Antibodies to OX-2/CD200 and uses thereof in inhibiting immune responses

Abstract

This disclosure provides methods and compositions for inhibiting immune responses. The disclosure also provides methods and compositions for inhibiting graft rejection and promoting or prolonging graft survival.

Description

FIG. 6 demonstrates
F. Flow Cytometry

One hundred μL it of cell suspensions (mOKT3-activated CD3+ cells or purified CD56+ NK cells) prepared as described above were distributed to wells of a 96-well round bottom plate (Falcon, Franklin Lakes N.J.; cat #353077). Cells were incubated for 30 minutes at 4° C. with the indicated combinations of the following fluorescein isothiocyanate (FITC)-, Phycoerythrin (PE)-, PerCP-Cy5.5-, or allophycocyanin (APC)-conjugated antibodies (all from Becton-Dickinson, San Jose, Calif.); anti-human CD25-FITC (cat #555431); anti-human CD3-APC (cat #555335); anti-human CD200-PE (cat #552475); anti-human CD8-PerCP-Cy5.5 (cat #341051); anti-human CD4-APC (cat #555349); anti-human CD5-APC (cat #555355) and anti-human CD56-APC (cat #341025). Isotype controls for each labeled antibody were also included. After washing cells twice with FACS buffer (1800 rpm centrifugation for 3 minutes), cells were resuspended in 300 μL of PBS (Mediatech, Herndon, Va.; cat #21-031 -CV) and analyzed by flow cytometry using a FACSCalibur machine and CellQuest Software (Becton Dickinson, San Jose, Calif.).

As shown in FIG. 5, activated T cells show high CD200 expression on their surface. Activated T cells are efficiently killed in the presence of hB7V3V2-G1 but not hB7V3V2hG2G4 when NK cells are used as effector cells (FIG. 6). These data demonstrate that anti-CD200 antibodies with effector function can eliminate activated T cells. Such an antibody can be of therapeutic use in the transplantation setting or for the treatment of autoimmune diseases.

In addition to regulatory T cells, plasmacytoid dendritic cells have been shown to play a negative immunoregulatory role in human cancer (Wei S, et al., Cancer Res. 2005 Jun. 15; 65(12):5020-6). Combination of a therapy eliminating plasmacytoid dendritic cells with anti-CD200 therapy can therefore be advantageous.

EXAMPLE 2

Anti-CD200 mAb Prevents Acute Allograft Rejection in a Mouse Cardiac Transplantation Model

The calcineurin inhibitors, such as cyclosporine A (CsA) and tacrolimus, are known to have narrow therapeutic ranges. Even at therapeutic doses, these drugs carry a considerable risk for nephrotoxicity (Seron, D., and F. Moreso. 2004, Transplant Proc 36:257S). Treatment with subtherapeutic levels of either CsA or tacrolimus results in significantly lower incidence of nephrotoxicity but at the same time shows marked graft rejection (Seron, D., and F. Moreso, 2004. Transplant Proc 36:257S; Dunn et al., 2001, Drugs 61:1957; Scott et al. 2003 Drugs 63:1247). The limitations and side effects of current therapy regimens indicate that it is of value to search for novel drugs that reduce the requirement of CsA and have synergy with low dose CsA to prevent acute rejection and prolong graft survival.

The present study examined graft survival in a C57BL/6-to-BALB/c fully MHC-mismatched mouse heart transplantation model. Each experimental group consisted of five animals. Treatments were administered as follows:

