Antibody quality is defined by many parameters including affinity, specificity, function, and stability. Improving any of these characteristics can result in a better therapeutic. Therefore, we have developed robust antibody improvement strategies that leverage the modular nature of our heavy and light chain libraries.
Our heavy and light chain libraries are created separately and afford the opportunity to find improved chains by holding one chain constant during screening. This strategy can preserve specificity and when combined with antigen titration, can lead to the selection of antibodies with higher affinity. We have successfully used this approach to achieve double digit improvements to marginal antibodies and several fold improvements to antibodies with high initial affinity.
In addition, opportunities exist to leverage what is known about the primary amino acid sequence of both heavy and light chain candidates to improve the quality of any given antibody. It is well understood that the complementarity determining regions (CDR’s) within heavy and light chains comprise the primary points of contact with the antigen. Therefore, it is most efficient to focus antibody improvement efforts on the amino acid sequence within these regions.
Vaccinex uses its knowledge and experience to select one or more amino acids to mutagenize and create new recombinant libraries in vaccinia. These libraries can then be employed to screen for antibodies with improved binding properties.
Figure 1. A parental antibody with reasonable initial affinity was subjected to further affinity improvement by substitution with alternative heavy or light chains independently. Presented are three examples of replacement antibodies that retain the binding characteristics of the parent but have higher affinity in the range of 2 to 10 fold.
Figure 2. Our screening strategy involves holding one chain constant and introducing a library of substitute complementary chains. Selection is achieved by expressing antibody on the surface of cells and introducing labeled antigen at reduced concentrations. Cells that express antibodies with improved affinity are separated by FACS. The virus within these cells is harvested and amplified and can be used in subsequent rounds of selection until a suitably enriched positive population of antibodies is identified.
Figure 3. Heavy chain CDR’s are depicted in yellow and light chain CDR’s are in blue. Regions in grey provide the framework upon which the CDR's are supported. The space filling model illustrates the way in which CDR’s from both heavy and light combine in three dimensions to create a binding pocket. Focused efforts to modify amino acids within these CDR’s has led to the successful improvement of antibodies with marginal initial affinity.