Modulation, shedding, and serum titers of the chronic lymphatic leukemia-associated antigen: characterization and clinical correlations

The fate of the common chronic lymphatic leukemia antigen (cCLLa), a leukemia-associated antigen selectively expressed by clonal cells in chronic lymphatic leukemia (CLL) was examined in 31 patients. cCLLa was detected by immune precipitation assay in extracts of metabolically labeled CLL culture cells, in CLL cell culture supernatants, and in patients' sera. In vitro shed membrane cCLLa was comparable in all patients (n = 15) on a per cCLLa-positive cell basis but was independent of cCLLa density (r = .46), absolute lymphocyte count ([ALC] r = .46) and stage (r = .44). In contrast, serum cCLLa (n = 31) correlated with absolute cCLLa-positive cell count (r = .92) and to a lesser extent with stage (r = .67), but was independent of cCLLa density (r = .47). cCLLa modulation was assessed from changes in membrane density estimated by radioreceptor assay before and after in vitro exposure to anti-cCLLa monoclonal antibody (MoAb) CLL2. Immune precipitation studies of metabolically labeled CLL cells showed that modulated cCLLa was internalized as judged by its detection within modulated cells but not in their supernatants. Intact cCLLa-CLL2 complexes were not detected within the modulated cells nor in their supernatants. Regeneration of modulated cCLLa was rapid with return to baseline density levels within 24 hours of antibody removal. Modulation was specific and depended on exposure time, medium temperature, and on antibody titer; correlated with extent of disease (versus absolute lymphocyte count, r = .79; versus stage, r = .66; n = 22); was independent of cCLLa density or affinity (r = .44 to r = .57; n = 11); and was unaffected by T cells or by monocytes (n = 14).     

Monoclonal antibodies against the chronic lymphatic leukemia antigen cCLLa: characterization and reactivity

Monoclonal antibodies (MoAbs) were developed against the cCLLa, a 69-kilodalton leukemia-associated antigen expressed on malignant cells of B-type chronic lymphatic leukemia (B-CLL) and its variants: prolymphocytic (PLL) and hairy cell leukemias (HCL). Two hybridomas yielded approximately 2 and approximately 7.5 mg/mL of IgG2a kappa and IgM kappa, respectively. Monoclonal surface immunoglobulin-bearing cells of all B-CLL patients studied (n = 30) reacted with the MoAbs (r greater than .99) regardless of stage or lymphocyte count. This suggests that the malignant clone in CLL can be identified and its size monitored by using our MoAbs. In contrast, normal B lymphocytes, a large panel of normal, reactive and neoplastic cells, and malignant cell lines failed to react with either MoAb as judged by indirect immunofluorescence and by flow cytometry. Only two patients (one with non-Hodgkin's lymphoma, the other with acute myeloblastic leukemia) exhibited a small cell subset reactive with the MoAbs. cCLLa specificity was suggested by selective target cell reactivity and competitive inhibition-absorption and confirmed by immunoprecipitation. MoAbs IgG2a kappa and IgM kappa appeared to share antigenic determinants and were moderate and avid complement binders inducing 100% and 40% target cell lysis, respectively. cCLLa density on malignant CLL and HCL cells was estimated by equilibrium binding studies using the IgG2a kappa MoAb at 1.7 and 9 X 10(6)/cell, respectively. The restricted expression of the cCLLa and the specificity and cytolytic activity of the anti-cCLLa MoAbs support these antibodies as probes for the classification of lymphoproliferative diseases and for the specific diagnosis and treatment of B-CLL and its variants.     

