W9 - Targeted deletion of non-neutralizing HIV germinal center B cells using antigen-drug conjugates

Introduction: : In an effort to redirect the immune response to HIV vaccination away from a non-neutralizing response to one characterized by a broadly neutralizing antibodies (bnAbs), we have designed antigen-drug conjugates capable of depleting non-neutralizing germinal center B cells. Using HIV antigens targeting VRC01 bnAb precursors, or epitope knockouts mutated to target non-neutralizing B cells, we screened four different cytotoxic payloads and found a single bioactive chemotherapeutic, SGD-1910, for antigen-drug conjugates. We show DAR-, dose-, and affinity-dependent deletion of target germinal center B cells using antigen-drug conjugates in vitro, ex vivo, and in vivo in both humanized mice and adoptive transfer models.

Materials and

Methods: : VRC01-class antigens eOD-60mer or MD39-VGT3.1 were modified using bioconjugation chemistries to bear cytotoxic small molecule payloads forming antigen-drug conjugates (AgDCs) characterized by immunoassays and spectroscopy. Toxicity and specificity of AgDCs were tested in vitro against glVRC01 Ramos B cells, ex vivo applied to primary VRC01+ B cells from homozygote VRC01HHLL mice, in an adoptive transfer model of CD45.2+ VRC01 B cells, and in VH1-2 humanized mice. After priming with either eOD-60mer or MD39-VGT3.1, mice were injected on day 6 with antigen-matched AgDCs and inguinal lymph nodes were harvested for flow cytometric germinal center analysis at various timepoints after AgDC treatment.

Results, Conclusions, and Discussions:: Antigen drug conjugates bearing four different FDA-approved chemotherapeutic payloads were toxic against antigen-specific VRC01+ Ramos B cells in vitro, however primary VRC01+ splenocytes were found to be resistant to all payloads except one: SGD-1910, a PBD dimer. In vivo, antigen drug conjugates bearing SGD-1910 were observed to be highly potent in deleting antigen-specific B cells in two different humanized mouse models of HIV vaccination, where AgDC toxicity was directly correlated with drug-to-antigen ratio (DAR), dose, and affinity of the antigen to target B cell receptors. In VH1-2 humanized mice, off-target, non-neutralizing germinal center B cells were targeted using epitope knockout AgDCs. Selective elimination of distracting GC B cells has the potential to remodel the output of vaccine induced immunity in cases like HIV where immunodominance thwarts protective responses.

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