B Cell Engineering for a Regulated, Potent and Evolving Response to HIV

Broadly Neutralizing antibodies (bNAbs) are an effective class of antibodies against HIV and their administration to infected individuals is associated with good clinical outcomes. However, high cost of treatment and repeated injections are predictable. Moreover, viral escape limits the therapeutic effect of injected bNAbs.

To tackle these issues, we engineer B cells to be bNAbs expressers in an endogenously regulated manner. These cells would undergo differentiation to germinal center B cells, enhancing the response by Class Switch Recombination (CSR) and Somatic Hypermutation (SHM), facilitating affinity maturation and countering pathogen escape. The cells would then differentiate to plasma cells or memory B cells, allowing long term potency and systemic control.

Using CRISPR/Cas9 and Adeno Associated Vectors (AAV), we introduce the anti-HIV broadly neutralizing antibody 3BNC117 at the immunoglobulin heavy chain (IgH) locus of B cells. The introduced cassette is regulated by a derivative of a murine IgH promoter that is active only upon on-target integration in proximity to the endogenous enhancers, as we first demonstrated using a GFP reporter gene.

For therapeutic cassettes, the light chain is encoded in full followed by the variable segment of the heavy chain, separated by a 2A peptide. Downstream of the variable heavy chain, we inserted a splice donor, which allows splicing with the endogenous constant segment. Flow cytometry and ELISA showed bNAb expression as a B cell receptor and as secreted immunoglobulins, respectively, in both cell culture and activated splenic B lymphocytes.

Importantly, adoptive transfer of the engineered cells into syngeneic mice allowed antigen-induced activation upon immunization. Donor specific cells were tracked with the CD45.1 cell surface marker in CD45.2 recipient mice. In germinal centers (GC) following immunization, all donor specific cells were binding to the HIV antigen gp120, demonstrating the strong selection for the specific humoral response. Strikingly, most of the gp120 binding cells in GCs were donor derived, demonstrating immunodominance of the engineered cells over the endogenous natural response. We followed the serological response using an anti-idiotypic antibody to quantify antibody titers. Interestingly, the bNAb would underdo CSR in-vivo, as demonstrated by IgA isotype in serum and in GCs. The total antibody response rose much further following boost immunization implying immunological memory and affinity maturation.

Uniquely, our method enables antigen-induced bNAb secretion that may be further enhanced by affinity maturation, class switch recombination, and the retention of immunological memory. B cells could thus be engineered as a living and evolving drug to counteract pathogen escape.