An antigen common to chronic lymphocytic and hairy cell leukemia cells not shared by normal lymphocytes or by other leukemic cells

Rabbit xenoantisera and mouse monoclonal antibodies prepared against human chronic lymphocytic leukemia (CLL) B cells were found to react against a single polypeptide chain with a mol wt of 69 kd found on leukemic cells of all CLL (N = 40) and B type hairy cell leukemia (HCL) patients (N = 9) examined. This common CLL-associated antigen (cCLLa) was not detectable on circulating T or B lymphocytes, thymocytes, lymph node and splenic lymphocytes, or bone marrow leukocytes from normal persons. In addition, the cCLLa was not detectable on cultured T or B lymphoblastoid cell lines or on malignant cells from other forms of lymphocytic or myelocytic leukemia. Non-Hodgkin's lymphoma cells also failed to express the antigen. Autologous cultured lymphoblastoid cell lines were established from residual normal B cells from a CLL patient whose cells were used to generate one of the antisera. Absorption of the antibody with these cultured polyclonal B cells did not affect the anti-CLL activity, which suggests that the cCLLa is not HLA related. Unlike the T cell differentiation complex gp65-71, the cCLLa was not expressed on fetal or cord blood lymphocytes or on mitogen-stimulated normal lymphocytes and was distinct from the antigen recognized by the LEU-1 antibody in spite of their similar mol wt. The cCLLa was also determined to be unrelated to the human T cell leukemia lymphoma virus (HTLV-1). One of the monoclonal antibodies generated against the cCLLa was a complement binding IgG which exhibited highly selective cytotoxic activity against 100% of cells bearing the cCLLa. Such an antibody might prove clinically useful in early diagnosis and treatment of CLL and HCL.     

Blood kinetics, tissue distribution, and radioimaging of anti-common chronic lymphatic leukemia antigen (cCLLa) monoclonal antibody CLL2 in mice transplanted with cCLLa-bearing human leukemia cells

The blood kinetics and biodistribution of anti-common chronic lymphatic leukemia antigen (cCLLa) monoclonal antibody (MoAb) CLL2 were assessed in mice bearing cCLLa+ tumors. The cCLLa is a 69-Kd glycoprotein antigen expressed selectively by malignant B cells in human CLL, hairy cell leukemia (HCL), and prolymphocytic leukemia. Immunoreactive 125I-CLL2 (5 micrograms/mouse, specific activity 4.3 microCi/micrograms) was injected intravenously in mice bearing HCL-derived EH xenografts, and blood kinetics and biodistribution were ascertained up to 16 days postinjection. Radioimages were also obtained up to 72 hours after injecting 10 micrograms/mouse (specific activity 50.1 microCi/micrograms) of 125I-CLL2. Distinct 125I-CLL2 blood kinetics were observed in EH engrafted compared with tumor-free mice including: a longer 125I-CLL2 T 1/2 (153 hours v 72 hours), and a considerably greater blood clearance (173 mg/h v 54.7 mg/h) with biexponential rather than monoexponential configuration; and a greater volume of antibody distribution (31,483 mg v 5,729 mg). These data suggest more rapid tissue uptake by grafted tumours. Preferential 125I-CLL2 uptake by EH tumours relative to normal tissues was observed beginning 24 hours postinjection (mean ratio, 4.2) with average peak tumor 125I-CLL2 levels of 428.7 pg/mg. 125I-CLL2 uptake selectivity by EH tumor cells was also supported by: (1) negligible 125I-CLL2 uptake by cCLLa- Molt-4 xenografts (average 29.1 pg/mg 24 hours postinjection); (2) background uptake of cCLLa-irrelevant MoAb 131I-LEU1 by CD5- EH xenografts (average 31.4 pg/mg 48 hours postinjection); and (3) by scintigraphy. The EH xenograft mouse model might be useful to ascertain preclinically the anti-tumor effect of anti-cCLLa MoAbs and of their conjugated derivatives.     

The biological and clinical relationship between CD5+23+ monoclonal B-cell lymphocytosis and chronic lymphocytic leukaemia

A CD5(+)23(+) monoclonal B-cell population is detectable in approximately 3% of the general adult population. The phenotype of the monoclonal CD5(+)23(+) B cells is identical to chronic lymphocytic leukaemia (CLL) with respect to a large number of proteins in addition to the standard diagnostic markers used to identify CLL. Studies in CLL families and direct assessment of genetic features indicate a close biological association between indolent CLL and the CLL-phenotype cells detected in individuals with a normal blood count. Patients with a CLL-phenotype monoclonal B-cell lymphocytosis (MBL) often have increasing CLL cell counts with time and some progress to a stage requiring treatment. Analysis of intraclonal variation in the immunoglobulin heavy chain gene suggests a process of clonal diversification rather than clonal selection in the early stages of disease progression. CLL-phenotype MBL is detectable in approximately 10% of cases referred for investigation of a lymphocytosis and future studies should be directed towards the detection of factors which identify MBL patients at risk of disease progression.     

