Antibody Affinity Maturation in Antigen-Distal Residues
Cisneros III, Alberto
:
2019-03-28
Abstract
The FV region of an antibody consists of the heavy chain (HC) and light chain (LC) variable domains whose association is maintained by a series of conserved, non-polar interactions. During chronic infections, somatic mutations are induced, often in the HC/LC interface. Sequence variation in these interactions allows the HC and LC domains to inhabit a range of orientations relative to one another. Thus, I hypothesize that these interface mutations are critical to orient and rigidify the HC/LC interface to arrange the paratope for optimal interaction with the antigen, thereby affecting antigen binding affinity allosterically. To test this hypothesis, I measured the HC/LC orientation of a set of broad and potent human HIV neutralizing antibodies. The HC/LC interface of these antibodies contained a large number of mutations and achieved unusual relative orientations compared to other human antibodies. I expressed and characterized a panel of recombinant HIV CD4 binding site antibodies as the fully matured variant and compared these with variants mutated to the HC/LC interface of the inferred unmutated common ancestor antibody. I found that HC/LC interface reverted antibodies have a reduced affinity, confirming that introduction of somatic mutations in the HC/LC interface was one of the critical steps in affinity maturation. I then used the Rosetta software suite to examine the mechanisms through which these mutations affect binding affinity. I determined to what extent the mutations were critical in altering the relative orientation of HC/LC domains to a conformation that is competent to bind the antigen. I further determined whether the mutations excluded alternative HC/LC conformations that would be incompetent to bind the antigen. These findings suggest that somatic mutations in the HC/LC interface, distant from the antigen/antibody contact region, play a critical role in affinity maturation of HIV antibodies by pre-configuring the bound conformation of the antibody in the orientation required for high affinity recognition of the antigen. Thus, optimization of HC/LC interface could serve as an important tool for maximizing antibody/antigen binding affinity without altering antigen contact residues.