In vivo killing of primary HIV-infected cells by peripheral-injected early memory-enriched anti-HIV duoCAR T cells

Abstract

HIV-specific chimeric antigen receptor-T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell-like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).

Keywords: AIDS/HIV; Immunotherapy; Therapeutics.

Figure 1. Optimization of anti-HIV duoCAR T cells for clinical translation.

(A) Illustration of the anti-HIV duoCAR T cell (Created with BioRender.com). (B) Schematic of the anti-HIV duoCAR LV constructs evaluated in preclinical studies. MSCV+W is the original anti-HIV duoCAR D13 vector, which contains the MSCV promoter and WPRE. The MSCV+W duoCAR vector was modified for clinical use by excising WPRE (MSCV-ΔW) followed by replacement of the MSCV promoter with the MND promoter (MND-ΔW). A vector identification (ID) tag is engineered upstream of the 3′SIN/LTR for qPCR detection of vector-marked cells. LV titers are indicated to the right of each duoCAR vector in transducing units per mL (TU/mL). (C–E) In vitro killing efficacy of MSCV+W, MSCV-ΔW, and MND-ΔW duoCAR T cells against autologous PBMCs infected with an HIV-LucR IMC expressing the 396-R1_F6_20 (Clade A), CH077 (Clade B), or Du151.2 (Clade C) HIV-1 Env glycoprotein. Magnitude of HIV-1 infection 7 days after challenge quantified via Renilla luciferase (LucR) activity (y axis; RLU, relative light units). Data are shown as mean ± SEM of 2 donors tested in triplicate. Statistical analysis performed by 1-way ANOVA followed by Tukey’s multiple-comparison post hoc test. (F) Long-term killing efficacy of duoCAR T cells after repeated challenge with Env⁺ GFP⁺ target cells. DuoCAR T cells were challenged with fresh Env⁺ GFP⁺ target cells (E:T ratio = 0.3:1) on Day 0* and subsequently on Day 7* (2nd challenge) and Day 13* (3rd challenge). Asterisks in y axis labels indicate date of challenge. Magnitude of duoCAR-mediated killing expressed as percent remaining Env⁺GFP⁺ target cells in the cocultures (y axis). Data are shown as mean ± SEM (n = 3 donors). Statistical analysis performed by 2-way ANOVA followed by Dunnett’s multiple-comparison post hoc test. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

Figure 2. In vivo localization and potent anti-HIV efficacy of i.v.-administered anti-HIV duoCAR T cell therapy in humanized HIV-infected mice.

(A) Illustration of the hu-spl-PBMC-NSG model of HIV-1 infection. The safety and efficacy of i.v.-administered anti-HIV duoCAR T cell therapy were evaluated in PBMC-humanized NSG mice after 17–18 days of intrasplenic HIV-1 infection. (B) T cell memory phenotype of the preinfusion anti-HIV duoCAR T cell product manufactured on the CliniMACS Prodigy device (n = 2 donors). T_SCM (CD45RA⁺CCR7⁺) and T_CM (CD45RA^–CCR7⁺) cell populations were determined by flow cytometry. T_SCM were delineated from T_N (naive T cells [CD45RA⁺CCR7⁺CD95^-]) cells by the presence of CD95⁺ on CD45RA⁺CCR7⁺ cells (gating strategy shown in Supplemental Figure 4). (C) Quantification of HIV-1 viral load via Renilla luciferase (LucR) activity in the spleens of humanized mice after 17–18 days of HIV-1 infection. (D) Quantification of cell-associated total HIV-1 DNA in the spleens of humanized mice after 17–18 days of HIV-1 infection. Results are expressed as HIV-1 Gag DNA copies per 1 million β-actin copies. ND, not detected. One of the mice in the MND-ΔW duoCAR T cell–treated group had insufficient cells for HIV DNA analysis; therefore, only 5 samples were evaluated for this group. (E and F) Persistence and biodistribution profile of intravenously administered anti-HIV duoCAR T cells in the blood and major organs of humanized mice after 17–18 days of HIV-1 infection. Data are expressed as CAR DNA copies per 1 million polypyrimidine tract binding protein 2 (PTBP2) copies. Data are shown as mean ± SD of samples tested (n = 5–9 mice). Statistical analysis was performed by 1-way ANOVA followed by Dunnett’s multiple-comparison post hoc test. *P < 0.05.

