Reducing the size of the latent HIV reservoir and controlling subsequent viral rebound by immune engineering could lead to a sustained viral remission in HIV-infected individuals in the absence of ART. CTLs could reduce the size of the reservoir by recognizing and killing reactivated reservoir cells. However, cellular exhaustion and the presence of CTL-resistant viruses may undermine their effectiveness. We have tested a new approach to reservoir reduction where convertibleCAR-T cells (cCAR-Ts) programmed with multiple HIV-specific broadly neutralizing antibodies (bNAbs) are deployed.
cCAR-Ts utilize a mutated, inert form of the NKG2D receptor. Orthogonal MIC ligands that bind to inert NKG2D but not wild-type NKG2D are fused to antibodies to generate bispecific MicAbodies for directing cCAR-T targeting and activation. cCAR-Ts can therefore be readily redirected by altering the antibody component of the MicAbody and furthermore, MicAbodies can be multiplexed. 4 bNAbs were engineered as MicAbodies and tested for their ability to kill tonsil, spleen, or blood cells infected with GFP-tagged R5 or X4-tropic or transmitted/founder viruses. Specificity of infected cell killing was monitored by loss of GFP+ vs GFP- cells. Reactivated CD4 T cells from HIV-infected individuals on ART were assayed for loss of cell-associated viral RNA in the presence cCAR-Ts either armed or not armed with bNAbs. The platform was checked in vivo, in NSG mice model of cancer, by measuring size reduction of cancer tumors.
In the presence of bNAb-MicAbodies, CD8 cCAR-Ts effectively killed HIV-infected, but not uninfected, cells from tonsil, spleen and blood. Killing was strictly dependent on the presence of bNAb-MicAbodies targeting HIV Env. Multiplexing of four MicAbodies increased the breadth of killing. cCAR-T cells also reduced by more than half the inducible reservoir present in blood of HIV-infected individuals on ART. Administration of cCAR-Ts cells in a mice cancer model, demonstrated highly effective in vivo killing.
An attractive feature of cCAR-Ts is that it is a modular platform that not only allows for multiplexing of MicAbodies, but also targeted delivery of kill switches if needed or cytokines for cCAR-T rejuvenation. This platform could be an important tool for reducing and controlling the size of the latent HIV reservoir.