Differential diagnosis of chronic lymphocytic leukemia and diffuse well-differentiated lymphoma using monoclonal antibodies

Diffuse well-differentiated lymphocytic lymphoma (DWDL) with leukemic change and chronic lymphocytic leukemia (CLL) are not significantly different in morphology, immunophenotype and clinical course. Two monoclonal antibodies (MoAbs), designated L20 and A01, were established with respective reactivity against DWDL and CLL cells. The L20 MoAb reacted with both DWDL and CLL cells, and 20-50% of normal B cells. The second MoAb, A01, reacted not only with CLL cells but also with 33% of non-T, non-B cells. Immunoprecipitation patterns of L20 and A01 MoAbs against 125I-surface-labeled BALL and Daudi cell extracts revealed respective bands with molecular weights of 86 and 66 kilodaltons. These results showed that DWDL and CLL cells are different in nature, and that these two MoAbs can be used to distinguish DWDL from CLL cells.     

Clonal cell surface structures related to differentiation, activation and homing in B-cell chronic lymphocytic leukemia and monoclonal lymphocytosis of undetermined significance

Cell surface structures related to differentiation, activation and "homing" were identified on the leukemic cell clone in blood of 64 patients with a monoclonal B-cell lymphoproliferative disorder. Patients were selected with regard to clinical signs and symptoms of the disease. 39 patients had progressive chronic lymphocytic leukemia of B-cell type (B-CLL): 16 with lymph node enlargement and 14 with progressive lymphocytosis as the most prominent symptom, respectively. 1 patient had an isolated splenomegaly and 8 had symptoms from enlarged lymph nodes, lymphocytosis and/or splenomegaly. 25 patients had an isolated monoclonal B-cell lymphocytosis in blood and bone-marrow but no other signs or symptoms of the disease. The lymphocytosis in these patients was considered to be of "undetermined significance" and the term B-cell lymphocytosis of undetermined significance (B-MLUS) was used. Patients with a prominent lymphadenopathy and/or splenomegaly had CD22+ leukemic cells while in patients with a progressive lymphocytosis the B-cell clone expressed Leu-8. Thus, CD22 might be related to the homing capacity of B lymphocytes for lymphnodes and spleen, while Leu-8 might define a circulating B-cell subset. In B-MLUS about 50% of the monoclonal B cells co-expressed Leu8 which is consistent with a more differentiated phenotype compared to B-CLL with progressive lymphocytosis. The CD22 expression was mostly low in B-MLUS although a few patients showed high values. The expression of receptors for growth factors (CD23, CD25, CD71) was higher in B-CLL compared to B-MLUS patients (p less than 0.001), which is consistent with a difference in lymphocyte activation stage and/or response to growth factors.     

Four ricin chain A-based immunotoxins directed against the common chronic lymphocytic leukemia antigen: in vitro characterization

The common B-chronic lymphocytic leukemia (B-CLL) antigen (cCLLa) appears to be ideal for targeted immunotherapy in that it is the most prevalent and disease-restricted marker in B-CLL. To assess this potential, we developed four immunotoxins (ITs) of anti-cCLLa monoclonal antibody CLL2m (an IgG2a kappa), using ricin chain A (RTA) or its deglycosylated derivative (dgA), each conjugated to either the whole IgG molecule or its Fab' fragment. Each IT was tested in vitro for specificity and cytotoxic activity (assessed by protein synthesis inhibition [PSI] and by cell kill [CK] in the clonogenic assay) against B-CLL cells. RTA-based anti-CD5 ITs and enriched normal B and T lymphocytes were used as controls. Each IT exhibited antigen-specific, dose-dependent activity. Thus, whereas B-CLL cells exhibited dose- dependent PSI and CK (whether the B-CLL clone was CD5+ or CD5-), normal B (cCLLa-/CD5-) and T lymphocytes (cCLLa-/CD5+) remained unaffected. IT potency was independent of toxin glycosylation, but was slightly influenced by antibody valence; divalent ITs were twice as potent as monovalent ITs (IC50, 2.3 v 7.1 x 10(-11) mol/L; CK, 2.6- v 2.0-log reached with 524 v 1,072 IT molecules bound/cell, respectively). In the presence of ammonium chloride or Verapamil, IT-induced CK was enhanced 10- to 80-fold. These data suggest that the cCLLa is a promising target for IT-based immunotherapy of B-CLL in vivo and ex vivo.     

