Development of a bispecific tetravalent antibody for treating Hodgkińs Disease
Authors: Melvyn Little, Fabrice Le Gall, Karin Hoffmann, Stefan Knackmuss, Vera Mick, Uwe Reusch
Journal: Cancer Research (2008)

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA

4905     Introduction : To recruit natural killer cells for targeting and lysing Reed-Sternberg cells in Hodgkins disease with high efficacy, we have developed a bispecific tetravalent homodimer antibody comprised solely of Ab variable domains. It has a molecular weight of 105kDa, which is well above the threshold for first pass renal clearance. Two binding sites are directed against CD30 on RS cells and two sites are directed against the A isoform of the FcγIII receptor (CD16A), which is mainly present on the surface of NK cells and macrophages. Experimental Procedures : The anti CD30 moiety of the homodimer was derived from the variable domains of a bispecific anti CD30/anti CD16 mouse mAb that had already showed some promising results in clinical trials (Blood (1997) 89:2042; Clin. Cancer Res. (2001), 7:1873). To isolate a human antibody that was specific for the A isoform of the FcγIII receptor on NK cells, we screened Affimeds human Ab libraries using phage display. The heavy and light chain variable domains of both antibodies were genetically engineered for the expression of a four domain product arranged in the order VH anti-CD16A, VL anti-CD30, VH anti-CD30, VL anti-CD16A. The linkers between the domains were made fairly short to prevent intra domain pairing and favor dimerisation with the formation of a functional homodimer. The efficacy of this molecule for recruiting NK cells to lyse CD30+ target cells was tested both in vitro and in vivo using the CD30+ cell lines L540CY and KARPAS-299, respectively. Summary of results : We succeeded in isolating a human antibody against the CD16 antigen that was highly specific for the A isoform displayed on NK cells. This was very surprising since there are only three amino acid differences between the A and B isoforms. A bispecific tetravalent antibody comprising the variable domains of this antibody for recruiting NK cells and the variable domains of a murine anti-CD30 for targeting RS cells was able to induce the specific lysis of CD30+ cells at picomolar concentrations in the presence of peripheral blood lymphocytes. Significant lytic activity was also seen in the presence of serum even though IgGs compete for the same or a nearby binding site on the FcγIII receptor. An in vivo NOD/SCID mouse model using a Winn Assay with human PBMCs demonstrated that the antibody was able to diffuse into the site of the sc tumor and significantly enhance the action of NK cell tumor lytic activity. Using groups of ten mice, none of the antibody plus PBMC treated mice developed a tumor whereas nine out of 10 of the PBMC treated mice developed tumors. Conclusion : A tetravalent bispecific antibody in a homodimeric format comprised only of antibody variable domains from a murine anti-CD30 and a human anti-CD16A antibody proved to be very effective for recruiting NK cells to specifically lyse malignant CD30+ cells. This chimeric antibody is now being further developed for production under GMP conditions and clinical testing.