Bernd Dörken

Removal of cells from a malignant B-cell line from bone marrow with immunomagnetic beads and with complement and immunoglobulin switch variant mediated cytolysis.

In this report we describe the generation of complement (C) fixing IgG2b CD19 and CD22 monoclonal antibodies (mAbs) by the isolation of immunoglobulin (Ig) class switch variants using a simple and efficient method for the selection of spontaneously mutating hybridomas. The aim of this study was to compare the efficacy of C-mediated cytolysis vs immunomagnetic (IB) depletion of tumor cells from mixtures of malignant B cells and normal bone marrow. In a clonogenic assay employing the B-cell lines Namalwa and OCI.LY1, we found that the use of immunomagnetic beads warranted a highly efficient tumor cell removal independent of the Ig isotype of the mAbs used. Elimination of up to 4 log was achieved using a cocktail consisting of CD19, CD20, CD22 and CD37 B-cell mAbs. A less efficient killing of 2 log was obtained by C lysis using IgM mAbs, while only about 1 log tumor cell elimination was obtained using IgG2b mAbs. Immunomagnetic purging, besides being more effective than C-mediated cytolysis, is easier to handle and more rapid.

HLA B27 and defects in the T‐cell system in Whipple's disease

Abstract. The cellular immune system was tested in nine patients with Whipples disease. Three patients had active disease, and six had been in remission for up to 10 years. Intradermal delayed hypersensitivity reactions to candidin, trichophytin, tuberculin and varidase, T‐cell counts as determined by E‐rosettes, allogeneic stimulation of lymphocytes in the mixed lymphocyte culture, and mitogenic activation of lymphocytes by concanavalin A, phytohaemagglutinin and by pokeweed mitogen, were tested in the patients and compared with control subjects. HLA typing was performed in all patients. The reaction to tuberculin and varidase, the T‐cell counts and the activation of lymphocytes by concanavalin A were significantly reduced in patients with active disease and in patients during remission. The reaction to candidin and trichophytin was poor even in the controls. The mean results of the mixed lymphocyte culture, phytohaemagglutinin, and pokeweed mitogen activation tests were not significantly different from the controls. In patients with active disease the mixed lymphocyte culture reaction and the T‐cell counts were less than in patients in remission. The results suggest a persistent defect of T‐cells in patients with Whipples disease, a defect that is more severe in patients with active disease. The finding of HLA B27 in four of the nine patients supports the hypothesis of primary rather than secondary impairment of the cellular immune system in Whipples disease.

Mitoxantrone and high-dose cytarabine as salvage therapy for refractory non-Hodgkin's lymphoma

Mitoxantrone (Novantrone, NO) and high‐dose cytarabine (Ara‐C, AC) have each been shown in mono‐therapy trials to be active in non‐Hodgkins lymphoma (NHL). In the current study, a combination of the two drugs (NOAC) was administered to 31 patients with advanced NHL refractory to modern sequential chemotherapy regimens. Ara‐C was administered at 3 g/m2 as a 3 hour infusion every 12 hours on day 1 (2 doses) and mitoxantrone at 10 mg/m2/day on days 2 and 3. Of the 18 patients with high‐grade malignant NHL, six have attained a complete remission (CR) and two, a partial remission (PR). One CR and 5 PRs were achieved among the other 13 patients with intermediate or low‐grade NHL. The median time to relapse (TTR) of patients achieving CR was 7 months with a range from 4 to 17 months. Myelosuppression with subsequent infections was the major toxicity of this regimen. The median duration of severe neutropenia (< 0.5/nl) was 9 days with a range of 0 to 27 days and the median duration of severe thrombocytopenia (< 20/nl), 5 days with a range of 0 to 35 days. Infectious complications during cytopenia was seen in 45.3% of the courses administered and fever of unidentified origin was seen in 42.3%. About 63% of the patients were hospitalized for intravenous antibiotic or antimycotic treatment. Other side effects were mild and included nausea, stomatitis, and transient tachycardia of > 100/min. Thus, this regimen was active in refractory NHL with poor prognosis, and the toxic side effects were not excessive. Evaluation of the activity of this regimen at higher dose levels of Ara‐C is warranted.

Identity of HML‐1 Antigen on Intestinal Intraepithelial T Cells and of B‐ly7 Antigen on Hairy Cell Leukaemia

Immunoprecipitation of radioiodinated hairy cell leukaemia (HCL) cell lysates with monoclonal antibody (MoAb) HML‐1. originally reported to recognize intraepithelial T cells, and with MoAb B‐ly7, originally reported to react with HCL. led to identical biochemical characteristics. In SDS PAGE under reducing conditions, a major band of 143 kDa. a broad band ranging from 112 to 122 kDa. and two additional faint bands of 175 and 100 kDa could be determined. Deglycosylation of N‐linked sugar moieties by treatment of immunoprecipitates with endoglycosidases indicated that the two main protein cores of the antigen are predominantly if not exclusively glycosylated by complex and hybrid types of oligosaccharide chains. Competitive binding inhibition demonstrated that both MoAb are directed against different epitopes. Immunohistochemically. the staining patterns obtained with both MoAb in normal tissues, in T‐and B‐cell lymphomas, and in HCL were identical except for a single case of HCL which was HML‐1−/B‐ly‐7+, We conclude that MoAb HML‐1 and B‐1y7 recognize the same antigen.

