Preclinical Antitumor Activity of an Antibody Against the Leukocyte Antigen CD48’

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 therapeutic 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.

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. Collected tissues were sectioned for histological stain cells were then washed with PBS, and the pellets were mixed with and immunohistochemistry. Histological stain was performed 150 pA of PPD [1 mg/mI p-phenylenediamine in 90% (v/v) glycerol using H&E. For immunohistochemistry, the sections were in- and 10% (v/v) PBS (pH 8.0)] as an anti-fading agent and placed on cubated with normal rabbit serum to block any endogenous a glass slide and covered with a circular coverslip. To examine peroxidase activity and then incubated with anti-CD2O (Dako, antibody internalization, confocal laser scanning microscopy Carpintena, CA) or HuLy-m3 for 2 h at 37#{176}C.After washing, (CLSM) was performed using a Sarastro 2000 CLSM (Molecular the sections were chased by rabbit anti-mouse immunogbobulin- Dynamics, Sunnyvale, CA), with a plan apochromat X60/l.40 NA HRP (Silenus, Melbourne, Victoria, Australia) and counter- oil immersion lens and an argon-ion class H laser. Optical sections stained with hematoxylin and Scotts blue. After a final wash, the (usually 0.3-mm intervals) through FITC-labeled cells were cap- sections were dehydrated and mounted in Eukitt. tured using a 50-mm fixed pinhole, with excitation at 488 nm, a CD34/CD48 Expression by Flow Cytometric Analysis of 510-nm beam splitter, and a 510-nm barrier filter. Image process- Human PBMCs. Human peripheral blood (10 ml from pa- ing was performed using a Silicon Graphics Personal Iris 4D 35 tients and 50 ml from healthy individuals) was obtained from work station. The cell viability was examined by trypan blue healthy individuals or CLL and NHL patients of St. Vincent’s exclusion. Hospital, Sydney, Australia and was collected into EDTA tubes. PBMCs were prepared on a Ficoll gradient. Viable cells were counted by trypan blue exclusion and resuspended in PBS to a RESULTS final concentration of 1 X 108 cells/mI Establishment of a B-Cell Lymphoma Model in scid For flow cytometric analysis, anti-CD34-FITC (Becton Mice. To determine whether antibodies against CD48 could Dickinson, San Jose, CA), anti-CD45-PerCP (Becton Dickin- mediate an antitumor effect in vivo, we established a model of son). and biotinylated anti-CD48 or isotype control antibodies B-cell lymphoma in scid mice. Groups of five scid mice re-

were incubated with 5.0 X 106 cells for 30 mm at 4#{176}C,and ceived iv. injections of the human Raji B-cell lymphoma cell washed once with 2 ml of wash media (PBS/l% BSA); strepta- line. In all experiments, the injection of Raji cells resulted in the vidin-phycoerythrin was then added for 30 mm at 4#{176}C.Cells development of hind leg paralysis in 100% of untreated animals. were washed twice, fixed in 500 il of fixing buffer containing The onset of paralysis was reproducible and depended on the

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.. . .,... 4 .;. - ... .. ,,. ... :.-_ ‘ ...J .. ,,...... - (. I . ‘ . :- . - Fig. 1 Liver sections from a scid mouse in- A, oculated with human Raji cells. H&E- stained section containing an aggregate of Raji cells. B, a section stained with an isotype control murine antibody and anti-mouse/peroxi- .1 dase labeled antibody. C, a section stained with murine anti-human CD2O antibody and anti-mouse/peroxidase-labeled antibody.

