Interactions between the crystallizable fragment (Fc) domain of antibodies and a plethora of cellular Fc receptors (FcRs) or soluble proteins form a critical link between humoral and innate immunity. ...In particular, the immunoglobulin G Fc domain is critical for the clearance of target cells by processes that include (
a
) cytotoxicity, phagocytosis, or complement lysis; (
b
) modulation of inflammation; (
c
) antigen presentation; (
d
) antibody-mediated receptor clustering; and (
e
) cytokine release. More than 30 Fc-engineered antibodies aimed primarily at tailoring these effects for optimal therapeutic outcomes are in clinical evaluation or have already been approved. Nonetheless, our understanding of how FcR engagement impacts various immune cell phenotypes is still largely incomplete. Recent insights into FcR biology coupled with advances in Fc:FcR structural analysis, Fc engineering, and mouse models that recapitulate human biology are helping to fill in existing knowledge gaps. These advances will provide a blueprint on how to fine-tune the Fc domain to achieve optimal therapeutic efficacy.
The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following SARS-CoV-2 infection are unknown. Proteomic deconvolution of the IgG ...repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor-binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an N-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multi-donor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.
The pharmacokinetic properties of antibodies are largely dictated by the pH-dependent binding of the IgG fragment crystallizable (Fc) domain to the human neonatal Fc receptor (hFcRn). Engineered Fc ...domains that confer a longer circulation half-life by virtue of more favorable pH-dependent binding to hFcRn are of great therapeutic interest. Here we developed a pH Toggle switch Fc variant containing the L309D/Q311H/N434S (DHS) substitutions, which exhibits markedly improved pharmacokinetics relative to both native IgG1 and widely used half-life extension variants, both in conventional hFcRn transgenic mice and in new knock-in mouse strains. engineered specifically to recapitulate all the key processes relevant to human antibody persistence in circulation, namely: (i) physiological expression of hFcRn, (ii) the impact of hFcγRs on antibody clearance and (iii) the role of competing endogenous IgG. DHS-IgG retains intact effector functions, which are important for the clearance of target pathogenic cells and also has favorable developability.
IgG antibodies mediate the clearance of target cells via the engagement of Fc gamma receptors (FcγRs) on effector cells by eliciting antibody-dependent cellular cytotoxicity and phagocytosis (ADCC ...and ADCP, respectively). Because (i) the IgG Fc domain binds to multiple FcγRs with varying affinities; (ii) even low Fc:FcγRs affinity interactions can play a significant role when antibodies are engaged in high avidity immune complexes and (iii) most effector cells express multiple FcγRs, the clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIaV158 and hFcγRIIIaF158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIaV158. Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIaV158 allotype with a somewhat lower KD than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIaV158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIaF158 variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, Fc3aV-formatted Rituximab and Herceptin perform ADCP toward CD20- and Her2-expressing cancer cells, respectively, at a level comparable to that of the respective wild-type antibodies. We further show that hFcγRIIIa activation plays a significant role on ADCC by human peripheral monocytes. Our data highlight the utility of Fc3aV and other similarly engineered exquisitely selective, aglycosylated Fc variants toward other hFcγRs as tools for the detailed molecular understanding of hFcγR biology.
We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (Vsub.H:Vsub.L) amplicons are ...expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.
Fc-mediated effector functions are important for the clearance of pathologic cells by therapeutic IgG antibodies through two mechanisms: via the activation of the classical complement pathway and ...through the binding to Fcγ receptors (FcγRs) which mediate clearance of targeted cells by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by effector cells such as macrophages, NK cells, and other leukocytes subsets. Complement activation results in direct cell killing through the formation of the membrane attack complex (MAC, complement-dependent cytotoxicity or CDC) and in the deposition of complement opsonins on pathogen surfaces. The latter are recognized by complement receptors on effector cells in turn triggering complement-dependent cell cytotoxicity and phagocytosis (CDCC and CDCP, respectively). Little is known about the role of CDCC and CDCP on therapeutic antibody function because on the one hand, IgG isotype antibodies bind to both FcγR and C1q to activate the complement pathway, and on the other, immune cells express complement receptor as well as FcγRs. We engineered IgG1 Fc domains that bind with high affinity to C1q but have very little or no binding to FcγR. To this end, we employed display of IgG in E. coli (which lack protein glycosylation machinery) for the screening of very large libraries (>2 × 10
) of randomly mutated human Fc domains to isolate Fc variants that bind to C1q. Herein we introduce and describe the method.
Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective ...binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.
We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (V
:V
) amplicons are expressed in ...a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.
T-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and ...surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV). However, TCRs are more difficult to express and engineer than antibodies, and advanced methods are needed to enable their widespread use. Here, we engineered the human CMV–specific TCR RA14 for high-affinity and robust soluble expression. To achieve this, we adapted our previously reported mammalian display system to present TCR extracellular domains and used this to screen CDR3 libraries for clones with increased pMHC affinity. After three rounds of selection, characterized clones retained peptide specificity and activation when expressed on the surface of human Jurkat T cells. We obtained high yields of soluble, monomeric protein by fusing the TCR extracellular domains to antibody hinge and Fc constant regions, adding a stabilizing disulfide bond between the constant domains and disrupting predicted glycosylation sites. One variant exhibited 50 nm affinity for its cognate pMHC, as measured by surface plasmon resonance, and specifically stained cells presenting this pMHC. Our work has identified a human TCR with high affinity for the immunodominant CMV peptide and offers a new strategy to rapidly engineer soluble TCRs for biomedical applications.
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