Novel CD4-Based Bispecific Chimeric Antigen Receptor Designed for Enhanced Anti-HIV Potency and Absence of HIV Entry Receptor Activity

Abstract

Adoptive transfer of CD8 T cells genetically engineered to express "chimeric antigen receptors" (CARs) represents a potential approach toward an HIV infection "functional cure" whereby durable virologic suppression is sustained after discontinuation of antiretroviral therapy. We describe a novel bispecific CAR in which a CD4 segment is linked to a single-chain variable fragment of the 17b human monoclonal antibody recognizing a highly conserved CD4-induced epitope on gp120 involved in coreceptor binding. We compared a standard CD4 CAR with CD4-17b CARs where the polypeptide linker between the CD4 and 17b moieties is sufficiently long (CD4-35-17b CAR) versus too short (CD4-10-17b) to permit simultaneous binding of the two moieties to a single gp120 subunit. When transduced into a peripheral blood mononuclear cell (PBMC) or T cells thereof, all three CD4-based CARs displayed specific functional activities against HIV-1 Env-expressing target cells, including stimulation of gamma interferon (IFN-γ) release, specific target cell killing, and suppression of HIV-1 pseudovirus production. In assays of spreading infection of PBMCs with genetically diverse HIV-1 primary isolates, the CD4-10-17b CAR displayed enhanced potency compared to the CD4 CAR whereas the CD4-35-17b CAR displayed diminished potency. Importantly, both CD4-17b CARs were devoid of a major undesired activity observed with the CD4 CAR, namely, rendering the transduced CD8(+) T cells susceptible to HIV-1 infection. Likely mechanisms for the superior potency of the CD4-10-17b CAR over the CD4-35-17b CAR include the greater potential of the former to engage in the serial antigen binding required for efficient T cell activation and the ability of two CD4-10-17b molecules to simultaneously bind a single gp120 subunit.

Importance: HIV research has been energized by prospects for a cure for HIV infection or, at least, for a "functional cure" whereby antiretroviral therapy can be discontinued without virus rebound. This report describes a novel CD4-based "chimeric antigen receptor" (CAR) which, when genetically engineered into T cells, gives them the capability to selectively respond to and kill HIV-infected cells. This CAR displays enhanced features compared to previously described CD4-based CARs, namely, increased potency and avoidance of the undesired rendering of the genetically modified CD8 T cells susceptible to HIV infection. When adoptively transferred back to the individual, the genetically modified T cells will hopefully provide durable killing of infected cells and sustained virus suppression without continued antiretroviral therapy, i.e., a functional cure.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Schematic representation of CAR constructs. The extracellular targeting moieties consist of the indicated components. In each CAR construct, the targeting segment is linked via a tripeptide spacer (AAA) to the identical hinge, transmembrane (TM), and intracellular signaling domain of CD28 followed by the intracellular signaling domain of CD3-zeta. The numbers represent the lengths (in amino acids) of the linker joining the CD4 moiety to the scFv of the indicated CAR. The 139 and DDY3 CARs are controls containing scFvs against irrelevant antigens.

FIG 2

Detection of CARs on transduced T cells. CD3+ cells (FITC-labeled anti-CD3 antibody, clone HIT3a) were gated on CD8+ APC-labeled anti-CD8 antibody (clone SK1). The CD4 CAR (top four panels) was detected with PE-labeled anti-CD4 antibody (clone RPA-T4); the 139 CAR was detected with protein l-biotin followed by PE-streptavidin. Data on the x and y axes are based on relative fluorescence intensities (arbitrary units).

FIG 3

Regulated expression of JR-FL Env and its effect on activation of CAR-transduced PBMCs. (A) Western blot analysis of whole-cell lysates of HeLa Tet-Off cells showing the level of JR-FL Env expression in the presence of various concentrations of doxycycline. A lysate from untransfected cells (UT) served as a negative control; CHO-env cells, stably expressing HIV-1 IIIB Env, served as a positive control. Only the relevant region of the blot is shown. (B) IFN-γ production in cocultures of Env-expressing HeLa target cells and CAR-transduced effector PBMCs. Three populations of target cells were tested that had been prepared by growing HeLa Tet-Off cells in the presence of 0, 2, or 20 ng/ml of doxycycline. The effector cells expressing the 139 CAR and untransfected (UT; Env-negative) target cells served as negative controls. The error bars represent standard deviations of the results derived from duplicate samples.

