CD200R agonist antibodies and uses thereof

Abstract

The present invention relates to anti-human CD200R agonist antibodies, and uses thereof for treating diseases such as atopic dermatitis, chronic spontaneous urticaria, allergy, asthma, scleroderma, IBD, SLE, MS, RA, GvHD, or psoriasis.

Claims

1. An antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises a HCDR1, HCDR2, and HCDR3, and the LCVR comprises a LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of the HCDR1 is SEQ ID NO: 1, the amino acid sequence of the HCDR2 is SEQ ID NO: 2, and the amino acid sequence of the HCDR3 is SEQ ID NO: 3, the amino acid sequence of the LCDR1 is SEQ ID NO: 4, the amino acid sequence of the LCDR2 is SEQ ID NO: 5, and the amino acid sequence of the LCDR3 is SEQ ID NO: 6, and wherein the antibody binds human CD200R.

2. The antibody of claim 1, comprising a HCVR and a LCVR, wherein the amino acid sequence of the HCVR is SEQ ID NO: 7 and the amino acid sequence of the LCVR is SEQ ID NO: 8.

3. The antibody of claim 2, wherein Xaa at position 1 of SEQ ID NO: 7 is glutamine.

4. The antibody of claim 2, wherein Xaa at position 1 of SEQ ID NO: 7 is pyroglutamic acid.

5. The antibody of claim 2, comprising a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of the HC is SEQ ID NO: 9 and the amino acid sequence of the LC is SEQ ID NO: 10.

6. The antibody of claim 5, wherein Xaa at position 1 of SEQ ID NO: 9 is glutamine.

7. The antibody of claim 5, wherein Xaa at position 446 of SEQ ID NO: 9 is glycine.

8. The antibody of claim 5, wherein Xaa at position 1 of SEQ ID NO: 9 is glutamine and Xaa at position 446 of SEQ ID NO: 9 is glycine.

9. The antibody of claim 5, wherein Xaa at position 1 of SEQ ID NO: 9 is glutamine and Xaa at position 446 of SEQ ID NO: 9 is absent.

10. The antibody of claim 5, wherein Xaa at position 1 of SEQ ID NO: 9 is pyroglutamic acid and Xaa at position 446 of SEQ ID NO: 9 is absent.

11. A pharmaceutical composition comprising i) an antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises a HCDR1, HCDR2, and HCDR3, and the LCVR comprises a LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of the HCDR1 is SEQ ID NO: 1, the amino acid sequence of the HCDR2 is SEQ ID NO: 2, and the amino acid sequence of the HCDR3 is SEQ ID NO: 3, the amino acid sequence of the LCDR1 is SEQ ID NO: 4, the amino acid sequence of the LCDR2 is SEQ ID NO: 5, and the amino acid sequence of the LCDR3 is SEQ ID NO: 6, and ii) one or more pharmaceutically acceptable carriers, diluents, or excipients.

12. The pharmaceutical composition of claim 11 wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of the HC is SEQ ID NO: 9 and the amino acid sequence of the LC is SEQ ID NO: 10.

13. The pharmaceutical composition of claim 12 wherein Xaa at position 1 of SEQ ID NO: 9 is glutamine and Xaa at position 446 of SEQ ID NO: 9 is absent.

14. The pharmaceutical composition of claim 12, wherein Xaa at position 1 of SEQ ID NO: 9 is pyroglutamic acid and Xaa at position 446 of SEQ ID NO: 9 is absent.

Description

EXAMPLE: ANTIBODY EXPRESSION AND PURIFICATION

(1) Anti-human CD200R agonist antibodies of the present invention can be expressed and purified essentially as follows. An appropriate host cell, such as HEK 293 or CHO, can be either transiently or stably transfected with an expression system for secreting antibodies using an optimal predetermined HC:LC vector ratio (such as 1:3 or 1:2) or a single vector system encoding both the HC and the LC. Clarified media, into which the antibody has been secreted, may be purified using any of many commonly-used techniques. For example, the medium may be conveniently applied to a Mab Select® column (GE Healthcare), or KappaSelect column (GE Healthcare) for Fab fragment, that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column may be washed to remove nonspecific binding components.

(2) The bound antibody may be eluted, for example, by pH gradient (such as 20 mM Tris buffer, pH 7.0 to 10 mM sodium citrate buffer, pH 3.0, or phosphate buffered saline pH 7.4 to 100 mM glycine buffer, pH 3.0). Antibody fractions may be detected, such as by SDS-PAGE, and then may be pooled. Further purification is optional, depending on intended use. The antibody may be concentrated and or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, multimodal, or hydroxyapatite chromatography. The purity of the antibody after these chromatography steps is between about 95% to about 99%.

(3) Notably, the C-terminal glycine of Antibody I-4P or the C-terminal lysine of Antibody I-IgG1 may be truncated post-translationally. Additionally, the N-terminal glutamine of Antibody I-4P or Antibody I-IgG1 may be converted to pyroglutamic acid.

(4) The product may be held refrigerated, immediately frozen at −70° C., or may be lyophilized. Amino acid SEQ ID NOs for exemplified humanized antibodies of the present invention are shown below in Table 1.

(5) TABLE-US-00001 TABLE 1 Amino acid sequences of exemplified anti-human CD200R agonist antibodies. Antibody SEQ ID NOs Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Antibody I SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO: 6 Antibody HCVR LCVR Antibody I SEQ ID SEQ ID NO: 7 NO: 8 Antibody HC LC Antibody I-4P SEQ ID SEQ ID NO: 9 NO: 10

EXAMPLE: ANTIBODY I-4P BINDS HUMAN AND CYNOMOLGUS MONKEY CD200R

(6) Surface Plasmon Resonance (SPR) at 37° C. is performed to determine the binding kinetics and affinity of Antibody I-4P to human CD200R, cynomolgus monkey CD200R, and cynomolgus monkey CD200RLa (herein also referred to as the cynomolgus monkey “activating form”).

