Membrane-proximal external region is a superior target for mediating effector activity of HIV-1 specific chimeric antigen receptor modified T cells.

The use of chimeric antigen receptor modified-T (CAR-T) cells in adoptive immunotherapy has been popularized through recent success in the field of cancer treatment research. CD4{zeta} CAR, which targets HIV-1-infected cells, has been developed and evaluated in patients. Though well-tolerated for over a decade, efficacy was disappointingly limited. This result encourages us to develop a novel CAR more effective than CD4{zeta} CAR. To quantitatively compare anti-HIV-1 activity of different CAR constructs in a highly sensitive and reproducible manner, we developed a multicolor flow cytometry method for assessing anti-HIV-1 effector T-cell activity. "Target" Jurkat cells inducibly expressing an HIV-1HXBC2 envelope protein and "Non-target" control cells were genetically labeled with red and blue fluorescent protein, respectively, and co-incubated with human primary T cells transduced with anti-HIV-1 "Effector" CARs at various Effector vs Target cell ratios. Absolute cell numbers of each population were collected by MACSQuant Analyzer and used for calculation of relative cytotoxicity. We successfully ranked the cytotoxicity of three previously reported single chain-antibody CARs and six newly developed single-domain antibody CARs in comparison to CD4{zeta} CAR. Interestingly, three CARs--10E8, 2E7, and 2H10--which demonstrate high cytotoxic activity were all known to target the membrane-proximal external region. Use of this novel assay will simplify assessment of new CAR constructs and in turn accelerate the development of new effective CARs against HIV-1. Author SummaryAdoptive immunotherapies that utilize autologous T cells expressing a desired antigen-specific CAR aim to elicit directed immune responses. In recent years, CAR immunotherapies have been promoted extensively in B cell malignancy treatments. The HIV-1-targeting CAR, known as CD4{zeta}, was developed over 20 years ago and has been widely and longitudinally tested in patients. However, its effectiveness was hindered by poor survival and functionality of the transduced cells. To conduct quantitative evaluation of newly designed anti-HIV-1 CARs, we developed a novel multicolor flow-based assay for HIV-1-specific cytotoxicity, enabling sensitive and quantitative assessment in a high-throughput fashion. This assay would be also useful in screening HIV-1-targeting immune receptors--including CARs and T cell receptors--and other immunotherapeutic drugs such as anti-HIV-1 antibodies.