 95, respecti ely . Exemplary antibodies that bind human CD200R1 with higher affinity than human CD200, and which are provided in the invention and described herein are antibodies comprising

the VH of SEQ ID NO: 10 and. the VL of SEQ ID NO: 11; the VH of SEQ ID NO: 10 a d the VL of SEQ ID NO: 33; the VH of SEQ ID NO : 10 and the VL of SEQ ID NO : 56; the VH of SEQ ID NO : 10 and the VL of SEQ ID NO : 57; the VH of SEQ ID NO : 10 and the VL of SEQ ID NO : 58; the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 59; the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 60; the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 61; the VH of SSQ ID O : 10 and the VL of SEQ ID NO : 62; the VH of SEQ ID O : 10 and the VL of SEQ ID NO : 63; the VH of SEQ D NO: 10 and the VL of SEQ ID NO: 64; the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 33; the VH of SEQ ID NO: 41 and the VL of SEQ ID NO: 33; the VH of SEQ D NO: 43 and the VL of SEQ ID NO: 33; the VH of SEQ ID NO: 44 a d the VL of SEQ ID NO: 33; the VH of SEQ ID NO: 45 and. the VL of SEQ ID NO: 33; the VH of SEQ ID NO: 46 and. the VL of SEQ ID NO: 33; the VH of SEQ ID NO: 47 a d the VL of SEQ ID NO: 33; the VH of SEQ ID NO : 48 and the VL of SEQ ID NO : 33; the VH of SEQ ID NO : 49 and the VL of SEQ ID NO : 33; the VH of SEQ ID NO : 51 and the VL of SEQ ID NO : 33; the VH of SEQ ID NO: 55 and the VL of SEQ ID NO: 33;

C) T.

the VH of SEQ ID NO: 42 and the VL of SEQ ID NO: 65.

Another embodiment of the invention, is ειΐι isoleited agonistic antibody that specifically binds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4) , comprising a light chain complementarity determining region (LCDR) 1 (LCDR1) 2 (LCDR2 ) and 3 (LCDR3) of SEQ ID NOs : 149, 24 and 150, respectively .

Another embodiment of the invention is an isolated agonistic antibody that specifically binds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4), comprising a light chain complementarity determining region (LCDR) 1 (LCDR1) 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs: 149, 24 and 150, respectively, and a heavy chain complementarity

determining region (HCDR) 1 (KCDR1 ) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 20, 154 and 22 respectively.

Agonistic antibodies of the invention comprising thi HCDR1, HCDR2 , HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 20, 154, 22, 149, 24 and 150 bind human CD200R1 with bind, human CD200R1 with higher affinity and may ago ize CD200R1 inhibitory singaling with higher potency than human CD200. In some embodiments, the the antibodies of the invention bind human CD200R1 with an affinity constant (K _D ) of

about 1.7xl0 ^""3 M or less;

about lxlO ^"9 M or less; or

between about 1x10 ^"* M and about lxl0 ^{" 0} M, when the K _D is measured using E ^J roteOn XPR36 using a protocol described in Example 2.

In some embodiments, the antibodies of the invention compr i se

the HCDR1 of SEQ ID NO: 20;

the HCDR2 of SEQ ID NO: 21, 50, 78, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or 141;

the HCDR3 of SEQ ID NO: 22

the LCDR1 of SEQ ID NOs : 23, 86, 87 or 88;

the LCDR2 of SEQ ID NO: 24; and

the LCDR3 of SEQ ID NOs: 25, 40 or 83,

In some embodiments, the antibodies of the invention comprise the HCDR1, the HCDR2 , the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of

SEQ ID NOs: 20, 21, 22, 23, 24 and 25, respectively;

SEQ ID NOs: 20, 21, 22, 23, 24 and 40, respectively;

SEQ ID NOs: 20, 21, 22, 86, 24 and 83, respectively;

SEQ ID NOs: 20, 21, 22, 87, 24 and 83, respectively; or

SEQ ID NOs: 20, 21, 22, 88, 24 and 83, respectively. In some embodiments, the antibodies of the invention comprise

the VH of SEQ ID NOs: 12 and the VL of SEQ ID NOs: 13;

the VH of SEQ ID NOs: 12 and the VL of SEQ ID NOs: the Vfl of SEQ ID NOs : 12 and the VL of SEQ ID MOs : 66;

the VH of SEQ ID NOs: 12 and the VL of SEQ ID NOs: 67 ; or

the VH of SEQ ID NOs: 12 and the VL of SEQ ID NOs: 68.

The antibodies of the invention activate the

CD200R.l-mediated inhibitory activity in the cell.

The terra ^W CD200R1-mediated inhibitory activity" as used he ein refers to the activity of a cell tha is reduced or inhibited upon CD200R1 agonist such as CD200 or agonistic CD200R1 antibody binding to CD200R1. Such exemplary inhibitory activities are inhibition of LE ^J S~ induced cytokine secretion such as IL-6 by differentiated U937 cells. Other exemplary CD200R1-mediated inhibitory activities include inhibition of mast cell degranulation or cytokine release, inhibition of IL-13 and ΙσΞ-induced histamine and/or leukotriene release from basophils, or phospho ylation, of accesso y protein. Dok2.

The antibodies of the invention can be tested for their ability to agonize CD200R1-mediated inhibitory activity using for example cell lines overexpressing human CD200R1. For example, U937 lymphoma cells overexpressing human CD200R1 are differentiated into macrophages, and the ability of the antibodies of the invention to suppress LPS'-induced IL-6 secretion when compared to the IL-6 secretion by the same cells in the absence of the antibody is assessed. Amount of secreted IL-6 can be measured using standard ELISA, and EC ₅₀ values for the activation of the i h.ibito r y activi ty (e.g. s ppressio of cell activity) can be calculated using standard methods. The agonistic CD200R1 antibodies of the invention typically inhibit cell activity by about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The antibodies of the invention bind human CD200R1 having the sequence of SEQ ID NO: 4 with a dissociation constant (K _D ) of about 1x10 ^" ' M or less, about 5x10 ^" " M or less, abou 1x10 ^"8 M or less, about. 5xl0 ^~9 M or less, about lxlO ^"9 M or less, about lxl 0 ^"10 M or less, about 1x10 ^"11 M or less, or about lxlO ^"12 M or less. The antibodies of the invention cross-react with cyno CD200R1 and bind cyno CD200R1 with a dissociation constant (K _D ) of about lxlO ^"7 M or less, about 5x10 ^~ M or less, about lxl 0 ^"8 M or less, about 5xl0 ^"9 M or less, about lxlO ^"9 M or less, about lxlO ^"10 M or less, about lxlO ^"11 M or less, or about lxl 0 ^"12 M or less .

The affinity of an antibody to human CD200R1 having the sequence of SEQ ID NO: 4 or cyno CD200R1 having the sequence of SEQ ID NO: 5 can be measured expe imentally using any suitable method. Such methods may utilize ProteOn XPR36, Biacore 3000 or KinExA instrumentation, ELISA or competitive binding assays known to those skilled in the art. Exemplary methods are those described in Example 2 under "ProteOn receptor binding studies". The measured affinity of a particular antibody/ CD200R1 interaction can vary if measured under different

conditions (e.g., osmolarity, pH) . Thus, measurements of affinity and other binding pa ameters (e.g. , K _D , K _or „ K _off ) are made with standardized conditions and a standardized buffer, such as the buffer and conditions described in Example 2. Skilled in the art will appreciate that the internal error for affinity measurements for example using Biacore 3000 or ProteOn (measured as standard deviation, SD) can typically be within 5-33% for measureme ts withi the typical limits of detection. Therefore the term "about" reflects the typical standard deviation in the assay. For example, the typical SD for a K _D of 1x10 ^" ' M is up to +0.33x10 ^"9 M.

Antibodies of the invention may agonize cyno CD200R1-- mediated inhibitory signaling in cells overexpressing cynoraolgus CD200R1 or in endogenous cynoraolgus cells in assays as described when testing agonistic properties of antibodies against human CD200R1,

Antibodies of the invention optionally bind he activating receptor on cynoraolgus cells, cyno CD200R1L (SEQ ID NO: 6) with an affinity constant (K ) of about lxl 0 ^~6 M or more; i.e. the antibodies of the invention optionally do not bind cynoraolgus CD200R1L within the detection limits of the assa (s) . Lack of binding to CD200R1L by the antibodies of the i vention can be measured using for example ProteOn using assays described herein for example in Example 2 under "ProteOn receptor binding studies".

One embodiment of the invention, is an isolated monoclonal antibody agonist or fragment thereof that specifically binds human CD200R1 having the sequence of SEQ ID NO: 4, wherein the antibody competes for binding to the human CD200R1 with an isolated antibody comprising: the heavy chain variable region (VH.) amino acid sequence of SEQ ID NO: 10 and the light chain variable region (VL) amino acid sequence of SEQ ID NO: 11;

the VH amino acid sequence of SEQ ID NO: 12 and the

VL amino acid sequence of SEQ ID NO: 13;

the VH ami o acid seque ce of SEQ I NO: 10 and the

VL amino acid sequence of SEQ ID NO: 33;

the VH amino acid sequence of SEQ ID NO: 34 and the

VL amino acid sequence of SEQ ID NO: 33; or

the VH amino acid sequence of SEQ ID NO: 12 and the

VL amino acid sequence of SEQ ID NO: 35.

In some embodiments, the antibodies of the invention compete for binding to human CD200R1 (SEQ ID NO: 4) with an isolated antibody comprising the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 33.

In some embodiments, the antibodies of the invention. compete for binding to human CD200R1 (SEQ ID NO: 4) with an isolated antibody comprising the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 35.

The antibodies "compete for binding to the human CD200R1" when an antibody or ragment thereof of the i vention (reference an ibody) inhibi s bi ding of other antibodies or fragments thereof (test antibody) to CD200R1 having the sequence of SEQ ID NO: 4 in a standard competitive binding assay. Competition may be partial or comple e, e.g. the re erence antibody may partially inhibit binding of the test antibody to CD200R1; for example by 50%, 60%, 70%, 80%, 85%, 90% or 95%.

Competition between binding to human CD200R1 having the sequence of SEQ ID NO: 4 of a test antibody with antibodies of the invention comprising certain VH and VL sequences can be assayed in vitro using well known methods. For example, binding of MSD Sulfo-Tag™ NHS-ester labeled test antibody to human CD200R1 having the sequence of SEQ ID NO: 4 in the presence of an unlabeled reference antibody can be assessed by elect ochemi luminescent sandwich assay to demonstrate competition. Alte natively, real-time label -free competitive binding assays using Biacore, ProteOn (BioRad) or Octet (ForteBio, Menlo Park, CA) can be used. The ability of a test antibody to inhibit the bindi g of the antibody comprising the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 11, the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 13, the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 33, the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 33, or the VH of SEQ DI NO: 12 and the VL of SEQ ID NO: 35 to human CD200R1 having the sequence of SEQ ID MO: 4 demonstrates that the test antibody competes with these antibodies for binding to human CD200R1. The ability of a test antibody to inhibit the binding of the antibody comprising the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 33 to human CD200R1 having the sequence of SEQ ID NO: 4 demonstrates that the test antibody competes with the reference antibody for binding to human CD200R1.

Antibodies that compete for binding to human CD200R1 (SEQ ID NO: 4) with an isolated, antibody comprising the VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 33 may be made using methods described herein, and assaying the ability of the antibody of VH of SEQ ID NO: 10 and the VL of SEQ ID NO: 33 to inhibit binding of the resulting an ibody clones to CD200R1.

Another embodiment of the invention is an isolated monoclonal antibody agonist or fragment thereof that specifically binds human CD200R1 having the sequence of SEQ ID NO: 4, wherein the antibody binds human CD200R1 within CD200R1 amino acid residues 11-20, 36-42 and/or 112-116 of SEQ ID NO: 4.

Another embodiment of the invention is an isolated monoclonal antibody agonist or fra.gment thereof that specifically binds human CD200R1 having the sequence of SEQ ID NO: 4, wherein the antibody binds human CD200R1 at least within CD200R1 amino acid residues 11-20 and 112-116 of SEQ ID NO: 4. "At least within CD200R1 amino acid residues 11-20 and 112-116" means that the CD200R1 antibody binds a.t least one residue residing within the amino acid, stretc of residues 11-20 of SEQ ID NO: 4, and at least one residue residing within the amino acid stretch of residues 112-116 of SEQ ID NO: 4, The antibody may bind additional residues within the residues 11-20 and 112-116, and additional residues outside of residues 11-20 and 112-116 of SEQ ID NO: 4.

In some embodi ents, the antibody binds CD200R1 at least at residues Lib, D112 and F115 of SEQ ID NO: 4.

In some embodiments, the antibody binds CD200R1 at least at residues Nil, K14, V15, L16, A17, E18, V19, N20, P36, 138, N42, D112, G113, N114, F115, and H116 of SEQ ID NO : 4. An exemplary antibody that binds human CD200R1 within CD200R1 amino acid residues 11-20 and 112-116 of SEQ ID O : 4 is H2RM147 having YH of SEQ ID NO: 10 and YL of SEQ ID NO: 33. Based, on crystal structure analyses, the main epitope residues bound by H2RM147 are L16, D112 and F115 of CD200R1 of SEQ ID NO: 4, based on the number of contacts between these residues and the antibody heavy and light chain residues.