(7) Biacore® T100 instrument (GE Healthcare, Piscataway, N.J.), Biacore reagents and Scrubber2 Biacore® Evaluation Software (Biologics 2008) are used for the SPR analysis of Antibody I-4P binding. A CM4 chip (Biacore P/N BR-1006-68) is prepared using the manufacturer's EDC/NHS amine coupling method (Biacore P/N BR-1000-50). Briefly, the surfaces of all 4 flow cells (FC) are activated by injecting a 1:1 mixture of EDC/NHS for 7 minutes at 10 μL/minute. Protein A (Calbiochem P/N 539202) is diluted to 100 μg/mL in 10 mM acetate, pH 4.5 buffer and immobilized for approximately 400 RU onto all 4 flow cells by 7 minute injection at a flow rate of 10 μL/minute. Un-reacted sites are blocked with a 7-minute injection of ethanolamine at 10 μL/minute. Injections of 2×10 μL of glycine pH 1.5 are used to remove any non-covalently associated protein. Running buffer is 1× HBS EP+(Biacore P/N BR-1006-69).

(8) Human, cynomolgus monkey (cyno), and cynomolgus monkey activating CD200 receptors are purified using IMAC and size exclusion chromatography. Mouse CD200R is generated by Factor Xa cleavage from a mouse CD200R Fc fusion protein made in house. The final polishing step for the mouse CD200R receptor is size exclusion chromatography.

(9) For human and cyno CD200R binding, antibodies are diluted to 2.5 μg/mL in running buffer, and approximately 150 RU of Antibody I-4P is captured in flow cells 2 through 4 (RUcaptured). FC1 is the reference flow cell; therefore, no antibody is captured in FC1. Human and cyno CD200R are diluted to 500 nM in running buffer and then two-fold serially diluted in running buffer to 3.9 nM. Duplicate injections of each concentration are injected over all FC's at 50 μL/minute for 250 seconds followed by a 1200 second dissociation phase. Regeneration is performed by injecting 15 μL of 10 mM glycine pH 1.5 at 30 μL/minute twice over all FC's. Reference-subtracted data is collected as FC2-FC1, FC3-FC1, and FC4-FC1. The measurements are obtained at 37° C. The affinity (K.sub.D) is calculated using a “1:1 (Langmuir) binding” model in BIA Evaluation.

(10) For cyno activating CD200R binding, antibodies are diluted to 2.5 μg/mL in running buffer, and approximately 150 RU of Antibody I-4P is captured in flow cells 2 through 4 (RUcaptured). FC1 is the reference flow cell. Cyno activating CD200R is diluted to 8.1 μM in running buffer and then 2 fold serially diluted in running buffer to 63.2 nM. Duplicate injections of each concentration are injected over all FC's at 50 μL/minute for 250 seconds followed by a 1200 second dissociation phase. Regeneration is performed by injecting 15 μL of 10 mM glycine pH 1.5 at 30 μL/min twice over all FC's. Reference subtracted data is collected as FC2 FC1, FC3 FC1, and FC4-FC1. The measurements are obtained at 37° C. The affinity (K.sub.D) is calculated using the steady state equilibrium analysis with the Scrubber 2 Biacore® Evaluation Software.

(11) For mouse CD200R binding, antibodies are diluted to 2.5 μg/mL in running buffer, and approximately 150 RU of Antibody I-4P is captured in flow cells 2 through 4 (RUcaptured). FC1 is the reference flow cell. Mouse CD200R is diluted to 10 μM in running buffer and then 2 fold serially diluted in running buffer to 78 nM. Duplicate injections of each concentration are injected over all FC's at 50 μL/minute for 250 seconds followed by a 1200 second dissociation phase. Regeneration is performed by injecting 15 μL of 10 mM glycine pH 1.5 at 30 μL/min twice over all FC's. Reference subtracted data are collected as FC2 FC1, FC3 FC1, and FC4-FC1. The measurements are obtained at 37° C. The affinity (K.sub.D) was calculated using the steady state equilibrium analysis with the Scrubber 2 Biacore® Evaluation Software.

(12) Following procedures essentially as described above, the following data were obtained. As shown in Table 2, Antibody I-4P binds human CD200R and cynomolgus monkey CD200R with an affinity in the nM range, and Antibody I-4P binds the CD200RLa activating receptor with an affinity in the μM range. Antibody I-4P binds mouse CD200R with an affinity of >10 μM.

(13) TABLE-US-00002 TABLE 2 Affinity of Antibody I-4P to Human, Cyno, Cyno Activating, and mouse CD200 Receptors Measured Using Surface Plasmon Resonance (SPR) at 37° C. Receptor Average K.sub.D Std. Dev. Antibody I-4P Human 5.6 nM 1.2 Cynomolgus monkey 2.3 nM 0.1 Cynomolgus monkey activating 2.5 μM 0.4 *Mouse CD200R >10 μM  n = Assay was performed three times; *n = 1 time assayed

(14) These data demonstrate that Antibody I-4P binds the CD200RLa activating receptor and mouse CD200R with reduced affinity compared to Antibody I-4P affinity to human CD200R and cynomolgus monkey CD200R.

(15) Despite substantial engineering to overcome significant problems associated with lack of cross-reactivity between human and cyno CD200R, isomerization under stressed conditions (driven by primarily by an aspartic acid residue in LCDR1 (LC D28)), and a non-native disulfide bond between HC CDR1 and CDR2, Antibody I-4P demonstrated a favorable binding profile. For instance, a heavy chain and light chain CDR residue saturation mutagenesis procedure using mammalian cell expression was used to determine CDR changes that closed the affinity gap between human and cyno CD200R. This procedure was also used to find a residue replacement for LC D28 without compromising affinity. A second CDR library was screened using a phage-based process, which led to the discovery of non-predicted and non-germline replacement residues for the non-native disulfide without compromising antigen binding affinity.

EXAMPLE: IN VITRO BINDING OF ANTIBODY I-4P TO CD200R EXPRESSED IN CELLS

(16) CD200R is a member of the “paired receptor family”, which means that a close homologue with opposite, activating activity exists. This form has not been identified in humans, but low level mRNA transcripts have been described in whole blood and testis of cynomolgus monkeys (herein referred to the cynomolgus monkey “activating form” or “cynomolgus monkey CD200RLa”). Therefore, the cynomolgus monkey activating form could present a safety concern during toxicology studies in cynomolgus monkeys.

