The chronic lymphocytic leukemia antigen (cCLLa) as immunotherapy target: pharmacokinetics and biodistribution of two divalent, ricin-based immunotoxins in xenografted athymic mice.

The chronic lymphocytic leukemia (CLL) antigen (cCLLa) is potentially suitable for targeted immunotherapy given its restriction to clonal CLL cells and lack of expression by normal lymphocytes. In order to assess the pharmacokinetics and biodistribution of two potent anti-cCLLa immunotoxins (ITs) were examined in the mouse model. The IgG fraction of anti-cCLLa monoclonal antibody CLL2m was conjugated with 125I-labeled intact (RTA) or deglycosylated (dgA) ricin chain A, injected intravenously into athymic mice engrafted with cCLLa-expressing human tumors, and monitored over 120 hours. Blood concentrations of CLL2m/125I-RTA and CLL2m/125I-dgA were best fit to biexponential equations but the latter exhibited a lower alphaT1/2 and betaT1/2 (4.1 and 102 min vs 5.9 and 126 min), a smaller volume of distribution (5.1 g vs 9.7 g), and a lower blood clearance (2.2 g/hr vs 4.6 g/hr). Both ITs exhibited preferential tumor uptake that followed distinct kinetics: rising tumor uptake for 2 hrs post-injection (while tissue uptake decreased), reaching tumor/non-tumoral tissue uptake ratios up to 16.9; and slower dissociation rates of tumor- vs tissue-bound ITs (>45% vs <20% remaining tissue-bound 6 hrs post-injection, respectively). Non-specific liver uptake was not prominent for either IT. In vivo IT deconjugation reached 50% approximately 12 hours pos-injection. The pharmacokinetics and biodistribution data in the mouse model suggest that ricin-based anti-cCLLa ITs are suitable for use in human trials.

The chronic lymphocytic leukemia antigen (cCLLa) as immunotherapy target: assessment of LD50 and MTD of four ricin-based anti-cCLLa immunotoxins (ITs) in Balb/c mice.

The chronic lymphocytic leukemia (CLL) antigen (cCLLa) is a promising immunotherapy target given its disease-restricted expression, its highest prevalence among CLL surface antigens, and its lack of expression by normal T- and B-lymphocytes. The objectives of this study were to assess the 50% lethal dose (LD50) and the maximum tolerated dose (MTD) in Balb/c mice of four anti-cCLLa immunotoxins (ITs) derived from the intact monoclonal antibody (MoAb) or its Fab fraction, each conjugated to either ricin chain-A (RTA) or its deglycosylated derivative (dgA). The IgG fraction of anti-cCLLa monoclonal antibody CLL2m and its Fab fraction were conjugated to RTA or dgA to generate four ITs: IgG/RTA, IgG/dgA, Fab/RTA and Fab/dgA. Progressive concentrations of each IT (ranging between 2.60 mg/kg and 100.00 mg/kg) were injected intravenously into groups of 5 mice each. After injection, mice were monitored daily for 10 days for survival. Observed mortality data in each group were matched to those in Weil's tables for estimating LD50 (mg/kg) from the moving average interpolation method. Estimated LD50 (in mg/kg) were: IgG/RTA, 13.33; Fab/RTA, 25.53; IgG/dgA, 55.33; Fab/dgA, 55.33. Their respective MTD (mg/kg), defined as the highest dose level survived by all mice, were 8.78, 13.17, 29.63 and 29.63. Depending on the animal-to-human extrapolation method used, the calculated LD50 and MTD in humans ranged from 1.2 mg/kg and 0.8 mg/kg (IgG/RTA), to 55.6 mg/kg and 36.9 mg/kg (IgG/dgA and Fab/dgA), respectively. The following conclusions are drawn. 1. Antibody valence exerted little influence on either the LD50 or the MTD; 2. The LD50 to MTD ratios were approximately 2:1; 3. dgA-derived ITs were approximately one half as toxic as their RTA-derived counterparts; and 4. Extrapolation of LD50 and MTD mouse data to humans resulted in dose levels comparable to or exceeding those reported in most IT human trials. These data suggest the suitability of anti-cCLLa ITs for clinical immunotherapy trials.

