In vivo engineering of chimeric antibody reprogrammed (CAR) B cells with fully tunable antibody response

The longstanding paradigm in vaccine development has been the introduction of immunogens that culminates in generation of pathogen-binding antibodies (Ab). While the precise format of the antigen presentation differs, all vaccines rely on somatic hypermutation of immunoglobulin genes in B cells to generate Ab with greater affinity, avidity or anti-pathogen activity. Unfortunately, the natural variations in our immune system leads to highly variable Ab responses, both in magnitude of the induced Ab response and the breadth/potencies of the specific Ab clones generated, resulting in variable efficacy. Here, we seek to overcome these shortcomings by directly reprogramming circulating B cells to secrete specific potent Ab of interest, including stable integration of Ab-encoding transgenes into circulating B-cells. The resulting chimeric antibody reprogrammed (CAR) B cells can produce Ab with magnitude, affinity and effector functions that can all be controlled with molecular/genetic specificity. We refer to this as in vivo engineering of CAR-B cells. The key to realizing this vision is a delivery platform that can facilitate highly specific transduction of B- cells in vivo. We have engineered lentivirus (LV) vectors that effectively transduce circulating immune cells. Our first-generation system achieved highly specific transduction of circulating T-cells even at very low virus:cell ratio, and generated substantial CAR-T cells in the circulation and in tumor of immune-deficient NSG mice with highly aggressive BV173 lymphoma, leading to effective suppression of the tumor and prolonged survival. We have since improved upon our first-generation system with a second-generation LV system incorporating the Nipah virus fusion protein. Nipah LV (NLV), coupled with a proprietary combo of transduction enhancers (TE), efficiently and specifically transduced non-activated B-cells in human PBMCs. In pilot studies, a single injection of NLV+TE into NSG mice with circulating human PBMCs lead to effective transduction of >0.5% of all circulating B-cells. We have also demonstrated we can harness CRISPR/Cas9 to insert Ab transgene into a well conserved site in B cells, leading to functional B cells that can react to antigens. In this proposal, we seek to perform key enabling studies that substantiate our proposed CAR-B strategy. In Aim 1, we will engineer NLV that targets different B-cell populations, and identify the combination of NLV and TE that maximizes transduction of key human B-cells. We will then assess delivery of transgene encoding for a potent RSV neutralizing Ab (nAb) either by non-specific integration or site-specific integration. We will then advance into animal studies in Aim 2, where we will assess transduction efficiency and specificity in NSG mice infused with circulating human PBMCs. We will quantify for the levels of the nAb in serum over time, and assess protection against infection by respiratory syncytial virus (RSV). If successful, our work will advance an alternative strategy for achieving highly tunable immune protection in cases where specificity of the variable domain or Ab isotype is essential, or in individuals who do not respond to traditional vaccination.