Other exemplary antibodies that bind hum.an. CD200R1 within CD200R1 amino acid residues 11-20 and 112-116 are affinity-matured variants of H2RM147, whose YH and YL sequences are shown in Tables 5 and 7. Affinity

maturation of antibodies typically involves amino acid substitutions in the CDRs or in the Vernier zone

(framework, regions that underline the CDRs) . The matured variants are selected by panning the combinatorial libraries, which may contain up to 10 ^i! mutants. The cap on the size of the library limits the number of variable positions to 6-7 i all 20 amino acids are allowed in each position. The majority of the paratope residues are preserved in each combinatorial library, which ensures that the binding epitope is also preserved. Several crystallographic studies of the parent and matured an ibodies have shown that the epitope is always preserved during affinity maturation (e.g. Fransson et al . , J. Mol . Biol. 2010, 398:214-231; Gustchina et al . , PLoS Pathog . 2010, 6:el001182; La Porte et al . , MAbs 2014; 6:1059- 1068) .

Antibodies that bind, human CD200R1 within CD200R1 amino acid residues 11-20 and 112-116 may be made for example by immunizing mice with peptides having the sequences of residues 1-20 and 112-116 of SEQ ID NO: 4, immunizing mice with CD200R1 chimeric mouse protein that has human CD200R.1 sequences at residue positions 11-20 and 112-116, or panning phage display libraries with wild type human CD200R1, and cross-screening the resulting hits with CD200R1 variants that have substitutuions at each or several residue positions within the reidues 11-20 and 112-116 of human CD200R1 using methods described herein.

An ibodies whose VH or YL amino acid sequences di fer irisubstariti ally from those shown in Table 2, Table 5, Table 7, Table 12 and Table 16, or in SEQ ID NO: 154 are encompassed within the scope of the invention. Typically, this involves one or more conservative amino acid, substitutions with an amino acid having similar charge, hydrophobic, or stereochemical characte istics in. the antigen-binding site or in the framework without adversely altering the properties of the antibody. Conservative substitutions may also be made to improve antibody properties, for example stability or affinity. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions can be made to the VH or VL sequence. In some embodiments, 1, 2, 3, 4, 5 or 6 amino acid

substitutions can be made to the VH or VL sequence. For example, a "conservati e amino acid, substitution" may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al (1998) Acta Physiol. Scand. Suppl .

643:55-67; Sasaki et al (1998) Adv. Biophys. 35:1-24) . Desired amino acid substitutions can be determined by those skilled in the art at the time such substit tions are desired. For example, amino acid substitutions can be used to identify important residues of the molecule sequence, or to increase or decrease the affinity of the molecules described herein. The following eight groups contain amino acids tha are conservative amino acid substitutions for one another: 1) Alanine (A), Glycine (G) ; 2) Aspartic acid (D) , Glutamic acid (E) ; 3) Asparagine (M) , Glutamine (Q) ; 4) Arginine (R) , Lysine (K) ; 5) Isoleucine (I), Leucine (L) , Methionine (M) ,

Valine (V) ; 6) Phenylalanine (F) , Tyrosine (Y) , Tryptophan (W) 7) Serine (S) , Threonine (T) ; and. 8) Cysteine (C) ,

Methionine (M) (see, e.g., Creighton, Proteins (1984) ) .

However, the antibodies of the invention comprising a

HCDR3 that is a variant of HCDR3 of SEQ ID NO: 16

( LYWYVLSSSRLDY) have a W3F, 3H or 3Y substitution at residue position 3, when that position is substituted.

Exemplary such antibodies are engineered variants of

HRM226, such as H2RM286, H2RM288 and H2RM302 shown in

Table 12.

Amino acid substitutions can be done for example by PGR mutagenesis (US Pat. No. 4,683,195) . Libraries of variants can be generated using well known methods, for example using random (N K) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp) and screening the libraries for variants with desi ed properties.

In some emboidments , the antibody of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VH

is at least 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 10;

comprises 1, 2, 3, 4, 5 or 6, 7, 8 or 9

substitutions when compared to the VH of SEQ ID NO: 10; or

comprises the amino acid sequence of SEQ ID NO: 152. Figure 7A shows the amino acid squences of such exemplary VH chains, and Figure 7C snows the amino acid sequence of SEQ ID NO: 152. SEQ ID NO: 152 is a consensus VH sequence of affinity-matured variants of antibody K2RM147. SEQ ID NO: 152 :

EVQLLESGGGLVQPGGSLRLSCAASGFTFx ₁ x _? Yx ₃ Mx ₄ WVRQAPGKGLEWVSAIx _{5 e} ;X-; GGxeTYYADSVKGRFTISRD SKNTLYLQMNSLRAEDTAVYYCAKLYxgYVLSSSRLDY ^" WGQGTLVTVSS wherein

is S or D;

A _? is S or M;

is A or G;

V , is s, N or Q;

v _c is s, D, T or Q;

is G or A;

X 1 s S, N, A o E

s 1 s S, Y or T; and

In some emboidments, the antibody of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL) , wherein the VL

is at least. 95%, 96%, 97%, 98% or 99% identical to the VL of SEQ ID NO: 33;

comprises 1, 2, 3, 4, 5 or 6 substitutions when compeired to the VH of SEQ ID NO: 33; or

comprises the amino acid sequence of SEQ ID NO: 153. Figure 7B shows the amino acid squences of such exemplary VL chains, and Figure 7D shows the amino acid sequence of SEQ ID NO: 153. SEQ ID NO: 153 is a consensus VL sequence of affinity-matured variants of antibody K2RM147.

SEQ ID NO: 153

EIVLTQSPGTLSLSPGERATL5CRASQSVXiSX ₂ YLA YQQKPGQAPRLLIYGASX ₃ RA TGIPDRFSGSGSGTDFTLTISP.LEPEDFAVYYCX-jQYGXsXePLTFGQGTKVEIK; wherein

is S or N;

x ₂ is s, Y, D or

, is s, T or N;

5 1 s s, A, or ana

Xg i s s, A or

In some embodiments, the antibody of the invention comprises the VH of SEQ ID NO: 152 and the VL of SEQ ID NO: 153. E era 1a ry such antibodies a e antibodies

H2RM147, H2RM185, H2RM187, H2RM189, H2RM190, H2RM191, H2RM193, H2RM195, H2RM198, H2RM200, H2RM204, H2RM218, H2RM219, H2RM225, H2RM226, H2RM227, H2RM279, H2RM280, H2RM281, H2RM282, H2RM286, H2RM288 add H2RM302.

In some embodiments, the antibody of the invention compirses the VH and the VL, wherein he VL

is at least 95%, 96%, 97%, 98% or 99% identical to the VL of SEQ ID NO: 35;

comprises 1, 2, 3, 4, 5, 6, 7 or 8 substitutions when compared to the VL of SEQ ID NO: 35; or comprises the amino acid sequence of SEQ ID NO: 151. Figure 6A shows the amino acid squences of such exemplary VL chains, and Figure 63 shows the amino acid sequence of SEQ ID NO: 151. SEQ ID NO: 151 is a consensus VL sequence of affinity-matured variants of antibody H2RM211.

SEQ ID NO: 151

DIQMTQS PSSLSASVGDRV ITCRASX _: X ₂ lX _: .SX LA YQQKPEKAPKSLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHX ₅ X ₆ YPRTFGQGTKVEIK;

wherein

X] is Q, G, E or S;

X ₂ is G, ? or Q;

X ₃ is S, D or Ξ;

X ₄ is W or A;

X ₅ is D or N; and

X ₆ is A or S.

The LCDR3 H91 substitution increases affinity to human CD200R1 nearly 300-fold.

In some embodiments, the antibody of the invention compirses the VH and the VL, wherein the VH

is at least 95%, 96%, 97%, 98% or 99% identical to the VH of SEQ ID NO: 12;

comprises 1, 2, or 3 substi utions when compared, to the VL of SEQ ID NO: 12; or

comprises the amino acid sequence of SEQ ID NO: 154.

SEQ ID NO: 154 is a consensus VH sequence of designed antibody N2RM266 VH (SEQ ID NO: 12) variants to minimize potential antibody instability risk. Example 8 describes exem l ry des igned a riants . SEQ ID NO: 154 :

QVQLVESGGGVVQPGRSLRLSCAASGFT SSYGMHWVRQAPGKGLEWVAVIX _] YX ₂ X;jS NKYYADSVKGRFTISRDNSK TLYLQM SLBAEDTAVYYCAREAYGSGSYYSDYWGQG TLVTVSS ; wherein

Xi is A, E, F, G, H, I, K, L, P, R, S, T, V, Y or D;

X ₂ is G, A, D, E, F, H, I, K, L, N, Q, R, S, T, V or Y; a d

X ₃ is A, D, E, F, G, H, I , K, L, N, P, Q, R, S, T, V OR Y, In some embodiments., the antibody of the invention comprises the VH of SEQ ID NO: 154 and the VL of SEQ ID NO: 151. Exemplary such a ibodies are antibodies

H2RM211, H2RM266, H2RM267, H2RM268 and H2RM269.

The generated variants can be assayed for their binding to human and cyno CD200R1 and for their agonistic activity using assays described herein.

Although the embodiments illustrated in the Examples comprise pairs of variable regions, one from, a heavy chain and one from a light chain, a skilled artisan will recognize that alternative embodiments may comprise single heavy or light chain variable regions. The single variable region can be used to screen for variable domains capable of forming a two-domain specific antigen-binding fragment capable of, for example, binding to human CD200R1 having the sequence of SEQ ID NO: 4. The screening may be accomplished by phage display screening methods using for example hierarchical dual combinatorial approach disclosed in Int. Pat. Publ . No. WO92/01047. In. this approach, an individual colony containing either a H or L chain clone is used to infect a complete library of clones encoding the other chain. (L o H) , and the resulting t o-chain specific ant igen-bind g domain is selected in accordance with phage display techniq es as described. The efore, the individual VH and VL polypeptide chains are useful in identifying additional antibodies specifically binding human CD200R1 having the sequence of SEQ ID NO: 4 using the methods disclosed in Int. Pat. Publ. No. WO92/01047. The individual VH or VL chains of antibodies of the invention may be re-combined in matrix assays with other VL or VH chains of antibodies of the invention. The resulting new antibodies may be tested for their

functionality in binding human and cy.no CD200R1 and their agonistic activity using assays described herein.

The individual CDRs, KCDR1 , HCDR2 , KCDR3 , LCDR1, LCDR2 and/or LCDR3 may be grafted onto VH and/or VL chains of non-parental antibodies . The non-parental antibody may be ano her a ibody described herei , o a non-- related antibody. The non-parental antibody may be non-human species, such as mouse or rat; in this case the resulting antibodies are humanized antibodies. CDR greifting may be done using standard molecular biology methods. For example, one or more CDRs of antibodies isolated from the phage panning (H2RM147 and its affinity-matured variants) may be grafted onto antibody K2RM211 and its affinity- matured variants; which antibodies were generated using human transgenic animals, and vice versa, generating additional repertoire of antibodies that can be tested for their binding to human CD200R1 and their potency in agonizing CD200R1-mediated inhibitory signaling.

Antibodies of the invention may be made using a va iety of technologies for generating monoclonal antibodies. For example, the hybridoma method of Kohler and Milstein, Nature 256:495, 1975 can be used. In the hybridoma method, a mouse or other host animal, such as a hamster, rat or monkey, is immunized with human CD200R1 and/or cyno CD200R1 protein or fragments of these proteins, such as an extracellular portion of human CD200R1, followed by fusion of spleen cells from immunized animals with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and. Practice, pp.59-103 (Academic Press, 1986)) . Colonies arising from single immortalized hybridoma cells are screened for production of antibodies with desired properties, such as specificity of binding, cross- reactivity or lack thereof, and affinity for the antigen.

Various host an mals can. be used to produce

antibodies against human CD200R1, For example, Balb/ ^' c mice may be used to generate mouse anti-human CD200R1 antibodies . The antibodies made in Balb/c mice and other non-human animals can be humanized using various

technologies to generate more human-like sequences.

Exemplary humanization techniques including selection of human acceptor frameworks are known to skilled in the art and include CDR grafting (U.S. Pat. No , 5, 225, 539) , SDR grafting (U.S. Pat. No. 6,818,749), Resurfacing (Padlan, Mol Immunol 28:489-499, 1991), Specificity Determining Residues Resurfacing (U.S. Pat. Putal . No. 2010/0261620) , human-adaptation (or human framework adaptation) (U.S. Pat. Publ. No. US2009/0118127) , Superhumanization (U.S. Pat. No. 7,709, 226) and guided selection (Osbourn et al (2005 Methods 36:61-68, 2005; U.S. Pat. No. 5,565,332) .

Humanized an ibodies can be further optimized to improve their selecti ity o affinity to a desired antigen by incorporating altered framework support residues to preserve binding affinity (backmutations ) by techniques such as those disclosed as described i Int. Pat. Publ. No. WO90/007861 and in Int. Pat. Publ. No. W092/22653.

Transgenic mice carrying human immunoglobul in (Ig) loci in their genome can be used to generate human antibodies against a target protein, and are described in for example Int. Pat. Publ. No. WO90/04036, U.S. Pat. No. 6,150,584, Int. Pat. Publ. No. 099/45962, Int. Pat. Publ. No. WO02/066630, Int. Pat. Publ. No. O02/43478, Lonberg et al (1994) Nature 368:856-9; Green et al (1994) Nature Genet. 7:13-21; Green & Jakobovits (1998) Exp. Med.