A simple technique for the rapid enrichment of class and subclass hybridoma switch variants. A 1000-fold enrichment in half the time, for half the cost.

Switching parental hybrids in vitro to downstream switch variant clones producing more desirable monoclonal antibodies (MoAbs) requires either labor intensive and time consuming subcloning techniques, or fluorescence activated sorting of the desired clones. We tested the hypothesis that enrichment of downstream switch variant clones might be achieved by selective lysis of upstream hybridoma cells followed by expansion of the enriched downstream clone. Using a parental hybridoma with surface and secretory IgM, we attempted to enrich downstream switch variant clones producing class (IgG) and subclass (IgG1 or IgG2a) MoAbs. Enrichment of downstream IgG, IgG1 and IgG2a MoAb-producing switch variants was achieved by single or repeated antibody-dependent, complement-mediated lysis of the upstream IgM-bearing parental hybridoma cells followed by limited subcloning. Two exposures of parental hybridoma cells to lysis followed by plating at 100 cells/well enriched the frequency of switch variants up to 1235-fold, enabling the development of IgG1 or IgG2a-producing subclones exhibiting high yield antibody production. Using this protocol, production time and costs were reduced by > 50% when compared to the standard technique. This novel technique for the rapid isolation and expansion of switch variant clones should be ideal for most laboratories, particularly those without access to cell sorting capabilities.

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.

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.

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).

Immunophenotypic diagnosis of clinical and preclinical chronic lymphatic leukemia by using monoclonal antibodies against the cCLLa, a CLL-associated antigen.

The clinical usefulness of monoclonal antibodies (MoAbs) against the cCLLa, an antigen restricted to B-chronic lymphatic leukemia (CLL) and its variants, was ascertained in 65 patients with overt CLL and 25 individuals with unexplained mild lymphocytosis. Healthy volunteers (n = 25) and patients with malignant and nonmalignant disorders (n = 58) served as controls. The following observations were made in CLL. (a) Anti-cCLLa MoAbs identified neoplastic CLL cells as judged by the high correlation (r = .985) between monoclonal surface immunoglobulins (Slgs) and cCLLa expression in all patients, and dual-label flow cytometry studies showing cCLLa expression by monoclonal Slg-bearing B-CLL cells but not by normal B lymphocytes. (b) The size of the circulating cCLLa-positive clone paralleled the degree of lymphocytosis (r = .987) and was associated with reciprocal (r = .893) relative T lymphopenia. Ten patients with borderline lymphocytosis exhibited a subset of monoclonal Slg/cCLLa-positive cells ranging from 16% to 45% of the total. These patients were indistinguishable from those with CLL in terms of age, clone lineage, and reciprocal relative T lymphopenia. Two patients have progressed to overt CLL within 19 months, but eight have not (observation time, 18 to 82 months). These data suggest that anti-cCLLa MoAbs are sensitive probes useful to identify and monitor cCLLa clones during their clinical and preclinical phases.

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.

Cytochemical, cytogenetic, immunophenotypic and tumorigenic characterization of two hairy cell lines.

Two cell lines (EH and HK) were derived from two patients with hairy cell leukemia (HCL). Both patients exhibited a clinical picture characteristic of HCL, including splenomegaly, cytopenias, and tartrate-resistant acid phosphatase (TRAP)-positive "hairy" lymphocytes in blood and marrow. EH and HK were demonstrably of B lineage, as judged by cytochemistry and immunophenotype, including expression of B1, B2, and LEU-12 antigens and of monoclonal surface immunoglobulins (slgs). Monoclonality was confirmed by clonal karyotype abnormality demonstrated in cell line HK. HCL parentage suggested by cytochemistry and electron microscopy was confirmed by the immunophenotypic observation that HCL lines expressed antigens alpha S-HCL1, alpha S-HCL3, and cCLLa. While alpha S-HCL1 and alpha S-HCL3 are nonspecific, their co-expression is characteristic of HCL cells. The cCLLa is a novel 69-kd membrane HCL-associated polypeptide antigen not shared by circulating normal T or B lymphocytes nor by malignant cells from unrelated lymphoid or nonlymphoid malignancies. The doubling time of EH and HK was 24 and 36 hours, respectively. While HK included a small subset of Epstein-Barr virus (EBV) nuclear antigen-positive cells, EH cells were homogeneously negative for the presence of this antigen. Both cell lines were consistently implantable in irradiation-preconditioned immunodeficient mice giving rise to primary tumors and widespread metastasis.

