A Trispecific Anti-HIV Chimeric Antigen Receptor Containing the CCR5 N-Terminal Region

Abstract

Anti-HIV chimeric antigen receptors (CARs) promote direct killing of infected cells, thus offering a therapeutic approach aimed at durable suppression of infection emerging from viral reservoirs. CD4-based CARs represent a favored option, since they target the essential conserved primary receptor binding site on the HIV envelope glycoprotein (Env). We have previously shown that adding a second Env-binding moiety, such as the carbohydrate recognition domain of human mannose-binding lectin (MBL) that recognizes the highly conserved oligomannose patch on gp120, increases CAR potency in an in vitro HIV suppression assay; moreover it reduces the undesired capacity for the CD4 of the CAR molecule to act as an entry receptor, thereby rendering CAR-expressing CD8 + T cells susceptible to infection. Here, we further improve the bispecific CD4-MBL CAR by adding a third targeting moiety against a distinct conserved Env determinant, i.e. a polypeptide sequence derived from the N-terminus of the HIV coreceptor CCR5. The trispecific CD4-MBL-R5Nt CAR displays enhanced in vitro anti-HIV potency compared to the CD4-MBL CAR, as well as undetectable HIV entry receptor activity. The high anti-HIV potency of the CD4-MBL-R5Nt CAR, coupled with its all-human composition and absence of immunogenic variable regions associated with antibody-based CARs, offer promise for the trispecific construct in therapeutic approaches seeking durable drug-free HIV remission.

Evolving Strategies to Eliminate the CD4 T Cells HIV Viral Reservoir via CAR T Cell Immunotherapy

“…binding lectin (MBL) recognizing the highly conserved oligomannose patch on gp120 and CD4 ligand by addition of a third antigen recognition domain against a distinctly conserved region on Env. The group employed a polypeptide sequence derived from the N-terminus of the HIV coreceptor CCR5 to develop the trispecific CAR to demonstrate enhanced in vitro anti-HIV potency compared to the bispecific CAR (108). Binding of either CAR in dual or tandem systems to its associated antigen is sufficient to stimulate full T cell activation.…”

Chimeric antigen receptor engineered cells and their clinical application in infectious disease

“…added a third targeting domain against a conserved Env determinant and constructed the trispecific CD4‐MBL‐R5Nt CARs. Compared with CD4‐MBL CARs, they had particularly higher anti‐HIV potency, lower immunogenicity and minimized CD4 receptor‐mediated virus entry activity in vitro 61 …”

“…The second generation CD4 CAR had a co‐stimulation domain of CD28 or 4‐1BB 16,57 . Some groups also added extracellular domains such as MBL and R5Nt to construct multi‐targeting CARs 60,61 . In order to protect CD4 CAR‐T cells from HIV infection, researchers developed a variety of methods such as using truncated CD4 or using shRNA or ZFN to inactivate CCR5 and/or CXCR4.…”

“…The first method is to change the CD4 moiety of CAR to minimize CD4‐mediated virus entry. For example, using D1D2CAR, 65 bispecific CD4‐MBL CAR 60,86 or trispecific CD4‐MBL‐R5Nt CAR 61 could help CAR‐T cells resist HIV infection. The second method is to disrupt co‐receptors.…”

Chimeric Antigen Receptor-Modified Immune Cells for Eradication of HIV Reservoirs

“…[25] To further improve the bispecific CD4-MBL CAR, a third targeting moiety against a conserved Env determinant was added to the N -terminal region of the HIV coreceptor CCR5. [26] Compared with CD4-MBL CAR, the tri-specific CD4-MBL-R5Nt CAR showed enhanced anti-HIV efficacy in vitro and undetectable HIV entry receptor activity. At present, there are many CARs specific for different antigens.…”