188:483-95; Lonberg and Huszar (1995) Int. Rev. Immunol. 13 : 65-93; Bruggemann et al (1991) Eur. J. Immunol.

21:1323- 1326; Fishwild et al (1996) Nat. Biotechnol .

14:845-851; Mendez et al (1997) Nat. Genet. 15:146-156; Green (1999) J. Immunol, Methods 231:11-23; Yang et al (1999) Cancer Res, 59:1236-1243; Bruggemann and Taussig (1997) Curr, Opin, Blotechnol . 8:455-458; Int. Pat. Publ . No. WO02/043478) . The endogenous immunoglobulin loci in such mice may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the mouse genome using homologous or non ^¬ homologous recombination, using t a nschromosomes , or using minigenes . Companies such as Regeneron

(http: // www regeneron com) , Harbour Antibodies

(http : / / www harbourantibodies com) , Open Monoclonal Technology, Inc. (OMT) (htt : //_www_omtinc_net ) , KyMab (http:// www kymab com), Trianni (http:// www.trianni com) and. Ablexis (http:// www ablexis com) can be engaged to provide human antibodies directed against a selected antigen using technologies as described above.

Human antibodies can be selected from a phage display library, where the phage is engineered to express human i munojlobul ins or portions thereof such as Fabs, single chain antibodies (scFv) , or unpaired or pai ed antibody variable regions (Knappik et al (2000) J. idol, Biol, 296:57-86; Krebs et al (2001) J, Immunol. Meth, 254:67-84; Vaughan et al (1996) Nature Biotechnology 14:309-314;

Sheets et al (1998) PITAS (USA) 95:6157-6162; Koogenboom and Winter, (1991) J. Mol . Biol. 227:381; Marks et al (1991) J. Mol, Biol. 222:581) . The antibodies of the invention may be isolated for example from phage display library expressing antibody heavy and light chain variable regions as fusion p oteins with bacteriophage pIX coat protein, as described in Shi et al (2010) J. Mol. Biol. 397:385-96 and Int. Pat. Publ. No. WO09/085462 ) . The antibody libraries are screened for binding to human CD200R1 extracellular domain and the obtained positive clones are further characterized, the Fabs isolated from the clone lysates, and expressed as full length IgGs .

Such phage display methods for isolating human, antibodies are established in the art. See for example: U.S. Patent Kos, 5,223,409; 5,403,484; and 5,571,698 to Ladner et al . ; U.S. Patent. Nos . 5, 427, 908 and 5, 580, 717 to Dower et al . ; U.S. Patent. Nos. 5, 969, 108 and 6, 172, 197 to McCafferty et al,; and U.S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al .

Preparation of antigens and monoclonal antibody production can be performed using any suitable technique, such as recom i ant protein production. e antigens can. be administered to an animal in the form, of purified protein, or protein mixtures including whole cells or cell or tissue extracts, or the antigen can be formed de novo in the animal's body from nucleic acids encoding said antigen or a portion thereof.

The antibodies of the invention may be human or humanized .

The antibodies of the invention may be synthetic or recombinant .

The antibodies of the invention may be of IgA, IgD, IgE, IgG or IgM type. The antibodies of the invention may be of IgGl, IgG2, IgG3, IgG4 isotype.

Immune effector properties of the antibodies of the invention may be enhanced or silenced through Fc

modifications by techniques known to those skilled in the art. For example, Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC) , antibody-dependent cell-mediated cytotoxicity (ADCC) , phagocytosis, down regulation of cell surface receptors (e.g., B cell receptor; BCR) , etc. can be provided and/or controlled by modifying residues in. the Fc responsible for these activities. Pharmacokinetic properties could also be enhanced by mutating residues in the Fc domain that extend antibody half-life (Strohl (2009) Curr Opin

Biotechno 1 20:685-91) . Exemplary Fc modifications are IgG4 S228P/L234A/L235A ( SEQ ID NO: 159), IgG2

M252Y/S254T/T256E (Dall'Acqua et al (2006) J. Biol. Chem . 281:23514-24; or IgG2

V234A/G237A/P238S/K268A/V309L/A330S/P331S ( SEQ ID NO: 96) (Intl. Pat. PubI . No. WOll/066501) , or those described in US. Pat. No. 6,737,056 (residue numbering according to the EU numbering) .

SEQ ID NO: 159

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAG GPSWLFPPKPKDTLMISRTPEVTCWVOVSQEDPEVQFNWYVDGVEVHNAKT PREE QFNSTYRVVSVLTVLilQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL SRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 96

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSWTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAA SSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVKNAKTKPREEQ FNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWES GQPENNYKTTPPMLDSDGSFFL SKLT VDKSRWQQGNVFSCSV HEALHNHYTQKSLSLSPGK

Additionally, antibodies of the invention can be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation or non- naturally occurring covalent modification such as the addition of polyethylene glycol moieties (pegylation) and lipidation. Such modi ications may occur in vivo or in vi tro . For exarap1 e , the antibodi.es o f the invention can be conjugated to polyethylene glycol (PEGylated) to improve their pharmacokinetic profiles. Conjugation can be carried out by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics while not

interfering with function (Knigh st al (2004) Platelets

15:409-18; Leong et al (2001) Cytokine 16:106-19; Yang et al (2003) Protein Eng. 16:761-70) .

Antibodies or f agments thereof of the invention modified to improve stability, selectivity, cross- reactivity, affinity, immunogenicity or other desirable biological or biophysical property are within the scope of the invention. Stability of an antibody is influenced by a number of factors, including (1) core packing of individual domains that affects their intrinsic stability, (2) protei /protei interface i teractions that have impact upon the HC and LC pairing, (3) burial of polar and charged residues, (4) H-bonding network for poleir and charged, residues; and. (5} surface charge and polar residue distribution among other intra- and inter-molecular forces (Worn et al {2001} J. Mol . Biol. 305:989-1010) . Potential structure destabilizing residues may be identified based upon the crystal structure of the antibody or by molecular modeling in certain cases, and the effect of the residues on antibody stability can. be tested by genera ing and. evaluating variants harboring mutatio s in the identified residues. One of the ways to increase antibody stability is to raise the thermal transition midpoint (Tm) as measured by differential scanning calorimetry (DSC) . In general, the protein Tm is correlated with its stability and inversely correlated wi h its s sceptibility to unfolding and denaturation in solution and the degradation processes that depend on the tendency of the protein to unfold (Remmele et ai (2000} Biopharm 13:36-46) . A number of studies have fo nd correlation between the ranking of the physical stability of formulations measured as thermal stability by DSC and physical stability measured by other methods (Gupta et al (2003) AAPS PharmSci 5E8; Zhang et al (2004) J. Pharm. Sci . 93:3076-89; Maa et al (1996) Int. J. Pharm. 140:155-68; Bedu-Addo et al (2004) Pharm. Res. 21:1353-61; Remmele et al (1997) Pharm. Res. 15:200-8) . Formulation studies suggest that a Fab Tm has implication for long-term physical stability of a corresponding mAb . Differences in amino acids in either framework or within the CDRs a.y effect, the thermal stability of the Fab domain (Yasui et al (1994) FEBS Lett. 353:143-6) . CD200R1 antibodies of the invention can be engineered into bispecific antibodies which are also encompassed within the scope of the invention. The VL and/or the VH regions of the antibodies of the invention can be engineered using published methods i o single chain bispecific antibodies as structures such as TandAb® designs (Int. Pat. Publ . No. WO99/57150; U.S. Pat. Publ . No. US2011/0206672) or into bispecific scFVs as structures such as those disclosed in U.S. Pat. No. US, 586, 9620; Int. Pat. Publ. No. W095/15388, Int. Pat. Publ. No. W097/14719 or Int. Pat. Publ, No. WO11/036460.

The VL and/or the VH regions of the antibodies of the invention may be engineered into bispecific full length antibodies, where each antibody arm binds a distinct antigen or epi ope. Such bispecific antibodies may be made by modulating the CH3 interactions between the two antibody heavy chains to form bispecific antibodies using technologies such as those described in U.S. Pat. No. 7,695,936; Int. Pat. Publ. No. O04/111233; U.S. Pat. Publ. No. 2010/0015133; U.S. Pat. Publ. No. 2007/0287170; Int. Pat. Publ. No. O2008/119353; U.S. Pat. Publ. No. 2009/0182127; U.S. Pat. Publ. No. 2010/0286374; U.S. Pat. Publ. No. 2011/0123532; Int. Pat. Publ. No. WO2011/131746; Int. Pat. Publ. No. WO2011/143545; or U.S. Pat. Publ. No. 2012/0149876.

For example, bispecific antibodies may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two

monospecific homodimeric antibodies and forming the bispecific heterodiraeric antibody from two parent monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in Intl. Pat. Publ. No. WO2011/131746. In the methods, the first monospecific bivalent antibody (e.g., anti-CD200Rl antibody of the invention) and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions may optimally be restored to non-reducing. Exemplary reducing agents that may be used are 2- me rcaptoethylamine (2-MBA) , dithiothreitol (DTT) , dith ioeryth i ol (DTE) , glutathione, tris (2-carboxyethyl) phosphine (TCBP) , L-cysteine and beta- mercaptoethanol , preferably a reducing agent selected from the group consisting of: 2- mercaptoethylamine ,

dithiothreitol and tris (2-carboxyethyl) phosphine . For example, incubation for at least 90 rain at a temperature of at least 20° in the presence of at least 25 mM 2-ΜΞΑ or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.

Exemplary CH3 mutations that may be used in a first heavy chain and in a second heavy chain of the bispecific antibody are K409R and/or F405L,

Additional bispecific structures into which the VL and/or the VH regions of the antibodies of the invention may be incorporated are for example Dual variable Domain Immunoglobulins (DVD) (Int. Pat. Publ . No. WO2009/134776) , or structures that include various dimerization domains to connect the two antibody arms with different specificity, such as leucine zipper or collagen dimerization domains (Int. Pat. Publ . No. WO2012/022811, U.S. Pat. No.

5,932,448; U.S. Pat. No. 6,833,441) . DVDs are full length antibodies comprising the heavy chain having a structure VHl-linker-VH2-CH and the light chain having the structure VLl-linker-VL2-CL; linker being optional.

Another embodiment of the invention is an isolated monoclonal antibody agonist or fragment thereof tha specifically binds human CD200 receptor 1 (CD200R1) having the sequence of SEQ ID NO: 4 having certain VH and VL sequences., wherein the antibody VH is encoded by a first synthetic polynucleotide and the antibody VL is encoded by a second synthetic polynucleotide. The synthetic polynucleotide may be a complementary deoxynucleic acid (cDNA), or may be codon optimized for expression in suitable host. Codon optimization is a well known technology .

In some embod ments, the synthetic polynucleotides comprise the sequences of SEQ ID Os : 29, 30, 31, 32, 155, 156, 157 and 158.

Another embodiment of the invention is an isolated synthetic polynucleotide encoding any of the antibody heavy chain, variable regions or the an ibody light chain variable regions of the invention. Certain exemplary synthetic polynucleotides are disclosed herein, however, other synthetic polynucleotides which, given the

degeneracy of the genetic code or codon preferences in a given expression system, encode the antibodies of the invention are also within the scope of the invention.

Exemplary synthetic polynucleotides are for example polynucleotide sequences shown in SEQ ID NOs: 29, 30, 31, 32, 155, 156, 157 and 158. The synthetic polynucleotide sequences encoding a VH or a VL or a fragment thereof of the antibody of the invention can be operably linked to one or more regulatory elements, such as a promoter or enhancer, that allow expression of the nucleotide sequence in the intended host cell. The synthetic polynucleotide may be a cDNA.

In some embodiments, the antibody VH and the antibody VL are encoded by the first synthetic

polynucleotide and the second synthetic polynucleotide comprising the sequence of SEQ ID NOs:

155 and 156, respectivel

157 and 158, respectively;

29 and 30, respectively; or 31 and 32, respectively.

