Apoptosis Induction by Hypercross-Linking of the Surface Antigen CD5 with Anti-CD5 Monoclonal Antibodies in B Cell Chronic Lymphocytic Leukemia DP Cioca and K Kitano
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Leukemia (2002) 16, 335–343 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Apoptosis induction by hypercross-linking of the surface antigen CD5 with anti-CD5 monoclonal antibodies in B cell chronic lymphocytic leukemia DP Cioca and K Kitano Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Nagano, Japan We evaluated cells from 24 patients with B cell chronic lympho- are secreted by malignant B cells and can sequester the MoAb cytic leukemia (B-CLL) to determine apoptosis induced by CD5 away from the malignant cells.11 These difficulties can be hypercross-linking. Following the CD5 hypercross-linking with anti-CD5 monoclonal antibodies (MoAbs), we identified 10 avoided by using MoAbs that recognize antigens more univer- patients where CD5 hypercross-linking induced apoptosis sally expressed on the surface of B cells. One such antigen is (group A) and 14 patients whose cells were resistant to the anti- the surface antigen CD20, against which a humanized anti- CD5 MoAbs (group B). The programmed cell death pathway of CD20 MoAb, Rituximab (Genentech, San Francisco, CA, USA; the cells from patient group A was caspase-3 and poly (ADP- IDEC Pharmaceuticals, San Diego, CA, USA) has been applied ribose) polymerase (PARP)-dependent, involved a reduction of 12 ⌬⌿ to B-CLL patients. However, clinical trials proved only a lim- the mitochondrial transmembrane potential and a down- 13 regulation of the anti-apoptotic Bcl-2, Mcl-1 and iNOS proteins. ited activity of Rituximabin B-CLL. Another target antigen Early activation-associated molecules such as CD25 and CD69 is the surface molecule CD52, Campath-1H being a were expressed at higher levels than in controls after 6 h of humanized anti-CD52 MoAbwhich proved to beeffective in culture with anti-CD5 MoAb. The expression of CD5 and of patients with B-CLL.14 CD72, the ligand for CD5, were significantly lower in group A CD5 is a monomeric 67-kDa class I transmembrane glyco- compared with group B. Anti-CD20 MoAb had similar activity protein which belongs to the receptor cysteine-rich domain with anti-CD5 MoAb and the combination of the two MoAbs seemed to be additive. In this study, it is suggested that the family of polyanion binding receptors. The surface marker cells from some B-CLL patients can be induced into pro- CD5, which was primary described as a T cell marker, was grammed cell death by CD5 hypercross-linking with anti-CD5 identified on certain B cell tumors and reported in a human MoAbs. leukemia study.15 Subsequently, it was identified on a subset Leukemia (2002) 16, 335–343. DOI: 10.1038/sj/leu/2393 of normal B cells16 designated as the B-1 subset and which is Keywords: apoptosis; B-CLL; CD5 hypercross-linking; caspase-3; found mostly in the peritoneal and pleural cavities.17 monoclonal antibodies Therefore, it seems logical to assess the effects of anti-CD5 MoAbs in B-CLL since expression of CD5 on tumor cell sur- face is a hallmark of the disease, and because previous studies Introduction have shown that CD5 ligation induces apoptosis in normal B cells without affecting T cells.18 B cell chronic lymphocytic leukemia (B-CLL) represents the In this study, we identified a group of B-CLL patients whose quintessential example of a human malignancy in which the cells are induced into programmed cell death by anti-CD5 clonal excess of B cells is caused principally by defects that MoAbs. Our findings may help correlate the clinical hetero- prevent cell turnover due to programmed cell death rather geneity of B-CLL with the developmental stages at which B than by alterations in cell cycle regulation. In the vast majority cells display different responses to various stimuli, and may of patients, B-CLL cells gradually accumulate not because provide a definition of different B-CLL subsets. they are dividing more rapidly than normal, but because they are surviving too long.1 Many factors have been described to determine this abnormal extended life span due to defective Materials and methods apoptosis of the B-CLL cells, among them bcl-2, bax and bcl- 2,3 xL gene over-expression, increased serum levels of