Bert J. E. G. Bast

Immunotherapeutic potential of bispecific antibodies.

Abstract Bispecific antibodies (BsAbs) offer therapeutic potential by targeting tumors or pathogens as well as cytotoxic effector and/or antigen-presenting cells. A recent meeting ∗ ∗The Vth World Conference on Bispecific Antibodies was held at Volendam, The Netherlands, on 25–28 June 1997. focused on current issues in the BsAb field.

Efficacy of an anti-CD138 immunotoxin and doxorubicin on drug-resistant and drug-sensitive myeloma cells

Multidrug resistance is an increasing problem in the treatment of cancer. We evaluated in vitro the effect of an anti‐CD138 plasma‐cell‐specific immunotoxin (IT, B‐B4‐SO6) in combination with the chemotherapeutic drug doxorubicin on drug‐sensitive and drug‐resistant variants of the multiple‐myeloma (MM)‐derived cell line RPMI8226 and freshly isolated malignant‐myeloma cells. Drug‐resistant RPMI8226 cells were still sensitive to the IT, although to a lesser extent than drug‐sensitive cells. In the clonogenic assay, using 10 nM B‐B4‐SO6, at least 5 logs kill was found for drug‐sensitive RPMI8226 cells, vs. 2.5 logs kill for the drug‐resistant RPMI8226 cells. When a sub‐optimal dose of 1 nM IT was combined with 3 ng/ml doxorubicin, which was toxic for drug‐sensitive but not for drug‐resistant cells, an additive effect was found for drug‐sensitive RPMI8226 cells. The IT did not influence the sensitivity of resistant cells for doxorubicin. We therefore speculate that this type of IT, may be of more value in combination with primary chemotherapy. The effect of B‐B4‐SO6 on malignant‐myeloma cells of patients was investigated in a viability assay. Both drug‐sensitive and drug‐resistant cells from MM patients were sensitive to B‐B4‐SO6. After 2 days, a 50% kill of malignant cells was found when 10 nM IT were used. Doxorubicin was effective only on sensitive cells, and there was a tendency for an additive effect in the combination of these cells. Int. J. Cancer 83:571–576, 1999.

Determination of the growth fraction in monoclonal gammopathy with the monoclonal antibody Ki-67

The monoclonal antibody Ki‐67 reacts with a nuclear antigen that is present only in proliferating cells. The proportion of Ki‐67 positive cells may therefore serve as a reliable measurement for the growth fraction in normal and neoplasmic cell populations. We have tested the significance of the MoAb Ki‐67 in the classification of monoclonal gammopathy and compared the results with the plasma cell labelling index. In benign monoclonal gammopathy the percentage of Ki‐67 positive plasma cells (median 1.6%) was significantly lower than in untreated multiple myeloma (median 9.6). Among the patients with more than 10% Ki‐67 positive plasma cells there were some very short survivors.

Determination of the plasma cell labelling index with bromodeoxyuridine in a double fluorescence technique

Summary. A new technique is described in which plasma cells actively synthesizing DNA can be identified in a heterogeneous cell population such as bone marrow. This method uses bromodeoxyuridine (BrdU) and a fluorescent monoclonal antibody against BrdU in combination with cytoplasmic staining for immunoglobulin (Ig). In 26 patients with monoclonal gammopathy (MG) the plasma cell labelling index (LI) as determined by this immunofluorescent method was compared with the tritiated thymidine (3H‐TdR) LI. No difference in sensitivity was found between the two methods. As the determination of the plasma cell LI with the BrdU/anti BrdU method is easy to perform and results can be obtained within 4 h, this immunofluorescent method seems to be an attractive alternative to the laborious time‐consuming classic 3H‐TdR LI.

Comparison of various in vitro assays for efficacy screening of immunotoxins.

Before using immunotoxins in vivo, their efficacy is evaluated in in vitro assays. In this study we compare six different assays for the evaluation of immunotoxins: protein and DNA synthesis inhibition assay, chromium release assay, cell line colony assay, limiting dilution assay and clonogenic assay. All assays except the chromium release assay show specificity of the immunotoxins in appropriate concentrations. The protein and DNA synthesis inhibition assays are easy to perform and, therefore, suitable for initial screening, while the clonogenic assay seems to be the best one for immunotoxin efficacy determination.

Mitogenic stimulation of malignant B cells CLL: diminished in-vitro stimulation with anti-CR1 antibodies.

Peripheral B lymphocytes of five CLL patients were tested in a radioimmunoassay to determine the density of the C3b receptor (CR1) and the cells were assayed for their ability to mature into IgM secreting cells after in-vitro culture with a combination of Pokeweed Mitogen (PWM) and antibodies directed against CR1. Despite the presence of normal amounts of CR1 on the leukemic B cells, crosslinking of these receptors by anti-CR1 antibodies stimulated only a fraction of the leukemic cell population to differentiate into IgM secreting cells. These results add to the partial functional impairment of CLL-B cells.