SEQ ID NO: 29: H2RM190 VH H2RH81 cDNA

GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCC

5 TGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCA GGCGCCGGGCAAAGGCCTGGAATGGGTGAGCGCGATTAGCGGCAGCGGCGGCAGCACC TATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACAGCAAAAACA CCCTG ATCTGCAGA GAACAGCCTGCGCGCGGAAGA ACCGCGG G ATTATTGCGC GAAACTCTATTGGTATGTTCTCTCTTCTTCGAGGTTGGACTATTGGGGCCAGGGCACC

10 CTGGTGACCGTGAGCAGC

SEQ ID NO: 30; H2RM190 VL H2RL185 cDNA

GAAATTGTGC GACCCAGAGCCCGGGCACCC GAGCC GAGCCCGGGCGAACGCGCGA CCCTGAGCTGCCGCGCGAGCCAGAGCGTCAACAGCTACTATCTTGCGTGGTATCAGCA

15 GAAACCGGGCCAGGCGCCGCGCC GCTGATTTATGGTGCATCCTCCCGTGCGACCGGC

ATTCCGGATCGCTTTAGCGGCAGCGGTTCCGGCACCGATTTTACCCTGACCATTAGC C GCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCATCAATATGGTGCAAGTCCACT TACCTTTGGCCAGGGCACCAAGG GGAG TCAAG

20 SEQ ID NO: 31; H2RM211 VH H2RH102 cDNA

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGAC TCTCCTGTGCAGCGTCTGGA TCACCT CAGTAGCTATGGCATGCACTGGGTCCGCCA GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAA TAGTATGCAG-ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA

25 CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGC

GAGAGAGGCTTATGGTTCGGGGAGTTATTATGAGGACTACTGGGGCCAGGGAACCCT G GTCACCGTCTCCTCA

SEQ ID NO: 32; H2RM211 VL H2RL191 cDNA

30 GATATTCAGATGACTCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCA CCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAA ACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTC CCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCC TGCAGCC GAAGAT TTGCAAC TATTACTGCCAACAGTA AATAGTTACCCTCGGAC

35 GTTCGGCCAAGGGACCAAGGTGGAAA C AA

SEQ ID NO: 155 H2RM226 VH H2RH111

GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCC TGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCTCCTATGGCATGCAATGGGTGCGCCA

40 GGCGCCGGGCAAAGGCCTGGAATGGGTGAGCGCGATTGACGGCGCCGGCGGGTC ACT

TACTACGCGGATAGCGTGAAAGGCCGCTTTACCATTTCACGAGATAACAGCAAAAAC A CCCTGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCGC GAJLACTCTATTGGTATGTTCTCTCTTCTTCGAGGTTGGACTATTGGGGCCAGGGCACC CTGGTGACCGTGAGCAGC

Δ ^Γ · SEQ ID NO: 156 H2RM226 VL PH9L1

GAAA GTGCTGAGCCAGAGCGCGGGGAGCCTGAGGC GAGGCCGGGGGAACGCGCGA CCCTGAGCTGCCGCGCGAGCCAGAGCGTGAGCAGCAGCTATCTGGCGTGGTATCAGCA GAAACCGGGCCAGGCGCCGCGCCTGCTGATTTATGGCGCGAGCAGCCGCGCGACCGGC ATTCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCC GCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAGTATGGCAGCAGCCCGCT GAGCTTTGGCCAGGGCACC AAG GG AAT AAA

SEQ ID NO: 157 H2RM266 VH H2RH102

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA C TCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCA GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAA TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGC GAGAGAGGCTTATGGTTCGGGGAGTTATTA GAGGACTACTGGGGCCAGGGAACCCTG

GTCACCGTCTCCTCA

SEQ ID NO: 158 H2RM266 VL H2RL222

GATATTCAGATGACTCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCA CCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAA

ACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGT C CCATCAAGGTTCAGCGGCAG GGATCTGGGACAGATTTCACTCTCACCATCAGCAGCC TGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGCATGATGCTTATCCTCGTAC GTTCGGCCAAGGGACCAAGG GGAAA CAAA

Another embodi merit of the invention is a vector comprising the synthetic polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the synthetic polynucleotide of the invention into a given organism or genetic background by any means. For example, synthetic polynucleotides encoding light and heavy chain variable regions of the antibodies of the invention, optionally linked to constan regions, are inserted into expression vectors. The light and heavy chains can be cloned in the same or separate expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector (s) that ensure the expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters (e.g. naturally associated or heterologous promoters). enhancer elements, and transcription termination

sequences., and eire chose to be compatible with the host cell chosen, to express the antibody. Once the vector has been, incorporated, into the appropriate host, the host is mai tai ed under conditions suitable for high level expression of the proteins encoded by the incorporated synthetic polynucleotides .

Suitable expression vecto s are t picall replicable i the host organisms either as epi somes or as an integral part of the host chromosomal D A . Expression vectors typically contain selection markers such as a picillin, hygro ycin, tetracycline, kanamycin or neomycin resistance to permit detection of those cells transformed with the desi ed DMA sequences.

Suitable promoter and enhancer elements are known in. the art. For expression in a bacterial cell, exemplary promoters include lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, exemplary promoters include light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse meta.llothionein-I promoter; and various art-known tissue specific promoters. For expression in a yeast cell, an exemplary promoter is constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2 promoter, a P.H05 promoter, a CUP1 promoter, a GAL7 promoter, a MBT25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a. LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia) . Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant constructs. The following vectors are provided by way of example.

Bacterial: pSs, phagescript ., PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNHl 8a, pNH46a (Stratagene, La Jolla, Calif., USA) ; pTrc99A, pK 223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden) . Eukaryotic: p Lneo, pSV2cat, pOG , PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia) .

Another embodiment of the invention is a host cell comprising the vector of the invention. The term "host cell" refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modi icatio s may occ r in s cceedi g generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such hos cells may be eukaryotic cells, prokaryotic ceils, plant cells or archeal cells.

Escherichia coli, bacilli, such as Bacillus

subtilis, and other enterobacteriaceae, such as

Salmonella, Serratia, and various Pseudomonas species are examples of prokaryotic host cells. Other microbes, such as yeast, are also useful for expression. Saccharomyces

(e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origins.

Mammalian eukaryotic cells include immortalized cell lines suc as hybridomas or myeloma cell lines such as SP2/0

(American Type Culture Collection (ATCC) , Manassas, VA, CRL-1581) , NSO (European Collection of Cell Cultures (ECACC) , Salisbury, Wiltshire, UK, ECACC No. 8D110503) , FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196) . Other useful cell lines include those deri ed f om Chinese Hamster Ovary (CHO) cells such as CHO-K1SV {Lonza Biologies, alkersville, MD) , CHO-K1 (ATCC CRL-61) or DG44.

Another embodiment of the inventio is a method of producing an antibody of the Invention comprising cu11u r i ng the host ce11 o f the invention and recovering the antibody produced by the host cell. Methods of making antibodies and purifying them are well known in the art. Once synthesized (either chemically or recombinantly) , the whole antibodies, their dlmers, individual l ght and heavy chains, or other antibody fragments such as VH or VL, can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification. (Springer-- Verlag, Μ.Ϊ., (1982)} , A subject antibody can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99%, or more, pure, e.g., free from contaminants such as cell debris, macromolecules other than a subject antibody, etc.

Another embodiment of the invention is a method for producing an isolated monoclonal eiiitibody agonist or fragment, thereof tha specifically binds human CD200 receptor 1 (CD200R1) having the sequence of SEQ ID NO: 4, comprising :

incorporating the first synthetic polynucleotide encoding the VH of the antibody and the second synthetic polynucleotide encoding the VL of the antibody nto an expression vector;

t ansformi g a host cell with the expression vector; culturing the host cell in culture medium under conditions wherein the YL and the VH are expressed and fo m, the antibody; and

recove ing the a tibody from t e host cell or cult re medi ,

Another embodiment of the invention is a monoclonal antibody agonist or fragment thereof that specifically binds human CD200 receptor 1 (CD200R1) having the sequence of SEQ ID NO: 4 comprising the VH of SEQ ID NOs : 10, 12 or 34 and the VL of SEQ ID NOs: 11, 13, 33 or 35 produced by a method comprising:

incorporating a first synthetic polynucleotide encoding the VH of SEQ ID NOs: 10, 12 or 34 and a second synthetic polynucleotide encoding the VL of SEQ ID NOs: 11, 13, 33 or 35 into an expression vector;

transforming a host cell with the expression vector;

culturing the host cell in culture medium under conditions wherein the VH of SEQ ID NOs: 10, 12 or 34 and the VL of SEQ ID NOs: 11, 13, 33 or 35 are expressed, and conjugated togethe to form an antibody; a d

recovering the CD200R1 agonistic antibody comprising the VH of SEQ ID NOs: 10, 12 or 34 and the VL of SEQ ID NOs: 11, 13, 33 or 35 from the host cell or culture medium,

Another embodiment of the invention is an isolated agonistic antibody that specifically bi ds human CD200 receptor 1 (CD200R1) (SEQ ID NO: 4) comprising the VH and the VL of SEQ ID NOs: 12 and 35 or 34 and 33, produced by a method comprising:

incorporating into an. expression vector

a first synthetic polynucleotide encoding the VH of

SEQ ID NOs: 12 and a second synthetic polynucleotide encoding the VL of SEQ ID NOs: 35; or

a first synthetic polynucleotide encoding the VH of

SEQ ID NOs: 34 a d a second synthetic polyn cleotide encoding the VL of SEQ ID NOS: 33;

t ansformi g a host cell with the expression vector; culturing the host cell in culture medium under conditions wherein the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 35 or the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 33 are expressed; and

recove ing the antibody comprising

the VH of SEQ ID NO: 12 and the VL of SEQ ID

NO: 35; or

the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 33 from, the host cell or culture medium. The synthetic polynucleotides encoding certain VH or VL sequences of the invention are incorporated into vectors using standard molecular biology methods. Host cell transformation, culture, antibody expression and purification are done using well known methods.

Methods of Treatment

Agonistic antibodies specifically binding human CD200R1 having the sequence of SEQ ID NO: 4 disclosed herein may be utilized to modulate the immune system and. are suitable for treating or preventing a spectrum of CD200R1 -mediated conditions .

The term *CD200R1-raediated condition" as used herein refers to a condition resulting at least partially from inappropriate reduction in CD200R1-induced inhibito y myeloid or T cell activity, i.e. overactivity of the CD200R1 expressing cells. CD200R1 -mediated conditions are therefore associated with improper function of myeloid cells (e.g., mast cells, dendritic cells, neutrophils, monocytes, or macrophages ) and T cells.

"I flammatory condition" as used herein refers to acute or chronic localized or systemic responses to harmful stimuli, such as pathogens, damaged cells, physical injury or irritants, that are mediated in part by the activity of cytokines, chemokines, or inflammatory cells (e.g., neutrophils, monocytes, lymphocytes, macrophages, mast cells, dendritic cells, neutrophils) and is characterized in most instances by pain, redness, swelling, and impairment of tissue function.

The term. ^W CD200R1 -mediated inflammatory condition" as used herein, refers to an inflammatory condition resulting at least partially from, inappropriate reduction in

CD200Rl--induced inhibitory myeloid or T cell activity. Exemplary CD200R1 -mediated inflammatory conditions are pulmonary diseases such as asthma, COPD and pulmonary exace bations and inflammatory bowel diseases.

The lung and gut, the sites exposed to abundant antigenic material, must maintain a high threshold of immune 'ignorance' to innocuous antigens to avoid inflammatory disease, and at the same time must maintain the ability to respond to the antigen representing a serious threat. Immune homeostasis is maintained through the balance of positive and negative inflammatory signals and pathways. The mechanisms for maintaining homeostasis may also be eipplied to resolution of inflammation.

CD200R.1 has been identified as one of the mechanisms required for the homeostasis and resolution of

inflammation by negatively regulating myeloid and T cell activity .

Airway and intestinal myeloid cells such as

macrophages express higher level of CD200R1 than their systemic counterparts (S elgrove et al, Nat Immunol 2008 Sep; 9 (9) :1074-83), consistent with their role in maintaining homeostasis in the lung and gut. During viral (eg. influenza) infection or asthma/COPD exacerbation, the level of CD200R1 may be up- egulated to control

exacerbated inflamma ion and tissue damage (Snelgrove et al., Nat Immunol 2008 Sep; 9 (9) :1074-83) . Their agonistic ligand, CD200, has been shown to be

significantly reduced or lost in the lung after

in ratracheal instillation of LPS (Ya-Fen et al., J Anat . Mar 2010; 216(3) : 407-416. , 2010) or nebulized allergen challenge in rats, which, may release rayeloid/T cell from restrain thus leading to activation of inflammatory response. Furthermore, by inhibiting p38 and ERK

activation, CD200R agonists may contribute to reducing or reversing steroid insensi t.ivity, which is an issue for treatment of severe asthma and COPD. Corticosteroid- resistant asthma is associated with classical macrophage activation, induction of LE ^J S signaling pathways and i creased p.38 activa ion (Goleva e ai . , J Allergy Clin Immunol; 122 (3) : 550-9, 2008; Bhavsar et al . , Thorax; 63 (9) : 784-90, 2008) , and in mice it has been shown that macrophages and TLR4/MyD88 drive steroid-resistant airway hyper-reactivity (Yang et al . , J Immunol; 182 (8} :5107- 15, 2009) . CD200R1 agonists can reduce LPS-induced cytokine production in macrophages, basophil and mas ceil degranulation, and T cell activation; i vivo

administration of CD200R agonists has been reported to reduce lung and intestinal i flammation in animeil models, The existing data suggests a application of CD200R agonists in treatment of pulmonary diseases (asthma, COPD, pulmonary exacerbations) and inflammatory bowel diseases.

Exemplary CD200R1-mediated conditions are autoimmune diseases such as rheumatoid arthritis (RA) ,

osteoarthritis, osteoporosis, inflammatory fibrosis (e.g., scleroderma., lung fibrosis, and cirrhosis), inflammatory bowe1 disorders (e.g., Crohn ' s disease a d u 1cerative colitis), inflammatory pulmonary conditions such as asthma and including allergic asthma, allergies, chronic

obstructive pulmonary diseeise (COPD), multiple sclerosis, psoriasis, uveitis and Lichen, planopilaris (LPP) .

The methods of the invention may be sed to treat an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.

The antibodies of the i vention, are useful in the preparation of a medicament for such treatment, wherein the medicament is prepared for administration in dosages defined herein.

