Background:
In most cases, the cellular immune response is unable to durably control HIV replication. Chimeric antigen receptor (CAR) T cell therapy provides an avenue to engineer a more potent T cell response. Broadly neutralizing antibodies (bNAbs) targeting conserved regions of HIV Envelope may further facilitate the creation of a CAR T cell therapy effective against the majority of HIV isolates.
Methods:
A panel of 4-1BB costimulated CAR T cells expressing 18 distinct bNAb-based CARs were generated against all well-characterized epitopes on HIV Env: V1/V2 apex, V3-glycan supersite, CD4 binding site, gp120-gp41 interface, fusion peptide, silent face, and MPER. CAR T products manufactured from primary human T cells were screened for potency via degranulation when cocultured with Env-expressing K562 (K.Env) cells, as well as K.Env killing assays and HIV suppression assays in the GXR25 cell line using the Incucyte SX5 live-cell imaging system. To map escape pathways and determine CAR efficacy, donor-matched CAR T products were adoptively transferred into HIV-infected, BLT humanized mice. CAR expansion and HIV viral loads were monitored weekly in the blood by flow cytometry and qRT-PCR, and HIV env sequences were amplified from plasma at >6-weeks post infection using nested PCR and deep sequenced to identify putative escape mutations.
Results:
Potency hierarchy, as measured by degranulation potential, K.Env killing kinetics, and effective suppression of virus replication in vitro was heterogeneous among bNAb CAR constructs, but clustered between epitope specificities. Notably, Env epitopes most distal (V1/V2 apex, V3-glycan supersite) from the plasma membrane were associated with greater killing potency. Deep sequencing of HIV env derived from bNAb CAR T-treated HIV-infected humanized mice identified bNAb CARs with overlapping versus orthogonal associated escape pathways. This data guided the design of bNAb CAR combinations hypothesized to restrict escape. Using this approach, we demonstrate that a triple combination of V1/V2 apex, V3-glycan supersite, and CD4 binding site-directed CARs were able to durably suppress acute viremia to undetectable levels in humanized mice.
Conclusions:
Distinct in vitro assays identified bNAb CARs with superior potency (PGT128, PGDM1400). HIV escape from individual bNAb CARs can be restricted when CARs associated with orthogonal escape pathways are combined, and restriction of HIV escape leads to durable suppression of HIV replication in vivo.