• • Anti-CD200 mAb: 100 μg/mouse/day, days 0-14, i.p.
  • Rapamycin (Rapa): 2 mg/kg/day, days 0-13, orally
  • Cyclosporine A (CsA):
    • Low dose/long-term treatment: 5 mg/kg/day, days 0-endpoint, s.c.
    • High dose/long-term treatment: 15 mg/kg/day, days 0-endpoint, s.c.
    • High dose/short-term treatment: 15 mg/kg/day, days 0-28, s.c.
      The anti-CD200 mAb used was OX90mG2a, a chimeric antibody derived from OX90, a rat anti-mouse CD200 mAb obtained as a hybridoma from the European Collection of Cell Cultures (ECACC No. 03062502; see Hoek et al., Science 290:1768-1771 (2000)). The rat antibody was genetically modified to contain the rat heavy chain variable regions fused to a murine IgG2a constant region and the rat light chain variable region fused to a murine kappa constant region. The antibody used to obtain the data for Table 1 was obtained from a different antibody preparation than the preparation from which the antibody in Table 2 was obtained. Table 2 additionally includes data using an antibody called OX90NE. Antibody OX90NE is a rat anti-mouse CD200 antibody that has been engineered to have decreased effector function. This was accomplished by mutating four amino acid residues of OX90mG2a heavy chain (the light chains are identical). The sequences of OX90mG2a and OX90NE are shown in FIG. 10. The various OX90 antibodies are blocking antibodies, as shown in FIG. 11. Cells of the A20 line, which express high levels of murine CD200, were incubated with 20 μg/mL 12B4 (control) or OX90 variant antibodies for 30 minutes at 4° C. Cells were washed and incubated with 10 μg/mL CD200R1-Fc conjugated with R-PE (Invitrogen/molecular probes, catalog Z 25155) for 30 minutes at 4° C. Binding was analyzed by flow cytometry. As shown, OX90hG2G4, OX90NE and OX90mG2a efficiently block the binding of R-PE labeled CD200R1-Fc to CD200 on A20 cells, whereas the control antibody 12B4 does not block binding.
      Graft Histology

At necropsy, heart tissue samples were fixed in 10% buffered formaldehyde, embedded in paraffin and sectioned for hematoxylin and eosin (H&E) staining. The microscopic sections were examined in a blinded fashion for severity of rejection by a pathologist (B.G.). Criteria for graft rejection included the presence of vasculitis, thrombosis, hemorrhage and lymphocyte infiltration and were scored as: 0, no change; 1, minimum change; 2, mild change; 3, moderate change; or 4, marked change compared to normal tissues.

Immunohistochemistry

Four micrometer sections were cut from cardiac frozen tissue samples embedded in Tissue-Tek Optimum Cutting Temperature (O.C.T.) gel (Skura Finetek, Torrance, Calif.), mounted on gelatin-coated glass microscope slides and stained by a standard indirect avidin-biotin immunoperoxidase method using an Elite Vectastain ABC kit (Vector Laboratories Inc., Burlingame, Calif.). Specimens were evaluated for the presence of CD4⁺ and CD8⁺ T cells using a biotin-conjugated rat anti-mouse CD4 mAb (clone YTS 191.1.2, Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) and a biotin-conjugated rat anti-mouse CD8 mAb (clone 53-6.7, BD Biosciences, Franklin Lakes, N.J.), respectively. Intragraft monocyte/macrophage infiltration was detected with a biotin-conjugated rat anti-mouse Mac-1 mAb (Cedarlane). Mouse IgG and IgM deposition was detected in grafts using biotin-conjugated goat anti-mouse-IgG and goat anti-mouse-IgM, respectively (Cedarlane). For identification of complement deposition, tissue sections were sequentially incubated with polyclonal goat anti-mouse C3 or anti-mouse C5 sera (Quidel, San Diego, Calif.), biotinylated rabbit anti-goat IgG (Vector Laboratories), and HRP-conjugated-streptavidin (Zymed Laboratories, South San Francisco, Calif.). Slides were washed with phosphate-buffered saline (PBS) between the antibody incubation steps and examined under light microscopy. Negative controls were performed by omitting the primary antibodies. Antibody reactivity was evaluated in five high-powered fields of each section using tissue samples from five animals per treatment group. The intensity of staining was graded from 0 to 4+ according to the following: 0, negative; 1+, equivocal; 2+, weak; 3+, moderate and 4+, intensive staining.

TABLE 1
Experimental Groups and Survival Data
	Individual survival	MST ± SD
Treatment	(days)	(days)
1)	Untreated	8, 8, 9, 9	8.5 ± 0.6
		(Historical data)
2)	CsA (Low dose/long-term)	9, 10, 10, 10, 11, 11	10.1 ± 0.3
		(Historical data)
3)	CsA (High dose/long-term)	15, 16, 16, 17	16 ± 0.8
		(Historical data)
4)	OX90mG2a	8, 9, 9, 9, 10, 11	9
5)	OX90mG2a + CsA (High	>100 × 4	>100
	dose/long-term)
6)	OX90mG2a + CsA (High	56 (B), 71 (B), 75 (B)	71
	dose/short-term)
7)	OX90mG2a + CsA	53, 54, 54, >76 (A), >76	>76
	(Low dose/long-term)	(A), >81 (A-), >81 (A-)
8)	OX90mG2a + Rapa	>100 × 6	>100
* The degree of pulsation is scored as: A, beating strongly; B, mild decline in the intensity of pulsation; C, noticeable decline in the intensity of pulsation; or D, complete cessation of cardiac impulses.
MST = Mean Survival Time; SD = Standard Deviation.

