Abstract B113: Protective serum responses by CRISPR/Cas9-edited primary B cells expressing antibodies of choice
Authors: Hartweger Harald , Jankovic Mila , Nussenzweig Michel C. 
Published: February 2019

Abstract
Attempts to create an effective, traditional vaccine against several pathogens such as HIV have so far failed and current research suggests that in the case of HIV protective serum responses mediated by broadly-neutralizing antibodies (bNAbs) will only be achieved by multiple, sequential immunizations. Identifying, producing and implementing a regimen consisting of a series of immunogens that lead to a relatively consistent, protective immune response in a heterogeneous population will be highly challenging. We are developing an alternative approach in which autologous primary B cells are edited to express an antibody of choice, in this case a bNAb against HIV. Following adoptive transfer the individual is immunized with a cognate antigen to activate the edited B cells and generate a protective, humoral immune response to HIV. This method can be adapted for any combination of high-affinity antibody and antigen. We have developed a CRISPR/Cas9-based method to target the endogenous IgH and IgK loci in mice. Both loci must be targeted in order to avoid producing B cells with chimeric and potentially self-reactive receptors. In addition, both of the bNAb encoding chains must be coordinately expressed. To do so we assemble an RNP consisting of Cas9 and single stranded DNA (ssDNA) encoding IgK followed by a P2A self-cleaving oligopeptide and the VDJ exon. A separate Cas9/guide complex is included to ablate the endogenous IgK locus. Since the heavy chain variable region is knocked into the endogenous locus in between the VDJ and constant regions normal isotype switching can occur and only on-target integrations can express the full BCR of choice. B cell survival is dependent on Ig expression and therefore all of the B cells that have lost Igk expression and do not properly integrate the bNAb will die. Using this approach, we have created cells carrying a synthetic intermediate of the anti-HIV bNAb 3BNC60. Transfer of 3BNC60 synthetic intermediate-edited cells into congenically marked wild-type mice followed by immunization with a cognate Env-derived antigen showed that the edited cells were able to start an immune response and create 3BNC60 synthetic intermediate serum antibody with the predicted epitope specificity of different isotypes. One of the potential benefits to this approach is that it would not be limited to a single monoclonal. B cells could be edited to express different antibodies and then be transferred together. Our initial experiments show that the approach is feasible but the process must be optimized to become practical; particularly, the number of correctly edited B cells needs to be increased for efficient transfer. We are also working on translating these experiments from mice to macaques in order to do protection experiments.We believe the approach presented here would have wide-ranging implications for both research and public health. It boasts several advantages compared to traditional vaccination, particularly in the context of HIV as it skips bNAb evolution, can potentially combine more than one type of bNAb and, as opposed to a sequential immunization, could potentially be achieved in a two-visit procedure (blood draw followed by injections). Since the approach uses primary B cells that are modified in their endogenous loci, which then mature into plasma cells in vivo, and not other cell types ectopically expressing an antibody, the immune response should in principle be boostable, long-lived and allow for bNAb diversification by somatic hypermutation. As opposed to an immunogen series in a sequential immunization, our approach is not limited to a particular bNAb and relies only on a functional bNAb and antigen combination as opposed to an entire series of antigens. Lastly, this approach is not limited to HIV and could be applicable to other diseases in which constant antibody intervention is indicated.
Citation Format: Harald Hartweger, Mila Jankovic, Michel C. Nussenzweig. Protective serum responses by CRISPR/Cas9-edited primary B cells expressing antibodies of choice [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B113.