Therapeutic effects of monoclonal antibody g250, interferons and tumor necrosis factor, in mice with renal‐cell carcinoma xenografts

Abstract

Because renal-cell carcinoma (RCC) is considered relatively resistant to radio- and chemotherapy, RCC patients may benefit from new treatment modalities, e.g. immunotherapy. In vitro and in vivo studies suggest that combinations of cytokines such as interferon gamma or interferon alpha (IFN-gamma, IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) may act synergistically. In this study we tested whether a monoclonal antibody (MAb) G250, reactive with a RCC-associated antigen, showed anti-tumor effects in vivo in nude mice with established s.c. human RCC xenografts, and also whether this MAb could enhance the anti-tumor effect of combinations of IFNs and TNF-alpha. Treatment with combinations of IFN-alpha/TNF-alpha or IFN-gamma/TNF-alpha, or with MAb G250 alone, resulted in a significant inhibition of tumor growth. Treatment with MAb G250, in combination with IFN-gamma/TNF-alpha, did not result in an improve anti-tumor effect as compared to that of either treatment alone. In contrast, MAb G250 combined with IFN-alpha/TNF-alpha resulted in a significantly enhanced anti-tumor response. In one experiment, 3 out of 10 mice showed complete tumor regression, with no recurrence after 90 days. Large numbers of infiltrating macrophages were found surrounding viable and necrotic tumor tissue after treatment with G250 combined with IFN-alpha/TNF-alpha. These results suggest that combination therapy, consisting of IFN-alpha, TNF-alpha and MAbs, may have therapeutic value in the treatment of RCC.

Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma

“…The G250 antigen is an attractive target for antibodymediated therapy, because of its nearly exclusive expression in renal carcinoma cells. Studies with the G250 MAb and the murine bispecific G250/OKT-3 (anti-CD3) MAb have shown preferential localisation of these antibodies to the tumour (Oosterwijk et al, 1993;van Dijk et al, 1991) and antitumour effects in RCC-xenografted nude mice (van Dijk et al, 1994). The G250/anti-CD3 MAb was chimerised to decrease immunogenicity of the bispecific antibody in patients, because studies in patients with murine anti-tumour antibodies and bispecific antibodies are limited by the development of HAMA responses.…”

“…The absence of a third peak representing parental anti-CD3 MAb was not the result of the loss of either the anti-CD3 MAb heavy or light chain, since RT-PCR analysis showed the presence of mRNA for all four antibody chains. The chromatographic separation method has been used previously to purify bispecific OVTL3/OKT-3 MAb ( van Ravenswaay Claasen et al, 1993), murine bispecific OC/TR (Warnaar et al, 1994), chimeric bispecific OC/TR (Coney et al, 1996) and murine bispecific G250/OKT-3 MAb (van Dijk et al, 1989), and was shown to separate all combinations of heavy and light chains into distinguishable peaks. Therefore, we expect peak 2 of the chimeric G250/anti-CD3 MAb separation to contain IgG molecules composed of the parental heavy and light chains.…”

“…Depending on the combination of parental antibodies, preferential association of heavy and light chains may occur, resulting in the absence of some heavy and light chain combinations. In addition, the profiles of the chromatograms of different clones producing the same bispecific OC/TR MAb have been shown to differ greatly, as a result of different rates of synthesis of the antibody chains (Warnaar et al, 1994). In both the murine OC/TRproducing hybridoma and the chimeric OC/TR-producing hybridoma, clones were found that produced only one parental antibody and the bispecific MAb.…”

“…Chimeric IgGI was purified from the concentrated supernatant on a protein A-Sepharose column using 0.1 M citrate buffer at pH 3.0 for antibody elution. The bispecific antibody fraction was separated from parental antibody by high pressure liquid chromatography (HPLC) on Mono S -Sepharose using a sodium chloride gradient from 0 to 0.2 M in 25 mM sodium acetate, pH 5.0, a matrix and procedure that had previously been described (van Ravenswaay Claasen et al, 1993;Warnaar et al, 1994;van Dijk et al, 1989 human IgG MAb as detecting antibody to determine the total amount of antibodies with anti-CD3 specificity present in the culture supernatant of the transfectants (Figure 2b and d). Subsequently, the capacity of culture supernatant of the transfectants producing chimeric bispecific cG250/anti-CD3 MAb to trigger activated human CD8+T cells for lysis of target cells that express the G250 antigen was analysed in a cytotoxicity assay.…”

“…Whole immunoglobulin molecules and F(ab)2 fragments of the G250 MAb localised preferentially in tumours, both in model studies with RCC xenografted nude mice and RCC patients, and were suitable for tumour visualisation in mice (Van Dijk et al, 1988 and in patients (Oosterwijk et al, 1993). Therapeutic effects of murine G250 IgG2a were demonstrated by tumour growth inhibition studies in nude mice xenografted with RCC (Van Dijk et al, 1994). The G250 antigen can therefore be considered a suitable target for antibody-mediated immunotherapy.…”