Monoclonal antibodies against the human lymphocyte differentiation antigen CD 76 bind to gangliosides

Two monoclonal antibodies, HD 66 and CRIS‐4, by which the new CD 76 B‐cell‐associated cluster was defined, bound to several gangliosides (sialic acid containing glycolipids) of different polarity. One of the gangliosides recognized by HD 66 could be identified as NeuAcα2‐6Galβl‐4GlcNAcβl‐3Galβl‐4Glc‐βl‐lCer. This antigen was enzymatically synthesized. Sialidase treatment of the ganglioside antigens abolished binding of HD 66 and CRIS‐4.

A comparison of membrane marker phenotypes in hairy-cell leukemia and phorbol-ester induced B-cll cells using monoclonal antibodies.

The neoplastic cells from 14 cases of hairy-cell leukemia were investigated in order to determine their membrane phenotype on the basis of their reactivity with a large panel of B-, T-, myeloid/monocytic- and non-lineage restricted monoclonal antibodies. The data were compared to those from monoclonal antibody studies on phorbol-ester (TPA) induced cells from 10 patients with B-type chronic lymphocytic leukemia. The study has so far revealed further evidence for the B-cell nature of hairy-cells leukemia and demonstrates a developmental link between the two cell types, suggesting that hairy-cells represent a more advanced differentiation stage along the B-cell lineage.

Monoclonal antibody uptake in B-cell lymphomas: Experimental studies in nude mouse xenografts

Accumulation of radiolabelled monoclonal antibodies (mAb) in human B-lymphoma xenografts was found to result in two distinct patterns. The basic elements leading to these patterns were elucidated by autoradiographic and immunohistological analysis applied to the nude mouse xenografts BJAB and OCI.LY1. With BJAB, accumulation occurred exclusively in peripheral cell layers of the lymphoma nodule, while central areas were not accessible irrespective of mAb dose. This feature was the consequence of an inefficient transport across intratumoral vessels together with peripheral mAb supply through a subcapsular pseudosinus. With OCI.LY1, intratumoral vessels showed generalized leakiness. Furthermore, interstitial transport was operative to a fair extent, such that in early images multiple sites of mAb extravasation were obvious, which coalesced during the course of prolonged uptake. The pattern of peripheral mAb uptake resulted in a low overall tumour uptake, while multifocal uptake yielded substantial accumulation values.

Ligand-dependent modulation of membrane phospholipid metabolism in ConA-stimulated lymphocytes

Abstract Human peripheral lymphocytes were activated by ConA in serum-free culture medium, supplemented by BSA. Incorporation of [3H]thymidine into DNA, of [3H]uridine into RNA and of oleate or acetate into membrane phospholipids was investigated. DNA synthesis could be completely inhibited by αMM or by anti-ConA-IgG. Fab and F(ab)2 fragments of the anti-ConA were equally active. When αMM or anti-ConA was added to cultures at different times after stimulation with ConA, incorporation of [3H]thymidine into DNA (measured after 72 h) could be prevented up to 6–8 h completely and up to 20–30 h partially. Incorporation of [3H]uridine into RNA could be arrested at any time of the culture up to 40 h at the level it had reached but did not reverse to the level of unstimulated cells for a long time. In contrast, incorporation of oleate into lecithin returned to the level of unstimulated cells within 2–3 h after removal of ConA. This suggests that the activation of the phospholipid turnover in stimulated cells is a direct consequence of the presence of the mitogen at the membrane and thus may be a critical initial event in lymphocyte activation.

An immunoenzymatic staining assay (ISA) for the rapid screening of monoclonal antibodies detecting membrane and cytoplasmic antigens.

We report a rapid and very sensitive immunoenzymatic staining assay (ISA) for the determination of the fine specificity of monoclonal antibodies directed against cellular antigens. Reactivity is analysed at the single cell level by light microscopy using alkaline phosphatase-conjugated second and third antibodies on cells bound to Terasaki plates. Reactive cells can be defined by their morphology even in preliminary screening procedures. Only small numbers of cells are necessary for this assay which is comparable to radioimmunoassays in its sensitivity. Plates with fixed cells can be stored at 4 degrees C so that a permanent library of various cell types is always immediately available for use. The test is also suitable for human blood cell phenotyping. Moreover, a simple modification of the procedure by short pretreatment of the cells with the detergent Brij allows the detection of antigens within the cytoplasm. The importance of evaluating the cytoplasmic compartment in order to define antibody specificity and study the distribution of different cytoplasmic and membrane antigens is emphasized.

Long-term disease-free survival following autologous bone marrow/blood stem cell transplantation in 89 patients with acute leukemia.

Myeloablative consolidation therapy followed by transplantation of the patient’s prior harvested and cryopreserved bone marrow has been shown to be successful for the treatment of acute leukemia. Yeager et al. [1] recently reported data on autologous marrow graft harvested in second or subsequent complete remission (CR) of acute myelogenous leukemia (AML), treated ex vivo with the active cyclophosphamide (CY) derivative 4-HC, and eventually retransfused into patients with “high-risk” AML after myeloablative treatment with busulfan (BU) and CY. The 43% actuarial disease-free survival (DFS) in these patients was clearly superior to that with conventional treatment, which is 5% under such “high-risk” conditions [2].