Clinical and cell surface marker characterization of the early phase of chronic lymphocytic leukemia.

The immunologic surface markers on lymphocytes and clinical characteristics of 35 patients with established (stages 0-4) CLL with absolute lymphocyte counts greater than 15,000/cu mm were compared to those of a group of 25 patients with CLL in an early or preleukemic phase (counts of less than 15,000/cu mm). We found a monoclonal B cell proliferation in most cases in the latter group, in spite of the paucity of clinical and laboratory findings. Furthermore, early CLL can readily be distinguished from benign lymphocytosis by surface marker criteria. In untreated CLL, surface marker characteristics are stable with time and predominantly reflect expansion of clones expressing only B cell markers; however, small increase of blood T cells are often seen. Surface markers are a simple and clinically useful tool for definding and characterizing the preleukemic phase of CLL and its ultimate progression to established CLL.     

General population low-count CLL-like MBL persists over time without clinical progression, although carrying the same cytogenetic abnormalities of CLL

Monoclonal B-cell lymphocytosis (MBL) is classified as chronic lymphocytic leukemia (CLL)-like, atypical CLL, and CD5(-) MBL. The number of B cells per microliter divides CLL-like MBL into MBL associated with lymphocytosis (usually detected in a clinical setting) and low-count MBL detected in the general population (usually identified during population screening). After a median follow-up of 34 months we reevaluated 76 low-count MBLs with 5-color flow cytometry: 90% of CLL-like MBL but only 44.4% atypical CLL and 66.7% CD5(-) MBL persisted over time. Population-screening CLL-like MBL had no relevant cell count change, and none developed an overt leukemia. In 50% of the cases FISH showed CLL-related chromosomal abnormalities, including monoallelic or biallelic 13q deletions (43.8%), trisomy 12 (1 case), and 17p deletions (2 cases). The analysis of the T-cell receptor β (TRBV) chains repertoire showed the presence of monoclonal T-cell clones, especially among CD4(high)CD8(low), CD8(high)CD4(low) T cells. TRBV2 and TRBV8 were the most frequently expressed genes. This study indicates that (1) the risk of progression into CLL for low-count population-screening CLL-like MBL is exceedingly rare and definitely lower than that of clinical MBL and (2) chromosomal abnormalities occur early in the natural history and are possibly associated with the appearance of the typical phenotype.     

Role of T-cell antigens in the cytolytic activities of large granular lymphocytes (LGLs) in patients with LGL lymphocytosis

By analyzing surface antigens and cytolytic functions of proliferating large granular lymphocytes (LGLs), three types of T cell LGL lymphocytosis were delineated. The first, most commonly encountered type exhibited CD3+4-8+16+, WT31+ phenotype, low or undetectable non- major histocompatibility complex (MHC)-restricted cytotoxicity, and moderate to strong antibody-dependent cellular cytotoxicity (ADCC) and lectin-dependent cellular cytotoxicity (LDCC). Because these LGLs carried T cell antigen receptor (Ti) recognized by WT31 monoclonal antibody (MoAb), and treatment with anti-Ti, anti-CD3 MoAbs and phytohemagglutinin elicited non-MHC-restricted cytotoxicity, they may have developed from populations of in vivo primed cytotoxic T lymphocytes with unknown antigen specificity. The second, rare type of LGL lymphocytosis exhibited CD3+4-8-16+, WT31 phenotype, and strong non- MHC-restricted, ADCC and LDCC cytotoxicities. These cells were probably derived from the lymphocytes of the same phenotype found in small numbers in normal peripheral blood. Because anti-CD3 MoAb inhibited non- MHC-restricted cytotoxicity of the LGLs, a Ti not detected by WT31 MoAb, but putatively present seemed to serve as a specific receptor for target tumor cell recognition. The third type of LGL lymphocytosis showed CD3+4+8-16+, WT31+ phenotype, and lacked cytolytic activities and parallel tubular arrays. These LGLs probably evolved from cells with the same characteristics selectively located in the germinal centers of lymphoid tissues. Taken together, in patients with LGL lymphocytosis, T cell-associated antigens expressed on LGLs were shown to be involved in the regulation of LGL-mediated cytolytic activities. In addition, studies of surface antigens and the effects of MoAbs and lectins on cytolytic activities may be useful in clarifying the normal counterpart of LGLs from which leukemic or reactively proliferating LGLs originate.     

