A Complement-Optimized EGFR Antibody Improves Cytotoxic Functions of Polymorphonuclear Cells against Tumor Cells

Abstract

Complement-dependent cytotoxicity (CDC) has been suggested to be an important mechanism of action of tumor-targeting Abs. However, single unmodified epidermal growth factor receptor (EGFR)–targeting IgG1 Abs fail to trigger efficient CDC. For the current study, we generated a CDC-optimized variant of the EGFR Ab matuzumab (H425 wt) by introducing amino acid substitutions K326A/E333A (H425 mt). This Ab was then used to elucidate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert Ab-dependent cell-mediated cytotoxicity (ADCC). H425 mt, but not H425 wt, significantly induced complement deposition, release of anaphylatoxins, and CDC against distinct tumor cell lines, whereas no differences in ADCC by MNC or PMN were detected. Notably, stronger cytotoxicity was induced by H425 mt than by H425 wt in whole blood assays and in experiments in which MNC or PMN were combined with serum. Although MNC-ADCC was not affected by C5 cleavage, the cytotoxic activity of PMN in the presence of serum strongly depended on C5 cleavage, pointing to a direct interaction between complement and PMN. Strong cell surface expression of C5a receptors was detected on PMN, whereas NK cells completely lacked expression. Stimulation of PMN with C5a led to upregulation of activated complement receptor 3, resulting in enhanced complement receptor 3–dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR Abs may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in Ab-based tumor therapy.

Immune Effector Functions of Human IgG2 Antibodies against EGFR

“…Frustrated phagocytosis leading to tumor cell death (trogocytosis/trogoptosis) is a documented mechanism of tumor cell killing by macrophages (42) and has recently been described as PMN's major tumor cell killing mechanism (29). Individual EGFR antibodies do not trigger CDC against human tumor cells, unless they are specifically Fc engineered (43,44). However, combinations of two noncross-blocking EGFR antibodies of human IgG1 isotype were demonstrated to trigger significant CDC (25).…”

Potent Therapeutic Activity Against Peritoneal Dissemination and Malignant Ascites by the Novel Anti-Folate Receptor Alpha Antibody KHK2805

“…Furthermore, increased complement activation was seen in SCID mice compared with Nude or BALB/cJ mice [35]. Consequently, the complement-enhanced ADCC [12], [13] in peritoneal cavity may be a mechanism underlying the effects of KHK2805 in vivo . Regarding ADCC mechanisms, Srivastava et al reported that the anti-EGFR antibody cetuximab markedly enhanced the induction of Th1-polarizing cytokines such as CXCL10 (IP-10) in cancer-NK-DC cells co-culture [36].…”

“…Natsume et al found that the engineered constant Fc region as human IgG1/IgG3 chimeric isotypes with nonfucosylated oligosaccharides (113F[−f]) possess ADCC and CDC dual-enhanced cytotoxic functions [11]. This approach is expected to be useful for generating potent therapeutic antibodies, since complement-enhanced ADCC has also been suggested as a synergistic effect of ADCC and CDC [12], [13]. However, few studies have explored generating antibodies with such dual enhancement for application to cancer treatment.…”

Neuromyelitis Optica: Deciphering a Complex Immune-Mediated Astrocytopathy

“…CDC could be critical for demyelination in NMO lesions due to the combined production of anaphylatoxins (C3a, C4a, and C5a) and opsonins (C3b, C4b, and C1q) that recruit inflammatory cells (63), enhance polymorphonuclear-mediated ADCC (64), and facilitate phagocytosis (65) (Fig. 1).…”