IN VITRO GERMINAL CENTERS FOR MAB DIRECTED EVOLUTION AND IMMUNOGEN TESTING

Using CRISPR-cas9, we engineer specific antibody variable genes into the heavy and lambda immunoglobulin (Ig) loci of a human B cell lymphoma cell line. Activation-induced cytidine-deaminase (AID) which is expressed in this line, mutates these new genes, generating variant B cell receptors. Variants with desired binding properties can be selected using various methods such as FACS. These variants can be sequenced, expressed as mABs, and characterized (1A). We are applying this ‘in vitro germinal center’ to perform directed evolution to improve potential therapeutic monoclonal antibodies (mABs) against HIV, Coronavirus, and Flavivirus. We are also using it to test “germline targeting immunogens” designed to elicit HIV bnAbs from their naive B cell precursors. For example, we have recently demonstrated that a ‘germline targeting immunogen’ series developed by another group, could select variants from an engineered B cell line expressing the inferred VRC01 precursor BCR that were capable of binding to a near native HIV envelope trimer lacking an N-linked glycan at position 276. These variants could not be further evolved to  accommodate this glycan however (1B). We are currently developing modified B cell lines for improved performance (i.e. inducible AID that will generate higher levels of somatic mutation).