(17) To determine if Antibody I-4P binds to cell-expressed, membrane-bound CD200R from cynomolgus monkey, human, and the activating form cynomolgus CD200RLa, flow cytometry is used. CHO cells are transfected with human CD200R (SEQ ID NO: 15), cynomolgus monkey CD200R (SEQ ID NO: 16), or the cynomolgus monkey activating form (SEQ ID NO: 17) and are selected for high expression. Cells (2.sup.e5) are suspended in 1.sup.e6/50 μL in PBS for each cell line and FL4 dye (MultiCyt® Proliferation and Encoder FL4 dye) is added. The FL4 dye is diluted 1:5000 for cells expressing human and cyno CD200R, 1:700 for cells expressing the cynomolgus monkey activating form, and 1:50 for untransfected cells. The dye is mixed with the cells and the mixture is incubated at 4° C. for 30 minutes in the dark. The cells are washed twice with 10 mL of PBS and spun down at 1200 RPM for 5 minutes. The cells are then mixed in FACS buffer at 8.sup.e5 cells/50 μL/well.

(18) The cells are incubated for 30 minutes at room temperature with antibody titrations made in FACS buffer. The cells are washed once with FACS buffer and 100 of PE-conjugated anti human-Fc antibody at a 1:1000 dilution is added to each well for 15 minutes in the dark at 4° C. Cells are washed three times and then resuspended in 150 μL of FACS buffer. Sytox blue (2 μL/well) is added, cells are transferred to a FACS plate, and run on a Fortessa LSRII cytometry instrument (BD Biosciences). Data is analyzed using FlowJo (FlowJo, LLC) software.

(19) Following procedures essentially as described above, the following data were obtained. Antibody I-4P binds to cynomolgus monkey CD200R and human CD200R. Antibody I-4P binds to the cynomolgus monkey activating form similar to binding to untransfected control cells. These data demonstrate that there is no binding of Antibody I-4P to the cynomolgus monkey activating form; therefore, there may be a reduced safety concern during toxicology studies in cynomolgus monkeys.

EXAMPLE: ANTIBODY I-4P IS A CD200R AGONIST

(20) To demonstrate the agonist activity of Antibody I-4P, a human monocyte cell line U937 (ATCC, CRL1539.2), is transfected with the cDNA for human CD200R. Cytokine production, including IL-8, from these cells can be induced by immune complexes (IC) that bind and activate Fcγ Receptors. For IC stimulation, human IgG1 isotype control antibody is coated to a high-binding plate overnight. The next day, 4×10.sup.5 CD200R-expressing U937 cells/well are incubated with different concentrations of Antibody I-4P for 1 hour on ice before added to the pre-coated plate for IC stimulation and incubated at 37° C. for 24 hours. After 24 hours the cells are spun down, the supernatant is removed, and the IL-8 concentration measured using MSD kit (Mesoscale Diagnostics).

(21) Following procedures essentially as described above, the following data were obtained. As shown in Table 3, the reduction of IC-induced IL-8 with Antibody I-4P as percent inhibition compared to isotype control at the corresponding concentration. The relative IC.sub.50 is based on a four parameter logistic fit of the slope of percent inhibition over concentration. The average IC.sub.50 from 3 independent experiments was determined to be 0.2 μg/mL±0.02 μg/mL.

(22) TABLE-US-00003 TABLE 3 Concentration-dependent inhibition of immune-complex induced IL-8 secretion in cells expressing human CD200R. Antibody I-4P average % IL-8 (μg/ml) inhibition SEM 0.01 −2.0 3.1 0.03 2.3 2.9 0.1 14.0 6.0 0.3 24.0 4.2 1 47.1 2.8 3 55.7 2.9 10 67.8 3.3 30 76.2 4.0

(23) These data demonstrate that Antibody I-4P is able to inhibit IC-induced IL-8 production in a concentration-dependent manner.

(24) The ability of CD200R agonist antibodies with different isotype backbones to agonize CD200R and inhibit immune-complex stimulated IL-8 release from human CD200R-expressing U937 cells is also examined. For stimulation, human IgG1 isotype control antibody is coated at 10 μg/ml to a high-binding plate overnight. The next day, 4×10.sup.5 CD200R-expressing U937 cells/well are incubated with different concentrations of Antibody IgG4PAA (the two leucine to alanine substitutions (SLL228PAA) are known to disrupt hydrophobic interactions with FcγRs to eliminate residual effector function) or Antibody I-4P for 1 hour on ice before added to the pre-coated plate for IC-stimulation followed by an incubated at 37° C. for 24 hours. The cells are spun down, the supernatant is removed, and the IL-8 concentration measured using MSD kit (Mesoscale Diagnostics) according to manufacturer's instructions. The IL-8 concentrations are converted to percent inhibition relative to isotype control. The IL-8 concentration are plotted versus the antibody concentration, and a 4 parameter logistic model is used to fit percent inhibition versus log concentration using R statistical software. According to procedures essentially as described above, the following data (shown in Table 4) were obtained.

(25) TABLE-US-00004 TABLE 4 Concentration-dependent reduction in IL-8 production Antibody IgG4PAA IgG4SP μg/ml avg % IL-8 inhibition SEM* avg % IL-8 inhibition SEM* 0.01 −3.2 6.0 15.4 3.7 0.03 −5.0 5.6 35.8 3.6 0.1 −10.4 10.1 44.0 3.0 0.3 15.0 5.3 80.0 2.8 1 16.9 3.9 73.8 1.8 3 35.5 4.1 82.0 2.6 10 45.4 1.7 87.1 1.4 30 53.5 3.2 86.4 1.5 *Standard error of the mean These data demonstrate that IgG4PAA has weaker inhibitory activity (IC.sub.50 = 1.45 μg/ml) compared to Antibody I-4P (IC.sub.50 = 0.07 μg/ml).

EXAMPLE: FCγ RECEPTOR BINDING IS REQUIRED FOR AGONISM IN VIVO

(26) Clustering Through Fcγ Receptor in the Lipid Raft can Increase the Inhibitory potency on inflammatory cells. In order to identify whether Fcγ Receptor interaction is beneficial for agonism through CD200R, two mouse CD200R antibodies are engineered; one to ablate any Fcγ Receptor binding (mIgG2aAA) and one to have functional Fcγ Receptor binding (mIgG2a). Both molecules are tested in two independent models of induced inflammatory disease in mice; contact dermatitis and CD40-induced colon inflammation model.

