Interaction with Fc gamma RIIB Is Critical for the Agonistic Activity of Anti-CD40 Monoclonal Antibody

Division of Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, United Kingdom.
The Journal of Immunology (Impact Factor: 4.92). 08/2011; 187(4):1754-63. DOI: 10.4049/jimmunol.1101135
Source: PubMed


A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.

Download full-text


Available from: Mark Cragg
  • Source
    • "For instance, CD40-signaling induced by soluble CD40L (sCD40L) was potentiated ~10-fold upon secondary cross-linking of CD40L into higher order multimers [15-17]. In line with this, CD40 signaling induced by anti-CD40 antibodies critically depends on the presence of Fc-receptor positive cells [18]. Based on these crosslinking requirements for CD40/CD40L signaling, CD40L has also been evaluated in a proof-of-concept study with a fibroblast activation protein (FAP)-targeted scFv:CD40L fusion protein. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Stimulation of CD40 can augment anti-cancer T cell immune responses by triggering effective activation and maturation of antigen-presenting cells (APCs). Although CD40 agonists have clinical activity in humans, the associated systemic activation of the immune system triggers dose-limiting side-effects. To increase the tumor selectivity of CD40 agonist-based therapies, we developed an approach in which soluble trimeric CD40L (sCD40L) is genetically fused to tumor targeting antibody fragments, yielding scFv:CD40L fusion proteins. We hypothesized that scFv:CD40L fusion proteins would have reduced CD40 agonist activity similar to sCD40L but will be converted to a highly agonistic membrane CD40L-like form of CD40L upon anchoring to cell surface exposed antigen via the scFv domain. Targeted delivery of CD40L to the carcinoma marker EpCAM on carcinoma cells induced dose-dependent paracrine maturation of DCs ~20-fold more effective than a non-targeted control scFv:CD40L fusion protein. Similarly, targeted delivery of CD40L to the B cell leukemia marker CD20 induced effective paracrine maturation of DCs. Of note, the CD20-selective delivery of CD40L also triggered loss of cell viability in certain B cell leukemic cell lines as a result of CD20-induced apoptosis. Targeted delivery of CD40L to cancer cells is a promising strategy that may help to trigger cancer-localized activation of CD40 and can be modified to exert additional anti-cancer activity via the targeting domain.
    Full-text · Article · Apr 2014 · Molecular Cancer
  • Source
    • "In contrast to these activating FcγRs that function as immunostimulatory receptors, inhibitory FcγRIIb is reported to function as an immunomodulatory receptor (Li and Ravetch, 2011; White et al., 2011). The inhibitory receptor FcγRIIb is the only IgG Fc receptor expressed on B-cells and plays a critical role in regulating B-cell homeostasis (Heyman, 2003; Nimmerjahn and Ravetch, 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Engaging inhibitory FcγRIIb by Fc region has been recently reported to be an attractive approach for improving the efficacy of antibody therapeutics. However, the previously reported S267E/L328F variant with enhanced binding affinity to FcγRIIb, also enhances binding affinity to FcγRIIa(R131) allotype to a similar degree because FcγRIIb and FcγRIIa(R131) are structurally similar. In this study, we applied comprehensive mutagenesis and structure-guided design based on the crystal structure of the Fc/FcγRIIb complex to identify a novel Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131). This novel variant has more than 200-fold stronger binding affinity to FcγRIIb than wild-type IgG1, while binding affinity to FcγRIIa(R131) and FcγRIIa(H131) is comparable with or lower than wild-type IgG1. This selectivity was achieved by conformational change of the CH2 domain by mutating Pro to Asp at position 238. Fc variant with increased binding to both FcγRIIb and FcγRIIa induced platelet aggregation and activation in an immune complex form in vitro while our novel variant did not. When applied to agonistic anti-CD137 IgG1 antibody, our variant greatly enhanced the agonistic activity. Thus, the selective enhancement of FcγRIIb binding achieved by our Fc variant provides a novel tool for improving the efficacy of antibody therapeutics.
    Full-text · Article · Jun 2013 · Protein Engineering Design and Selection
  • Source
    • "It is therefore possible that in vivo FcR-expressing cells would provide cross-linking stimulus similar to secondary Abs in vitro inducing cell death in the tumor cell. It has been shown that the in vivo activity of the agonistic CD40 mAb depends on cross-linking provided by FcγRIIb demonstrating that cross-linking by FcγRs can be an important mechanism of action of mAb therapy [79] [80]. For CD20 mAb therapy, expression of FcγRs are needed as shown by FcRγ-chain KO mice, but whether this is required for active signaling or whether FcγR are purely serve as a scaffold to provide cross-linking was unclear. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Therapeutic monoclonal antibodies (mAbs) that target the CD20 antigen on B cells are successfully used in the clinic for the depletion of B cells to treat various forms of cancer and autoimmune diseases. The first CD20 mAb, approved by the FDA in 1998, was rituximab (RTX) and since then it has been widely used to treat more than one million patients thus far. The success of RTX has led to a general interest in the mechanism of action of CD20 mAbs. CD20 mAbs can induce tumor killing via various mechanisms, such as direct induction of apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent lysis (CDC). Although we now understand these mechanisms better, it is still unclear which of these mechanisms is the most important for in vivo RTX action. Not every patient respond to RTX treatment and eventually the overwhelming majority will experience a relapse. Therefore, there is an urgent need to improve the efficacy of CD20 mAbs. This review aims to summarize our current understanding on the mechanism of action of CD20 mAbs.
    Full-text · Article · Dec 2012 · American Journal of Cancer Research
Show more