The human B cell repertoire constitutes a source of antibodies capable of recognizing virtually any antigen (Ag). This is the result of a complex B lymphocyte maturation process. Newly produced B cells express B cell receptors (BCRs) generated by random somatic recombination of V (Variable), D (Diversity) and J (Junction) gene segments and which generally have a low affinity for their cognate Ag . After exposure to an Ag, naïve B cells with Ag-specific BCRs undergo somatic hypermutation (SHM) catalyzed by the enzyme Activation induced cytidine deaminase (AID) [2,3,4]. This enzyme is targeted to the Ig-loci in B cells and deaminates cytosines, thus provoking point mutations, insertions and deletions in the variable domains of both the heavy and light chains. This process ultimately leads to antibody diversification and is followed by the selection of a matured B cell repertoire with higher affinity and specificity for the Ag. This allows the overall diversity of the BCR / antibody molecules to reach theoretically about 1013 different receptors in humans . The repertoire thus constitutes an almost unlimited resource of antibodies.
For several decades, monoclonal antibodies (mAb) have been crucial tools in the treatment of diseases such as autoimmune diseases and cancer, or for the control of graft rejection. It is important to generate fully human mAbs because they have a lower risk of immune response induction in humans than the mouse, chimeric or humanized mAbs generally used hitherto. Various methods have been developed for isolating antibodies directly from a natural repertoire of human B lymphocytes. In general, they derive from two main approaches. The first of these is the high-throughput screening of mAb produced by B cell cultures or plasma cells [6, 7]. This is a very effective method for obtaining mAb against Ag to which an individual is exposed naturally or by vaccination. However, many Ag of therapeutic interest are not encountered sufficiently frequently naturally, or exploitable in vaccine strategies in humans, to profit from this type of methodology. The second technique consists in isolating single Ag-specific B cells using fluorescent-tagged Ag, followed by cloning of their immunoglobulin genes and expression of recombinant antibodies in a cell line. This technique allows interrogation of both the immune/matured B cell repertoire and the naïve/germline repertoire of an individual with respect to any Ag available in purified form [8,9,10]. There is a limitation to the interrogation of a naive B cell repertoire however: the generally limited affinity of the corresponding recombinant antibodies, requiring identification of mutations that enhance affinity while maintaining specificity.
Antibody optimization currently relies heavily on the use of libraries generated by mutagenesis of antibody chains using error-prone PCR or degenerate primers. Libraries are screened using techniques such as ribosome, phage, yeast or mammalian display . Co-expression of AID and antibody or non-antibody genes in various mammalian cell lines has also been used to initiate a mutagenic process mimicking SHM [12,13,14,15,16,17,18,19,20]. This approach circumvents the need to construct mutant libraries, but does not allow targeting of the AID enzyme to sequences encoding the antibody. In B cells, AID is targeted to the immunoglobulin locus by complex mechanisms not yet fully elucidated .
We wanted to develop a simple strategy for AID-targeting to antibody sequences in non-B cells to obtain mutated antibodies with increased affinity. Various CRISPR Cas9-based approaches using guide RNAs to target base editors such as APOBEC or AID fused to dead Cas9 (dCas9) to specific DNA sequences have been described recently [22, 23]. These approaches generally lead to mutations limited to a small part of the sequences corresponding to the guide RNA binding site. A variant approach (CRISPR-X) uses a complex containing dCas9 and a guide RNA containing bacteriophage MS2 coat protein binding sites to recruit a coat-AID fusion to DNA . This leads to more extensive mutagenesis covering a window of approximately 100 bp around the guide RNA binding site.
In this work, we present a CRISPR-X based strategy for targeted in cellulo affinity maturation of low affinity human mAbs. We apply it to a low affinity mAb named A2Ab against HLA-A*02:01 which shows some crossreactivity against other HLA-A alleles. A2Ab was isolated from circulating B cells of a naïve individual using a procedure recently developed by our group [8, 10]. We used CRISPR-X with multiplexed guide RNAs to target AID to the VDJ segment encoding the A2Ab heavy chain variable domain in HEK 293 cells co-expressing the light chain. This directed-mutagenesis approach, combined with mammalian surface expression display and a very sensitive Ag-associated magnetic enrichment process, allowed us to identify mAbs with increased affinity and a sharpening of their specificity for HLA-A*02:01. Overall we describe a novel procedure for generation of high-affinity/optimized human mAbs that is applicable to both naïve and mature circulating human B cells, raising the possibility of generation of private antibodies from a particular individual.