(27) Contact dermatitis model: The ability of anti-human CD200R agonist antibodies to treat contact dermatitis may be determined by an in vivo mouse model performed essentially as described as follows (see e.g. Tolstrup et al., Anti-inflammatory effect of a retrovirus-derived immunosuppressive peptide in mouse models, BMC Immunology 2013, 14:51). Male 12 week-old C57B1/6J mice are anesthetized, their abdomens are shaved, and 100 μL of 3% oxazalone in ethanol is applied to the shaved area. Seven days after sensitization, CD200R agonist antibody IgG2a or IgG2aAA is administered at 0.1, 1, or 10 mg/kg subcutaneously (SC), or an isotype control mIgG2a is administered at 10 mg/kg SC for comparison. Four hours after antibody administration, mice are anesthetized, baseline ear thickness is measured with calipers, and ears are challenged with 10 μL of 2% oxazalone in ethanol on each side of both ears. Twenty-four hours post-challenge, ear thickness is again measured. The hypersensitivity reaction is assessed by measuring the difference between ear thickness pre- and 24 hours post-challenges. Statistical differences from isotype control are determined using a 1-way ANOVA with Dunnett's post post test (GraphPad Prism).

(28) CD40-induced colon inflammation model: The ability of anti-human CD200R agonist antibodies to treat CD40-induced colon inflammation model may be determined by an in vivo mouse model performed essentially as described as follows. Female 14 week-old RAG2N12 (B6.129S6-Rag2tm1Fwa N12; Taconic) mice are injected with 100 μg/mouse anti-CD40 antibody (BioXcel clone FGK4.5) to induce colon inflammation. One hour post-induction of disease, CD200R agonist antibody IgG2a, IgG2aAA, or isotype control antibody is administered subcutaneously at 0.1, 1, or 10 mg/kg. Animals are sacrificed six days later and colon inflammation is determined by measuring the length and the weight of the colon. The colon length-to-weight ratio is used to determine colon inflammation. Statistical differences from isotype control are determined using a 1-way ANOVA with Dunnett's post post test (GraphPad Prism).

(29) Following procedures essentially as described above, the following data were obtained.

(30) TABLE-US-00005 TABLE 5 Ear inflammation as measured in the contact dermatitis model by change in ear thickness (mm) Ear thickness (mm) mIgG2a mIgG2aAA Isotype control 0.200 ± 0.05  0.200 ± 0.05  10 mg/kg 0.118 ± 0.03**  0.160 ± 0.03* 1.0 mg/kg 0.113 ± 0.06** 0.170 ± 0.04 0.1 mg/kg 0.176 ± 0.05  0.178 ± 00.3 *p < 0.05; **p < 0.001. n = 5/group

(31) TABLE-US-00006 TABLE 6 Colon inflammation as measured in the CD40-induced colon inflammation model by weight-to-length ratio (mg/cm) Colon length to weight ratio (mg/cm) mIgG2a mIgG2aAA Isotype control 37 ± 1.1 37 ± 1.1  10 mg/kg  26 ± 0.8** 33 ± 0.7 1.0 mg/kg  29 ± 1.6* 36 ± 1.9 0.1 mg/kg 32 ± 2.6 36 (n = 1) n = 5/group, *p < 0.05; **p < 0.001.

(32) These data demonstrate that compared to isotype controls, the antibody with full effector function (mIgG2a) exhibited an immune suppressive function in both models. However, the Fcγ Receptor null variant (mIgG2aAA) was much less potent in the contact dermatitis model and had little to no effect in the colon inflammation model. The difference in activity was not due to depletion of CD200R-expressing cells, as the Fcγ Receptor-competent IgG2a antibody was demonstrated in an independent experiment not to deplete CD200R expressing cells in mice (data not shown).

(33) These data demonstrate that Fcγ Receptor binding is required to provide optimal agonism to the CD200R to mediate an anti-inflammatory signal.

EXAMPLE: ANTIBODY I BINDING TO FCγ RECEPTORS

(34) To determine if the antibody Fc affects the binding characteristics of Antibody I-4P to Fcγ receptors, the binding of Antibody I-4P, Antibody I-IgG1, and Antibody I-4PAA, to the human FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa receptor extracellular domains (ECDs) is measured by SPR at 25° C. Antibody I-IgG1 and Antibody I-4PAA have the same CDRs as Antibody I-4P. Antibody I-IgG1 has identical HCVR, LCVR, and LC as Antibody I-4P, but Antibody I-IgG1 has a HC whose amino acid sequence is given by SEQ ID NO: 11. Antibody I-4PAA differs from Antibody I-4P by having a SLL228PAA mutation in the HC.

(35) Biacore® T100 instrument and Biacore® 3000 (GE Healthcare, Piscataway, N.J.), Biacore® reagents and Scrubber2 Biacore® Evaluation Software (Biologics 2008) are used for the SPR analysis of antibody binding. A CM5 chip (Biacore® P/N BR-1006-68) is prepared using the manufacturer's EDC/NHS amine coupling method (Biacore® P/N BR-1000-50). Briefly, the surfaces of all 4 FCs are activated by injecting a 1:1 mixture of EDC/NHS for 7 minutes at 10 μL/minute. Protein A (Calbiochem P/N 539202) is diluted to 100 μg/mL in 10 mM acetate, pH 4.5 buffer and immobilized for approximately 400 RU onto all 4 flow cells by 7 minute injection at a flow rate of 10 μL/minute. Un-reacted sites are blocked with a 7-minute injection of ethanolamine at 10 μL/minute. Injections of 2×10 μL of glycine pH 1.5 are used to remove any non-covalently associated protein.

(36) The FcγR ECDs-FcγRI (CD64), FcγRIIA_131R, and FcγRIIA_131H (CD32a), FcγRIIIA_158V, FcγRIIIA_158F (CD16a), and FcγRIIb (CD32b; inhibitory receptor) (see e.g. Bruhns et al., Blood. 2009 Apr. 16; 113(16):3716-25) are produced from stable CHO cell expression according to methods well-known in the art and purified using IgG Sepharose and size exclusion chromatography.