Inflammatory pulmonary condition is an. example of a CD200R1 -mediated inflammatory condition. Exemplary i flammatory pulmonary conditions include infection- induced pulmonary conditions including those associated with viral, bacterial, fungal, parasite or prion

inflections ; a 11 ergen-induced pulmonary conditions;

pollutant-induced pulmonary conditions such, as asbestosis, silicosis, or berylliosis; gastric aspiration-- induced pulmonary conditions, immune dysregulation, inflammatory conditions with genetic predisposition such as cystic fibrosis, and physical trauma-induced pulmonary

conditions, such as ventilator injury. These inflammatory conditions also include asthma, emphysema, bronchitis, chronic obstructive pulmonary disease (COPD) , sarcoidosis, histiocytosis, lymphangiomyomatosis , acute lung injury, acute respiratory distress syndrome, chronic lung disea.se, bronchopu1monary dys 1 asia, commun i t. -acqui red ne monia , nosocoraia1 pneu onia , vent ilator-associated pne onia, sepsis, viral pneumonia, influenza infection,

parainfluenza infection, rotavirus infection, human metapneumovirus infection, respiratory syncitial virus infection and Aspergillus or other fungal infections.

E¾erap1ary infection -associated. i nf1amma.tory d.i seases may include viral or bacterial pneumonia, including severe pneumonia, cystic fibrosis, bronchitis, airway

exacerbations and acute respiratory distress syndrome (ARDS) . Such infection-associated conditions may involve multiple infections such as a. primary viral infection and a secondary bacterial infection, Dysregulated CD200R1 signaling may play a role in the lung immune homeostasis (Snelgrove et al (2008) Nat. Immunol. 9:1074-83) .

Asthma is an inflammatory disea.se of the lung that is characterized by airway hyperresponsiveness {"AHR"), bronchoconsfriction, whee ing, eosi ophilic o neutrophilic inflammation, mucus hypersecretion,

subepithelial fibrosis, and elevated IgE levels. Patients with asthma experience "exace rbations " , a worsening of symptoms, most commonly due to microbial infections of the respiratory tract (e.g. rhinovirus, influenza virus, Haemophilus influenza, etc.) , Asthmatic attacks can be triggered by environmental factors (e.g. ascarids, insects, animal s (e.g., cats, dogs, rabbits, mice, rats, hamsters, gu nea pigs and birds}, fungi, air poll tants (e.g., tobacco smoke), irritant gases, fumes, vapors, aerosols, chemicals, pollen, exercise, or cold air. Apart from asthma, several chronic inflammatory diseases affecting the lung are characterized by neutrophil infiltration to the airways, for example chronic

obstructive pulmonary disease (COPD) , bacterial pneumonia and cystic fibrosis (Linden et al (2000) Eur. Respir. J. 15:973-7; Rahman et al (2005) Clin. Immunol. 115:268-76) , and diseases such as COPD, allergic rhinitis, and cystic fibrosis are characteri ed by airway hyperresponsi veness (Fahy and 0' Byrne (2001) Am. J. Respir. Crit. Care Med. 163 : 822-3) ,

Commonly used animal models for asthma and airway inflammation include the ovalbumin challenge model, met acholi e sensitization models and sensitization with Aspergillus fumigatus (Hessel et al (1995) Eur. J.

Pharmacol. 293:401-12, 1995) , Inhibition of cytokine and chemokine production from cultured human bronchial epithelial cells, bronchial fibroblasts or airway smooth muscle cells can also be used as in vitro models. The admi istration of agonistic antibodies of the present invention to any of these models can be used to evaluate the use of the agonists to ameliorate symptoms and alter the course of asthma, airway inflammation, COPD and the J. i k.e .

Ar thrit is , inc 1uding osteoarthritis , rheumatoid arthritis, arthritic joints as a result of injury, and the like, are common inflammatory conditions, which would benefit from the therapeutic use of anti-inflammatory proteins, such as the agonistic antibodies of the present invention. Reduced inhibitory signal via CD200R1 may perpetuate inflammation and further tissue damage in the inflamed joint. Several animal models for rheumatoid arthritis are known. For example, in the collagen-induced arthritis (CIA) model, mice develop chronic in lammatory arthritis that closely resembles human rheumatoid arthritis. Administration of CD200R1 antibodies to this model resulted in attenuated disease and pathology

(Gorczynski et al (2002) Clin. Immunol, 104:256-64) . The administration of agonistic antibodies of the present invention to the CIA model can be used to evaluate the use of the agonists to ameliorate symptoms of arthritis.

Gastrointestinal inflamination is inflammation of a mucoseil layer of the gastrointestinal treict, and

encompasses eicute and chronic inflammatory conditions. Acute inflammation is generally characterized by a sho t time of onset and infiltration or i flux of neut ophils. Chronic inflammation is generally characterized by a relatively longer period of onset and infiltration or influx of mononuclear cells. Mucosal layer may be mucosa of the bowel (including the small intestine and large intestine), rectum, stomach (gastric) lining, or oral cavity. Exemplary chronic gastrointestinal inflammatory conditions are inflammatory bowel disease (I3D), colitis induced by environmental insults (e.g., gastrointestinal inflammation (e.g., colitis) caused by or associated wit

(e.g., as a side effect) a therapeutic regimen, such as administration of chemotherapy, radiation therapy, and the like), infections colitis, ischemic colitis, collagenous or lymphocytic colitis, necrotizing enterocolitis, colitis in conditions suc as chronic granulomatous disease or celiac disease, food allergies, gastritis, i fectio s gastritis or enterocolitis (e.g., Helicobacter pylori- infected chronic active gastritis) and other forms of gastrointestinal inflammation caused by an infectious agent .

In lammatory bowel disei3.se (IBD) includes a group of chronic inflammatory disorders of generally unknown etiology, e.g., ulcerative colitis (UC) and Crohn's disease (CD) . Clinical and experimental evidence suggest that the pathogenesis of IBD is m l ifactorial involving susceptibility genes and environmental factors. In inflammatory bowel diesase, the tissue damage results from an inappropriate or exaggerated immune response to antigens of the gut microflora. Several animal models for inflammatory bowel diseases exist. Some of the most widely used models are the 2,4, 6-trinitrobenesulfonic acid/etha.noI. (TNBS) -induced colitis model or the oxazalone model, which induce chronic inflammation and ulceration in the colon (Neurath eu aJ . , Intern. Rev. Immunol 19:51-62, 2000) . Another model uses dextran sulfate sodium (DSS), which induces a.n acute colitis manifested by bloody diarrhea, weight loss, shor ening of the colon and mucosal ulceration with neutrophil infiltration, DSS-induced colitis is characterized histologically by infiltration of inflammatory cells into the lamina propria, with lymphoid hyperplasia., focal crypt damage, and epithelial ulceration (Hendrickson e al . , Clinical Microbiology Reviews 15:79- 94, 2002) . Another model involves the adoptive transfer of naive CD45RB ^high CD4 T cells to RAG or SCID mice. In this model, donor naive T cells attack the recipient gut causing chronic bowel inflammation and symptoms simila to human inflammatory bowel diseases (Read and Powrie, Curr, Protoc. Immunol. Chapter 15 unit 15.13, 2001) . The administration of agonistic antibodies of the present invention to any of these models can be used to evaluate the potential ef icacy of these agonists to amelio ate symptoms and alter the course of diseases associated with inflammation in the gut, such as inflammatory bowel disease. Several treatment options for I3D are available, for example anti-TNF- antibody therapies have been used for a decade to treat Crohn's disease (Van Assche et al . , Eur, J. Pharmacol . Epub Oct 2009) . However, a. significant percentage of patients are refractory to the current treatments (Hanauer et al . , Lancet 359:1541-1549, 2002; Hanauer et al . ,

Gastroenterology 130:323-333, 2006), and thus new therapies targeting refractory patient populations are eeded .

Administration./Pharmaceutical Compositions

The invention provides for pharmaceutical

compositions comprising the CD200R1 agonistic antibodies of the invention and a pharmaceutically acceptable carrier. For therapeutic use, the CD200R1 agonistic antibodies of the invention may be prepared as

pharmaceutical compositions containing a therapeutical ly effective amount of the CD200R1 agonistic antibody as an actice ingredient in a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0,4% saline and 0.3% gl cine can be used. These solutio s are sterile and generally free of particulate matter. They may be sterilized by conventional, well- known sterilization techniques (e.g., filtration) , The compositions may contain pharmaceutically acceptable auxi iary substances as required to approximate

physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the molecules or antibodies of the invention in such pharmaceutical formulation, may vary widely, i.e., from less than about 0.5%, usually to at least about 1% to as much as 15 or 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight and will be selected primarily based, on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and

formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Reming on: The Science and Practice of Pharmacy, 21 ^st' Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical

Manufacturing pp 691-1092, See especially pp. 958-989,

The "therapeutically effective amount" of the CD200R1 antibodies of the invention effective in the treatmen of CD200R1 -mediated conditions may be determined by standard research techniques. For example, the dosage of the CD200R1 antibodies of the invention that will be effective in the treatment of inflammatory diseases such as asthma or IBD can be determined by administering the CD200R1 antibodies to relevant, animal models well known in. the art and as described he rei .

In vitro assays may optionally be employed to help identify optimal dosage ranges. Selection of a particular effective dose can. be determined (e.g., via clinical trials) by those skilled, in the art based upon the consideration of several factors. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known by the skilled artisan. The precise dose to be employed in the formulation will also depend on the route of administration, and the severity of disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-respon.se curves derived from in vitro or animal model- test systems. The antibodies of the invention may be tested for their efficacy and effective dosage using any of the models described herein.

The mode of administration for therapeutic use of the antibody of the invention, may be any suitable route such as parenteral administration, e.g., intradermal,

intramuscular, intraperitoneal, intravenous or

subcutaneous, pulmonary, trans ucosal {oral, intranasal, i travaginal , rectal) or other means appreciated by the skilled, artisan, as well known in the art.

The CD200R1 antibodies may be administered to a patient by any suitable route, for example parentally by intravenous (i.v.) infusion or bolus injection,

intramuscularly or subcutaneously or intraperitoneally . i.v. in sion ma be given over for, example, 15, 30, 60, 90, 120, 180, or 24 0 minutes, or from. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

The dose given, to a patient having a CD200Rl-mediated disease is sufficient to alleviate or at least partially arrest the disease being t eated ("therapeuticall effective amount") and may be sometimes 0.005 mg/kg to about 100 mg/kg, e.g. about 0.05 mg/'kg to about 20 mg/kg or about 0,1 mg/kg to about 20 mg/kg, or about 1 mg to about 20 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/ ^' kg or about 24 mg/kg , or, e.g. , about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 500, 4 00 , 300 , 250 , 200 , or 100 mg/'m ² . Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may be administered to treat CD200R-1 mediated diseases, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be given.

The administration of the CD200R1 antibodies of the invention may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose. For example, the CD200R1 agonistic antibodies may be administered at 0.1 mg/kg, at 1 mg./kg, a.t. 5 mg/kg, at 8 mg./kg or at 16 mg/kg a.t. weekly interval for 8 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every two 'weeks for an additional 16 weeks , followed by administra ion at 8 mg/ kg or at 16 mg/kg every four weeks by intravenous infusion.

The CD200R1 antibodies of the invention may be administered by maintenance therapy, such as, e.g ., once a week for a period of 6 months or more.

For example, the CD200R1 agonistic antibodies may be provided as ει daily dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, o any co bination thereof .

The CD200R1 antibodies of the invention may also be administered prophylactically in order to reduce the risk of developing, delay the onset of, and/or reduce the risk or recurrence of the CD200R1-mediated disease.

For example, a pharmaceutical composition comprising the CD200R1 antibody of the invention for intravenous infusion may bs made up to contain about 200 ml of sterile Ringer's solution, and about 8 mg to about 2400 mg, about 400 mg to a.bout 1600 mg, or abo t. 400 mg to about 800 mg of the CD200R1 antibody for adm.inistrat.ion to a 80 kg patient. Methods for preparing parenteral.].y administrable compositions are well known and are described in more detail in, for example, "Remington's Pharmaceutical Science", 15th ed., Mack Publishing Company, Eastern, PA.

Thus, a pha rmaceut cal composition of the invention for intramuscular injection could be prepared to contain 1 ml sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more

preferably, about 5 mg to about 25 mg, of an antibody of the 1 en t i o .

The antibodies of the i vention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and protein preparations and art-known 1 yoph i 1 i ation and reconst i t t.ion techniques can be employed.

The present invention will now be described with reference to the following specific, non-limiting examples .

Example 1. Identification of CD200R1 antibodies

CD200R1 antibodies were identified using phage display libraries and transgenic mice expressing human immunoglobulin loci.

Phage panning

Phage libraries displaying Fab variants on coat protein pIX were panned against biotinylated extracellular domain of human CD200R1 (SEQ ID NO: 7) .

SEQ ID NO:7 (human CD200R1 extracellular domain) LCMDEKQITQNYSKVLAEV TSWPVKMATNAVLCCPPIALRNLI I ITWEI ILRGQPSC TKAYK ETNETKETNCTDERITWVSRPDQNSDLQIRTVAITHDGYYRCIMV PDGNFH RGYHLQVLVTPEVTLFQNRNRTAVCKAVAGKPAAHIS I PEGDCATKQEYWSNGTVTV KSTCK EVHNVSTVTCHVSHLTGNKSLYIELLPVPGAKKSAKL

The phage libraries have been described elsewhere (Shi et al (2010) J. Mol. Biol. 397:385-96/ Int. Pat.

Publ . No. WO09/085462) . Three rounds of selections were carried out followed by phage ELISA to verify enrichment of binders to the antigen used for panning. Individual Fab clones rom the phage panning we e sc eened for binding to human and cynomolgus CD200R1 BCD (SEQ ID NO: 7 AND 8, respectively), and the variable heavy (VH) and variable light (VL) domains of identified Fab hits were sub-cloned into mammalian expression vectors for full IgG production. Select antibodies were affinity matured by d iversi i: ica.t.ion of selec residues in the CDR.s in the VL and/or VH domain and panned stringently during additional rounds of panning.

