Optimizing Antibodies to Efficiently Target Influenza Virus Infection

Antibody-based therapies are not new, as the use of these molecules to combat human diseases dates back to the 1890s. Antibodies are a key component of the immune system adaptive branch that recognizes a foreign molecule called antigen and mobilize various immune processes to neutralize the disease-causing agent. Nowadays, nanotechnology allow us to design and produce new classes of enhanced antibodies with better properties than the natural ones. 

Antibody structure comprises two major regions: an antigen-binding fragment (Fab) that contains a variable antigen-binding region different in every antibody, and a crystallizable fragment (Fc), a conserved structure which is similar in different antibodies and engages with other elements of the immune system and a plethora of Fc receptors present on immune and non immune cells [1]. The most common antibody that is secreted during viral infections is called immunoglobulin G (IgG), and engages a subfamily of Fc receptors called FcγRs. Three different activating FcγRs have been described: FcγRI, FcγRIIa and FcγRIIIa; whereas only one inhibitory FcγR (FcγRIIb) has been found. After IgG binding to FcγRs, the balance of activating to inhibitory engagement determines the responses of cells types that have both types of FcγRs. 

Antibody-mediated immune responses against viral pathogens. Yu X, et al. Engineered antibodies to combat viral threats. Nature. 2020 Dec;588(7838):398-399. doi: 10.1038/d41586-020-03196-2

IgG can mediate pleiotropic effects as a result of the diversity of Fc binding molecules that engage the Fc domain. Natural IgG heterogeneity contribute to the efficacy of polyclonal IgG responses against viral infections, and provides a mechanism for the recognition of diverse viral antigens and triggering of several effector pathways. Furthermore, recent advances in nanotechnology and bioengineering allows for the development of antibodies that selectively bind to specific neutralizing viral epitopes coupled to Fc modifications to facilitate the engagement of specific FcγRs and optimize the potency of these therapeutic agents. 

Previous studies demonstrated that antibody enhancement affinity for FcγRIIIa results in an improved antibody-dependent cell phagocytosis of tumor cells by macrophages, as well as an enhanced antibody-dependent cell cytotoxicity by natural cells [2]. It is possible that enhancing the ability of the Fc domain to engage and activate FcγRIIIa would also lead to an increased therapeutic efficacy of protective antiviral antibodies. 

At present antibodies have been mainly used to combat cancer and autoimmunity, with a great medical impact. These advances have been followed by the use of antibodies acting as immune-checkpoint blockers that help to unleash immune responses against tumor cells. In a recent work published in Nature [3], Bournazos et al. tried to elucidate the mechanisms underlying antibody action by engineered IgG against influenza antigens with modified FcγRs. Antibodies against influenza virus epitopes from hemagglutinin (HA) and neuraminidase (NA) have been shown to confer broad and potent antiviral activity against diverse influenza strains, requiring Fc effector activity to provide full protection from lethal viral challenge [4]. Although previous studies demonstrated that these antibodies depend on activating, but not inhibitory, FcγRs for activity [5, 6], the cell types and specific FcγRs that contribute to the antiviral activity of these antibodies remain unknown. 

Improved protective activity of engineered IgGs with increase FcγRIIa affinity. Different Fc variants will bind with different affinity to each FcγR. Bournazos S, et al. Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection. Nature. 2020 Dec;588(7838):485-490. doi: 10.1038/s41586-020-2838-z. 

Authors used a mouse model in which only human FcγRs are expressed, in a pattern that recapitulates the ones seen in human tissues as accurately as possible despite the inherent limitations and differences in the absolute FcγR expression levels between humans and the FcγR humanized mice. Mice were treated with IgG anti-influenza antibodies which Fc is expressed as a series of variants with selective binding affinity to specific human FcγRs before lethal challenge with influenza virus. Mice treated with IgGs targeting HA (hereafter FY1 [5]) showed enhanced protection when the Fc is modified to engage the FcγRIIa (GA variant [7]), alone or in combination with enhanced FcγRIIIa binding (GAALIE variant [8]). They found that enhancing FcγRIIIa alone does not provide enhanced protection, compared with the wild-type human IgG, and the administration of a FcγRIIa blocking antibody reduce the ability of IgG GA variants of FY1 to protect FcγR humanized mice against lethal influenza challenge. No protection is observed when the Fc is modified to abrogate FcγR binding (GRLR variant) or engineered to engage the inhibitory FcγRIIb (V11 variant [9]). Quantification of anti-influenza IgG levels in mice serum after infection revealed comparable levels among the different Fc domain variants, indicating that the observed effects could nor be attributed to differential antibody half-lives or in vivo stability. Authors tested engineered IgGs against other viral epitopes with the GA or GAALIE variants, which showed an enhanced protective activity compared with their wild-type IgG counterparts, thus suggesting that the FcγR mechanisms by which anti-influenza IgG confers protection against infection are conserved among antibodies with differential in vitro neutralization potency and epitope specificity. 