(37) For FcγRI binding, antibodies are diluted to 2.5 μg/mL in running buffer (1× HBS-EP+(Biacore® P/N BR-1006-69), and approximately 150 RU of each antibody is captured in flow cells 2 through 4 (RUcaptured). FC1 is the reference flow cell, therefore, no antibody is captured in FC1. FcγRI ECD is diluted to 200 nM in running buffer and then two-fold serially diluted in running buffer to 0.78 nM. Duplicate injections of each concentration are injected over all FCs at 40 μL/minute for 120 seconds followed by a 1200 second dissociation phase. Regeneration is performed by injecting 15 μL of 10 mM glycine pH 1.5 at 30 μL/minute over all FCs. Reference-subtracted data is collected as FC2-FC1, FC3-FC1, and FC4-FC1. The measurements are obtained at 25° C. The affinity (K.sub.D) is calculated using either steady state equilibrium analysis with the Scrubber 2 Biacore® Evaluation Software or a “1:1 (Langmuir) binding” model in BIA Evaluation.

(38) For FcγRIIa, FcγRIIb, and FcγRIIIa binding, antibodies are diluted to 5 μg/mL in running buffer, and approximately 500 RU of each variant is captured in flow cells 2 through 4 (RUcaptured). FC1 is the reference flow cell. Fcγ receptor ECDs are diluted to 10 μM in running buffer and then 2 fold serially diluted in running buffer to 39 nM. Duplicate injections of each concentration are injected over all FCs at 40 μL/minute for 60 seconds followed by a 120 second dissociation phase. Regeneration is performed by injecting 15 μL of 10 mM glycine pH 1.5 at 30 μL/min over all FCs. Reference-subtracted data is collected as FC2-FC1, FC3-FC1, and FC4-FC1. The measurements are obtained at 25° C. The affinity (K.sub.D) is calculated using the steady state equilibrium analysis with the Scrubber 2 Biacore® Evaluation Software.

(39) Following procedures essentially as described above, the following data as shown in Table 7 were obtained.

(40) TABLE-US-00007 TABLE 7 In Vitro Binding Parameters of Antibody I-4P, Antibody I-IgG1, and Antibody I-4PAA to Human Fcγ Receptor ECDs Measured Using SPR at 25° C. Sample Human Ligand Average KD Std Dev* IgG1 Control Antibody Fcγ RI  56.1 pM 2.2 IgG4 PAA Control Antibody Fcγ RI 229.0 nM 11.5 Antibody I-IgG1 Fcγ RI  48.9 pM 2.2 Antibody I-4PAA Fcγ RI 273.3 nM 12.6 Antibody I-4P Fcγ RI 369.3 pM 9.2 IgG1 Control Antibody Fcγ RIIA_131H 0.5 μM 0.0 IgG4 PAA Control Antibody Fcγ RIIA_131H >10 μM  Antibody I-IgG1 Fcγ RIIA_131H 0.5 μM 0.0 Antibody I-4PAA Fcγ RIIA_131H >10 μM  Antibody I-4P Fcγ RIIA_131H 3.9 μM 0.3 IgG1 Control Antibody Fcγ RIIA_131R 0.6 μM 0.0 IgG4 PAA Control Antibody Fcγ RIIA_131R >10 μM  Antibody I-IgG1 Fcγ RIIA_131R 0.6 μM 0.0 Antibody I-4PAA Fcγ RIIA_131R >10 μM  Antibody I-4P Fcγ RIIA_131R 1.7 μM 0.1 IgG1 Control Antibody Fcγ RIIb 2.8 μM 0.1 IgG4 PAA Control Antibody Fcγ RIIb >10 μM  Antibody I-IgG1 Fcγ RIIb 2.8 μM 0.1 Antibody I-4PAA Fcγ RIIb >10 μM  Antibody I-4P Fcγ RIIb 2.2 μM 0.1 IgG1 Control Antibody Fcγ RIIIA_158V 0.2 μM 0.0 IgG4 PAA Control Antibody Fcγ RIIIA_158V 8.9 μM 1.1 Antibody I-IgG1 Fcγ RIIIA_158V 0.2 μM 0.0 Antibody I-4PAA Fcγ RIIIA_158V >10 μM  Antibody I-4P Fcγ RIIIA_158V 4.3 μM 0.4 IgG1 Control Antibody Fcγ RIIIA_158F 1.0 μM 0.1 IgG4 PAA Control Antibody Fcγ RIIIA_158F >10 μM  Antibody I-IgG1 Fcy RIIIA_158F 0.9 μM 0.1 Antibody I-4PAA Fcy RIIIA_158F >10 μM  Antibody I-4P Fcy RIIIA_158F >10 μM  Assay was performed three independent times. *Standard deviation was not determined for measurements >10 μM.

(41) Table 7 summarizes the affinity (K.sub.D) of Antibody I-IgG1, Antibody I-4PAA, and Antibody I-4P to the human FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa receptor ECDs as measured by SPR. The binding characteristics of Antibody I-4P demonstrate binding to the Fcγ receptors with affinities that are substantially in between the binding affinities of IgG1 control/Antibody I-IgG1 and IgG4 PAA control/Antibody I-4PAA. For example, the data demonstrate that Antibody I-4P has reduced binding to FcγRIIIa receptor ECD compared to Antibody I-IgG1 (which can be attributed to cytokine release in the whole blood assay) but still has a higher binding affinity to FcγRI and FcγRIIb receptor ECDs compared to Antibody I-4PAA.

(42) The binding characteristics demonstrated by Antibody I-4P to FcγRs are thought to contribute to enhanced in vivo efficacy without causing significant cytokine release.

EXAMPLE: IGG1 FC MUTANTS BINDING TO FCγ RECEPTORS

(43) IgG1 antibodies are known to induce cytokine release. To determine the mechanism for IgG1-induced cytokine release, IgG1-Fc mutations are generated. These CD200R antibodies have different CDRs from Antibody I. The antibodies in Table 8 (IgG1, no mutations, P331S, P331S+S267G, A330S+P331S+S267G, A330S+S267G, K322A, K322A+S267G, and N325S+L328F+S267G) have identical CDRs with one another. The S267G antibody has different CDRs from the other antibody mutants and Antibody I-4P.

(44) An S267G mutation is generated to reduce FcγRIII binding (EU numbering: see, e.g., Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); and Shields R L et al., High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to Fc gamma R. 2001 J. Biol. Chem. 276, 6591-6604).

