Preclinical Antitumor Activity of an Antibody Against the Leukocyte Antigen CD48’
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Vol. 4, 895-900, April 1998 Clinical Cancer Research 895 Preclinical Antitumor Activity of an Antibody against the Leukocyte Antigen CD48’ Haiping Sun, Belinda J. Norris, Kerry Atkinson, and patients with low-grade lymphoma are generally incurable James C. Biggs, and Glenn M. Smith2 (1 ). Monoclonal antibodies have been used in a number of clinical trials for the treatment of leukemia and lymphoma (2). Cooperative Research Centre (CRC) for Biopharmaceutical Research, Ltd. [H. S., B. J. N., G. M. S.] and Department of Hematology, St. Monoclonal antibodies may be valuable in the treatment of Vincents Hospital [K. A., J. C. B.], Darlinghurst, New South Wales, relapsed patients because they act by different mechanisms than Australia 2010 chemotherapy to deplete malignant cells. In general, the thera- peutic effect of monocbonal antibodies that are not coupled to toxins or radioisotopes depends on the recruitment of host ABSTRACT effector mechanisms, including complement activation, ADCC, We have evaluated the antitumor activity of a murine and phagocytosis of antibody-coated malignant cells. In a Phase antibody (IgG2a) against the leukocyte antigen CD48. CD48 I/lI study, 15 patients with relapsed B-cell lymphoma were is expressed on T and B lymphocytes, monocytes, and a wide treated with an anti-CD2O chimeric antibody. Forty-seven % of range of lymphoid malignancies. To assess the therapeutic patients responded to treatment for at least 2 months, and some potential of an anti-CD48 antibody, we established a repro- remained in remission for over 7 months (3). ducible model of human B-cell (Raji) leukemia/lymphoma in CD48 is a Mr 47,000 glycophosphatidylinositol-linked gly- C.B17/scid mice, where untreated mice develop hind leg coprotein that is expressed on T and B lymphocytes, monocytes, paralysis due to tumor engraftment. Using this model, the and a wide range of lymphoid malignancies but not on other murine anti-CD48 antibody HuLy-m3 was shown to mediate tissues (4-6). The biological function of CD48 in humans is a strong in vivo antitumor effect. Long-term survival (>1 still not clear. In mice, CD48 is a high-affinity ligand ofCD2 (7) year) of scid mice was obtained after treatment with three but is a low-affinity ligand of human CD2 (8, 9). The DNA 200-pig i.v. doses of anti-CD48 antibody on days 0, 2, and 4 sequence of CD48 is known (10-12). An anti-CD48 antibody after i.v. injection of tumor cells. In contrast, mice treated was used in the treatment of patients with B-cell CLL. Transient with an isotype control antibody developed hind leg paral- clinical responses were observed in these patients using a mu- ysis after 34 ± 3 days. A strong antitumor response was still rime 1gM anti-CD48 monocbonal antibody, WM63 (13). The observed when a dose of 20 tg of HuLy-m3 antibody was lack of a sustained clinical response with the WM63 antibody used. also examined a During preclinical investigations, we was due to the inability of the 1gM antibody to mediate effective number of properties of the CD48 antigen. CD48 is present cell-mediated cytotoxicity. We have extended this study by at high levels on the surface of T and B cells, but most further characterizing the CD48 antigen and have demonstrated (>95%) CD34-positive cells do not express CD48. Anti- that a murine IgG2a antibody against CD48 can mediate strong CD48 antibodies are maintained on the surface of antigen- in vivo antitumor effects in scid mice. Our studies suggest that positive cells for extended periods (>24 h). These properties antibodies of an appropriate isotype against CD48 may be useful suggest that anti-CD48 antibodies may be useful in the in the treatment of lymphoid leukemias and lymphomas, poten- treatment of a number of diseases including lymphoid leu- tially as adjuvant immunotherapy in the conditioning regimens kemias and lymphomas. for hematopoietic stem cell transplantation and in the palliative treatment of T- and B-cell leukemias and lymphomas. INTRODUCTION Although first remissions are achievable in most lym- MATERIALS AND METHODS phoma patients with chemotherapy, recurrence is inevitable in Cell Lines. The hybridoma cell line secreting the murine the majority of patients. Presently, only 40% of patients with anti-CD48 monoclonal antibody HuLy-m3 was a obtained from intermediate- or high-grade NHL3 achieve long-term survival, Dr. Mauro S. Sandrin, The Austin Research Institute, Mel- bourne (5), and was cultured in RPMI 1640 with either 10% FBS or 10% bovine IgG-free serum (Starrate, Bethungra, New South Wales, Australia), 2 mi glutamine at 37#{176}Cina 5% CO2 Received 9/5/97; revised 1 1/13/97; accepted 1/8/98. incubator. The Raji cell line was obtained from American Type The costs of publication of this article were defrayed in part by the Culture Collection and cultured in RPM! 1640, 2 mrvi glutamine, payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to 10% FBS at 37#{176}C,and 5% CO2. indicate this fact. Production and Purification of Antibodies. The anti- I This work was supported by the Australian Government Cooperative CD48 antibody (IgG2a, Ka l0 M ) was purified from Research Program. 