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injected cell dose. For example, groups of five scid mice injected the murine 1gM anti-CD48 antibody WM63 on days 0, 2, and 4 with 1 X 106 or S X 10” cells developed paralysis after 19 ± 2 days after tumor cell injection gave no enhanced survival compared or 34 ± 3 days, respectively. Mice were sacrificed at the onset of with treatment with an isotype control antibody. paralysis. Upon autopsy, mice were found to have disseminated Effect of Anti-CD48 Antibody Dose on Tumor Engraft- tumor in various organs including liver, kidney, ovary, spleen, ment. Groups of five scid mice injected with 1 X l0 Raji lung, and lymph node. Grossly, the neoplastic infiltrates frequently cells on day 0 were treated with 20 or 200 pg of murine formed distinct, white, variably sized nodules. Immunohistochem- anti-CD48 antibody or 200 ig of an isotype control antibody on istly confirmed the presence of distinct nodules of human tumor days 0, 2, and 4. The time to hind leg paralysis is shown in Fig. cells (Fig. 1). These cells stained positive with antibodies specific 3. Mice treated with isotype control antibody developed hind leg for human CD2O. Staining with anti-CD48 antibodies was always paralysis 37 ± 8 days after tumor cell injection. All mice treated relatively weak, probably due to denaturation of the CD48 during with 200 p.g of antibody achieved enhanced survival, whereas a immunohistochemistry. Bone marrow from the femur of sacrificed majority (three of five) of mice treated with 20 p.g of antibody mice contained 20-60% human cells, but no human cells were also had enhanced survival. ever detected in the peripheral blood. Properties of the CD4S Antigen: Estimation of CD34/ Antitumor Activity of Anti-CD48 Antibodies. Groups CD48 Cells. The percentage of CD34 cells expressing of five scid mice injected with Raji cells on day 0 were treated CD48 was determined by triple-label flow cytometry using with 200 p.g of the murine anti-CD48 antibody HuLy-m3 or an antibodies against CD34, CD45, and CD48. CD34 cells were isotype control antibody on days 0, 2, and 4. The time to hind initially selected, then CD48/CD45 cells were examined. The leg paralysis is shown in Fig. 2. Treatment of mice injected with PBMCs from two healthy donors, two CLL patients, and two 1 X 106 Raji cells with anti-CD48 antibody resulted in a 40% NHL patients were examined. Less than 5% of CD34 cells in increase in the time to hind leg paralysis. Four of five mice healthy individuals and patients with CLL and NHL were found injected with a cell dose of 5 X 10” cells and treated with the to express CD48. All samples examined had a level of around same antibody dose achieved survival greater than 1 year, after 4% (3-5%) of CD34 cells being CD48. which the mice where sacrificed. Those treated with an isotype Number of Anti-CD48 Antibody Binding Sites/Cell. control antibody developed hind leg paralysis after approxi- The number of anti-CD48 antibody binding sites/cell was esti- mately 32 days. These results indicate that the HuLy-m3 anti- mated using Quantum Simply Cellular Microbeads and flow body can mediate a strong antitumor effect in vivo. Treatment of cytometry. Healthy individuals and CLL patients had similar scid mice injected with 1 X l0 Raji cells with 200-i.g doses of numbers of binding sites with -40,000 ± 10,000 binding sites

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Days after tumor injection Days after tumor cell injection Fig. 3 The effect of different anti-CD48 antibody doses. scid mice B were injected with 1 X l0 Raji cells, and groups of five mice received 100 injections of either 20 (#{149})or 200 ig (0) of anti-CD48 antibody or 200 ji.g of an isotype control antibody (W) on days 0, 2, and 4 after Raji cell injection.

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and degraded after - 1 h, as described previously (15, 16). In 1. contrast, cells labeled with the anti-CD48 antibody still showed 50 significant labeling after incubation for 24 h (Fig. 4, C and D). There was evidence of CD48 clustering after 30 mm, presum- ably due to the formation of caveolae (17). Permeabilization of 25 - cells prior to labeling anti-CD48 antibodies gave similar results. Experiments performed on PBMCs isolated from healthy mdi- viduals, CLL patients, and leukemic cell lines all gave similar results. Isotype control antibodies gave no detectable signal.