TABLE 2
Heart Graft Survival:
		Median
Groups	Individual Survival*	Survival (days)
1)	OX90mG2a	9, 10, 10, 11	10
2)	OX90mG2a + CsA (Low	13#, 13#, 14#, 31#,	75
	dose/long-term)	40**, 75, 78
3)	OX-90NE + CsA (Low	14#, 16#, 39, 39, 64,	64
	dose/long-term)	67, 68
4)	Isotype control (12B4) +	12, 12, 13, 14	12.5
	CsA (Low dose/long-term)
*The degree of pulsation is scored as: A, beating strongly; B, mild decline in the intensity of pulsation; C, noticeable decline in the intensity of pulsation; or D, complete cessation of cardiac impulses.
**Animal died with strong beating of heart graft
#As mentioned above, unexpected early rejection may be due to a possible problem with this batch of antibody.

The heart grafts in the Isotype control (12B4) group were rejected rapidly. Further, no difference was observed in the survival time between Isotype control (12B4)+CsA group and CsA monotherapy group. The data in Tables 1 and 2 demonstrate that anti-CD200 therapy has a strong effect in prolonging survival. This was seen both with an antibody having effector function and an antibody lacking effector function.

TABLE 3
Median scores of histological changes of heart allografts
at necropsy (study endpoint or at time of rejection)*
Groups	Vasc	Infar	Lymph	Throm	Hemo
1)	Untreated	3.0	3.0	3.0	4.0	3.0
	(POD8/endpoint)
2)	CsA (High dose/long-term,	2.0	1.0	2.0	3.0	2.0
	POD16/endpoint)
3)	CsA (low dose/short-term)	3.0	2.0	2.0	4.0	2.0
4)	OX90mG2a	2.0	1.0	2.0	3.0	2.0
	(POD9/endpoint)
5)	OX90mG2a + CsA	0.0	1.0	1.0	1.0	0.0
	(High dose/long-term,
	POD100)
6)	OX90mG2a +	2.0	0.0	2.0	0.0	1.0
	(High dose/short-term)
7)	OX90mG2a + CsA	1.0	1.0	2.0	1.0	1.0
	(Low dose/long-term)
8)	OX9ONE + CsA	2.0	1.0	2.0	1.0	2.0
	(Low dose/long-term)
9)	Isotype control	2.0	2.0	2.0	3.0	2.0
	(12B4) + CsA
	(Low dose/long-term)
*Median scores: 0 - normal; 1 - minimal change; 2 - mild change; 3 - moderate change; 4 - marked change.
POD = Post Operative Day.

In addition to survival and graft survival, circulating anti-donor antibody levels and the number of T cell populations in the spleen were measured by flow cytometry. Anti-CD200 mAb in combination with a high dose of CsA inhibits anti-donor antibody production in long-term surviving recipients (FIGS. 7A and 7B). Further, anti-CD200 mAb in combination with a high dose of CsA significantly downregulates splenic CD4+ and CD8+ T cell populations in long-term surviving recipients (FIG. 8).

Additionally, the following cell populations were measured by flow cytometry: CD3CD200; CD3CD200R; CD19CD200; CD19CD200R; CD11cCD200; and CD11cCD200R. These results are shown in FIGS. 9A-C.

Intragraft deposition of IgG, IgM, C3 and C5 and other intragraft cellular markers (such as CD4, CD8, and Mac-1) were measured in frozen graft sections. The results are shown in Tables 4-6.