Three new monoclonal antibodies that define a unique antigen associated with prolymphocytic leukemia/non-Hodgkin's lymphoma and are effectively internalized after binding to the cell surface antigen

Prolymphocytic leukemia (PLL) is closely related to chronic lymphocytic leukemia (CLL), but present with distinctive clinical/laboratory features and associated with much worse prognosis. In this study, we generated three new IgG1-kappa monoclonal antibodies (MoAbs), termed SN8, SN8a and SN8b, by use of an unconventional approach, ie, by using an isolated B PLL antigen preparation to immunize mice. These MoAbs, particularly SN8, showed a highly selective reactivity to B PLL and B non-Hodgkin's lymphoma (NHL) among various human leukemia-lymphoma specimens tested; eg, SN8 was capable of effectively distinguishing B PLL from B CLL as well as from hairy cell leukemia (HCL) cell specimens. The cell surface antigen defined by the three MoAbs was determined to be a covalently linked heterodimeric glycoprotein complex (gp49/40) consisting of a 49,000 dalton (alpha-chain) and a 40,000- dalton component (beta-chain). Epitope comparison showed that the epitope defined by SN8 (SN8 epitope) is in close proximity to SN8a epitope but in a distant position from SN8b epitope. Western blot analysis showed that both SN8 and SN8a epitopes are on the beta-chain, but SN8b epitope was not detected on either the alpha- or the beta- chain of the reduced antigen in the same analysis. Binding of either SN8 or SN8b to the cell surface gp49/40 did not cause significant downregulation of the antigen expression whereas binding of SN8a to the antigen caused small (approximately 20%) decrease in the antigen expression. Among the various normal peripheral blood cells, only a subpopulation (6.0% to 24.2% among different specimens derived from different donors) of B cells reacted with the SN8 series MoAbs; these MoAbs showed no significant reactivity against T cells, granulocytes, monocytes, erythrocytes, and platelets. Minimal or no significant reactivity (0 to 2.6% among different specimens) was detected against normal bone marrow cells. Ricin A-chain conjugates of the three MoAbs are all strongly effective for specific killing of SN8 antigen- expressing leukemia cells in the absence of any potentiators; furthermore, the addition of 10 mmol/L NH4Cl, a potentiator, enhanced strongly the cytotoxic activities of the SN8, SN8a, and SN8b conjugates. Thus, each of the three MoAbs was effectively internalized after binding to the cell surface antigen.     

EBV negative Richter's syndrome from a coexistent clone after salvage treatment with alemtuzumab in a CLL patient

Transformation of B cell chronic lymphocytic leukemia (B-CLL) to large cell lymphoma or Hodgkin's disease is known as a Richter's syndrome (RS). According to the literature, 1-10% of B-CLL patients develop this high-grade lymphoid malignancy. The relationship between the immunosuppressive effect of nucleoside analogues (NA) and monoclonal antibodies and the development of large cell transformation still remains a controversial issue. We describe a CLL patient who developed a large B cell lymphoma 94 months after diagnosis and 3 months after the start of alemtuzumab. The CLL immunophenotype was retained by the transforming cells although a different light chain was expressed. Molecular analysis of the immunoglobulin heavy chain confirmed that the CLL and the RS had a different clonal origin. Subsequent molecular analyses of stored samples showed that the clone with transforming capacity already appeared two years before the clinical appearance of the RS. We hypothesize that alemtuzumab promoted the uncontrolled growth of the latest clone by eradicating the initial B-CLL clone efficiently, and by inducing a strong T cell depletion with consequent impairment of the immunosurveillance. We also ruled out that the RS was EBV driven. In conclusion, we report a case of EBV negative RS after alemtuzumab as salvage therapy.     

