Blockade of CD200 in the Presence or Absence of Antibody Effector Function: Implications for Anti-CD200 Therapy This information is current as Anke Kretz-Rommel, Fenghua Qin, Naveen Dakappagari, of September 29, 2021. Roxanne Cofiell, Susan J. Faas and Katherine S. Bowdish J Immunol 2008; 180:699-705; ; doi: 10.4049/jimmunol.180.2.699 http://www.jimmunol.org/content/180/2/699 Downloaded from References This article cites 37 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/180/2/699.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Blockade of CD200 in the Presence or Absence of Antibody Effector Function: Implications for Anti-CD200 Therapy Anke Kretz-Rommel,* Fenghua Qin,* Naveen Dakappagari,* Roxanne Cofiell,† Susan J. Faas,† and Katherine S. Bowdish1* CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two Downloaded from related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200- G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved http://www.jimmunol.org/ in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti- CD200 immunotherapy of cancer patients. The Journal of Immunology, 2008, 180: 699–705. he immunosuppressive molecule CD200 (1, 2) is up-reg- (9), alemtuzumab (anti-CD52) in chronic B cell leukemia (10), ulated on B cells in all patients with B cell chronic lym- trastuzumab (anti-human epidermal growth factor receptor 2) in T phocytic leukemia (3), and the up-regulation of CD200 breast cancer (11), and bevacizumab (anti-vascular endothelial correlates with a negative prognosis in multiple myeloma (4) and growth factor) (12) and cetuximab (anti-epidermal growth factor acute myeloid leukemia (5). CD200 is a type 1a membrane protein receptor) (13) in colorectal cancer. Although these Abs have by guest on September 29, 2021 related to the B7 family of costimulatory receptors with two ex- shown clinical effectiveness for many years, the critical in vivo tracellular Ig superfamily domains, a single transmembrane region, mechanisms of action are still in question (14, 15) but most likely and a short cytoplasmic tail with no known signaling motifs (6). It include direct effects on target cells via signaling mechanisms and is normally expressed on thymocytes, T and B lymphocytes, some by blocking growth factors, as well as mediating effector function dendritic cells, neurons, kidney glomeruli, syncytiotrophoblasts, through the constant region (C region) of the Ab. and endothelial cells (7). We previously demonstrated that the Because CD200 does not have a signaling domain it appears that presence of human CD200 on Namalwa tumor cells (Burkitt’s anti-CD200 Abs do not directly affect targeted tumor cells, and we 2 lymphoma cell line) coinjected with human PBMCs (hPBMCs) demonstrated that the anti-CD200 mAbs, as expected, did not in- into NOD/SCID mice prevents hPBMCs from eradicating the can- hibit tumor cell proliferation or directly induce cell death (8). cer cells. Administration of antagonistic anti-CD200 Abs blocked Therefore, antagonistic anti-CD200 Abs are expected to exert their tumor growth in this model (8), suggesting possible use in cancer effect by blocking immune suppression and, depending on the therapy. C region of the Ab, by potentially mediating Ab-dependent A number of mAbs have been successfully used in cancer ther- cellular cytotoxicity (ADCC) or complement-dependent cyto- apy such as rituximab (anti-CD20) in non-Hodgkin’s lymphoma toxicity (CDC). Our previous studies demonstrated that in an im- mediate treatment model where the first dose of Ab was injected at the same time as the tumor cells and effector hPBMCs, the effects *Alexion Antibody Technologies, San Diego, CA 92121; and †Alexion Pharmaceu- ticals, Cheshire, CT 06410 of anti-CD200-IgG1 constant region (G1) compared with those of Received for publication July 26, 2007. Accepted for publication October 27, 2007. anti-CD200-IgG2/G4 fusion constant region (G2G4) were nearly indistinguishable and confirm that a blockade of CD200 immune The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance suppression by an effectorless anti-CD200 mAb is sufficient to with 18 U.S.C. Section 1734 solely to indicate this fact. achieve high antitumor activity. 1 Address correspondence and reprint requests to Dr. Katherine S. Bowdish, Alexion To gain more insight into the mechanism of action of our anti- Antibody Technologies, 3985 Sorrento Valley Boulevard, Suite A, San Diego, CA 92121. E-mail address: [email protected] CD200 Abs in the xenograft NOD/SCID human (hu)-mouse model and to provide more information in support of one Ab C region 2 Abbreviations used in this paper: hPBMC, human PBMC; ADCC, Ab-dependent cellular cytotoxicity; CDC, complement dependent cytotoxicity; C region, constant over the other in the context of anti-CD200 therapy in the clinic, region; G1, IgG1 C region; G2G4, IgG2/G4 fusion C region; hu, human; mOKT3, we evaluated both anti-CD200-G1 and anti-CD200-G2G4 Abs in mouse OKT3. two additional tumor models, a delayed treatment model and a Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 model where the tumor xenograft lacks CD200 expression. These www.jimmunol.org 700 ANTI-CD200-G1 ANTIBODY MEDIATES KILLING OF ACTIVATED T CELLS studies, along with supporting in vitro data, indicate that mecha- Isolation of human CD3ϩ T cells nisms of cell killing provide clues that may be critical to the suc- hPBMCs were obtained from normal healthy donors as described above cess of CD200 blockade in cancer. We demonstrate that a blockade and passaged over a human T cell enrichment column (HTCC-5; R&D of CD200 in the presence or absence of effector function has Systems) according to the manufacturer’s instructions to obtain purified ϩ implications for cancer therapy and support the use of anti- CD3 T cells. Eluted cells were washed, counted, and resuspended in CD200-G2G4 in the clinic. complete RPMI 1640 medium (Mediatech) containing 5% heat-inactivated single donor serum, 2 mM L-glutamine (Mediatech), 10 mM HEPES (Mediatech) and 1% penicillin/streptomycin (Mediatech). Materials and Methods Mice PHA activation of hPBMCs hPBMCs from healthy human donors were cultured in 6-well plates (Fal- Four- to six-week-old NOD.CB17-Prkdcscid/J (NOD/SCID) mice were con) at a concentration of 2 ϫ 106 cells/ml in complete medium in the obtained from The Jackson Laboratory. Animals were housed at Perry absence or presence of 5–10 ␮g/ml PHA (Sigma-Aldrich). Cells were Scientific. All procedures and protocols were approved by the Perry Sci- maintained for 72 h at 37°C in a humidified incubator containing 5% CO . entific Institutional Animal Care and Use Committee. 2 At the end of the culture period, the cells were either chromated for use as Anti-CD200 mAbs targets in ADCC or for flow cytometric analysis. ϩ Engineering of chimeric and humanized anti-CD200 mAbs with either an Activation of CD3 T cells with plate-bound mouse IgG1 (G1) or an IgG2/G4 fusion C region (G2G4) have been described OKT3 (mOKT3) previously (8) and designated chB7-G1 and chB7-G2G4 to denote chi- meric anti-CD200 Abs derived from anti-CD200 Fab B7 or hB7V2-G1 and The wells of 12-well plates were coated by overnight incubation at 4°C ␮ Downloaded from hB7V2-G2G4 to denote humanized variants derived from anti-CD200 Fab with 10 g/ml mOKT3 (Ortho Pharmaceuticals) diluted in PBS. Residual Ab was removed and the plates were gently rinsed with PBS.