(45) The S267G mutation is also combined with mutations that reduce C1q binding without significantly impacting FcγR-binding (K322A, A330S, and P331S; see e.g. Oganesyan V et al., 2008 Structural characterization of a human Fc fragment engineered for lack of effector functions. Acta Crystallogr. D Biol. Chrystallogr. 64, 700-704; Idusogie, E et al., 2000 Mapping of the C1q Binding Site on Rituxan, a Chimeric Antibody with a Human IgG1 Fc. J. of Immunology, 164(8) 4178-4184; and Tao M. H. and Morrison M. L. 1993 Structural features of human immunoglobulin G that determine isotype-specific differences in complement activation. J. of Exp. Med., 178(2), 661-667). Additional mutations that reduce FcγRIII and C1q binding while modulating binding to FcγRIIA and FcγRIIB are also generated (N325S+L328F; see e.g. Shang L et al., 2014 Selective antibody intervention of Toll-like receptor 4 activation through FcγR tethering. J. Biol. Chem. 289, 15309-18; Monnet E et al., 2017 Evidence of NI-0101 pharmacological activity, an anti-TLR4 antibody, in a randomized phase I dose escalation study in healthy volunteers receiving LPS. Clin Pharmacol Ther. 2017 101, 200-208). Fcγ receptor binding is determined by Biacore®, and IFNγ is determined by a multiplex assay based on the Mesoscale platform, both as described herein. C1q binding is determined by ELISA. For the ELISA, a 96 well microplate is coated with 100 μL/well of each antibody diluted in DPBS (Dulbecco's HyClone) with a concentration range of 10 μg/mL to 0.19 μg/mL. Testing is performed in duplicate wells. The plate is sealed and incubated overnight at 4° C. The coating reagent is removed from each well, and 200 μL/well of casein blocking reagent (Thermo) is added. The plate is sealed and incubated for 2 hours at room temperature (RT). Each well is washed 3 times with Wash Buffer (1× TBE with 0.05% Tween 20). 100 μL/well of Human C1q (MS Biomedical) at 10 μg/mL diluted in casein blocking reagent is added and incubated for 3 hours at RT. The plate is then washed three times with wash buffer before 100 μL/well of a 1:800 times dilution of Sheep anti-human C1q-HRP (Abcam #ab46191) in casein blocker is added and incubated for 1 hour at RT. The plate is washed 6 times with wash buffer, and 100 μL/well of TMB Substrate (Pierce) is added to each well and incubated for 7 minutes. 100 μL of 1 N HCl is added to each well to stop the reaction. Optical density is immediately measured using a colorimetric microplate reader set to 450 nm.

(46) Following procedures essentially as described above, the following data were obtained (N=1; Table 8).

(47) TABLE-US-00008 TABLE 8 FcγR and C1q binding and whole blood cytokine release measurements with IgG1 mutants. Feγ Feγ Feγ Feγ Feγ Feγ Whole Blood RI, RIIA_131H, RIIA_131R, RIIb, RIIIA_158V, RIIIA_158F, C1q IFNγ Mutation pM μM μM μM μM μM Elisa Release.sup.a Hu IgG1 55.2 0.71 1.03 4.2 0.28 2.59 ++ ND IgG1, no 46.4 0.69 1.04 4.23 0.27 2.12 +++ Yes mutations P331S 54.3 1.15 1.14 4.64 0.45 3.11 + Yes P331S + 142.4 5.2 0.77 4.38 2.08 >10 − No S267G A330S + 511.4 5.1 0.78 4.3 2.48 9.8 − No P331S + S267G A330S + 167.7 3.31 0.82 4.99 1.66 10.83 − No S267G K322A 30.5 0.98 0.82 3.41 0.28 2.58 − Yes K322A + 70.5 5.02 0.66 4.43 1.65 9.99 − No S267G N325S + 68.7 2.64 0.06 0.275 7.35 >10 − No L328F S267G 130.7 3.13 0.53 3.41 0.73 4.5 Yes Human 384.7 5.12 2.89 3.31 5.47 >10 − No IgG4P control antibody .sup.aAny cytokine release significantly above baseline levels within whole blood is recorded as ‘Yes’, however, the exact levels over baseline may vary.

(48) These data demonstrate that combining mutations that reduce C1q binding and alter FcγR binding leads to a lack of IFNγ release over baseline, which suggests a more desirable safety profile when administered to patients. For example, reducing C1q binding and reducing binding to FCγRIII (or FCγRI) results in a lack of IFNγ release over baseline.

EXAMPLE: IN VITRO CYTOKINE RELEASE

(49) Clinical toxicity, including cytokine release syndrome (CRS), has been associated with the administration of antibodies. CRS, one of the most severe adverse events associated with monoclonal antibodies, is characterized by high levels of immune cell activation and rapid systemic release of pro-inflammatory cytokines and can potentially be fatal. Importantly, preclinical models do not adequately predict the potential risk for CRS. Consequently, an in vitro cytokine release assay using human blood cells is developed to mitigate potential risks of CRS after antibody administration. Antibody, in particular IgG1 antibody, binding to Fcγ receptors can cause unwanted cytokine release.

(50) To determine whether Antibody I-4P or Antibody I-IgG1 induce cytokine release from unstimulated human whole blood, an in vitro cytokine release study is performed. Freshly collected whole blood from six healthy humans are incubated with 100 μg/ml of Antibody I-4P, Antibody I-IgG1, or control IgG1 antibody for 24 hours. The positive control is a homolog of Campath-1H (anti-CD52) IgG1 antibody known to cause cytokine release syndrome in clinic. The negative control is an hIgG1 antibody that does not cause cytokine release. Using a commercially available multiplex assay based on the Mesoscale platform, ten cytokines including IFN-γ, IL-2, IL-6, IL-13, IL-8, IL-12p70, IL-10, and TNF-α are measured in cell culture supernatants.

(51) Following procedures essentially as described above, the following data were obtained. As shown in Table 9, incubation of whole blood with 10 μg/ml positive control antibody resulted in robust cytokine production for 9 of the 10 cytokines analyzed in most donors. Incubation of whole blood with Antibody I-IgG1 induced a significant release of IFN-γ. Incubation of whole blood with 100 μg/ml Antibody I-4P or 100 μg/ml negative control IgG1 did not result in significant levels of any of the evaluated cytokines.