DISCUSSION We have shown that a murine IgG2a anti-CD48 antibody Days after tumor cell injection can mediate a potent in vivo antitumor effect and can give long-term survival (>1 yea) to scid mice injected with Raji Fig. 2 The effect of an anti-CD48 antibody on the survival ofscid mice inoculated with Raji cells (Kaplan Meier curves). scid mice received cells; if mice are treated with an isotype control antibody, they injections of either 1 X 106 Raji cells (A) or 5 X l0 Raji cells (B). develop hind leg paralysis after -34 days. The scid mice de- Groups of five mice received injections of either 200 .g of munne velop disseminated tumors and hind leg paralysis similar to anti-CD48 antibody, HuLy-m3 (0), or an IgG2a isotype control anti- other scid mouse models using human B-cell lines (18, 19). body () on days 0, 2, and 4 after Raji cell injection. However, the time course to hind leg paralysis is much more rapid with the Raji cell line in comparison with the other models. For example, 1 X 106 Daudi cells injected iv. into scid present per cell. The Raji B-lymphoma cell line expressed mice resulted in hind leg paralysis after an average of 34 days, higher levels of CD48 than CLL patients or healthy individuals whereas the injection of 1 X 106 Raji cells resulted in hind leg with -P200,000 binding sites/cell. paralysis after -20 days. Thus, the Raji cell line causes a more Internalization of Anti-CD48 Monocbonal Antibody. rapid form of disease. We report injecting scid mice with 1 X Antibody internalization was examined by confocal microscopy, 106 Raji cells, a similar cell dose as reported in other models, which was used to determine the location (surface or intracel- and the results of a lower cell dose, which allows survival of the lular) of labeled antibody. The anti-CD3 antibody, OKT3, untreated mice for - 1 month, a similar survival period for known to be readily internalized into cells, was used as a untreated animals as reported in the Daudi scid model (19). At positive control (15, 16). The results are shown in Fig. 4. Fig. 4, the higher Raji cell dose, treatment with anti-CD48 antibody A and B, show a rapid signal loss from cells labeled with OKT3 prolonged the time to hind leg paralysis by 40%. For the lower and incubated at 37#{176}C,with most of the label being internalized cell dose experiment where the mice developed hind leg paral-

Fig. 4 Internalization of anti-CD48 antibody HuLy-m3. PBMCs from a healthy donor were labeled with OKT3 (A and B) or anti-CD48 antibody (C and D) at 4#{176}C(A and C). then incubated at 37#{176}Cfor1 h (B) or 24 h (D). Cells were then fixed in 4% paraformaldehyde/PBS, labeled with anti-mouse-FITC, and analyzed by confocal microscopy.

ysis after 34 days, treatment with anti-CD48 antibody produced The therapeutic effect of the anti-CD48 antibody in scid long-term survival, with the majority of mice surviving over 1 mice is antigen specific as an isotype control antibody, which year after tumor cell injection. The surviving mice were sacri- does not bind to Raji cells and has no therapeutic effect. The ficed at this time. The effect of anti-CD48 antibody dose was cytotoxic effect of the anti-CD48 antibody is likely to depend on examined in the low cell dose model. The antibody dose could a number of features, including the slow rate of antibody- be reduced to 20 p.g/dose, with the majority of treated mice induced antigen modulation and the effective interaction of having long-term survival. We have used the murine anti-CD48 murine IgG2a with murine effector functions. Because scid mice antibody in scid mice because it would be predicted that the lack significant T- and B-cell responses, any effector response murine IgG2a isotype of this antibody would be the most would be either a direct toxicity of the monocbonal antibody on effective in mediating murine effector functions in mice (20). the tumor cells or the recruitment of other cells with cytotoxic A murine 1gM anti-CD48 antibody, WM63, has been used activity such as neutrophils, macrophages, or natural killer cells. in a pilot Phase I clinical trial (13). In this study, four CLL Because the anti-CD48 antibody has no direct cytotoxicity on patients were treated with up to 64 mg of antibody over 6 days. Raji cells,4 the recruitment of effector cells seems the most A transient reduction in the number of circulating leukocytes likely explanation of the observed antitumor effect. was observed. The 1gM anti-CD48 antibody, WM63, like the To evaluate the potential of targeting CD48 antigen for the 1gM anti-CD52 antibody, CAMPATH-1 (21), was capable of treatment of lymphoma and leukemia, we have characterized a strong complement activation in vitro but could cause only number of properties of CD48. CD34 represents an early marker transient reductions in circulating tumor cells. Clinical trials in hematopoietic cell development (25). It would be beneficial with different isotypes of the CAMPATH-l antibody suggested for a therapeutic agent to target leukemia or lymphoma cells but that mediation of ADCC was important for a strong antitumor not CD34 cells. We have shown that antibodies against CD48 effect in vivo (22, 23). Murine IgG2a and human IgGl antibod- will not target the majority of CD34 cells, because CD48 is ies have been shown to mediate ADCC and antitumor activity expressed on <5% of CD34 cells. Thus, if all CD48 cells were (20, 24). Our results for the treatment of scid mice injected with Raji cells with the murine anti-CD48 1gM antibody WM63 supports the importance of antibody isotype in mediating antitumor activity, because the 1gM antibody gave no enhanced survival over treatment with control antibody. 4 Unpublished results.