Detection of hyperreactive T cells in multiple myeloma by multivalent cross-linking of the CD3/TCR complex.

Cellular immunity was investigated in 43 patients with multiple myeloma (MM) by assessing 3HTdR uptake induced by monocyte-dependent [CD3 monoclonal antibodies (MoAbs), phytohemagglutinin (PHA)] and monocyte-independent (CD2 MoAbs, ionomycin + phorbolester) stimulations. The former were evaluated in peripheral blood mononuclear cells (PBMNC) and purified T cells; the latter were evaluated in purified T-cell preparations only. MM showed significantly lower PBMNC responses to PHA (P less than .001), soluble OKT3 (CD3) (P = .01), and immobilized OKT3 MoAbs (P = .01). On purification of T cells, MM responses were still defective to soluble T11(2) + T11(3) (CD2) MoAbs (P = .004), phorbol myristate acetate (PMA) plus ionomycin (P less than .001), but significantly higher to plastic-immobilized OKT3 (P = .004). In some MM, 3HTdR uptake, interleukin-2 (IL-2) receptor (CD25) expression, and IL-2 production were as high on stimulation with plastic-immobilized OKT3 as that observed in normal subjects under optimal conditions (ie, plastic-immobilized OKT3 plus accessory signals). CD3 hyperreactivity correlated with the number of CD8+ HLA-DR+ cells in MM T-cell preparations. MM patients with more than 10% CD8+ HLA-DR+ cells had significantly higher responses to immobilized OKT3 (P less than .001), but lower responses to T11(2) plus T11(3) (P = .01), and PMA plus ionomycin (P = .03) than patients with less than 10% CD8+ HLA-DR+ cells. Phenotyping of CD45RA (naive) and CD45R0 (memory) expressions in resting MM T cells showed a lower ratio of CD45RA to CD45R0 in both CD4 (P less than .05) and CD8 (P less than .001) subpopulations. These data indicate that (a) some MM T cells require significantly fewer accessory signals (if any) to express the IL-2 receptor fully, secrete IL-2, and proliferate on multivalent cross-linking of the CD3/TCR complex; and (b) this peculiar state of activation is associated with high HLA-DR expression in CD8+ lymphocytes.     

Clonal CD8+ and CD52– T cells are induced in responding B cell lymphoma patients treated with Campath-1H* (anti-CD52)

Abstract: Five patients with non-Hodgkin's lymphoma (NHL) and 4 patients with chronic lymphocytic leukaemia (CLL) were treated with the CDR-grafted (rat × human) monoclonal antibody (mAb) Campath-1H (anti-CD52). Tumour regression was noted preferentially in peripheral blood and in the bone marrow but lymph nodes were less affected. Normal blood B and T cells were profoundly reduced in all patients whereas CD16⁺ NK cells and CD14⁺ monocytes decreased marginally. In all responding CLL patients CD52-negative T but not B cells appeared during treatment and persisted for several months (4–19+) during unmaintained follow-up. Clonal T cells defined as a predominance of a single T cell receptor (TCR) V gene usage, in one case verified by TCR CDR3 fragment analysis and nucleotide sequencing, emerged within the CD52^–/CD8⁺ cell population during Campath-1H therapy in 2 CLL patients, both achieving a long-lasting remission. The increase in CD8⁺ T cell expansions (up to 23-fold) during unmaintained remission and follow-up suggest that the clonal CD8⁺ cells may represent regulatory T cells controlling the growth of the tumour B cell clone. Clonal T cells might thus be a target for an immune therapeutic intervention in B cell tumours.     

