The Use of Somatic Hypermutation for the Affinity Maturation of Therapeutic Antibodies

Abstract

The engineering of antibodies and antibody fragments for affinity maturation, stability, and other biophysical characteristics is a common aspect of therapeutic development. Maturation of antibodies in B cells during the adaptive immune response is the result of a process called somatic hypermutation (SHM), in which the activation-induced cytidine deaminase (AID) acts to introduce mutations into immunoglobulin (Ig) genes. Iterative selection and clonal expansion of B cells containing affinity-enhancing mutations drive an increase in the overall affinity of antibodies. Here we describe the use of SHM coupled with mammalian cell surface display for the maturation of antibodies in vitro and the complementarity of these methods with the mining of immune lineages using next-generation sequencing (NGS).

Keywords: Affinity maturation; Antibody; Complementarity-determining region (CDR); Fluorescence-activated cell sorting (FACS); Heavy chain (HC); Light chain (LC); Somatic hypermutation (SHM).

Fig. 1

Example sequences of heavy chain (above) and a light chain (below) used for surface display are shown. Distinct synthetic signal peptides (gray) support high-level expression and secretion to the cell surface for both the heavy chain and light chain. A mouse variable region of each chain (white) encodes three CDRs (boxed) that make specific side-chain contact (dark gray) with the antigen anchored by framework regions (FW). Labels for regions are shown below the DNA sequence, and Kabat numbering is shown above. These sites are encoded by codons that are commonly altered by AID to generate directed diversity that may improve binding. Mutations at these Kabat positions are recombined during in vitro SHM to derive affinity- matured variants. Variable regions are joined with full-length human IgG1 and IgK constant regions (gray), and the heavy chains are further joined to a juxta-membrane extracellular (white background/bold text), trans- membrane (gray background/bold text), and cytoplasmic region (white background/bold text) adapted from mouse MHC-1 protein H2kk [19]

Fig. 2

An example of a synthetic AID sequence is shown in which potential AID DNA hotspot motifs have been removed using the redundancy of the genetic code, mammalian codon preferences, and other good practice for assembling synthetic gene constructs for expression. The nuclear export sequence (underlined) can be altered at canonical leucine positions to increase AID activity

Fig. 3

An example FACS scatterplot in which cell populations expressing a variant antibody sequence containing AID-derived mutations that impart higher-affinity antigen binding are seen right shifted relative to the parental cell population. Cell staining allows antigen binding as a function of cell surface antibody expression to be carefully quantified. All emergent cell populations are captured for subsequent expansion, sequencing, and additional rounds of AID transfection and flow sorting