*Purpose: CD40L is a costimulatory type II membrane receptor for CD40. The binding of CD40L on T helper cells to CD40 on antigen presenting cells induces multiple downstream immune and inflammatory responses, including B and T cell clonal expansion, antibody production and the production of pro-inflammatory cytokines and chemokines. Hu5c8 is an IgG1 antibody with high affinity ‎binding to CD40L and was a promising candidate for organ transplant ‎and autoimmune disease, but unpredicted on-target ‎toxicity due to Fc effector and platelet activation, resulted ‎in thromboembolic events in humans. AT-1501 contains point substitutions in the Fc region of the antibody, wherein three cysteine residues are substituted with serine and one proline residue is substituted with serine. We analyzed the binding affinity of AT-1501 and verified both functional activity and lack of binding to FCγRs or C1q.

*Methods: Functional blocking of CD40L signaling was demonstrated by utilizing a HEK293 cell line expressing CD40 on the cell surface and by inhibition of downstream NFΚB signaling in a cell lysate assay. PAC-1 is a protein expressed on the surface of activated platelets and was analyzed verify lack of platelet activation. We used CD40L specific antibodies to block function.

*Results: AT-1501 and 5c8 have similar binding affinity to CD40L with an EC50 of 100 ng/mL. Unlike 5c8, the amino acid modifications in AT-1501 ablate Fc-effector function with no detectable binding to FCγRs or C1q. Pre-incubation of CD40L with AT-1501 blocked the binding to CD40 on the cell surface and inhibited NFκβ signaling. In our antibody competition studies incubation with AT-1501 demonstrated inhibition of AF488-5c8-(F(Ab’)2 binding to CD40L expressing HEK293 cells equivalent to the reference 5C8 antibody. Similarly, AT-1501 inhibited the binding of AF488-5c8-F(Ab’)2 to activated human peripheral blood lymphocyte cells. Positive control 5c8:sCD40L immune complexes (ICs) caused platelet activation leading to surface PAC-1 expression. Critically, PAC1 staining was undetectable in untreated platelets or platelets exposed to AT-1501:sCD40L ICs demonstrating the improved safety profile of AT-150

*Conclusions: Our results support further clinical development of AT-1501 for transplantation and autoimmune indications.

« Back to 2021 American Transplant Congress