Methods: We treated IgG3-producing 17H12 parent hybridomas with LPS and IL-4 to generate isotype variants which were subcloned by sib selection. This yielded an IgG1-producing clone which was sequenced and compared with the complementary-determining region (CDR) sequence of the parent. We then compared binding kinetics of the two mAbs to CR-Kp capsular polysaccharide by ELISA. Opsonophagocytosis by macrophages was compared between CR-Kp strains pre-opsonized with the IgG1 or IgG3 mAb. Finally, mice were infected intratracheally with CR-Kp pre-opsonized with either IgG1 or IgG3 mAbs and organ burdens were compared after 24 hours.
Results: Sequence analysis showed the IgG1 antibody sequence to be identical to the 17H12 IgG3 parent. Interestingly, the IgG1 antibody bound at nanomolar affinity, but 10-fold less than the parent, suggesting loss of affinity or avidity. IgG1-opsonized CR-Kp were phagocytized by macrophages 40-60% less than IgG3-opsonized CR-Kp. However, both antibodies performed comparatively in vivo, reducing bacterial burden in the lung, liver and spleen of intratracheally-infected mice by an average of 3 log.
Conclusion: The IgG1 isotype variant of mAb 17H12 appears to have inferior binding and in vitro efficacy when compared with its IgG3 parent, despite having the same CDR region. However, in vivo efficacy is unaffected in our model. Future studies plan to further analyze the differences in binding kinetics between these two antibodies, as well as their ability to bind pro and anti-inflammatory Fc Receptors and mediate the host response to CR-Kp infection.
B. C. Fries, None