Engagement of FcγRIIa by Fc-engineered monoclonal antibodies drive dendritic cell maturation and protective CD8+ T cell responses. Bournazos S, et al. Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection. Nature. 2020 Dec;588(7838):485-490. doi: 10.1038/s41586-020-2838-z.

Once they found the key FcγR mediating viral protection, they tried to assess which cells within the immune system used these receptors to exert their action. Depletion of neutrophils, had no effect on the antiviral activity of FY1 GAALIE variant (specific for FcγRIIa/FcγRIIIa binding), suggesting that these cells are unlikely to contribute to the observed FcγRIIa mediated antiviral protection. They studied the effect of FcγRIIa engagement on the functional activity of various dendritic cell (DC) subsets, and found that treatment with the FY1 GA variant before influenza challenge resulted in DC maturation, whereas the same FY1 expressed either as wild-type or with the GRLR variant (that abrogates FcγR binding) did not result in DC maturation. 

As these results suggest that an antiviral IgG modified to enhance DC maturation by FcγRIIa engagement can induce an adaptive response that results in the induction of protective T cell immunity, authors next characterized the T cell responses in the lungs of FcγR humanized mice treated with the engineered antibodies. GAALIE variant induced increased activation of both CD8+ (cytotoxic) and CD4+ (helper) T cells, whereas neither wild-type IgG nor the GRLR variant showed evidence of robust induction of T cell responses. Furthermore, depletion of CD8+ T cells but not CD4+ T cells resulted in the loss of enhancement of the GA or GAALIE Fc variants, demonstrating that the improved protection is mediated by induction of CD8+ T cells responses.

Evaluation of FY1 GAALIE variants in models of therapy or prevention of influenza infection. In the upper row, % weight and survival were assessed when using the engineered antibodies as a therapy, whereas in the lower row data depicts its use as a prophylactic agent. Bournazos S, et al. Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection. Nature. 2020 Dec;588(7838):485-490. doi: 10.1038/s41586-020-2838-z. 

As Fc-engineered variants with increased affinity for FcγRIIa can enhance adaptive T cell responses by activation of FcγRIIa-expressing DCs, it is important to determine whether such variants could also modulate disease pathogenesis through inappropriate amplification of host inflammatory responses that are elicited in response to virus infection. To assess it, authors analyzed the in vivo activity of IgG variants in FcγR-humanized mice with stablished influenza infection. They found that wild-type IgG was unable to rescue mice from lethal influenza infection, while GAALIE variants exhibited a dose-dependent therapeutic benefit. In addition, the stimulation of CD8+ T cell responses had no pathogenic consequences, providing meaningful and robust protection from stablished infection with no adverse effects.

This work paves the way for the application of engineered antibodies to combat viral infections by targeting specific branches of the immune system. Although influenza is one of the most common seasonal viruses that we must fight against, these molecules could be used with other relevant viruses. Furthermore, the knowledge regarding FcγR engagement will be useful for the application of engineered antibodies in the treatment of other relevant diseases. 


1. Nimmerjahn, F. and J.V. Ravetch, Fcgamma receptors as regulators of immune responses. Nat Rev Immunol, 2008. 8(1): p. 34-47.

2. Goede, V., et al., Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. N Engl J Med, 2014. 370(12): p. 1101-10.

3. Bournazos, S., et al., Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection. Nature, 2020. 588(7838): p. 485-490.

4. Corti, D., et al., A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science, 2011. 333(6044): p. 850-6.

5. Kallewaard, N.L., et al., Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes. Cell, 2016. 166(3): p. 596-608.

6. DiLillo, D.J., et al., Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. J Clin Invest, 2016. 126(2): p. 605-10.

7. DiLillo, D.J. and J.V. Ravetch, Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal Effect. Cell, 2015. 161(5): p. 1035-1045.

8. Weitzenfeld, P., S. Bournazos, and J.V. Ravetch, Antibodies targeting sialyl Lewis A mediate tumor clearance through distinct effector pathways. J Clin Invest, 2019. 129(9): p. 3952-3962.

9. Mimoto, F., et al., Engineered antibody Fc variant with selectively enhanced FcgammaRIIb binding over both FcgammaRIIa(R131) and FcgammaRIIa(H131). Protein Eng Des Sel, 2013. 26(10): p. 589-98.

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