Switching from IgG1 to IgG3 Isotype to Boost Complement-Activating Capacities of Therapeutic Antibodies – Impact of CD55 Expression on the Mode of Action

Introduction: Complement-dependent cytotoxicity (CDC) against tumor cells is supposed to contribute to clinical efficacy of therapeutic CD20 antibodies of IgG1 isotype. However, some tumor-targeting antibodies such as cetuximab do not trigger CDC. Due to its prominent molecular flexibility and its longer hinge region human IgG3 gains advantage over human IgG1 in promoting activation of the complement system. Here, we investigated the cytotoxic activity of IgG3 isotype switch variants of the therapeutic antibodies rituximab (C2B8) and cetuximab (C225).

Methods: CDC against tumor cells was assessed by 3 hours ⁵¹chromium release assays utilizing 25% v/v human serum. Antibody mediated deposition of C1q, C4b, C3b, factor Bb or C5b-9 on tumor cells as well as the release of anaphylatoxins C3a, C4a and C5a were analyzed by flow cytometry.

Results: Both IgG1 and IgG3 variants of rituximab or cetuximab demonstrated similar efficiency in CD20 or EGFR binding, respectively. In the case of CD20 antibodies, C2B8-IgG3 induced stronger CDC against CD20 low-expressing cells, especially against freshly isolated CLL cells, than C2B8-IgG1, while no difference between both isotypes was detected against CD20 high-expressing cells. Importantly, CDC was also significantly triggered in an autologous setting against freshly isolated CLL cells by the IgG3 but not by the IgG1 version of rituximab. Furthermore, in the case of EGFR-targeting antibodies, only anti-EGFR-IgG3 but not anti-EGFR-IgG1 triggered significant CDC against some, but not all tested cell lines. CDC triggered by anti-EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement-regulatory proteins (mCRPs) CD55 and CD59, but not CD46. Notably, anti-EGFR-IgG3 promoted strong C1q and C3b but relatively low C4b and C5b-9 deposition on target cells. Furthermore, anti-EGFR-IgG3 triggered C4a release on all tested cell lines, but failed to induce C3a and C5a release on CD55/CD59 highly expressing cell lines. RNAi-induced knock-down or over-expression of mCRPs revealed CD55 expression to be a pivotal determinant of anti-EGFR-IgG3 triggered CDC and to force a switch from classical complement pathway activation to C1q-dependent alternative pathway amplification.

Conclusions: In conclusion, an isotype switch from human IgG1 to human IgG3 is accompanied by improvement of complement activating capacities of CD20 as well as EGFR targeting antibodies. However, despite of its strong C1q fixing capacity, anti-EGFR-IgG3 lacks the ability to efficiently deposit C4b on targets’ cell surfaces and thus to initiate the complement cascade on CD55 high-expressing tumor cells. These novel findings revealed human IgG3 directed against CD20 or EGFR to encompass improved cytotoxic potential with low CD55 expression on tumor cells potentially being a promising biomarker for this novel approach.