Abstract 4601: Mechanisms of action for therapeutic antibody variants of human IgG3 isotype: Enhancing the CDC activity of cetuximab and rituximab
Authors: Schwanbeck Ralf , Skof Anna , Rösner Thies , Jansen Marco , Räuchle Anina , Kretschmer Anna , Scheidig Axel , Leusen Jeanette , Derer Stefanie , Valerius Thomas 
Published: July 2017

Objectives. Human IgG3 differs from other antibody isotypes by its extended hinge region and by its higher affinity for C1q. The ability of complement to promote lysis of antibody-opsonized cells is well-established and may represent a main mechanism in antibody mediated tumor therapy. The aim of this project was to investigate effector mechanisms of tumor- directed IgG3 antibodies in more detail.

Experimental procedures. Therapeutic antibodies against EGFR (cetuximab, 225) and CD20 (rituximab, C2B8) were produced as IgG1 and IgG3 isotype variants by co-transfecting CHO cells with the respective de novo synthesized VH and VL domains fused to the respective heavy chain containing vectors. A Fab fragment of C2B8-IgG3 was crystallized and analyzed by X-ray structure analysis at PETRA III (DESY, Hamburg, Germany). Based on the resolved structure selected mutations were introduced into the CH1 domain of IgG3 aiming to improve or abolish CDC. Purified antibodies were assessed for complement fixation in ELISA and on tumor cells. Complement- and antibody-dependent cellular cytotoxicity (CDC and ADCC) activities were measured using 51chromium release assays. The significance of complement regulatory factors on target cells was investigated by siRNA mediated knock-down. A peritoneal tumor model was used to investigate the in vivo efficacy of IgG3 in comparison to IgG1 antibodies.

Results. Switching isotypes from IgG1 to IgG3 did neither affect target antigen binding nor the proliferation inhibition by EGFR antibodies. Unexpectedly, the IgG3 antibodies were not very active in ADCC, in spite of their strong FcγRIII (CD16) binding affinity. However, for target cells expressing low antigen levels, the IgG3 isotype improved CDC against CD20 and enabled CDC against EGFR expressing tumor cells. The complement-regulatory factor CD55 in particular mitigated the IgG3 induced CDC activity. Structural comparison of Fab fragments of IgG1 and IgG3 demonstrated significant differences in the CH1 domains of the respective heavy chains, which contain the disulfide bond to the light chain and also harbor the docking interface for the complement factors C3b and C4b. Preliminary results from an peritoneal tumor model demonstrated that 225-IgG3 was effective in killing EGFR- expressing cells. This in vivo activity was abolished by cobra venom factor treatment.

Conclusion. Switching the isotype of a therapeutic IgG1 antibody to IgG3 represents a promising approach to enable complement-mediated killing of tumor cells. Protein engineering of the C3b/C4b interaction interface in the CH1 domain may constitute a novel strategy to modulate the CDC activity against tumor target cells. Together, these approaches may enhance the CDC efficacy of CD20- and EGFR- directed antibodies.

Citation Format: Ralf Schwanbeck, Anna Skof, Thies Rösner, Marco Jansen, Anina Räuchle, Anna Kretschmer, Axel Scheidig, Jeanette Leusen, Stefanie Derer, Thomas Valerius. Mechanisms of action for therapeutic antibody variants of human IgG3 isotype: Enhancing the CDC activity of cetuximab and rituximab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4601. doi:10.1158/1538-7445.AM2017-4601