2 To whom requests for reprints should be addressed, at CRC for Biopharmaceutical Research Ltd., 384 Victoria Street, Darlinghurst, NSW, Australia 2010. Phone: 61-29-295-8465; Fax: 61-29-295-8451. 3 The abbreviations used are: NHL, non-Hodgkin’s lymphoma; ADCC, PBMC, peripheral blood mononuclear cell; CLL, chronic lymphoid antibody-dependent cellular cytotoxicity; FBS, fetal bovine serum; leukemia. Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. 896 Antibody against Leukocyte Antigen CD48 hybridoma conditioned media or from ascites fluid produced in 1% sucrose and 0.5% paraformaldehyde in PBS, and if not either nude (BALB/c-nu/+) mice or BALB/c X CBA/CaH F1 profiled immediately, stored at 4#{176}Cwith the addition of 1 ml of mice using Protein A affinity chromatography (Pharmacia, Up- PBS/l% BSA. Fluorescence was estimated on both lympho- psala, Sweden). The purity ofthe antibody was assessed by 10% cytes and monocytes within 24 h of sample preparation on a SDS-PAGE, and the activity was confirmed by flow cytometry Coulter Epics flow cytometer. using human leukemic cell lines. For animal experiments, the Estimation of the Number of CD48 Molecules per Cell. antibody was further purified by ion exchange chromatography Human PBMCs from two healthy donors and two CLL patients and gel filtration. An IgG2a isotype control antibody (anti- were prepared as described above. Three drops of Simply Ccl- human TSH) was obtained from Bioqest (North Ryde, Sydney, lular Microbeads (Flow Cytometry Standards Corp., Research Australia) and repurified as above. Protein concentration was Triangle Park, NC) were added to 1 X 106 cells/sample, fol- estimated by absorbance at 280 nm (14). Biotinylation of anti- lowed by the addition of anti-CD48 or isotype control antibody bodies was carried out using NHS-biotin (Bio-Rad, Hercules, to a final concentration of 40 p.g/ml, and the mixture was CA: Ref. 14). Purified murine anti-CD48 antibody WM63 incubated for 30 ml at 4#{176}C.After washing, 10 il of FITC- (1gM) was obtained from Dr. Tony Henniker (Hematology, conjugated goat anti-mouse antibodies (Becton Dickinson) were Westmead Hospital, Sydney, Australia). added and incubated for 30 ruin at 4#{176}C.After washing, the cells sd! Mouse Model of Human Lymphoma and Treatment were resuspended in PBS/1%BSA and fixed in 0.5% paaform- with an Anti-CD48 Monocbonal Antibody. Six to eight- aldehyde and 1 % sucrose prepared in calcium- and magnesium- week-old female C.B 17/scid mice were obtained from Animal free PBS prior to flow cytometry. Flow cytometry was per- Resources Center (Canning Vale, Western Australia, Australia). formed on a Coulter Epics flow cyto-meter. Mice were housed in a specific pathogen-free facility. Groups of Antibody Internalization. Examination ofthe internaliza- five mice received iv. injections of 1 X 106 or 5 X l0 Raji tion of anti-CD48 monoclonal antibodies was performed by con- cells in RPMI 1640 (200 l) on day 0. Antibodies were injected focal microscopy. Human PBMCs were isolated as described i.v on days 0, 2, and 4. The antibody dose on day 0 was given above. For confocal microscopy, 5-10 X 106 cells were incubated approximately 30 mm after Raji cell injection. Mice were ob- with either OKT3, anti-CD48, or control antibodies (10-20 g/ served and weighed daily and sacrificed on the onset of hind leg 0.5-1.0 x 106 cells) in PBS for 60 ruin at 4#{176}C.After washing, the paralysis. Blood and tissue samples were taken for analysis. cells were resuspended in complete medium (RPMI 1640 with Bone marrow collected from one femur and blood were ana- 10% FBS and 2 msi glutamine) and incubated for intervals at 37#{176}C lyzed immediately, and collected tissues were either frozen in in a 5% CO2 incubator. After the appropriate incubation time, the liquid nitrogen or fixed in 4% paraformaldehyde in PBS. Bone cells were washed, and duplicate samples were fixed with either marrow and blood were analyzed by flow cytometry to deter- 4% paraformaldehyde in PBS for cell surface staining or 4% mine the percentage of human cells. Anti-mouse CD45-phyco- paraformaldehyde/0.l% Triton X-lOO in PBS for surface and in- erythrin (PharMingen, San Diego, CA) and anti-human CD45- tracellular staining for 30 mm at room temperature. After washing FITC (Becton Dickinson, San Jose, CA) were used to detect with PBS/l% BSA, the cells were incubated with FITC conjugated mouse and human leukocytes by flow cytometry as described anti-mouse or anti-human IgGl antibody for 30 mm at 4#{176}C.The above.