TABLE 4
Intragraft Deposition of Humoral Markers Detected by
Immunohistochemistry
Frozen sections of the grafts were collected and stained
(for the sacrificed animals only)
	Groups (Treatment)	IgG	IgM	C3	C5
1)	Untreated	4+	2+	3+	3+
2)	CsA (High dose/long-term)	3+	2+	3+	3+
3)	CsA (Low dose/long-term)	3+	2+	3+	3+
4)	OX90mG2a	2+	2+	3+	3+
5)	OX90mG2a + CsA (High	1+	1+	3+	2.5+
	dose/long-term, POD 100)
6)	OX90mG2a + CsA (High	2+	2+	3+	3+
	dose/short-term)
7)	OX90mG2a + CsA (Low	2+	2+	3+	3+
	dose/long-term)
8)	OX90NE + CsA	2+	2+	3+	3+
	(Low dose/longterm)
9)	Isotype control (12B4) + CsA	2+	2+	3+	3+
	(Low dose/long-term)
Staining intensity grades: 0 is negative, 1+ is equivocal, 2+ is weak, 3+ is moderate, and 4+ is intense.

TABLE 5
Intragraft Cellular Markers Measured by Immunohistochemistry
Frozen sections of the grafts were collected and stained (for the
sacrificed animals only)
	Groups (Treatment)	CD4	CD8	Mac
1)	Untreated	3+	2+	3+
2)	CsA (High dose/long-term)	2+	2+	3+
3)	CsA (Low dose/long-term)	2+	2+	3+
4)	OX90mG2a	2+	1+	3+
5)	OX90mG2a + CsA (High dose/long-term,	1+	1+	1+
	POD 100)
6)	OX90mG2a + CsA (High dose/short-term)	2+	2+	2+
7)	OX90mG2a + CsA (Low dose/long-term)	2+	1+	2+
8)	OX9ONE + CsA (Low dose/long-term)	2+	2+	3+
9)	Isotype control (12B4) + CsA (Low	2+	2+	3+
	dose/long-term)
Staining intensity grades: 0 is negative, 1+ is equivocal, 2+ is weak, 3+ is moderate, and 4+ is intense.

TABLE 6
Intragraft CD200 and CD200R Deposition Measured by
Immunohistochemistry
Frozen sections of the grafts were collected and stained (for the
sacrificed animals only).
	Groups (Treatment)	CD200	CD200R
1)	Untreated	3+	2+
2)	CsA (High dose/long-term)	3+	2+
3)	CsA (Low dose/long-term)	3+	2+
4)	OX90mG2a	2+	1+
5)	OX90mG2a + CsA	2+	1+
	(High dose/long-term, POD 100)
6)	OX90mG2a + CsA	2+	1+
	(High dose/short-term)
7)	OX90mG2a + CsA	2+	1+
	(Low dose/long-term)
8)	OX9ONE + CsA (Low	2+	1+
	dose/long-term)
9)	Isotype control (12B4) + CsA	3+	2+
	(Low dose/long-term)
Staining intensity grades: 0 is negative, 1+ is equivocal, 2+ is weak, 3+ is moderate, and 4+ is intense.

The data above demonstrate that an anti-CD200 mAb in combination with CsA significantly prolongs heart allograft survival in a mouse cardiac transplantation model. Importantly, anti-CD200 mAb significantly reduces the requirement of CsA in achieving long-term allograft acceptance.

EXAMPLE 3

Effect of Anti-CD200 mAb, OX90NE-AG, in Prevention of Acute Allograft Rejection

The OX90NE antibody described above was originally thought to lack effector function, however, it was later found that OX90NE still retained some effector function and thus further experiments were performed with a different antibody, OX90NE-AG that lacks effector function. The OX90NE-AG antibody is similar to the OX90NE antibody but includes one additional mutation which replaces the Asn 298 residue with Gln. The AG designates that the antibody is aglycosylated (the Asn298 can be glycosylated but the Gln298 cannot be glycosylated); the resulting antibody cannot mediate ADCC or CDC.

Similar to the experiments described above, the present study examined graft survival in a C57BL/6-to-BALB/c fully MHC-mismatched mouse heart transplantation model. Each experimental group consisted of five animals. Treatments were administered as follows:

• • OX90NE-AG: 100 μg/mouse/day, days 0-14, i.p.
  • Cyclosporine A (CsA): 15 mg/kg/day, days 0-endpoint, s.c.
    The results are shown below in Table 7.

TABLE 7

Experimental groups and survival results
Groups                       Individual survival days*

CsA + OX9ONE-AG variant      16 (A) × 5
(Sacrificed on POD16)
CsA + OX9ONE-AG variant      >90 (A), >90 (A), >90 (A), >90 (A),
(to be sacrificed on POD100) >90 (A)
High dose/long-term treatment

*The degree of pulsation is scored as: A, beating strongly; B, mild decline in the intensity of pulsation; C, noticeable decline in the intensity of pulsation; or D, complete cessation of cardiac impulses.
MST = Mean Survival Time; SD = Standard Deviation; POD = Post Operative Days.