(52) TABLE-US-00009 TABLE 9 Fold change relative to baseline (PBS control sample); MEDIAN ± SEM Antibody Antibody Negative Positive Cytokine I-IgG1 I-4P control control IFN-γ 10 ± 19  0.9 ± 0.08  0.8 ± 0.06 612 ± 431 IL-1β 1.8 ± 3   1.19 ± 2   1.04 ± 1.4  3 ± 5 IL-2 0.36 ± 0.14  1.7 ± 0.133  1.9 ± 0.86 1.33 ± 1.3  IL-4 0.96 ± 1.4  1.08 ± 0.42 0.83 ± 0.73 10 ± 24 IL-6 1.25 ± 1.8  1.17 ± 0.17 1.03 ± 0.13 15 ± 18 IL-8  1.1 ± 0.58  1.2 ± 0.08 1.25 ± 0.24 8.8 ± 5   IL-10 0.88 ± 0.11 1.25 ± 0.15 1.26 ± 0.3  3.9 ± 2.6 IL-12p70 0.97 ± 0.37 0.63 ± 0.19 0.49 ± 0.5   7 ± 11 IL-13 1.18 ± 0.27 1.18 ± 0.12  1.1 ± 0.24  5.5 ± 1.89 TNF-α 1.37 ± 0.4   1.1 ± 0.05 0.96 ± 0.07 20 ± 17

(53) These data demonstrate that Antibody I-4P does not cause significant cytokine release, and suggest a low risk of cytokine release in the clinic following administration of Antibody I-4P.

EXAMPLE: ANTIBODY I DOES NOT BLOCK BINDING OF CD200 TO CD200R

(54) Both CD200 and CD200R are cell-expressed molecules and contain two Ig-like domains. They interact through their NH2 terminal domains compatible with immunological synapse-like interactions occurring between myeloid cells and other CD200-expressing cells. To determine if Antibody I-4P binds CD200R in the presence of ligand, co-binding experiments on HEL92.7.1 cells, a human erythroblastoma cell line which expresses CD200R, are performed by flow cytometry. For the study, 2.sup.e5 cells are incubated (pre-treated) with 300 nM of CD200Fc (RD Systems; fusion protein of immunoglobulin 1 Fc region with CD200), Antibody I-4P, isotype control antibody, or PBS for one hour at room temperature. Cells are washed 3 times and incubated with Fc block (Miltenyi Biotec) for 20 minutes at room temperature. The cells are stained with various concentrations of AF647-labeled Antibody I-4P for one hour at room temperature and cells are then washed and suspended in FACS buffer for analysis by flow cytometry.

(55) The median fluorescence intensity (MFI) is determined for each concentration of AF647-labeled Antibody I-4P, and the MFI indicates the amount of binding in the presence of ligand. Following procedures essentially as described above, the data in Table 10 were obtained.

(56) TABLE-US-00010 TABLE 10 Antibody binding in the presence of CD200. Pre-Treatment Stain No Pre-treatment Isotype Antibody Antibody I-4P- No Pre-treatment Control I-4P CD200-Fc AF647 (ug/mL) (MFI) (MFI) (MFI) (MFI) 0 49.1 49.1 49.1 49.1 0.4 416 399 70.4 230 0.8 694 664 76.3 370 1.6 1184 1154 96.9 630 3.125 1979 1914 133 1068 6.25 3097 2987 200 1728 12.5 4216 4105 319 2641 25 5137 4916 496 3421 50 5651 5515 745 3957

(57) These data demonstrate that Antibody I-4P does not block CD200 ligand from binding human CD200R (human CD200-Fc data compared to isotype control and no pre-treatment data). The Antibody I-4P pre-treatment data serve as a control and demonstrate reduced labeled Antibody I-4P binding following pre-treatment with Antibody I-4P.

(58) The epitope for Antibody I-4P was determined to be close to the cell membrane on domain 2 of CD200R (data not shown).

EXAMPLE: ANTIBODY I-4P INHIBITS CONTACT HYPERSENSITIVITY IN HUMANIZED MICE

(59) To demonstrate the anti-inflammatory effects of Antibody I-4P, female huNOG-EXL mice (NOD.Cg-Prkdc.sup.scid Il2rg.sup.tm1Sug Tg(SV40/HTLV-IL3,CSF2)10-7Jic/JicTac) are purchased from Taconic Biosciences at 20 weeks of age and allowed to acclimate for more than 1 week. Mice are housed four mice per cage at 22° C. under a 12 h light:dark cycle and allowed food and water ad libitum. On day 0, mice are anesthetized with 5% isoflurane, their abdomens are shaved, and 100 μL of 3% oxazalone in ethanol is applied to the shaved area. Five days after sensitization, Antibody I-4P is administered at 1 or 10 mg/kg subcutaneously (SC); IgG4P isotype control is administered at 10 mg/kg SC for comparison. Four hours after antibody administration, mice are anesthetized with 5% isoflurane, ear thickness is measured with calipers, and ears are challenged with 10 μL of 2% oxazalone in ethanol on each side of both ears. The challenge procedure is repeated on days 10 and 14. The hypersensitivity reaction is assessed by measuring the difference between ear thickness pre- and 24 hours post-challenges.

(60) Statistics: Inflammation is determined by measuring the differences in ear thickness from pre- to 24 hours post challenges for each challenge. Percent inhibition is calculated from the mean ear thickness of the isotype controls set to 0% inhibition. Statistical differences from isotype control are determined using a 1-way or 2-way ANOVA with Dunnett's test where appropriate (GraphPad Prism).

(61) Following procedures essentially as described above, the following data were obtained. As shown in Table below, a single treatment with Antibody I-4P at 1 or 10 mg/kg SC 4 hours prior to the first challenge significantly ameliorated the inflammatory response after the 3rd challenge compared to isotype-treated mice.