SEQ ID NO: 8: cynomolgus CD200R1 extracellular domain

LCMDEKQITQNHSKVLAEV ISWPVQMARNAVLCCPPIEFR.NLIVITWEI ILRGQPSC TKTYRKDTNETKBTNCTDERITWVSTPDQNSDLQIHPVAITHDGyyRCIMATPDGNFH RGYHLQVLVTPEVTLFESRNRTAVCKAVAGKPAAQISWIPAGDCAPTEQEYWGNGTVT VKSTCHWEGHNVSTVTCHVSHLTGNKSLYIELLPVPGAKKSAKL iMKiutTii zatiotTi

Mice expressing human transgenic loci (Medarex KM mice; Int. Pat. Publ. No. WO2002/043478) were immunized with human CD200R1 ECD { SEQ ID NO: 7) by intraperitoneal injection in Sigma adjuvant system and boosted on days 8, 13, and 21 followed by a final subcutaneous boost on day 46 together with an an.t.i-CD40 mAb . Spleens, lymph nodes and sera were harvested on day 50 and sera were tested for anti-CD200Rl response by ELISA. Lymphocytes from spleens were fused with mouse myeloma cells and the resulting hybridoma supernatants were tested for binding to human CD200R1 and cynomolgus CD200R1 ECDs . Supernatants demonstrating high binding signal to human and cynomolgus CD200R1 BCD were identified and their variable domains cloned into mammalian expression vectors for full IgG production using standard methods and characterized further. Select antibodies were affinity matured by divercifyi g select, residues in the CDRs in the VL domain, cloned into phageraid vector for Fab display on ρΙΧ, and panned stringently during additional rounds of panning.

Example 2. Characterization of CD200R1 antibodies

Antibodies were cloned as IgG2 isotypes optionally having Fc substitutions

V234A/G237A/P238S/H268A/V309L/A330S/P331S (residue numbering according to the EU numbering) and tested for their binding to cynomolgus CD200R1L protein. Antibodies H2RM190, H2RM211, H2RM147, H2R.M226 and. H2RM266 that demonstrated no detectable binding to cyno CD200R1L extracellular amain (SEQ ID NO: 9) (at concentrations tested) were further characterized in various assays.

Table 2 shows the VH and VL sequences and Table 3 shows the CDR sequences of the antibodies. The VL of H2RM147 and H2RM226 ( PH9L1 ) is identical to the germline A27 gene.

SEQ ID NO: 9 cynomolgus CD200R1L extracellular domain

SCMDGKQMTQNYSK SAEGNISQPVLMDTNANLCCPPIEFRNLI IVWEI I IRGQPSC TKAYRKETNETKETNCTDKRITWVSTPDQNSDLQIHPVAITHDGYYRCIMATPDGNFH RGYHLQVLWPEVTLFQSRNRTAVCKAVAGKPAAQISWIP GDCAPTEHEYWGNGTVT VESMCiI GDHNA.STVTCHVSHLTGNKSLYIKLNSGLRTSGSPALDLL

SEQ ID nO: 6 cynomolgus CD200R1L full length mature protein

SCMDGKQMTQNYSKMSAEGNISQPVLMDTNANLCCPPIEFRNLIVIVWEI I IRGQPSC TKAYRKETNETKETNCTDKRITWVSTPDQNSDLQIHPVAITHDGYYRCI ATPDGNFK RGYHLQVLV PEV LFQSR RTAVCKAVAGKPAAQISWI PAGDC PTEHEYWGNGTVT VESMCHWGDHNASTVTCHVSHLTGNKSLYIKLNSGLR SGSPALDLLI ILYVKLSLFV VILVTTGFVFFQRINYVRKSL Table 2.

Table 3. H2RM266 HCDR1 SYGMH. 20

H2RM266 HDCR2 VI YDGSNKYYADSVKG 21

H2RM266 HCDR3 EAYGSGS YYEDY 22

H2RM266 LCDR1 RASQGISSWLA 23

H2RM266 LCDR2 AASSLQS 24

H2RM266 LCDR3 QQHDAYPRT 40

Inhibition of lipopolysaccharide (LPS) -induced.

interleukin~6 {IL-6) production in cells overexpressing full length human CD200R1 receptor {"U937 IL-6 secretion assay")

Ability of select anti-CD200Rl antibodies to activate human CD200R1 -mediated inhibitory activity was evaluated by assessing their ability to inhibit LPS-induced IL-6 production in cells overexpressing human CD200R1.

U937 cells (ATCC CRL-1593.2) were trans fect.ed to express full length human CD200R1 isoform d (SEQ ID NO: 4) and cultured in RPMI 1640 supplemented with 10% heat- inactivated fetal bovine serum (FBS) and 400 μg/ l

Geneticin. U937 cells in. log-phase growth were washed with pre-warmed RPMI 1640 supplemented, with 10% heat- inactivated FBS but without Geneticin (assay medium.) and re-suspended in assay medium containing 100 nM phorbol-12- myristate-13-acetate (PMA) to yield a 2.5 x 10 ⁵ cells/ml suspension. From this suspension 5 x 10 ^'1 cells per well (200 ul) were seeded in 96-well tissue culture assay plates and the cells were differentiated for 72 h. in a humidified incubator set at 37°C and 5% C0 ₂ . After differentiation, the spent PMA containing medium was aspirated and replaced with fresh pre-warmed assay medium and the cells inc bated for 2 b i a humidi ied incubator prior to treatment of cells. Differentiated U937 cells were treated with 100 μΐ of 2X concentration of CD200R1 agonist mAbs, or appropriate isotype controls, prepared in pre-warmed assay medium. This was followed by addition of 100 ul of 20 ng/ml (2X) lipopolysaccharide (LPS) solution. also prepared in pre- armed assay medium. The final volume per well was 200 μΐ . Control wells included differentiated U937 cells without, any treatment and dif erentiated U937 cells treated with 10 rig/ml LPS and were included in at least six replicates per assay plate for normalization of raw data into percent inhibition. The treated U927 cells were then incubated in a humidified incubator at 37° c and 5% C02. After 24 h ncuba on the supernatants were harvested for analysis . The amounts of human IL--6 in the harvested supernatants were measured using the Meso Scale Discovery (MSD) Human IL--6 Tissue Culture Assay Kit according to manufacturer protocol. The deita was exported into Microsoft Excel for data analysis and the resulting data visualized in GraphPad Prism 5.0. Pe cent inhibition was calcula ed using the formul :

Percent Inhibition - { [1 - ({X - NT) / (T-NT) )] X 100} where "X" is the relative electrochemiluminescent signal observed in cells treated with 10 ng/ml LPS and CD200R1 antibody; "NT" is the relative

electrochemiluminescent signal representing basal level of IL-6 in non-treated cells; and ^W T" is the relative electrochemiluminescent signal representing IL-6 levels in cells treated with 10 ng/ml LPS.

Figure 1A, IB and 1C shows inhibition of 10 ng/ml LPS- induced IL-6 release in differentiated U937 cells by antibodies H2RM190 and H2RM211 (Figure 1A) , H2RM147

(Figure IB) and H2RM226 and H2RM266 (Figure 1C) . These antibodies were thus demonstrated to be CD200R1 agonists.

"ProteOn receptor binding studies"

Affinity measurements using Surface Plasmon Resonance (SPR) were performed using a ProteOn XPR36 system

(SioRad) . A biosensor surface was prepared by coupling anti-IgG Fc antibodies (mixture of anti-mouse (Jackson. cat#315-005-046) and anti-human (Jackson cat#109-005-098) ) to the modified alginate polymer layer surface of a GLC chip (BioRad, Cat#176-5011) using the manufacturer's instructions for amine-coupling chemistry. Approximately 5,000 RU (response units) of antibody were immobilized in each, of six flow cells. The kinetic experiments were performed at 25°C in running buffer (DPBS+0.01%P20+100 g/ral BSA) . Serial dilutions of analyt.es (human CD200R1 ECD ( SEQ ID NO: 7), cynomolgus CD200R1 ECD ( SEQ ID NO: 8), or cynomolgus CD200R1L ECD ( SEQ ID NO: 9), from 900 nM to 11.1 nM were prepared in running buffer. About 200 RU of mAb were captured on flow cell 1 to 6 of the sensor chip. Capture of mAb was followed by five minutes injection

(association phase) of analyte at 50 μΐ,/min, followed by 60 or 15 minutes of buffer flow (dissociation phase) . The chip surface was regenerated by two pulses of 18 seconds injection of 100 mM H3PO4 (Sigma, Cat#7961) at 100 μΐ./min.

The collected data were processed using ProteOn Manager software. First, double reference subtraction of the data was performed by subtracting the curves generated by buffer i jection from the reference-- subtracted

(iriterspot) curves for analyte injections. Then kinetic analysis of the data was performed using "Langmuir 1:1" binding model wit Rmax-group and RI- local setting. The result for each mAb was reported in the format of a (On- rate) , Kd (Off-- rate) and K (equilibrium dissociation, constant) and are shown in Table 4. For antibody H2RM211, no binding was observed for cyno CD200R1L protein.

Affinity constant could not be determined at the analyte concen ations used for binding to cyno CD200R1L for antibody H2RM190.

Table .

Example 3. Epitope and paratope of anti~CD200Rl antibody H2RM147

The epitope and paratope of antibody H2RM147 (VH: SEQ ID NO: 10; VL : SEQ ID NO: 33} was identified using X-ray crystallography. The Fab fragment of mAb H2RM147 and the extracellular domain of human CD200R1 (residues 1-217 of SEQ ID NO: 4) were expressed in HEK 293F cells and in HEK 293S GnTI cells, respectively, and were purified using affinity and size-exclusion chromatography. The

CD200R1 : H2RM147 complex was incuba ed overnight at 4°C, concen rated, and separated from the uncomplexed species using size-exclusion chromatography. The comple was crystallized by the vapor-diffusion method from, solution containing 22% PEG 3350, 1.0 M sodium acetate, pH 4.5. The structure of the complex was determined at 2.8-Ά resolution .

The structure of CD200R1 : H2RM147 is shown in Figure 2A. H2RM147 binds to the -terminal domain of CD200R1.

The CD200R1 epitope bound by H2RM147 is

conformational. The epitope spans 3 segments on CD200R1, residues 11-20, 36-42, and 112-116 (residue numbering according ot SEQ ID NO: 4) . The epitope includes 16 residues that are in direct 4-h contact with the antibody: Nil, K14, V15, L16, A17, E18, V19, N20, P36, 138, N42, D112, G113, N114, F115 and H116 (residue numbering according to SEQ ID NO: 4) . Carbohydrate chains attached to residues Nil and N20 form part, of the epitope. Based on the number of contacts, the key residues of the epitope are L16, D112 and F115 (residue numbering according to SEQ ID NO: 4) .

Antibody residues in conteict with CD200R1 form the paratope. The paratope of H2RM147 i nvolved 5 CDRs and included residues S30, S31 and Ύ33 in LCDR1 ; Y92, G93, S94 and S95 in LCDR3; F27, T28, S31, Y32 in HCDR1; G56, S57, T58, Y59 in HCDR2 and Y102, V103, L104, S105, S106, S107 in HCDR3. Paratope residue numbering refers to residues in the VL of SEQ ID NO: 33 for LCDR1 and LCDR3 , and to residues in. the VH of SEQ ID NO: 10 for HCDR1, HCDR2 and HCDR3. Figure 2C shows the epitope and paratope residues.

The mechanism by which H2RM1 7 activates CD200R signaling involves a displacement of the N-terminal beta- strand of CD200R1 by HCDR3 of the antibody. The displaced and unfolded section of CD200R1 (residues 11-20 of SEQ ID NO: 4) is bound to H2RM147 between LCDR3 and HCDR2.

Crystal structure of the mouse CD200 :CD200R1 has been solved (Protein Data Bank entry 4BFI Hatherley et al , , Structure 2013; 21:820-832. Both H2RM147 and mouse CD200 bind overlapping epitopes at the N-terminal domain of CD200R1; and therefore H2RM147 is likely to oco pete with human CD200 for binding to human CD200R1.

Affinity maturation does not substantially change the overall geometry of the antibody-antigen interface.

Therefore, it is expected that the a.ffi i t -matu red variants of H2RM147 bind largely the same CD200R1 epitope than what H2RM147 binds to.

Figure 2B shows the epitope and paratope residues of H2RM147. Example . Affinity maturation of H2RM147

Antibody H2RM147, isolated from phage display libraries, was affinity matured, by diversifying the VL, or diversi yi g both the VH and the VL . The diversified positions were chosen based on amino acid solvent exposure and predicted proximity to the antigen. The diversity was introduced by solid phase gene synthesis. Antibodies H2RM185-H2RM204 were generated from the VL affinity maturation campaign, and thus these antibodies have the parental H2RM147 VH, H2RH81 ( SEQ ID NO: 10) and parental HCDR sequences (HCDR1 of SEQ ID NO: 14 ( SYAMS ) , HCDR2 of SEQ ID NO: 15 (AISGSGGSTYYADSVKG) and HCDR3 of SEQ ID NO: 16 (LY YVLSSSRLDY) . Table 5 shows the VH and the VL sequences of the parental and the VL affinity-matured, a ibodies. able 6 shows the light chain CDR sequences of the VL affinity-matured antibodies.