inter- Patients feron gamma, interleukin-2, 4, 6 and 13,4 interleukin-8,5 free 6 7 iC3b(the ligand for beta2 integrins), and alpha interferon. The twenty-four patients (12 men and 12 women) enrolled in B-CLL cells have a down-regulated expression of the this study were randomly chosen at hospitals from Nagano, apoptosis-inducer CD95 (Fas), a disrupted CD95-dependent Yamanashi and Saitama prefectures, Japan. The mean age of 8 9 apoptotic pathway, and a perturbed T cell/B cell interaction. the patients was 66.5 years (median 70.5, range 27 to 88 In an attempt to counteract the defective apoptosis, mono- years). B-CLL was diagnosed according to standard clinical clonal antibodies (MoAbs) were used in order to trigger an and laboratory criteria, and at the time of inclusion in our intracellular antiproliferative or apoptotic signal upon binding study, seven patients were in stage 0, seven patients were in 10 with their cognate antigen. The early immunotherapy stage I, two patients were in stage II, two patients were in reagents used targeted the immunoglobulin variable region stage III, and six patients were in stage IV according to Rai (idiotype) of the malignant B cell. However, these antibody classification.19 Other characteristics of the B-CLL patients are reagents proved to be impractical as treatment modalities, summarized in Table 1. because being idiotype-specific, they have to be synthesized individually for each patient, and also because target idiotypes Cell preparation and culture Correspondence: K Kitano, the Second Department of Internal Medi- cine, Shinshu University School of Medicine, 3–1-1 Asahi, Matsu- After informed consent, heparinized samples of peripheral moto, Nagano-ken, 390–8621, Japan; Fax: 81-263-32-9412 blood were obtained from each patient. Peripheral blood Received 14 February 2001; accepted 9 November 2001 mononuclear cells were obtained from the interphase cell Apoptosis by anti-CD5 in CLL DP Cioca and K Kitano 336 Table 1 Characteristics of B-CLL patients Patient Age/Sex Rai Disease WBC Lymphocytes CD5/CD19 CD23 CD25 Chromosome Previous No. stage duration (%) (%) (%) (%) analysis treatment (years) 1 73/F IV 2.3 68 200 91 91.1 86.7 24.3 ND — 2 71/M II 8.0 6810 66 89.4 87.9 44.8 46,XY,+12 CPM 3 78/M I 0.8 30 250 72 92.5 43.3 18.7 47,XY,+22 — 4 58/F IV 6.9 67 100 87 92.9 53.9 24.9 46,XX CPM 5 72/M I 0.2 40 580 53 86.5 34.5 13.0 46,XY,inv(12) — 6 27/M IV 0.4 23 800 92 94.8 69.3 2.4 46,XY — 7 55/M 0 4.0 23 100 83 92.0 72.7 49.6 46,XY — 8 71/M III 0.4 12 900 93 91.3 69.9 38.6 45,X,−Y CHOP 9 71/F IV 8.0 56 800 97 91.9 65.6 56.0 ND — 10 59/F I 4.5 20 400 79 94.9 46.8 31.6 ND CPM 11 64/F 0 9.2 29 500 87 73.5 31.8 12.6 ND CPM 12 45/M I 1.2 19 700 65 88.5 57.0 23.2 46,XY — 13 63/F I 2.9 31 100 81 93.1 95.7 36.8 46,XX,+12 — 14 72/M IV 6.3 15 400 91 82.4 49.4 20.3 ND — 15 66/M 0 2.0 17 590 56 91.9 38.5 25.5 46,XY PSL 16 49/M I 5.5 32 800 79 96.4 51.2 16.3 47,XY,t(1;13),inv(2),del(14) — 17 71/M I 10.1 35 700 79 89.5 61.2 24.5 ND — 18 88/F III 0.2 35 000 87 92.3 60.3 29.8 46,XX,inv(11)(p11;q21) — 19 76/F 0 6.1 31 100 87 92.1 37.6 11.3 ND — 20 45/F 0 0.8 42 500 85 93.3 67.7 41.4 46,XX — 21 72/F 0 8.2 55 660 86 97.9 49.8 32.6 46,XX CPM 22 70/F 0 1.4 13 450 68 96.1 38.6 25.5 46,XX — 23 71/F IV 7.2 13 440 75 94.1 69.4 11.2 ND — 24 70/M II 0.6 15 500 72 85.4 49.8 58.1 46,XY — WBC, white blood cell count (×106/l); +12, trisomy 12; CPM, cyclophosphamide; CHOP, cyclophosphamide/doxorubicin/ vincristine/prednisolone; PSL, prednisolone; ND, not determined. layer after Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) Immunofluorescence antibodies and reagents density gradient centrifugation and washed twice in PBS. B- CLL cells were purified by negative selection executed by removal of T and NK cells using immunomagnetic anti-CD2 The murine MoAbs used in this study were phycoerythrin (PE)- coated microbeads (Dynabeads M-450; Dynal, Oslo, conjugated anti-CD19 (HIB19, IgG1), fluorescein isothiocyan- Norway). Because anti-CD19 and anti-CD20 MoAbs were ate (FITC)-conjugated anti-CD23 (M-L233, IgG1), anti-CD24 20,21 reported to influence the apoptosis phenomenon, a posi- PE (ML5, IgG2), anti-CD25 FITC (M-A251, IgG2), anti-CD69 tive selection using microbeads linked to these antibodies was FITC (FN50, IgG1) and anti-CD72 FITC (J4–117, IgG2), pur- not considered suitable.