(62) TABLE-US-00011 TABLE 11 Delta ear thickness % inhibition of Treatment (mm) ± SEM isotype p-value Isotype control 0.108 ± 0.005 N/A Antibody I-4P 0.056 ± 0.008 47.9 ± 7.8 0.0001 10 mg/kg Antibody I-4P 0.064 ± 0.007 41.4 ± 6.4 0.0001  1 mg/kg

(63) TABLE-US-00012 SEQUENCES HCDR1 of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 1) KASGFSFSSGYYMA HCDR2 of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 2) LIGVGSGSLWYAQKFQG HCDR3 of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 3) ARHFALSDPFNL LCDR1 of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 4) QASESIDSYLL LCDR2 of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 5) KQASTLAS LCDR3 of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 6) QNYYDISSND Antibody HCVR of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 7) XVQLVQSGAEVKKPGASVKVSCKASGFSFSSGYYMAWVRQAPGQGLEWMGLI GVGSGSLWYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHFALSDP FNLWGQGTLVTVSS wherein Xaa at position 1 is either glutamine or pyroglutamic acid Antibody LCVR of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 8) EIVLTQSPDFQSVTPKEKVTITCQASESIDSYLLWYQQKPDQSPKLLIKQASTLASG VPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNYYDISSNDFGGGTKVEIK Antibody Heavy Chain of Antibody I-4P (SEQ ID NO: 9) XVQLVQSGAEVKKPGASVKVSCKASGFSFSSGYYMAWVRQAPGQGLEWMGLI GVGSGSLWYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHFALSDP FNLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLX wherein Xaa at position 1 is either glutamine or pyroglutamic acid; and Xaa at position 446 is either glycine or absent. Antibody Light Chain of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 10) EIVLTQSPDFQSVTPKEKVTITCQASESIDSYLLWYQQKPDQSPKLLIKQASTLASG VPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNYYDISSNDFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody Heavy Chain of Antibody I-IgG1 (SEQ ID NO: 11) XVQLVQSGAEVKKPGASVKVSCKASGFSFSSGYYMAWVRQAPGQGLEWMGLI GVGSGSLWYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARHFALSDP FNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGX wherein Xaa at position 1 is either glutamine or pyroglutamic acid; and Xaa at position 450 is either lysine or absent. DNA Encoding Heavy Chain of Antibody I-4P (SEQ ID NO: 12) caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcatctggattctcc ttcagtagcggctactacatggcatgggtgcggcaggcccctggacaagggcttgagtggatgggactgattggtgttggtagt ggtagcctatggtacgcgcagaagttccaaggccgggtcaccatgaccagggacacgtccacgagcacagtctacatggagct gagcagcctgagatctgaggacacggccgtgtattactgtgcgagacattttgctctgtctgatccctttaacttgtggggccagg gcacactcgtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctgctccaggagcacctccgaga gcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccag cggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttg ggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtc ccccatgcccaccctgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcat gatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtgg atggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcac cgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaa ccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaacc aggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggaaagcaatgggcagccggagaa caactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtg gcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctg ggt DNA Encoding Light Chain of Antibody I-4P and Antibody I-IgG1 (SEQ ID NO: 13) gaaattgtgctgactcagtctccagactttcagtctgtgactccaaaggagaaagtcaccatcacctgccaggccagtgagtcgat tgatagctatttactgtggtaccagcagaaaccagatcagtctccaaagctcctcatcaagcaggcatccactctggcatctgggg tcccctcgaggttcagtggcagtggatctgggacagatttcaccctcaccatcaatagcctggaagctgaagatgctgcaacgtat tactgtcaaaactattatgatattagtagtaatgatttcggcggagggaccaaggtggagatcaaacggaccgtggctgcaccatc tgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg ccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggaca gcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcaccca tcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc DNA Encoding Heavy Chain of Antibody I-IgG1 (SEQ ID NO: 14) caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcatctggattctcc ttcagtagcggctactacatggcatgggtgcggcaggcccctggacaagggcttgagtggatgggactgattggtgttggtagt ggtagcctatggtacgcgcagaagttccaaggccgggtcaccatgaccagggacacgtccacgagcacagtctacatggagct gagcagcctgagatctgaggacacggccgtgtattactgtgcgagacattttgctctgtctgatccctttaacttgtggggccagg gcacactcgtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggg gcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccag cggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttg ggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtga caaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaagga caccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactg gtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcag cgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccccca tcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgac caagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcag ccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctcc ctgtctccgggtaag Human CD200R (SEQ ID NO: 15) MLCPWRTANLGLLLILTIFLVAEAEGAAQPNNSLMLQTSKENHALASSSLCMDE KQITQNYSKVLAEVNTSWPVKMATNAVLCCPPIALRNLIIITWEIILRGQPSCTKA YRKETNETKETNCTDERITWVSRPDQNSDLQIRPVAITHDGYYRCIMVTPDGNFH RGYHLQVLVTPELTLFQNRNRTAVCKAVAGKPAAQISWIPEGDCATKQEYWSN GTVTVKSTCHWEVHNVSTVTCHVSHLTGNKSLYIELLPVPGAKKSAKLYIPYIILT IIILTIVGFIWLLKVNGCRKYKLNKTESTPVVEEDEMQPYASYTEKNNPLYDTTNK VKASQALQSEVDTDLHTL Cynomolgus monkey CD200R (SEQ ID NO: 16) MLCPWRTANLGLLLILAVFLVAEAEGAAQSNNSLMLQTSKENHTLASNSLCMDE KQITQNHSKVLAEVNISWPVQMARNAVLCCPPIEFRNLIVITWEIILRGQPSCTKT YRKDTNETKETNCTDERITWVSTPDQNSDLQIHPVAITHDGYYRCIMATPDGNFH RGYHLQVLVTPEVTLFESRNRTAVCKAVAGKPAAQISWIPAGDCAPTEQEYWGN GTVTVKSTCHWEGHNVSTVTCHVSHLTGNKSLYIELLPVPGAKKSAKLYMPYVI LTIIILTIVGFIWLLKISGCRKYNLNKTESTSVVEEDEMQPYASYTEKNNPLYDTTN KVKASQALQSEVGTDLHTL Cynomolgus monkey CD200RLa (SEQ ID NO: 17) MHTLGKMSASRLLISIIIMVSASSSSCMDGKQMTQNYSKMSAEGNISQPVLMDTN AMLCCPPIEFRNLIVIVWEIIIRGQPSCTKAYRKETNETKETNCTDERITWVSTPDQ NSDLQIHPVAITHDGYYRCIMATPDGNEHRGYHLQVLVTPEVTLFQSRNRTAVCK AVAGKPAAQISWIPAGDCAPTEHEYWGNGTVTVESMCHWGDHNASTMTCHVS HLTGNKSLYIKLNSGLRTSGSPALDLLIILYVKLSLFVVILVTTGFVFFQRINYVRK SL