Table 5.

able 6.

Affinity matureition diversifying both the VL and the VH yielded, antibodies H2RM218-H2RMM227 , Table 7 shows the VH and the VL sequences of select. VH and VH affinity- matured antibodies. Table 8 shows the KCDR sequences and Table 9 showns the LCDR sequences of the affinity-matured antibodies of Table 7 ,

Table 7.

Table 8

.An i ody HCDRl I-ICDR2 HCDR3

SEQ

SEQ ID SEQ ID

Sequence ID Sequence Sequence

NO : NO: NO :

AIDGNGGYTY LYWYVL3SS

I-I2RM218 NYAM3 69 71 16

YADSVKG RLDY

H2RM219 ΝΥΑΜΞ 69 AT3GNGGTTY 72. LYWYVLSSS 16 YADSVKG RLDY

AISGSGGSTY LY YVLSSS

H2RM225 SYGM 70 15 16

YADSVKG RLDY

AIDGAGGSTY LYWYVLSSS

H2RM226 SYGMQ 38 39 16

YADSVKG RLDY

AISGEGGSTY LYWYVLSSS

H2RM2.27 SY MS 14 73 16

YADSVKG RLDY

Table 9.

The resulting antibodies were characterized for their binding to human and cyno CD200R1, and for their ability to inhibit LPS- induced IL-6 production using methods described in Example 2,

Table 10 shows the affinities of the antibodies to human CD200R1. Table 11 shows the affinities of the antibodies to the cyno CD200R1. Figure 3 snows inhibition of 10 ng/ l LPS induced IL-6 release in differentiated U937 cells by antibodyes H2RM185 (M185) , H2RM187 (M187) , H2RM190 (M190), H2RM200 (M200) and H2RM204 (M204) .

CNT08937: isotype control H2RG14 is soluble CD200R1.

Several antibodies were identified from the affinity- maturation campaigns that had improved affinity to CD200R1 and improved potency when compared to the parental antibody, H2RM147. Table 10.

Antibody Human CD200R1 ECD

kal (1/Ms) kdl (1/s) K _D

H2RM147 1.29E+04 4.16E-05 3.24E-09

H2RM185 2.51EH-04 7 , 07E-05 2.82E--09

I-I2RM187 2.96E+04 6.38E--05 2.16E-09

H2RM189 2.10E+04 2.18E-05 1.04E-09

H2RM190 2.09EH-04 1.78E-05 8.53E--10

I-I2RM191 2.60E+04 6.57E--05 2.53E-09

H2RM193 2.28E+04 5.60E-05 2..45E-09

H2RM195 1.67EH-04 1.78E-05 1.06E-09

I-I2RM198 1.97E+04 5.I7E--05 2.62E--09

H2RM200 2.08E+04 5.2. E-05 2.52E-09

H2RM204 1.66EH-04 1.22E-05 7.37E--10

I-I2RM218 7.69E+03 1.97E--05 2.56E-09

H2RM219 1.10E+04 4.0.1.E-05 3.65E-09

H2RM225 1.94EH-04 3.28E-05 1.69E--09

I-I2RM226 6.07E+04 4.36E--05 7.18E-10

H2RM227 1.79E+04 4.0.3E-05 2.25E-09

Table 11.

Cyno CD200R1 ECD

Ant ibody

kal (1/Ms) kdl (1/s! K _D (M)

H2RM147 1.80E+04 2.49E-05 1.38E-09

H2RM185 3.28Ef04 5.68E--05 1.73Ξ-09

H2RM187 3.70E+04 3.52E-05 9.52Ξ-10

H2RM189 2.64E+04 1.08E-05 4.09E-10

H2RM190 3.21Ef04 1.49E--05 4.63E-10

H2RM191 3. 1E+04 3.65E-05 1.07Ξ-09

I-I2RM193 2.95E+04 0 _ / '""00 1.94Ξ--09

H2RM195 2.02Ef04 2.07E--05 1.02Ξ-09

H2RM198 2..78E+04 3.62E-05 1.30Ξ-09

I-I2RM200 4„ 48E+04 3.17E-05 7.07Ξ--10

H2RM204 2.48Ef04 1.32E--05 5.32E-10

H2RM218 3.04E+02. 8.03E-05 2.64Ξ-07

I-I2RM219 8„ 48E+03 2.60E-05 3.07Ξ-09

H2RM225 I .68Ef04 9.98E--05 5.9 Ξ-09

I-I2RM226 6.09E+04 5.77E-05 9.48E--10

H2RM2.27 1.09E+04 2.16E-04 1.97E-08 Example 5. Optimization of CD200R1 antibodies

H2RM226 (VH SEQ ID NO: 34; VL SEQ ID NO: 33) HCDR2 (AIDGAGGS YY DSVKG; SEQ ID NO: 39) contains a DG motif at HCDR2 residue positions 3 and 4, which can lead to isomerization and antibody heterogeneity and potentially a subsequent partial loss of activity, H2RM226 HCDR3

( LY YVLSSSRLDY; SEQ ID NO: 16) contains a tryptophan (W) at HCDR3 residue posi ion 3, which can be oxidi ed, resulting in antibody heterogeneity and po entially a subsequent partial loss of activity. Thus, variants were generated where HCDR2 DG motif was mutated to SG, TG, QG or DA, and the HCDR3 W was mutated to A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or Y, and the resulting antibodies were tested for their binding to human CD200R1 in supernatant format (variant VH was paired with the parental VL of SEQ ID NO: 33) . In this experiment, variants that had retained DG at HCDR2 and had HCDR3 W substituted to A, D, E, G, K, N, P, Q, R, S, T or V deraons r ted no or weak binding to human CD 200P,1.

Select generated H2RM226 variants were purified and tested for their affinity to human and cyno CD200R1. All generated variant VH chains were paired with the parental H2RM226 VL (SEQ ID NO: 33) . The VH has the LCDR1 of SEQ ID NO: 36 (RASQSVSSSYLA) , LCDR2 of SEQ ID NO: 18 (GASSPAT ) , and LCDR3 of SEQ ID NO: 37 (QQYGSSPLT) .

Table 12 s ows the VH and the VL sequences of select generated antibodies, and indicates their mutated residues at HCDR2 residue positions 3 and 4 and HCDR3 residue position 3.

Table 12.

Table 13 shows the HCDR sequences of the generated antibodies .

Table 13

HCDR1 HCDR2 HCDR.3

Ant body

SEQ SEQ SEQ

Sequence ID Sequence ID Sequence ID

NO: NO : NO:

AISGAGGSTYYADS LYWYVLSSSRLD

H2RM279 SYGMQ .38 74 16

VKG Y

AITGAGGSTYYADS LYWYVLSSSRLD

H2RM280 SYGMQ .38 75 16

VKG Y

AIQGAGGSTYYADS LYWYVLSSSRLD

H2RM281 SYGMQ .38 76 16

VKG Y

AIDAAGGSTYYADS LYWYVLSSSRLD

H2RM2.82 SYGMQ .38 142. 16

VKG Y

AIDGAGGS YYADS LYFYVLS5SRLD

H2RM2.86 SYGMQ .38 39 77

VKG Y

AIDGAGGS YYADS LYKYVLSSSRLD

H2RM288 SYGMQ 38 39 79

VKG Y

AIDGAGGSIYYADS LYIYVLSSSRLD

H2RM289 SYGMQ 38 39 80

VKG Y

AIDGAGGSIYYADS LYLYVLSSSRLD

H2RM291 SYGMQ 38 39 81

VKG Y

AIDGAGGSTYYADS LYYYVLSSSRLD

H2RM299 SYGMQ 38 39 82

VKG Y

AISGAGGSTYYADS LYFYVLSSSRLD

H2RM302 SYGMQ 38 74 77

VKG Y Table 14 shows the affinities of select H2RM226 variant antibodies to human CD200R1. Table shows the results of two duplicate measurements for H2RM226.

Table 15 shows the affinities of the select generated antibodies to cyno CD200R1. Table shows the results of two duplicate measurements for H2RM226.

Collectively., the experiments show that the DG motif in HCDR2 can be replaced by e.g. SG, TG, QG or DA and binding is still retained. The HCDR3 W is less torelable to subsitutui ons ; only F, H or Y s bstituted a ibodies retained binding to human CD200R1.

Table 14.

Table 15.

Cyno CD200R1 ECD

.An ibody

kal (l/Ms) kdl (1/s) K _D (M)

H2R 226 6.16E 04 3.45E-05 5.59E-10

H2RM2.26 6.20E+04 3.78E-05 6.09E-10

I-I2RM279 6.91E+04 2.75E-05 3.99E-10

H2RM280 5.78E 04 2.58E-05 4.46E-10

H2RM281 6.95E+04 5.75E-05 8.27E-10

I-I2RM282 5.59E+04 4.76E-05 8.52E-10

H2RM286 3.16E 04 5.36E-05 1.70E-09

H2RM302 3.43E+04 4.95E-05 1.44E-09 Example 6. Affinity-maturation of H2RM211

H2RM211 (VH SEQ ID NO: 12; VL SEQ ID NO: 13) was obtained by immuni ing mice expressing human

immunoglobulin loci with human CD200R1 ECD. H2RM211 was affinity-matured by diversifying the parental VL . The positions chosen for diversification was solvent exposed residues and predicted to be in close proximity of the antigen . The di erisification was done by PGR of overlapping degenerate oligonucleotides . Table 16 shows the VH and the VL sequences of select aff nity-matured antibodies . Table 17 shows the LCDR sequences of the affinity-matured variants. The generated antibodies have the parental H2RM211 VH (SEQ ID NO: 12) and HCDR1 of SEQ ID NO: 20 (SYGMH) , HCDR2 of SEQ ID NO: 21

(VI YDGSNKYYADSVKG) and HCDP.3 of SEQ ID NO: 22

(EAYGSGS YEDY) .

Table 16.

Table 17.

The generated antibodies were tested for their affinity towards human CD200R1 (Table 18) . Table 18.

*Mean and standard deviation of four independent

measurentents

Figure 4 shows shows inhibition of 10 ng/ml LPS induced IL-6 release in differentiated U937 cells by various antibodies. All a ffi nity-matured H2RM211 variants were more potent in activati g CD200R1 --mediated i hibitory signaling when compared to CD200--Fc (CD200ECD ^~ Fc is H2RG14 in the Figure) . H2RM211 is named HRM211 in the Figure. CNT08937 : isotype control.

Example 7.

Ability of the anti-CD200Rl antibodies to inhibit cyno CD200R1 functionality was assessed by measuring inhibition of LPS-induced interleukin-6 (IL-6) production in cells ove express i n.g full length cynom.olgus monkey CD200R.1 receptor.

U937 cells (ATCC CRL-1593.2) were transfected to express full length cynomolgus monkey CD200R1 ( SEQ ID NO: 5) and cultured in RPMI 1640 supplemented with 25 mM HEPES , 10% heat-inactivated fetal bovine serum (FBS) and 500 ug/ml Genet icin (culture medium) . U937 cells in log- phase g owth were re -suspended i cul re medi m

containing 100 nM phorbol-12-myristate-13-acetate (PMA) to yield a 5 x 10 ⁵ cells/ml suspension. From this suspension 5 x 10 ⁴ cells per well (100 μΐ) were seeded in 96-well tissue culture assay plates and the ceils were

differentiated for 72 h in a humidified incubator set at

«1 37°C and 5% C0 ₂ . After differentiation, the spent PMA containing medium was aspirated, cells were washed and replaced with 80 p.l/well fresh assay medium (culture medium without Gen.et.icin) prior to treatment of cells, Differentiated U937 cells were treated with 10 μΐ of 10X concentration of CD200R1 agonist mAbs or appropriate isotype controls, followed by addition of 10 μΐ of 10X centra ion (100 ng./m.l) of 1 ipopo.lysa.ccha ide (LPS) solution prepared in pre-warmed assay medium. The final volume per well was 100 μΐ . Control wells included differentiated U937 cells without any treatment (medium control) and differentiated U937 cells treated with 10 ng/ml LPS. The treated U927 cells were then incubated in a humidified incubator at 37 °C and 5% CO ₂ . After 24 b. incubation the supernatants were harvested for analysis. The amounts of human IL-6 in the harvested supernatants were measured using the Meso Scale Discovery (MSD) Human TL-6 Tissue Culture Assay Kit according to manufacturer protocol. The data was exported into Microsoft Excel for data analysis and the resulting data, visualized in

GraphPad Prism 5.0,

Figure 5 shows that antibodies PI2RM226 and H2RM266 inhibited LPS-induced IL-6 release in differentiated U937 cells expressing cyno CD200R1.

Example 8. Optimization of H2RM266.

The VH domain of H2RM266 is shown below. The CDRs are underlined. HCDR1 : SYGMH ( SEQ ID NO: 20); HCDR2 :

VI YDGSNKYYADSVKG (SEQ ID NO: 21) , H.CDR3 : EAYGSGSYYEDY ( SEQ ID NO: 22) .

H2RM266_VH_H2RH102 ( SEQ ID NO: 12)

OVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMH VRQAPGKGLEWAVI YDGSNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREAYGSGSYYEDYWGQGTL VTVSS The HCDR2 ( SEQ ID NO: 21) of H2RM266 contains a DG motif at residue positions 5 and 6, which can lead to isomerization and. antibody heterogeneity. HCDR2 of H2RM266 also contains a tryptophan (W) at residue position 3, which can be oxidized, leading to antibody

heterogeneicit .