All 10 mice used in this study were treated identically. Five mice were sacrificed after day 16 for the purpose of further analyses such as those shown in Tables 3-6. The other 5 mice remained alive at day 90 and will be sacrificed at day 100, at which point analyses similar to those found in Tables 3-6 will be performed for both groups of mice sacrificed at day 16 and day 100.

It will be understood that various modifications may be made to the embodiments disclosed herein. For example, as those skilled in the art will appreciate, the specific sequences described herein can be altered slightly without necessarily adversely affecting the functionality of the polypeptide, antibody or antibody fragment used in binding OX-2/CD200. For instance, substitutions of single or multiple amino acids in the antibody sequence can frequently be made without destroying the functionality of the antibody or fragment. Thus, it should be understood that polypeptides or antibodies having a degree of identity greater than 70% to the specific antibodies described herein are within the scope of this disclosure. In particularly useful embodiments, antibodies having an identity greater than about 80% to the specific antibodies described herein are contemplated. In other useful embodiments, antibodies having an identity greater than about 90% to the specific antibodies described herein are contemplated. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of this disclosure.

References

The following references are incorporated herein by reference to more fully describe the state of the art to which the present disclosure pertains. Any inconsistency between these publications below or those incorporated by reference above and the present disclosure shall be resolved in favor of the present disclosure.

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Claims

1. A method of inhibiting an immune response in a subject who has received or will receive a cell, tissue, or organ transplant, wherein said method comprises administering to the subject an effective amount of i) an agent which inhibits interaction between CD200 and CD200R and ii) an immunosuppressive or drug, wherein the agent is an anti-CD200 antibody, or antigen-binding fragment thereof, exhibiting reduced effector function.

2. The method of claim 1, wherein said immune response is a humoral response.

3. The method of claim 2, wherein said immune response is an antibody mediated response.

4. The method of claim 2, wherein said agent has no effector function.

5. The method of claim 1, wherein said immunosuppressive drug is cyclosporine A or rapamycin.

6. The method of claim 1, wherein said anti-CD200 antibody, or antigen-binding fragment thereof, is selected from the group consisting of: a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, and a de-immunized antibody or antigen-binding fragment thereof.

7. The method of claim 1, wherein said antigen-binding fragment is selected from the group consisting of: single-chain antibody, Fab, Fab′, F(ab′)₂, F(ab′)₃, Fd, Fv, domain antibody, and any fragment of an anti-CD200 immunoglobulin that confers specific binding to CD200.

8. The method of claim 1, wherein said immunosuppressive drug is a calcineurin inhibitor.

9. The method of claim 8, wherein said calcineurin inhibitor is selected from the group consisting of tacrolimus and cyclosporine A.

10. The method of claim 1, wherein said immunosuppressive drug is selected from the group consisting of: adriamycin, azathiopurine, busulfan, cyclophosphamide, cyclosporine A, fludarabine, 5- fluorouracil, methotrexate, mycophenolate mofetil, a nonsteroidal anti-inflammatory, sirolimus (rapamycin), and tacrolimus (FK-506).

11. The method of claim 1, wherein said immunosuppressive drug is an antibody selected from the group consisting of: muromonab-CD3, alemtuzumab, basiliximab, daclizumab, rituximab, and anti-thymocyte globulin.

12. The method of claim 1, wherein said subject is human.

13. The method of claim 1, wherein said method prevents graft rejection or promotes graft survival.

14. The method of claim 1, wherein said method comprises administering to said subject said agent and said drug prior to receiving said allograft cell, tissue or organ transplant.

15. The method of claim 1, wherein said agent is administered i) prior to said drug, ii) subsequently to said drug, or iii) simultaneously with said drug.

16. The method of claim 1, wherein said method comprises administering said agent during a rejection episode of said allograft cell, tissue or organ transplant.

17. The method of claim 13, wherein said graft rejection is an acute or a chronic humoral rejection of a grafted cell, tissue, or organ.

18. The method of claim 1, wherein said subject is a recipient of a hematopoietic cell or bone marrow transplant, an allogeneic transplant of pancreatic islet cells, or a solid organ transplant selected from the group consisting of: a heart, a kidney-pancreas, a kidney, a liver, a lung, and a pancreas.

