Arman Izadi- Institutionen för kliniska vetenskaper, Lund

Title: Enhancing Fc-mediated effector functions of monoclonal antibodies targeting SARS-COV-2 and Streptococcus pyogenes through antibody engineering

Main supervisor: Pontus Nordenfelt  

Reviewers: Marcus Järås, Senior lecturer, Associate professor, Division of Clinical Genetics
                     Magnus Paulsson, Associate professor, Clinical assistent, Infection Medicine

Abstract

Background

In infection medicine, there exist several pathogens where standard treatment is insufficient to reduce mortality. One such example is the gram-positive bacteria Streptococcus pyogenes, which can cause invasive skin infection; despite the bacterium being sensitive to antibiotics and the fact that it can infect even healthy and young humans, the mortality rate is over 10%. New therapeutics are needed to complement the standard of care for this disease. In the context of viral infections, the SARS-COV-2 virus paralyzed our societies when it emerged in the winter of 2020. Although adequate vaccines exist, there are some populations of patients who cannot generate an adequate immune response and need therapies when they succumb to COVID-19. Very few options exist for these patients since the previous use of monoclonal antibodies is not functioning due to mutations in the current circulating SARS-COV-2 variants. There is thus an unmet need for both these pathogens in terms of therapeutics and more preclinical research is needed to develop such therapies. In this thesis project, we have focused on engineering promising monoclonal antibodies to see if they can be further developed in the future to be used to treat patients with COVID-19 and invasive Streptococcal infections.

Aim

1. To develop a pipeline to engineer the Fc domain of monoclonal antibodies to enhance effector functions
2. Study the Fc-mediated opsonization of engineered monoclonals using a newly developed phagocytosis assay called PAN
3. Study the importance of non-neutralizing functions of anti-SARS-COV-2 mAbs in protection against the pathogen

Preliminary results

In previous work, 8 monoclonal antibodies against the spike protein of SARS-COV-2 were generated by the lab from convalescent patients who had undergone infection and survived from the first wave in March 2020. Using these antibodies to start with, we have exchanged their constant domains from IgG1 backbone to IgG3 using molecular recombination techniques relating to aim 1). Relating to aim 2) These antibodies expressed as IgG3 did show much stronger Fc-mediated phagocytosis, complement phagocytosis, and neutrophil phagocytosis (including complement and Fc-mediated phagocytosis). Interestingly, using the IgG3 antibodies in cocktails further enhanced their output compared to the best-performing IgG3 mAb, which was not seen with the IgG1 mAbs. Furthermore, in vivo studies revealed that Fc-mediated effector functions of antibodies that are not neutralizing strongly protect against lethal SARS-COV-2 infection. This paper, paper 1, was published in PNAS. Building on paper 1, we proceeded to investigate if the protective non-neutralizing mAbs are protective and retain function against the mutated SARS-COV-2 variants. This work is a preprint in revision, and in vitro data shows that our mAb candidates retain binding and function to the current circulating (listed as paper 3). Finally, paper 2 focuses on an antibody targeting the M protein of Streptococcus pyogenes. We observed that different subclasses of this antibody had different properties in binding and Fc-mediated opsonization of the bacteria. Interestingly the IgG3 antibody had 13-fold reduced binding but 6-fold higher function compared to the IgG1 original mAb. This increase in efficacy was revealed to be due to its longer hinge region, granting it great flexibility in 3D space which in theory facilitates interaction with Fc-receptors on phagocytes. Using this information, we engineered an IgG1 antibody but with a longer IgG3 hinge; this hybrid antibody had an IgG1 higher affinity and IgG3’s opsonic function. In vivo models show that this hybrid antibody was vastly superior to both parent IgG1 and IgG3 in protecting mice from system bacterial dissemination in a skin infection model. The hybrid IgG1-IgG3 construct was also shown to enhance anti-SARS-COV-2 mAbs in terms of Fc-mediated opsonization potently. This work has been accepted in principle in Nature Communications. The hybrid IgG1-IgG3 anti-sars-cov-2 mAbs are being further studied for a fourth manuscript at an early stage.

Significance

• We reveal a novel mechanism of protection against SARS-COV-2 by reporting the first instance of a protective non-neutralizing antibody against the RBD domain of the spike protein.
• We show how Fc-mediated effector functions against SARS-COV-2 and Streptococcus pyogenes can be enhanced by switching constant domains from IgG1 to IgG3 as well as by only elongating the hinge.
• Our work increases our understanding of which qualities are important for antibodies to be effective in vitro and in vivo against the SARS-COV-2 and S. pyogenes pathogens, respectively.

Accepted publications included in the thesis

1. Izadi, A. et al. Subclass-switched anti-spike IgG3 oligoclonal cocktails strongly enhance Fc-mediated opsonization. PNAS. 2023 Apr 11;120(15):e2217590120. doi: 10.1073/pnas.2217590120.
2. Izadi, A. et al. The hinge-engineered IgG1-IgG3 hybrid subclass IgGh₄₇ potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 monoclonals antibodies. Nature Communications. Accepted in principle in

Preprints in revision

Izadi, A., Godzwon, M., Ohlin, M. & Nordenfelt, P. Protective non-neutralizing mAbs targets conserved opsonic epitopes on SARS-