H2RM266 variants are generated wherein the DG motif in HCDR2 and/or the tryptophan (W) in HCDR2 are mutated. Table 19 shows the variants in which, the DG residues in HCDR2 are mutated. Table 20 shows the variants in which the W in HCDR2 is mutated. The resulting variant VH chains are co-expressed with the parental VL H2R 266 chain of SEQ ID NO: 35. The resulting variant VH chains are also co-epressed. with the VL chains shown in Tables 2, 5, 7, 12 and. 16. The resulting antibodies are tested for their binding to human and cyno CD200R1, and their ability to inhibit LPS-induceci IL-6 production is assessed as described in Example 2. Additional antibodies are generated that ave those DG and W substit tions that when prese in a ibodies, retai the antibody binding to CD200R1 and agonistic activity. Antibodies that

demonstrate similar or improved binding to human CD200R1 and similar or improved agonistic activity (inhibition of LPS-lnduced IL-6 secretion) are characterized further.

The obtai ed a ibodies are cloned as IgGl, IgG2, IgG3 or IgG4 isotypes using standard protocols . The obtained antibodies are optionally cloned as IgG2 isotypes having Fc substitutions

V234A/G237A/F238S/H268A/V309L/A330S/P331S (residue numbering according to the EU numbering) . SEQ

Parental HCDR2 HCDR2 variant

ID SEQ ID NO: sequence sequen ce

NO:

VIWYAGSNKYYADSVKG 97

VI YEGS KYYADSVKG 98

VIWYFGSNKYYADSVKG 99

VIWYGGSMKYYADSVKG 100

VI YHGSMKYYADSVKG 101

VI YIGSMKYYADSVKG 102

VIWYKGSNKYYADSVKG 103

VI YLGSNKYYADSVKG 104

VI YPGSNKYYADSVKG 105

VI YRGSNKYYADSVKG 106

VI YSGSNKYYADSVKG 107

VI Y GS KYYADSVKG 108

VIWYVGSNKYYADSVKG 109

VIWYYGSNKYYADSVKG 110

VI YDGSNKYYADSVKG 21 VIWYDASMKYYADSVKG 111

VI YDDSMKYYADSVKG 112

VIWYDESNKYYADSVKG 113

VIWYDFS KYY DSVKG 114

VIWYDHSNKYYADSVKG 115

VIWYDI SNKYYADSVKG 116

VI YDKSNKYYADSVKG 117

VIWYDLSNKYYADSVKG 118

VIWYD SNKYYADSVKG 119

VI YDQSNKYYADSVKG 120

VIWYDRSMKYYADSVKG 121

VI YDSSMKYYADSVKG 122

VI YDTSMKYYADSVKG 123

VIWYDVSMKYYADSVKG 124

VIWYDYSNKYYADSVKG 125

Table 20.

Example 9. Amino acid sequences of the VH and the VL regions

The antibody VH and VL were sequenced using standard methds . Table 21 shows the VH sequences of the VH chains of select antibodies generated. Table 22 shows the VL sequences of the VL chains of select antibodies.

Table 21.

MNSLRAEDTAVYYCAKLYWYVLS SSRLDYWGQGTLVTVS3

H2RH105 EVQLLESGGGLVQPGGSLRLSCAASGFTFDNYAMSWVRQAP 42

GKGLEWVSAISG GGTTYYADSVKGRFTISRDNSKNTLYLQ MMSLRAEDTAVYYCA.KLYWYVLSS SRLDYWGQGTLVTVSS

H2RH110 EVQLLESGGGLVQPGG3LRLSCAA3GFTFDSYGMNWVRQAP 43

GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAED AVYYCAKLYWYVLSS SRLDYWGQGTLV VSS

H2RH112 EVQLLESGGGLVQPGG3LRLSCAA3GFTFDSYAMSWVRQAP 44

GKGLEWVSAISGEGGSTYYADS KGRFTISRDMSKNTLYLQ MNSLRAEDTAVYYC KLYWYVLSS SRLDYWGQGTLVTVSS

H2RH116 EVQLLESGGGLVQPGG3LRLSCAA3GFTFSSYGMQWVRQAP 45

GKGLEWVSAISGAGGSTYYADS KGRFTISRDMSKNTLYLQ MNSLRAEDTAVYYCAKLYWYVLSS SRLDYWGQGTLVTVSS

H2RH117 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMQWVRQAP 4(5

GKGLEWVSAITGAGGSTYYADS KGRFTISRDMSKNTLYLQ MNSLRAED AVYYCAKLYWYVLSS SRLDYWGQGTLV SS

H2RH118 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMQWVRQAP 47

GKGLBWVSAIQGAGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKLYWYVLSS SRLDYWGQGTLVT SS

H2RH119 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMQWVRQAP 48

GKGLEWVSAIDAAGGS YYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKLYWYVLSS SRLDYWGQGTLVT SS

H2RH.123 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMQWVRQAP 49

GKGLEWVSAIDGAGGS YYADSVKGRFTISRDNSKNTLYLQ M SLRAEDTAVYYCAKLYFYVLSS SRLDYWGQGTLV VSS

H2RH125 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMQWVRQAP 1

GKGLEWVSAIDGAGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAED AVYYCAKLYHYVLSS SRLDYWGQGTLVTVSS

H2RH126 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMQWVRQAP 52

GKGLEWVSAIDGAGGSTYYADS KGRFTISRDMSKNTLYLQ MNSLRAEDTAVYYCAKLYIYVLSS SRLDYWGQGTLVTVSS

H2RH128 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMQWVRQAP 53

GKGLEWVSAIDGAGGSTYYADS KGRFTISRDMSKNTLYLQ MNSLRAEDTAVYYCAKLYLYVLSS SRLDYWGQGTLVT SS

H2RH136 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMQWVRQAP

GKGLEWVSAIDGAGGSTYYADS KGRFTISRDMSKNTLYLQ MNSLRAEDTAVYYCAKLYYYVLSSSRLDYWGQGTLVTVSS

H2RH137 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMQWVRQAP 55

GKGLEWVSAISGAGGS YYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKLYFYVLSS SRLDYWGQGTLVT SS

Table 22.

VL name VL amino acid sequence SEQ ID

NO :

H2RL185 EL ^' VL QS GTLSLS GERATLSCFASQSV SYYLAVJYQQKPGQAPRL 11

LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYGA SPLTFGQGTKVEIK

H2RL191 DIQMTQSPSSLSASVGDRVTITCRASQGIS5WLA YQQKPEKAPKSL 13

IYAASSLQSGVPSRFSG3GSGTDFTLTISSLQPEDFATYYCQQYN3Y PRTFGQGTKVEIK

PH9L1 EIVLTQSPGTLSLSPGERATLSCRASQSVSS3YLA.WYQQKPGQAPRL 33

LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGS SPLTFGQGTKVEIK

H2RL222 DIQ TQSPSSLSASVGDRV ITCRASQGISSWLA YQQKPEKAPKSL 35

IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHDAY PRTFGQGTKVEIK

H2RL153 EIVLTQ5PGTLSL5PGERATLSCRASQ5VSSSYLAWYQQKPGQAFRL 56

LIYGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYTCQQYGS APLTFGQGTKVEIK

H2RL178 El ^' VL QSPGTLSLSPGERATLSCFASQSVSSSYLAVJY'QQKPGQAPRL 7

LIYGASSBATGIPDRFSGSGSGTDFTLTISRLEPBDFAVYYCHQYGA SPLTFGQGTKVEIK

H2R.L1 2 EIVLTQSPGTLSLSPGER TL3CRASQSVKSYY ^" LAWYQQKPGQAPRL 58

LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGN APLTFGQGTKVEIK

H2RL165 EIVLTQSPGTLSLSPGERJiTLSCRASQSVSSDYLAWYQQKPGQAPRL 59

LIYGASNRATGIPDRF5GSGSGTDFTLTISRLEPEDFAVYYCQQ GS APLTFGQGTKVEIK

H2RL169 EIVLTQ3PGTLSL3PGERATL3CRASQ3VSSSYLAWYQQKPGQAPRL 60

LIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGS APLTFGQGTKVEIK

H2RL150 EIVLTQ5PGTLSL5PGERATL5CRASQ5VSSDYLAWYQQKPGQAPRL 61

LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGH SPLTFGQGTKVEIK

H2RL176 EIVLTQSPGTLSLSPGEPJVTLSCPASQSVS5SYLAWYQQKPGQAPRL 62

LIYGASTPJVTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGN SPLTFGQGTKVEIK

H2RL188 EIVLTQ5PGTLSL5PGERATLSCRASQ5VSSNYLAWYQQKPGQAFRL 63

LIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGH LPLTFGQGTKVEIK

K2RL147 EIVLTQSPGTLSLSPGEBATLSCRASQSVSSNYLAWYQQKPGQAPRL 64

LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGH SPLTFGQGTKVEIK

H2R.L192 EIVLTQ3PGTLSLSPGER TL3CRASQSVSSSY ^" LAWYQQKPGQAPRL 65

LIYGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGS SPLTFGQGTKVEIK

K2RL223 DIQMTQSPSSLSASVGDRVTITCRASGGIDSALAWYQQKPEKAPKSL 66

IYAASSLQSGVPSRFSGSGSGTDFTL I3SLQPEDFA.TYYCQQHNSY PRTFGQGTKVETK

H2RL224 DIQ TQSPSSLSASVGDRV ITCRASEPIDSALA YQQKPEKAPKSL 67

IYAASSLQSGVPSRFSG3GSGTDFTLTISSLQPEDFATYYCQQH 3Y PRTFGQGTKVEIK

H2RL225 DIQMTQSPSSLSASVGDRVTITCRASSQIESWLAWYQQKPEKAPK3L 68

IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQH SY PRTFGQGTKVEIK. Example 10. CD200R1 agonists inhibit cytokine producton induced by pulmanory exacerbator

Ability of the CD200R1 agonists to inhibit cigarette smoke extract (CSE) and nontypeable Haemophilus influenzae (M Hi) induced cytokine production was assessed by

measuring C-GSF, IL-lb, TNFa and IL-6 reiei3.se from ceils overexpressi j human CD200R1.

U937 cells (ATCC CRL-1593.2) were transfected to express full length human CD200R1 (U937-2C9) and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FSS) and 400 μσ/ml Genet icin (culture medi m) . U937-2C9 ceils in log-phase growth we e re- suspended into assay medium (culture medium w thout Geneticin) containing 100 nM phorbol-12-myristate-13- acetate (PMA) to yield a 5 x 10 ⁼ cells /'ml suspension.

From this suspension 1 x 10 ⁵ cells per well (200 μΐ) were seeded in 96-well tissue culture assay plates and the cells were differentiated for 72 h in a humidified incubator set at 37 °C and 5% CO _? . After differentiation (on the day of experiment) , the spent PMA containing medium was aspirated, cells were washed and replaced with 80 p.l/well fresh assay medium containing 2.5% CSE and incubated for 4 hr prio to treatments. A day before experiment, NTHi (a clinical strain isolated from COPD patient with bacterial exacerbation) cells were grown on chocolate agar plate and incubated at 37°C, 5% C0 ₂ for overnight. On the day of experiment, NTHi cells grown on agar plate were resuspended in assay medium containing 100 'ug/ml gentamicin and their concentration adjusted to ~2xl0'/ml colony forming units per ml (CPU /ml) according to previously determined OD550 vs. CFU standard curve. After 30 minute incubation at 37°C, gentamic n-treated N Hi cells (10 μΐ/well) were added to U937-2C9 cells, following addition of 10 ul of 10X concentra ion of testing articles: CD200R1 agonists CD200FC or H2RM147, or appropriate isotype control (final concentration 10xg/ml) , Fluticasone propionate (FP, final concentrat i on 0.01 ug/ml), ERK inhibitor (PD98059, final concentra on 20 μΜ) , p38 inhibitor (SB203680, final concentration 1 μΜ) , alone or in combination. The final volume per well was 100 μΐ . Control wells had no testing (M, medium control) . The treated U937- 2C9 cells were then incubated in a humidified incubator set at 37 C and 5% CO ₂ . After 20 h incubation the supernatants were harvested for analysis. The amounts of human cytokines in the harvested supernatants were measured using the Millipore human 26- plex kit according to manufacturer protocol. The data was ana1 zed i n. GraphPad Prisra 5.0.

Figure 8A shows inhibition NTHi induced G-CSF release in differentiated [J937 cells by CD200R agonists, FP, ERK or p38 inhibitor, alone or in combination.

Figure 8B shows inhibition NTHi induced IL-Ιβ release in differentiated U937 cells by CD200R agonists, FP, ERK or p.38 inhibitor, alone or in. combi ation.

Figure 8C shows inhibition NTHi induced IL-6 release in differentiated U937 cells by CD200R agonists, FP, ERK or p38 inhibitor, alone or in combination.

Figure 8D shows inhibition NTHi induced TNF-a release in differentiated U937 cells by CD200R agonists, FP, ERK or p38 inhibitor, alone or in combination.

The data demonstrated that using this assay system, release of some cytokines by the cells (eg. G-CSF, IL-Ιβ) were not inhibited by FP but were inhibited by CD200R agonists CD200-Fc and the agonistic antibody H2RM147.

CD200R agonists enhanced inhibitory effect of FE ^J on some other cytokines (eg. IL-6, TNF ) . ERK inhibitor showed similar profile to CD200R agonists in this assay.