Periodic Reporting for period 3 - ENGRAB (Antibody engineering by natural selection and by design)
Reporting period: 2023-11-01 to 2025-04-30
Antibodies represent a powerful defense mechanism due to their capacity to link specific antigen recognition with effector functions and are currently developed as drugs for prophylaxis and therapy of infectious diseases. The generation of antibody diversity represents a remarkable example of protein engineering that is coupled to a stringent mechanism of clonal selection in the immune response. In this project we developed an integrated bioinformatics platform to unravel the dynamics of antibody responses in humans and used it to dissect the mechanisms that drive antibody selection and maintain immunological memory. We also continued to investigate a new type of “receptor-based antibodies” that we discovered in malaria infected individuals. These antibodies are generated by insertions into the antibody genes of large DNA fragments encoding the pathogen receptors and therefore display potent and broad neutralizing activity. Based on these observations, we developed a new bispecific antibody format with the insertion of a second antigen binding site in the VH-CH1 elbow to increasing the antibody avidity through dual binding to the target molecules. Finally, we use a novel screening approach to identify antibodies that broadly neutralize human and animal coronaviruses with the aim of developing new therapeutics and improving vaccine design. In this context we are particularly interested to dissect the relative contribution of viral neutralization versus effector function in antibody-mediated protection from viral infection.
Using single-cell sequencing and isolation of specific antibodies, we found that the memory B cell repertoire was dominated by large IgM, IgA and IgG2 clonal families, whereas IgG1 families specific for recall antigens were small. Analysis of multiyear samples demonstrated stability of memory B cell families and revealed that a large fraction of recently generated plasmablasts derives from long-term memory B cell families and is found recurrently in the absence of antigenic stimulation. We also found that, within large clonal families, somatic mutations generate individual clones with distinct specificities for microbial pathogens or commensals. Surprisingly, the predicted unmutated ancestor did not bind to the same antigen suggesting that somatic mutations diversify the naive B cell repertoire before rather than after antigen encounter. Collectively, these studies provide a systematic description of the structure, stability and dynamics of the human memory B cell pool and support a new model for the maintenance of plasma cells and antibody levels (Figure 1).
Using two complementary approaches we further investigated receptor-based antibodies. On one hand, we searched for insertion of inhibitory receptors other that LAIR1 and found antibodies generated by insertion of LILRB1 domains that were mapped to distinct families of RIFINS. On the other hand, we used an unbiased sequencing approach to identify different classes of non-VDJ inserts in 80% of individuals at frequencies of 1 in 104 to 105 B cells.
To dissect the antibody response to SARS-CoV2 we used a new screening strategy and isolated from immune donors several monoclonal antibodies that bind to all human-infecting coronavirus spike proteins. All these antibodies recognize the fusion peptide of the S protein and acquire affinity and breadth through somatic mutations and two of them neutralize Omicron BA.1 and BA.2 viruses and reduce viral burden and pathology in vivo. Interestingly, the analysis of the unmutated ancestor indicates that affinity and breadth were acquired through somatic mutations. Structural and functional analyses showed that these antibodies bind to a cryptic epitope hidden in prefusion stabilized spike, which becomes exposed upon ACE2 binding. Using the structural information gained in this study we plan to engineer the S protein to better expose the fusion peptide region.
Using two complementary approaches we further investigated receptor-based antibodies. On one hand, we searched for insertion of inhibitory receptors other that LAIR1 and found antibodies generated by insertion of LILRB1 domains that were mapped to distinct families of RIFINS. On the other hand, we used an unbiased sequencing approach to identify different classes of non-VDJ inserts in 80% of individuals at frequencies of 1 in 104 to 105 B cells.
To dissect the antibody response to SARS-CoV2 we used a new screening strategy and isolated from immune donors several monoclonal antibodies that bind to all human-infecting coronavirus spike proteins. All these antibodies recognize the fusion peptide of the S protein and acquire affinity and breadth through somatic mutations and two of them neutralize Omicron BA.1 and BA.2 viruses and reduce viral burden and pathology in vivo. Interestingly, the analysis of the unmutated ancestor indicates that affinity and breadth were acquired through somatic mutations. Structural and functional analyses showed that these antibodies bind to a cryptic epitope hidden in prefusion stabilized spike, which becomes exposed upon ACE2 binding. Using the structural information gained in this study we plan to engineer the S protein to better expose the fusion peptide region.
The systematic analysis of memory B cells and circulating plasma blasts reveals two novel and unexpected aspects: i) the prevalence of large clonal families that diversify before antigen encounter as a characteristic of T-independent responses and ii) the recurrent production of plasma blasts from memory B cell families that, together with long-lived plasma cells, may contribute to maintenance of serum antibody levels.
The discovery of antibodies made by templated insertions is a breakthrough since it represents a new mechanism of antibody diversification. This study also led to the discovery that the malaria parasite targets human inhibitory receptors LAIR1 and LILRB1 using 200 polymorphic and clonally expressed RIFINS. Based on the most recent sequencing data, we expect to find other examples in response to pathogens other than malaria. The structural analysis of antibodies with LILRB1 inserts demonstrates the possibility to engineer antibodies in the VH-CH1 elbow.
The discovery of neutralizing antibodies with exceptional breadth that bind to the fusion peptide region of alpha and beta-coronaviruses sheds light on the remarkable plasticity of the S protein with implications to produce an improved vaccine capable of eliciting broad anti-coronavirus immunity.
The discovery of antibodies made by templated insertions is a breakthrough since it represents a new mechanism of antibody diversification. This study also led to the discovery that the malaria parasite targets human inhibitory receptors LAIR1 and LILRB1 using 200 polymorphic and clonally expressed RIFINS. Based on the most recent sequencing data, we expect to find other examples in response to pathogens other than malaria. The structural analysis of antibodies with LILRB1 inserts demonstrates the possibility to engineer antibodies in the VH-CH1 elbow.
The discovery of neutralizing antibodies with exceptional breadth that bind to the fusion peptide region of alpha and beta-coronaviruses sheds light on the remarkable plasticity of the S protein with implications to produce an improved vaccine capable of eliciting broad anti-coronavirus immunity.