Monoclonal antibody production to human and bovine 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase): high-specificity recognition in whole brain acetone powders and conservation of sequence between CNP1 and CNP2.

Monoclonal antibodies against human and bovine 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.

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.

Drug-induced liver injury. In vitro demonstration of hypersensitivity to both phenytoin and phenobarbital.

Fever, lymphadenopathy, exfoliative dermatitis, and evidence of drug-induced liver injury developed in a 16-year-old girl three weeks after beginning therapy with phenytoin and phenobarbital. This clinical syndrome can be caused by either of these structurally related drugs but has been more frequently attributed to phenytoin. In vitro studies disclosed marked reactivity of this patient's lymphocytes to concentrations of both drugs, which encompassed their measured serum levels. The demonstration of dual reactivity raises concerns about continuing administration of phenobarbital during an apparent phenytoin-induced reaction. Whether this potential risk is greater than the risk of stopping all anticonvulsant medications in a patient with a seizure disorder is not known and remains to be established.

Chronic lymphatic leukemia evolving into chronic myelocytic leukemia.

A diagnosis of chronic lymphatic leukemia (CLL) was made in an 83-year-old man on the basis of marked lymphocytosis (131.1 X 10(9)/L) and infiltration of the marrow (77%) by small lymphoid cells. The hemoglobin was 11.8 g/dL and the platelet count was 427.5 X 10(9)/L. With minimal and unsustained treatment, abrupt resolution of the lymphocytosis occurred with reciprocal emergence of a classic picture of chronic myelogenous leukemia (CML). During the hematologic evolution, blood lymphoid cells were isolated for surface marker, cytogenetic, and functional studies. E-rosette receptors, Fc-receptors, and membrane immunoglobulins were found on only 7.5%, 7.4%, and 10.0% of these cells. These cells also failed to express a CLL-associated antigen we have detected on approximately equal to 95% of the cells of all CLL patients studied, regardless of cell phenotype. In addition, the patient's lymphoid cells responded poorly to leucoagglutinin (LPHA) stimulation, showed increased spontaneous metabolic activity and exhibited the Philadelphia chromosome. These observations suggest that the patient's transient initial lymphocytosis was not due to CLL, but perhaps represented myeloid precursors in circulation prior to terminal differentiation.

Immunologically diagnosed malignancy in Sjögren's pseudolymphoma.

Studies of lymphocyte markers in a patient with Sjögren's syndrome who exhibited histologically benign lymphoproliferation in the lung revealed a malignant cell clone. T and B cells were quantitated according to their ability to form spontaneous rosettes with sheep erythrocytes and to fluoresce with fluorescein-conjugated antiserums, respectively. Circulating lymphocytes were 66 percent T cells (N = 58 +/- 2 per cent) and 14 percent B cells (N = 22+/- 1 percent), the latter exhibiting normal polyclonal distribution of membrane immunoglobulins. However, lymphocyte suspensions obtained from fresh lymph node and from biopsy specimens from a lymphoid lung nodule revealed 95 percent and 88 percent B cells, with 1 percent and 2 percent T cells, respectively. Moreover, when cryostat-frozen sections from both tissues were reacted with each of the heavy and light chain-specific antiserums, most cells demonstrated the presence of intracytoplasmic mu kappa immunoglobulin exclusively. Twenty-two months later, a clinically and histologically classic lymphoma developed. Repeat marker studies performed on cells freshly isolated and on frozen sections from the histologically malignant lymph node revealed persistence of the monoclonal marker on most cells.