19. The method of claim 1, wherein said method results in a decrease in the production of anti-donor antibodies.

20. The method of claim 13, wherein said graft rejection is an acute graft rejection in a graft recipient of cell, tissue or organ allo- or xenotransplant.

21. The method of claim 13, wherein said graft rejection is a chronic graft rejection in a graft recipient of cell, tissue or organ allo- or xenotransplant.

22. The method of claim 1, wherein said method promotes long-term graft survival, wherein said long-term graft survival is selected from the group consisting of:

(a) at least 6 months post transplant;

(b) at least 1 year post transplant; and

(c) at least 5 years post transplant.

23. The method of claim 22, wherein said method promotes accommodation of the graft.

24. The method of claim 1, wherein said agent is administered systemically.

25. The method of claim 1, wherein said agent is administered locally.

26. The method of claim 1, wherein said immune response is a primary response.

27. The method of claim 1, wherein said immune response is a secondary response.

28. A method of decreasing what constitutes an effective amount of an immunosuppressive or drug administered to a subject who has received or will receive an allograft, said method comprising administering to said subject i) said immunosuppressive or immunomodulatory drug and ii) an anti-CD200 antibody or antigen-binding fragment thereof which inhibits interaction between CD200 and CD200R and exhibits reduced effector function,

29. The method of claim 28 wherein said drug is cyclosporine A or rapamycin.

30. A method of inhibiting rejection of an allograft in an allograft recipient during a rejection episode, said method comprising administering to said allograft recipient an effective amount of an anti-CD200 antibody or antigen-binding fragment thereof which inhibits interaction between CD200 and CD200R and exhibits reduced effector function, wherein rejection of said allograft is inhibited.

31. The method of claim 1, wherein said cell, tissue or organ transplant is an allograft.

32. The method of claim 1, wherein said anti-CD200 antibody or antigen-binding fragment thereof exhibits: (a) no ADCC activity, (b) no CDC activity, or (c) no ADCC activity and no CDC activity.

33. The method of claim 1, wherein said anti-CD200 antibody comprises a variant Fc region derived from an IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD, or IgE antibody.

34. The method of claim 1, wherein said anti-CD200 antibody comprises a variant Fc constant region that has ADCC activity or CDC activity equal to or less than the ADCC activity or CDC activity the antibody would have if it had a Fc constant region comprising: (a) a glutamic acid substitution at amino acid position 236, (b) a glutamine substitution at amino acid position 298, and (c) an alanine substitution at amino acid positions 319, 321, and 323, wherein each substitution is relative to the amino acid sequence depicted in SEQ ID NO:28.

35. The method of claim 28, wherein said anti-CD200 antibody or antigen-binding fragment thereof exhibits: (a) no ADCC activity, (b) no CDC activity, or (c) no ADCC activity and no CDC activity.

36. The method of claim 28, wherein said anti-CD200 antibody comprises a variant Fc region derived from an IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD, or IgE antibody.

37. The method of claim 28, wherein said anti-CD200 antibody comprises a variant Fc constant region that has ADCC activity or CDC activity equal to or less than the ADCC activity or CDC activity the antibody would have if it had a Fc constant region comprising: (a) a glutamic acid substitution at amino acid position 236, (b) a glutamine substitution at amino acid position 298, and (c) an alanine substitution at amino acid positions 319, 321, and 323, wherein each substitution is relative to the amino acid sequence depicted in SEQ ID NO:28.

38. The method of claim 30, wherein said anti-CD200 antibody or antigen-binding fragment thereof exhibits: (a) no ADCC activity, (b) no CDC activity, or (c) no ADCC activity and no CDC activity.

39. The method of claim 30, wherein said anti-CD200 antibody comprises a variant Fc region derived from an IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD, or IgE antibody.

40. The method of claim 30, wherein said anti-CD200 antibody comprises a variant Fc constant region that has ADCC activity or CDC activity equal to or less than the ADCC activity or CDC activity the antibody would have if it had a Fc constant region comprising: (a) a glutamic acid substitution at amino acid position 236, (b) a glutamine substitution at amino acid position 298, and (c) an alanine substitution at amino acid positions 319, 321, and 323, wherein each substitution is relative to the amino acid sequence depicted in SEQ ID NO:28.