removed by antibody treatment, the remaining CD34 progenitor receptor for mouse CD2 and is involved in T cell activation. J. Exp. cells should expand and repopulate the depleted cell types. Med., 176: 1241-1249, 1992. A significant fraction of anti-CD48 antibodies remain on the 8. Arulanandam, A. R. N., Moingeon, P., Concino, M. F., Recny, M. A., Kato, K., Yagita, H., Koyasu, S., and Reinherz, E. L. A soluble surface of cells for at least 24 h. Cross-linking of a cell surface multimeric recombinant CD2 protein identifies CD48 as a low affinity antigen by a bivalent antibody can result in patching, capping, and ligand for human CD2: divergence of CD2 ligands during the evolution finally internalization ofthe antigen. The rates at which this process of humans and mice. J. Exp. Med., 177: 1439-1450, 1993. can occur vary from antigen to antigen. Some lymphoid differen- 9. Sandrin, M. S., Mouhtouris, E., Vaughan, H. A., Warren, H. S., and tiation antigens, such as CD3 and CD7, can be internalized in Parish, C. R. CD48 is a low affinity ligand for human CD2. J. Immunol., minutes, whereas for others such as CD45 and CAMPATh-l, the 151: 4606-4613, 1993. process may take hours. Glycosyl phosphatidyl inositol-anchored 10. Korinek, V., Stefanova, I., Angelisova, P., Hilgert, I., and Horejsi, V. The human leucocyte antigen CD48 (MEM-l02) is closely related to proteins are generally excluded from the clathrin-mediated endo- the activation marker Blast-l. Immunogenetics, 33: 108-112, 1991. cytic pathway and generally have a low turnover rate. The stability 11. Vaughau, H. A., Henning, M. M., Purcell, D. F. J., McKenzie, of anti-CD48 antibodies on the surface of cells is likely to play an I. F. C., and Sandrin, M. S. The isolation of cDNA clones for CD48. important role in the observed cytotoxic effects mediated by this Immunogenetics, 33: 1 13-1 17, 1991. antibody by allowing interaction of the antibody with effector cells. 12. Staunton, D. E., and morley-Lawson, D. A. Molecular cloning of the There is no significant difference in the expression level of lymphocyte activation marker Blast-l. EMBO J., 6: 3695-3701, 1987. 13. Greenaway, S., Henniker, A. J., Walsh, M., and Bradstock, K. F. A CD48 on the surface of PBMC from healthy donors and CLL pilot clinical trial of two munne monocbonal antibodies fixing human patients. Both sources of cells bound antibody equivalent to complement in patients with chronic lymphatic leukemia. Leuk. Lym- about 40,000 sites/cell. CD48 is induced to high levels of phoma, 13: 323-331, 1994. expression on EBV-infected B cells (26). This was confirmed by 14. Harlow, E., and Lane, D. Antibodies. A Laboratory Manual. Cold analysis of the human Raji cell line, which is EBV positive and Spring Harbor, NY: Cold Spring Harbour Laboratory, 1988. expresses --200,000 sites/cell. 15. Telerman, A., Amson, R. B., Romasco, F., Wybran, J., Galand, P., For clinical use, humanized antibodies have a number of and Mosselmans, R. Internalization of human T lymphocyte receptors. Eur. J. Immunol., 17: 991-997, 1987. improved characteristics over their original murmne version, includ- 16. Press, 0. W., Hansen, J. A., Fair, A., and Martin, P. J. Endocytosis and ing mediation of higher effector function, which is thought to degradation of mmmc anti-human CD3 monoclonal antibodies by nonnal correlate with high antitumor activity in vivo (22-24), longer serum and malignant T-lymphocytes. Cancer Res., 48: 2249-2257, 1988. half-life (27), and lower immunogenicity (27). In this study, we 17. Major, S., Rotheberg, K. G., and Maxfield, F. R. Sequestration of have shown that a murine antibody against CD48 can mediate a GPI-anchored proteins in caveolae trigged by cross-linking. Science (Washington DC), 264: 1948-1951, 1994. strong antitumor effect in vivo. Anti-CD48 antibodies may be 18. Kawata, A., Yoshida, M., Okazaki, M., Yokota. S., Barcos, M., and useful in the treatment of a number ofdiseases, including lymphoid Seon, B. K. Establishment of new SCID and