Abstract 4327: A novel protease resistant antibody platform with selectable cell killing functions.

Abstract

Abstract Invasive cancers express matrix metalloproteases, such as MMP-3 and MMP-7, which have the ability to cleave human antibodies in the lower hinge region. The human IgG lower hinge region contains two heavy chains, and cleavage occurs in a two-step process. First, one heavy chain is cleaved, resulting in a singly-cleaved intermediate. Cleavage of the second heavy chain liberates the Fc region from the antigen-binding region, resulting in an Fc fragment and a F(ab’)2 fragment. Previous work has demonstrated that the singly-cleaved intermediate accumulates on the surface of IgG1-opsonzied tumor cells in the presence of MMPs. The singly-cleaved intermediate is indistinguishable from the intact IgG counterpart when assessed by analytical techniques under native conditions and maintains normal antigen binding. However, the singly-cleaved intermediate displays a profound loss of Fc-mediated cell-killing functions both in vitro (e.g. ADCC and CDC) and with in vivo cell clearance, presumably because the lower hinge contains key recognition points for both FcγRs and the C1q component of complement. Singly-cleaved IgGs are of particular concern with regards to human IgG1 monoclonal therapeutic antibodies, because the proposed mechanisms of action for several anti-tumor mAbs in the clinic may be partially dependent on Fc effector functions, such as ADCC and CDC. For this reason, our group and others have proposed that cleavage and the subsequent inactivation of Fc-effector functions of human IgG antibodies may function as an immune evasion mechanism. To overcome the loss-of-function associated with human IgG1 cleavage, we aimed to develop a protease-resistant IgG1 platform which would still maintain Fc-mediated cell-killing functions. Mutation of the lower hinge of IgG1 provided protease-resistance, but also resulted in a loss of Fc-effector functions. Through specific mutations of amino acids in the CH2 region to enhance complement or Fcγ receptor binding, we were able restore or even enhance cell-killing functions selectively while maintaining protease-resistance. Therefore, we were able to generate protease-resistant antibodies with the ability to mediate either complement-dependent and/or antibody-dependent cellular cytotoxicity, which were also significantly resistant to inactivation by proteases associated with many invasive cancers. Citation Format: Michelle Kinder, Katharine Grugan, Keri Soring, William Strohl, Robert Jordan, Randall Brezski. A novel protease resistant antibody platform with selectable cell killing functions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4327. doi:10.1158/1538-7445.AM2013-4327