Transplantation of human hairy cell leukemia in radiation- preconditioned nude mice: characterization of the model by histological, histochemical, phenotypic, and tumor kinetic studies

Two cell lines (EH and HK) with hairy cell leukemia (HCL) immunophenotypes were recently derived from two HCL patients. Both cell lines were transplanted subcutaneously (2 x 10(5) or 2 x 10(6)/mouse) in male BALB/c nu/nu mice (n = 128) with a 97% success rate when coimplanted with nonproliferative HT-1080 fibrosarcoma cells (2 x 10(6)/mouse) in recipients preconditioned with total-body irradiation (200 R weekly for 3 weeks). Tumors appeared five to ten days postimplant and reached up to 25% of body weight after a mean survival of 8 weeks (range, 30 to 90 days). Tumor histology suggested large cell lymphoma. Cytochemically and immunophenotypically, tumor cells were indistinguishable from their parent cells. Species and lineage derivation of tumor cells was confirmed by antibody probes against the mouse histocompatibility antigen H-2, human T and B lymphocyte antigens, and the HCL-associated common chronic lymphocytic leukemia antigen (cCLLa). In order of decreasing frequency, metastases occurred in the spleen, lungs, pleura, lymph nodes, bone marrow, and kidneys. Up to 12% of circulating lymphoid cells in mice were cCLLa-positive, which suggested hematogenous tumor dissemination. This HCL xenotransplantation model might be useful in preclinical studies for exploring novel experimental therapies for the management of human HCL.     

Idiotypic cross-reactivity of immunoglobulins expressed in Waldenstr?m's macroglobulinemia, chronic lymphocytic leukemia, and mantle zone lymphocytes of secondary B-cell follicles

Monoclonal antibodies (MoAbs) specific for autoantibody-associated cross-reactive idiotypes (CRIs) of Waldenstr?m's IgM react frequently with the surface Ig (slg) expressed by leukemia cells of patients with chronic lymphocytic leukemia (CLL). Evaluation of the molecular basis for this cross-reactivity indicates that such CRIs are encoded by conserved antibody variable region genes (V genes) that have undergone little or no somatic hypermutation. We find that such anti-CRI MoAbs stain a subpopulation of cells within the mantle zones surrounding the germinal centers of normal human tonsil. In contrast, MoAbs specific for variable region subgroup determinants react with cells in both the mantle zones and germinal centers of secondary B-cell follicles. To test whether mantle zone B cells not reactive with existing anti-CRI MoAbs may express slg bearing as-yet-unrecognized CRIs present on Igs produced by neoplastic cells of some patients with Waldenstr?m's macroglobulinemia or CLL, we immunized mice with purified Waldenstr?m's IgM that have been characterized for their variable region subgroups using subgroup-specific antisera raised against synthetic peptides. The supernatants of hybridomas generated from the splenocytes of immunized mice were screened for their ability to stain a subpopulation of mantle zone lymphocytes in human tonsil. With this approach, two new anti-CRI MoAbs were identified, designated OAK1 and VOH3. OAK1 binds to a CRI present on a subset of kappa light chains of the VK1 subgroup. VOH3 recognizes a CRI determinant(s) present on a subset of antibody heavy chains of the VH3 subgroup. Flow cytometric analyses demonstrated that OAK1 specifically binds leukemia cells from 5 to 20 patients (25%) with kappa light chain expressing CLL. In addition, VOH3 reacted with the leukemia cells from 1 of 17 (6%) patients tested. The success of these methods demonstrates that the variable regions of the Igs produced by mantle zone B cells share idiotypic determinants with Igs expressed in B-cell CLL (B-CLL) and Waldenstr?m's macroglobulinemia.     

Rituximab and alemtuzumab induce a nonclassic,caspase-independent apoptotic pathway in B-lymphoid cell lines and in chronic lymphocytic leukemia cells

The monoclonal antibodies (MoAbs) alemtuzumab (anti-CD52) and rituximab (anti-CD20) produce objective clinical responses in patients with chronic lymphocytic leukemia (CLL). However, their mechanisms of action are not fully understood. Therefore, we investigated the mechanisms of lymphoma and CLL cell killing by two anti-CD20 antibodies (rituximab, B1) and by alemtuzumab. All antibodies induced complement-independent cell death in B-lymphoid cell lines Raji, Ramos, and Mec-1. The efficiency of cell killing was increased by the addition of human complement in Raji but not Ramos cells. Both alemtuzumab and rituximab also killed freshly isolated CLL cells, with a much stronger response for alemtuzumab (from eight of eight patients) compared to rituximab (from two of six patients). Cell morphology and Western blot analyses revealed that the antibody-induced cell death lacked some typical features of apoptosis such as chromatin condensation or poly-ADP-ribose polymerase (PARP) cleavage. Taken together, the results suggest that the tumor killing activity of these MoAbs is not only mediated by complement-mediated cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC), but also by a nonclassic, caspase-independent apoptotic pathway.     