FIG 4

Targeted killing of Env-expressing cells by CD4-based CARs. (A) Direct killing of target cell lines. Direct cytotoxicity assays were performed using a CytoTox-Glo kit and, as targets (T), CHO, the CHO-env stable transfectant, and Raji cells. Cocultures were performed for 4 h with T cells expressing the indicated CARs (effector cells [E]); the 139 CAR served as a negative control. Error bars (representing negligible values) indicate standard deviations of the results from triplicate samples. (B) Suppression of HIV-1 pseudovirus production from producer target cells. HEK293T target cells (T) transfected with plasmids directing production of HIV-1 infectious pseudovirus particles (Env from the QH0692 primary isolate) were cocultured with CAR-transduced PBMCs (effector cells [E]) at the indicated effector/target (E:T) ratios. The amounts of infectious pseudovirus released into the supernatants were quantified by incubation with SupT1-DCSIGNR cells. Pseudovirus production in the absence of effector cells was defined as 100%. Untransduced effector cells served as a negative control. Error bars indicate standard deviations of the results from quadruplicate samples. For each panel, the number of target cells is defined as 1.

FIG 5

Suppression of spreading HIV-1 infection in PBMCs by T cells expressing various CD4-based CARs. (A) BX08 (clade B) data show the average level of infection of PBMCs from multiple donors, normalized to an infection obtained with effectors expressing the 139 CAR at a 0.04:1 E:T cell ratio (defined as 100). (B) CD4-based CARs were tested against the indicated HIV-1 isolates: CAM0015BBY, clade CRF02_AG; JR-FL and AD8, clade B; and RW020 and KNH1135, clade A. The 139 CAR was used as a negative control. In cases where points fell below detectable levels, horizontal dotted lines are shown to indicate the limits of detection.

FIG 6

Accessibility of the 17b moiety on HIV-1 pseudovirions captured by HOS (CD4-negative) cells expressing the indicated CARs. Flow cytometry was performed to detect binding of anti-gp120 MAbs on captured virions; MAb 2G12 binding served as a positive control for virion capture; MAb 17b revealed accessibility to the CD4-induced 17b epitope. HOS cells without pseudovirions served as a control for staining specificity. Untransfected cells (UT) served as an additional negative control. Data on the x axes are based on relative fluorescence intensities (arbitrary units).

FIG 7

HIV-1 infection susceptibility of cells expressing CD4-based CAR variants. (A) Pseudovirus infectivity (Env from the Ba-L isolate) of HOS-CCR5 cells expressing CD4-based CARs. The error bars represent standard deviations of the results from quadruplicate samples. The extremely low relative light unit (RLU) values for both CD4-17b CARs and the CD4-35-DDY3 CAR were indistinguishable from background values as defined by value determined from the HOS-CCR5 negative-control cells (approximately 15 RLU). (B) Susceptibility of CD8+ T cells expressing the different CD4-based CARs to infection by cell-free HIV-1. CD8+ T cells obtained by negative selection and expressing the indicated CARs were challenged with cell-free HIV-1. Infection was analyzed by flow cytometry and staining for intracellular p24. (Upper panel) The CD8+ T cells were challenged with the Ba-L isolate and analyzed 3 days later. Untransduced (CAR-negative) cells served as a negative control. The percent value in each dot blot indicates the p24-positive population. Data on the x axes are based on relative fluorescence intensities (arbitrary units). (Lower panel) CAR-expressing CD8+ T cells were challenged with the AD8 isolate. Infection was assessed by the increase in intracellular p24 staining at h 0 (background) compared to h 72.