Figure 3. CyTOF analysis of preinfusion anti-HIV duoCAR T cell products reveals a T_CM/T_SCM phenotype primed for lymphoid tissue homing and effector function.

(A) Gating strategy to identify D8 anti-HIV duoCAR T cells (% CAR⁺ of total CD3⁺ T cells) by mass cytometry staining. (B) Frequency of summed T_SCM + T_CM phenotype (CCR7⁺) duoCAR T cells at D8 among HIV seronegative (HIV^–, CAR⁺_1, CAR⁺_2, CAR⁺_3; n = 3) and seropositive donors (HIV⁺, CAR⁺_4, CAR⁺_5; n = 2). (C) Heatmap showing the phenotype (scaled by % marker⁺) of D8 duoCAR T cells from the same HIV^– or HIV⁺ donors compared with untransduced (D0) T cell subsets from an HIV^– donor. (D) Expression of Granzyme B in D0 T cell subsets compared with CCR7⁺ or CCR7^– subsets from D8 CAR⁺ product. TSCM, stem cell memory; TCM, central memory; TEM, effector memory.

Figure 4. Anti-HIV duoCAR T cells recognize and potently kill HIV-infected monocytes.

(A–C) Purified and matured monocytes (A), PBMCs (B), or CD4⁺ T cells (C) from the same donor were infected with HIV_BaL-LucR virus for 2 days (monocytes) or 3 days (PBMCs and CD4⁺ T cells). HIV-infected cells were either untreated (infected) or treated with donor-matched untransduced (UTD) T cells or MND-ΔW duoCAR T cells (duoCAR) at an E:T ratio of 1:1 for an additional 3 days. Uninfected cells were used as negative controls in the assay. The magnitude of HIV-1 infection was quantified 3 days after infection by measuring Renilla luciferase activity and was expressed as relative light units (RLU). Data are shown as mean ± SEM. The percent HIV-1 suppression is shown above the bar graph for MND-ΔW duoCAR T cells and was calculated relative to infected cells either left untreated or treated with UTD control T cells. The study shown is from 6 independent experiments with cells from 6 different HIV-1 seronegative donors. Statistical analysis was performed by unpaired Student’s t test. Significance is considered P < 0.05. (D) Sensitivity of anti-HIV duoCAR T cell killing against monocytes, PBMCs, and CD4⁺ T cells. Percent killing of HIV-infected cells was calculated relative to UTD control T cells. Data are shown as mean ± SEM. Data show n = 4 HIV seronegative donors for all E:T ratios except 1:100, for which data show n = 3. Statistical analysis was performed by 2-way ANOVA followed by Tukey’s multiple comparison post hoc test.

Figure 5. MND-ΔW duoCAR T cells derived from PWH potently kill autologous cells infected with HIV in vitro.

(A and B) In vitro CAR T cell–mediated killing of CD8-depleted PBMCs isolated from PWH with acute or established HIV-1 superinfection (BaL Env, clade B). Each graph represents a different PWH donor, and the donor’s unique identifier is indicated at the top of its respective graph. HGLK001, HGLK002, HGLK005, and HGLK022 donors are PWH who are ART suppressed, with undetectable HIV-1 viral loads at the time of blood collection. Donor HGLK047 is a long-term nonprogressor with an undetectable HIV-1 viral load at the time of blood collection. For acute infection studies, autologous MND-ΔW duoCAR T cells or untransduced (UTD) control T cells were added shortly after spinfection of PBMCs with HIV-LucR (E:T = 1:1) and challenged for 3 days, followed by quantification of HIV-1 infection (LucR activity). For established infection studies, HIV-LucR spinfected PBMCs were cultured for 3 days to establish HIV infection followed by addition of autologous duoCAR T cells or UTD control T cells (E:T = 1:1). The cocultures were challenged for an additional 3 days, followed by quantification of HIV-1 infection (LucR activity). The y axis shows the magnitude of the HIV-1 infection as quantified via Renilla luciferase (LucR) activity and expressed as relative light units (RLU). Uninfected CD8-depleted PBMCs serve as a negative control for the assay (Luc^– PBMCs). CD8-depleted PBMCs superinfected with HIV-LucR serve as a positive control for the assay (Luc⁺ PBMCs). Data are shown as mean ± SD of triplicate sample wells tested. Statistical analysis was performed by 1-way ANOVA, followed by Tukey’s multiple comparison post hoc test; Luc^– PBMCs were not included in the statistical analysis. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.