137 FcγRIIa-specific DARPins as a novel tool toward specific gene delivery into HIV reservoir cells

Background

FcγRIIa (CD32a) has recently been identified as a cellular marker for the latent HIV-1 reservoir.¹ Its relevance as a marker has been challenged, due to technical problems complicated by T-B cell conjugates, and trogocytosis, as well as the lack of reliable antibodies, to distinguish the a and b isoforms. Thus, there remains a need for specific FcγRIIa-binders for diagnostic and therapeutic strategies, including targeted gene delivery. Receptor-targeted viral vectors can mediate highly selective gene delivery into target-receptor positive cells not only ex vivo but also in vivo.

Methods

Libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to human FcγRIIa using ribosomal display. Binders unable to discriminate between isoforms were eliminated by counter selection against FcγRIIb. Specific binders were identified on cell lines artificially expressing FcγRIIa or FcγRIIb and by surface plasmon resonance. The binding epitopes were localized through chimeric FcγRIIa a/b proteins expressed on SupT1 cells. The most promising DARPins were inserted into the glycoprotein of Nipah virus to generate FcγRIIa receptor-targeted viral vector. Targeting FcγRIIa receptor on pure or mixed expressing cell lines as well as primary human PBMC was monitored.

Results

The identified DARPins discriminated between FcγRIIa and FcγRII b in protein and cellular binding assays with no detectable off-target activities. Their affinities for human FcγRIIa were in the low nanomolar range. In flow cytometry, the DARPins detected FcγRIIa + cells even when they made up less than 1% of the cell population. The difference between FcγRIIa and FcγRIIb is due to a single amino acid moiety. DARPins detected FcγRIIa⁺ cells could discriminate this even when they made up less than 1% of the cell population in flow cytometry. Lentiviral vectors, displaying the DARPin mediated highly selective gene delivery into FcγRIIa + cells in the presence of a large surplus of FcγRIIb+ cells. On primary cells, between 0.03% – 1.26% (PBMC) and 0.14% – 3.46% of CD4⁺ T cells were transduced. Such frequencies are well within expectation for FcγRIIa⁺ cells among T lymphocytes.

Conclusions

In summary, well-characterized high-affinity selected FcγRIIa- DARPins have become available offering the potential to become unique tools for the detection of and drug delivery into the latent cellular HIV-1 reservoir. Their application for cell detection supports the presence of FcγRIIa-positive T cells.

Reference

Descours B, Petitjean G, López-Zaragoza JL, Bruel T, Raffel R, Psomas C, Reynes J, Lacabaratz C, Levy Y, Schwartz O, Lelievre JD, Benkirane M. CD32a is a marker of a CD4 T-cell HIV reservoir harboring replication-competent proviruses. Nature 2017 Mar 23; 543 (7646):564–567