Antibody-Targeted TNFRSF Activation for Cancer Immunotherapy: The Role of FcγRIIB Cross-Linking

Abstract

Co-stimulation signaling in various types of immune cells modulates immune responses in physiology and disease. Tumor necrosis factor receptor superfamily (TNFRSF) members such as CD40, OX40 and CD137/4-1BB are expressed on myeloid cells and/or lymphocytes, and they regulate antigen presentation and adaptive immune activities. TNFRSF agonistic antibodies have been evaluated extensively in preclinical models, and the robust antitumor immune responses and efficacy have encouraged continued clinical investigations for the last two decades. However, balancing the toxicities and efficacy of TNFRSF agonistic antibodies remains a major challenge in the clinical development. Insights into the co-stimulation signaling biology, antibody structural roles and their functionality in immuno-oncology are guiding new advancement of this field. Leveraging the interactions between antibodies and the inhibitory Fc receptor FcγRIIB to optimize co-stimulation agonistic activities dependent on FcγRIIB cross-linking selectively in tumor microenvironment represents the current frontier, which also includes cross-linking through tumor antigen binding with bispecific antibodies. In this review, we will summarize the immunological roles of TNFRSF members and current clinical studies of TNFRSF agonistic antibodies. We will also cover the contribution of different IgG structure domains to these agonistic activities, with a focus on the role of FcγRIIB in TNFRSF cross-linking and clustering bridged by agonistic antibodies. We will review and discuss several Fc-engineering approaches to optimize Fc binding ability to FcγRIIB in the context of proper Fab and the epitope, including a cross-linking antibody (xLinkAb) model and its application in developing TNFRSF agonistic antibodies with improved efficacy and safety for cancer immunotherapy.

Keywords: CD137 (4-1BB); CD40; FcγRIIB; TNFRSF; agonist; cancer; cross-linking; immunotherapy.

FIGURE 1

Expression and interaction of TNFSF and TNFRSF members. TNFRSF members (depicted in blue on the right) contain variable numbers of cysteine-rich domains (CRD) in their ligand-binding extracellular regions. TNFSF ligands (left side shown in green) are active primarily as non-covalently associated homotrimers or homodimers to facilitate the formation of TNFRSF trimer clustering and its downstream signaling activation. TNFRSF members regulate the immune system mainly through either stimulating cell proliferation and maturation or promoting apoptotic cell death via a death domain. Also depicted are the primary cell types expressing TNFRSF and TNFSF, although the list is not comprehensive to represent the complex expression profile of each molecule, and some cell populations such as NK cells or monocytes are not included. Both TNFSF and TNFRSF members are widely and dynamically expressed in different immune cell populations.

FIGURE 2

Scheme of oligomerization of TNFSF₃–TNFRSF₃ complexes. The cell membrane TNFSF (mem TNFSF, often associated as trimer) binds to TNFRSF via the interactions between the TNF homology domain (THD) and cysteine rich domain (CRD), resulting in the formation of TNFSF₃-TNFRSF₃ complex. However, the minimum TNFSF-TNFRSF complex is insufficient to trigger signaling of some TNFRSF members including CD40, CD137, OX40, and GITR. Secondary interaction of the initial trimeric complex leads to oligomerization of the ligand-receptor complexes. The clustering of multiple TNFRSFs is necessary to activate receptor intracellular signaling pathways.

FIGURE 3

Orthologous pairs of FcγRs between human and mouse and their cellular expression. (A) In human, three groups of FcγRs have been described: FcγRI, FcγRIIA/B/C, FcγRIIIA/B. Orthologous pairs identified in mouse include human FcγRI and mouse FcγRI, human FcγRIIA and mouse FcγRIII, human FcγRIIB and Mouse FcγRIIB, and human FcγRIIIA and mouse FcγRIV. All activating FcγRs, except FcγRIIIB, are associated with an immunoreceptor tyrosine-based activation motif (ITAM) either in the intracellular domain (FcγRIIA and FcγRIIC) or associated with the common FcRγ chain (FcγRI and FcγRIIIA). There is only one inhibitory FcγR (FcγRIIB) in human or mouse. FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular domain. (B) FcγRs are expressed solely or simultaneously at the membrane of the various immune cells. FcγRI, FcγRIIA/B and FcγRIIIA are found on macrophages; FcγRIIA/B on conventional DC (cDC), and FcγRIIB (the sole FcγR expressed) on B cells. When co-expressed, activating FcγRs generally expressed more abundantly with respect to the inhibitory FcγRIIB. Despite a similar expression profile of FcγRs in human and mouse, there are some differences between the two species: FcγRIIB is not expressed on human pDCs, but on mouse pDCs; while co-expressed on the same cells, the inhibitory FcγRIIB is relatively less than activating FcγRs in human, which is not so apparent in mouse. Size of symbols of FcγRs is drawn to reflect their relative expression levels.

FIGURE 4

xLinkAb working model: the role of FcγRIIB in the oligomerization of TNFSFR-IgG-FcγRIIB complexes. xLinkAb TNFRSF agonistic antibody Fab arms binding to TNFRSF is insufficient to cluster and activate TNFRSF target; xLinkAb IgG-TNFRSF complex engages FcγRIIB with its engineered Fc selectively for FcγRIIB binding, leading to the formation of oligomic TNFRSF-IgG- FcγRIIB complexes and clustering of TNFRSF and signaling activation. The initial step should be IgG-Fab binding to TNFRSF, which is followed by TNFRSF-IgG-Fc binding to FcγRIIB forming TNFRSF-IgG-FcγRIIB complex and secondary clustering of TNFRSF. Activation of TNFRSF signaling depends on the formation of the multivalent TNFRSFs-IgGs-FcγRIIBs complexes and TNFRSF super-clustering.