nude mouse models of leukemias and lymphoma. We are presently developing a human- human B leukemia/lymphoma and effective therapy of the tumors with ized version of this antibody for evaluation in clinical trials. immunotoxin and monocbonal antibody: marked difference between the SCID and nude mouse models in the antitumor efficacy of monocbonal antibody. Cancer Res., 54: 2688-2694, 1994. ACKNOWLEDGMENTS 19. Gretie, G. A., Richardson, J., Tucher, T., Jones, D., Uhr, J. W., and We would like to thank Mauro Sandrin and Ian McKenzie for Vitettaz, E. S. Disseminated or localized growth of a human B-cell supplying the HuLy-m3 hybridoma, Margaret Cooley for assistance tumor (Daudi) in SCID mice. mt. J. Cancer, 45: 481-485, 1990. with the flow cytometry, Peter French for assistance with the confocal 20. Herlyn, D., and Koprowski, H. IgG2a monocbonal antibodies inhibit microscopy, Nick Hawkins for assistance with the immunohistochem- human tumor growth through interaction with effector cells. J. Biol. istry, and Julie Ferguson for assistance with the animal experiments. Chem., 79: 4761-4765, 1982. 21. Dyer, M. J. S., Hale, G., Marcus, R., and Waldmann, H. Remission induced in patients with lymphoid malignancies using unconjugated CAM- REFERENCES PATh-l monocbonal antibodies. Leuk. Lymphoma, 2: 179-193, 1990. 1. Longo, D. L. Non-Hodgkin’s lymphoma. Curr. Opin. Haematol., 1: 22. Dyer, M. J. S., Hale, G., Hayhoe, F. G. J., and Waldmann, H. Effects 295-302, 1994. ofCAMPATH-l antibodies in vivo in patients with lymphoid malignancies: 2. Grossbard, M. L., Press, 0. W., Appelbaum, F. R., Bernstein, I. D., influence of antibody isotype. Blood, 73: 1431-1439, 1989. and Nadler, L. M. Monocbonal antibody-based therapies of leukemia 23. Bruggemann, M., Williams, G. T., Bindon, C. I., Clark, M. R., Walker, and lymphoma. Blood, 80: 863-878, 1992. M. R., Jefferis, R., Waldmann, H., and Neuberger, M. S. Comparison of the 3. Maloney, D. G., Liles, T. M., Czerwinski, D. K., Waldichuk, C., effector functions of human immunoglobulins using a matched set of Rosenberg, J., Grilbo-Lopez, A., and Levy, R. Phase I clinical trial using chimeric antibodies. J. Exp. Med., 166: 1351-1361, 1987. escalating single dose infusion of chimeric anti-CD2O monoclonal an- 24. Greenwood, J., and Clark, M. Effector functions of matched sets of tibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma. recombinant human IgG subclass antibodies. In: M. Clark (ed.), Anti- Blood, 84: 2457-2466, 1994. body Effector Functions, Protein Engineering of Antibody Molecules 4. Henniker, A. J., Bradstock, K. F., Grimsley, P., and Atkinson, M. K. for Prophylactic and Therapeutic Applications in Man (Section II), p. A novel non-lineage antigen on human leucocytes: characterization with 85. Nottingham, England: Academic Titles, 1993. two CD-48 monocbonal antibodies. Dis. Markers, 8: 179-190, 1990. 25. Baum, C. M., Weissman, I. L., Tsukamoto, A. S., Buckle, A. M., 5. Vaughan, H. A., Thompson, C. H., Sparrow, R. L., McKenzie, and Peault, B. Isolation of a candidate human haematopoietic stem-cell I. F. C. HuLy-m3-a human leucocyte antigen. Transplantation (Balti- population. Proc. Natl. Acad. Sci. USA, 89: 2804, 1992. more), 36: 446-450, 1983. 26. Thorley-Lawson, D. A., Schooley, R. T., Bhan, A. K., and Nadler, 6. Hadam, M. R. N10 Cluster Report: CD48. NK- and non-lineage L. M. Epstein-Barr virus supennduces a new human B cell differenti- antigens. Fourth International Workshop and Conference on Human ation antigen (Blast 1) expressed on transformed lymphoblasts. Cell, 30: Leucocyte Differentiation Antigens, p. 661-664, 1989. 415-425, 1982. 7. Kato, K., Koyanagi, M., Okada, H., Takanashi, T., Wong, Y. W., 27. Shin, S. U. Chimeric antibody: potential applications for drug Williams, A. F., Okumura, K., and Yagita, H. CD48 is a counter- delivery and immunotherapy. Biotherapy, 3: 43-53, 1991.

H Sun, B J Norris, K Atkinson, et al.

Clin Cancer Res 1998;4:895-900.

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