Abstract
BACKGROUND: New monoclonal antibodies (mAb) are being developed against infectious disease. However, antibody isotype is an important consideration, as different IgG variants interact with different Fc receptors and differ in avidity due to Fc structural differences. Our recent anti-capsular murine IgG(3) mAb 17H12 was shown to mediate protection against clade 2 ST258 carbapenem-resistant Klebsiella pneumoniae (CR-Kp). However, our previous studies showed an IgG(1) mAb to perform better than an IgG(3) mAb in mediating infection against a carbapenem-sensitive Kp isolate. Therefore, we sought to determine whether differences in antibody isotype contribute to differences in protection against CR-Kp infections. METHODS: We treated IgG(3)-producing 17H12 parent hybridomas with LPS and IL-4 to generate isotype variants which were subcloned by sib selection. This yielded an IgG(1)-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 IgG(1) or IgG(3) mAb. Finally, mice were infected intratracheally with CR-Kp pre-opsonized with either IgG(1) or IgG(3) mAbs and organ burdens were compared after 24 hours. RESULTS: Sequence analysis showed the IgG(1) antibody sequence to be identical to the 17H12 IgG(3) parent. Interestingly, the IgG(1) antibody bound at nanomolar affinity, but 10-fold less than the parent, suggesting loss of affinity or avidity. IgG(1)-opsonized CR-Kp were phagocytized by macrophages 40–60% less than IgG(3)-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 IgG(1) isotype variant of mAb 17H12 appears to have inferior binding and in vitro efficacy when compared with its IgG(3) 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. DISCLOSURES: All authors: No reported disclosures.