Expression of CD66 in non-Hodgkin lymphomas and multiple myeloma

Objectives: ⁹⁰Y‐labeled anti‐CD66b monoclonal antibody clone BW 250/183 was developed for treatment of tumors. The aim of the study was the analysis of CD66 expression in lymphoproliferative malignancies to expand the potential of anti‐CD66‐based therapy. Patients and methods: Bone marrow samples from 260 patients were examined for the expression of CD66 on tumor cells in 104 B‐chronic lymphocytic leukemias (B‐CLL), 28 mantle cell lymphomas (MCL), 22 follicular lymphomas (FCL), 15 marginal zone lymphomas (MZL), 12 lymphoplasmacytic lymphomas (LPL), 13 diffuse large B cell lymphomas (DLBCL), 4 T‐non‐Hodgkin lymphomas (T‐NHL), 3 B‐NHL not otherwise specified (B‐NHL NOS), 3 B acute lymphoblastic leukemias (B‐ALL), and in 56 multiple myelomas (MM) by flow cytometry. Results: Positive (≥ 20%) expression of CD66abce clone Kat4c was detected in 76% of B‐CLL and 76.8% of MM. The highest number of CD66abce‐positive samples was in MCL and LPL (96.4% of 28 and 91.7% of 12 patients, respectively). Expression of CD66b clone BW 250/183 was examined in 114 of 260 samples. Positive expression was detected in 23.3% of B‐CLL (6/35), 17.1% of MM (7/30), and 21.4% of MCL (3/14) samples. Conclusion: The expression of CD66b compared to CD66abce was lower in all measured samples. Use of radiolabeled anti‐CD66b antibody for the treatment of lymphoproliferative diseases appears to have limited preclinical substantiation.     

Autoantibody-associated cross-reactive idiotypes expressed at high frequency in chronic lymphocytic leukemia relative to B-cell lymphomas of follicular center cell origin

Using murine monoclonal antibodies (MoAbs) specific for immunoglobulin (Ig) cross-reactive idiotypes (CRI), we performed immunohistochemical analyses on frozen tissue sections and cytocentrifuge preparations of Ig-expressing malignant cells from patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin's lymphomas (NHL) of follicular center cell origin. Twenty percent (4/20) of the Ig kappa light chain- expressing CLL cells reacted with 17.109, a MoAb against a major CRI on human IgM autoantibodies that is encoded by a conserved Ig variable- region gene (V gene) of the V kappa IIIb sub-subgroup. Another MoAb specific for V kappa IIIb framework determinant(s) reacted exclusively with all the 17.109-reactive CLL cells. Only one of 20 kappa light- chain-expressing CLL cells reacted with 6B6.6, a monoclonal antibody specific for a CRI commonly found on rheumatoid factor (RF) paraproteins with light-chain variable regions of the V kappa IIIa sub- subgroup. Finally, greater than 20% (8/34) of all CLL reacted with G6, a MoAb specific for an Ig heavy chain-associated CRI present on several RF paraproteins. In contrast, these CRIs were expressed at significantly lower frequencies in NHL of follicular center cell origin. Only one of 30 NHL expressing kappa light chains reacted with the 17.109 MoAb. Also, in contrast to the concordance between the 17.109-CRI and V kappa IIIb framework determinant(s) in CLL, two lymphomas in addition to the 17.109-reactive lymphoma were recognized by the anti-V kappa IIIb framework MoAb. None of the NHL reacted with either the 6B6.6 or the G6 MoAbs. These results are the first to demonstrate that CLL and NHL differ with respect to the expression of autoantibody-associated CRIs. The data support the notion that NHL of follicular center cell origin differs from CLL in its utilization and/or somatic mutation of Ig variable-region genes. The physiological and immunotherapeutic implications of these findings are discussed.