Wim C. Vooijs

A plasmocyte selective monoclonal antibody (B-B4) recognizes syndecan-1.

We developed a new monoclonal antibody, B‐B4, which specifically identifies human plasma cells. It strongly reacts with all multiple myeloma cell lines and with malignant plasma cells of all tumour samples of the multiple myeloma patients tested. B‐B4 does not react with any peripheral blood, bone marrow or tonsil cells. Cloning of the B‐B4 antigen reveals that the monoclonal antibody recognizes syndecan‐1. It appears that the monoclonal antibody B‐B4 is a suitable marker for human plasmocyte identification among haemopoietic cells and a useful probe for the diagnosis of haematological malignancies. Furthermore, this monoclonal antibody can be used for depletions prior to CD34 grafting.

Resolution of cutaneous inflammation after local elimination of macrophages

We constructed an immunotoxin, composed of an antibody directed against the high-affinity IgG receptor CD64 and Ricin-A, with the aim of resolving chronic inflammation through elimination of activated macrophages. In vitro, this immunotoxin proved very efficient in inducing apoptosis in activated macrophages, leaving resting and low CD64-expressing macrophages unaffected. We examined the activity of our immunotoxin in a sodium lauryl sulfate (SLS)-induced cutaneous inflammation model, using transgenic mice expressing human CD64. Upon intradermal injection of the immunotoxin (IT), cutaneous inflammation resolved in 24 h. This was demonstrated histologically by clearance of all CD64-expressing macrophages, followed by clearance of other inflammatory cells. Clinical parameters associated with inflammation, such as local skin temperature and vasodilation, also decreased.

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.

Long-term bone marrow cultured stromal cells regulate myeloma tumour growth in vitro: studies with primary tumour cells and LTBMC-dependent cell lines

Long‐term bone marrow cultured stromal cells (LTBMC) produce IL‐6 after contact with tumour cells from multiple myeloma patients. We found that LTBMC could substitute for exogenous IL‐6 in the stimulation of bone marrow plasma cells from myeloma patients with active disease in short‐term cultures. In addition, tumour cells of some patients with inactive disease, which were unresponsive to exogenous IL‐6, were induced to IL‐6‐dependent growth after LTBMC co‐culture. To study the role of LTBMC in myeloma tumour growth in vitro, plasma cell lines UM‐2 and UM‐3 were selected. UM‐2 and UM‐3 grew in contact with LTBMC and proliferation was blocked by antibodies against IL‐6, IL‐6 receptor (IL‐6R, gp80, CD126) or the common signal transducing unit, gp130 (CD130). Culture with IL‐6 alone or combined with GM‐CSF resulted in cell death via apoptosis. The combination of IL‐6 with soluble gp80, however, maintained in vitro proliferation of UM‐2 and UM‐3 cells. These data imply that LTBMC regulate myeloma growth in vitro via production of IL‐6, possibly via induction of a functional IL‐6 receptor on the tumour cells.

Efficacy and toxicity of plasma-cell-reactive monoclonal antibodies B-B2 and B-B4 and their immunotoxins

Abstract Immunotherapy based on the delivery of toxic agents to the tumor site using monoclonal antibodies (mAb) may be a promising modality in the treatment of hematological malignancies. In the selection of mAb, both for ex vivo but even more for in vivo therapy, not only their reactivity to the neoplastic cells should be considered, but also reactivity to other body constituents. Here we describe the screening of two human plasma-cell-reactive mAb B-B2 and B-B4, which may be used for immunotherapy of multiple myeloma. Cross-reactivity of B-B2 and B-B4 was determined by immunohistochemistry on a series of tissues. This revealed for both B-B2 and B-B4 a strong staining of epithelial cells in various organs, e.g. lung, liver, skin, kidney and gut, while only a weak and diffuse staining was seen with endothelial cells. In bone marrow reactivity was only found with plasma cells and not with hemopoietic precursors (CD34+ cells). Immunotoxins from B-B2 and B-B4 were constructed by coupling them to the plant-derived ribosome-inactivating protein saporin. Both B-B2 and B-B4 immunotoxins appeared to be efficient in specific inhibition of protein synthesis in plasma cell lines (IC50 respectively 1 nM and 0.1 nm). The immunotoxins were also tested on epithelial cell line A431, on liver cell line HepG2 and on human umbilical vein endothelial cells. The epithelial cell line A431 was reactive with both B-B2 and B-B4, but was only inhibited by B-B4 immunotoxin. Cell line HepG2 was reactive with both mAb, but was not inhibited by either immunotoxin. The endothelial cells showed no reactivity with B-B2 and B-B4 and were not inhibited by either immunotoxin. Bone marrow treated with B-B2 and B-B4 immunotoxin did not show a decrease in colonies of hemopoietic precursor cells. Incubation of multiple-myeloma-derived bone marrow with these immunotoxin resulted in a clear decrease of the number of plasma cells.

B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkins disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration.