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.

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. From these data we conclude that B-B2 and B-B4 immunotoxin can be used for ex vivo bone marrow purging. Discrepancies were found between immunohistochemistry, binding assays and cytotoxicity assays with the mAb and the immunotoxin, which underlines the necessity for these various assays as a preclinical screening.

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.

Summary In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) 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.

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