CAR T cells, which are now associated with cancer therapy, were previously investigated as a treatment for HIV almost 30 years ago. Now wielding new technology and biological knowledge, Anthony-Gonda et al. report a series of multispecific anti-HIV CARs. These CARs target different portions of the HIV envelope protein and were able to eliminate diverse strains of HIV in vitro, even those that are resistant to potent broadly neutralizing antibodies. The CAR T cells are resistant to HIV infection and were able to control HIV in a humanized mouse model. The persistent surveillance capabilities of CAR T cells suggest that this therapy may one day be able to eradicate HIV in an infected person.

Adoptive immunotherapy using chimeric antigen receptor–modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1–based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4 + T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.

Here, we show potent in vitro and in vivo anti-HIV effects of multispecific anti-HIV duoCAR-T cells that simultaneously kill productively HIV-infected cells in our hu-spl-PBMC-NSG mouse model and protect the CAR-modified T cells from infection using HIV-1 viruses expressing diverse Env proteins. Coupled with the known long-term persistence and immunosurveillance properties of CAR-T cells [reviewed in ( 33 )], multispecific anti-HIV duoCAR-T cells could represent a viable approach for controlling viral loads and eliminating HIV-infected cells in HIV-positive patients in the absence of ART.

Previously, we described a humanized NSG mouse model (hu-spl-PBMC-NSG) that supports robust HIV infection after infection of intrasplenically injected peripheral blood mononuclear cells (PBMCs) with an Env-IMC-LucR virus that can be monitored by quantifying luciferase activity in the spleen of the mouse as early as 1 week after infection ( 32 ). We used this humanized mouse model to demonstrate that the LSEVh-LS-F fusion protein, which contains the same specificity as the CAR-T cells described herein, mobilized antibody-dependent cell-mediated cytotoxity activity and effectively eliminated cells infected with different infectious molecular clone (IMC) constructs expressing a broad range of Env glycoproteins in vivo via a natural killer cell–dependent mechanism ( 25 ). Despite its potency, this antibody displayed a short half-life, which renders it unsuitable as a therapeutic agent for long-term maintenance of viral suppression. Because gene-modified T cells including CAR-T cells have been shown to persist in patients for years ( 4 – 7 ), we postulated that development of a CAR-T LV construct expressing the very broad targeting specificities of mD1.22 and m36.4 would be a better therapeutic candidate for long-term control of HIV-1 infection in patients.

Here, we report the development of HIV-1–based lentiviral vectors (LVs) encoding multispecific anti-HIV duoCARs. The architecture of duoCAR has two CAR molecules consisting of multiple anti-HIV binders, including mD1.22, m36.4, and C46, expressed on the surface of T cells from a single LV. Primary human T lymphocytes were engineered with up to three functionally distinct HIV envelope-binding domains (mD1.22, m36.4, and C46) to form bispecific and trispecific targeting anti-HIV duoCAR-T cells. We hypothesized that CD8 + T cells engineered with LVs expressing multiple HIV envelope-binding CAR molecules would target and eliminate productively HIV-infected cells while protecting cooperating CD4 + CAR-T cells from HIV infection. The mD1.22 domain targets the highly conserved CD4-binding site on HIV gp120. In addition, the mD1.22 domain has been engineered to be molecularly compact with enhanced specificity and higher affinity for the HIV envelope glycoprotein and thus serves as an improved CD4-derived binding domain ( 27 ). The m36.4 domain is an affinity-matured, engineered human heavy chain–only (V H ) antibody domain ( 28 ) that binds to a discontinuous CD4-induced (CD4i) epitope on gp120 in the vicinity of the co-receptor binding site, similar to other co-receptor bNAb-binding sites, such as 17b ( 29 ). When combined to form a multivalent, bispecific soluble antibody, these domains synergistically neutralized diverse HIV strains in vitro ( 25 , 27 ). The C46 peptide inhibits HIV infection at the level of viral fusion and is similar to the U.S. Food and Drug Administration (FDA)–approved fusion inhibitor enfuvirtide or T20. When expressed on the surface of T cells as an anchored membrane-associated molecule, it potently abrogates HIV fusion to the T cell membrane ( 30 , 31 ).

An alternative approach to using the extracellular region of the CD4 receptor for targeting the HIV envelope glycoprotein is a single-chain variable fragment (scFv) derived from bNAbs. However, one major drawback to developing bNAb-based CARs has been that their scFv antigen-binding domain generally requires further engineering to account for reduced therapeutic effectiveness ( 17 ), and unlike the CD4 receptor, a single bNAb cannot fully neutralize all HIV isolates ( 18 , 19 ). Recent clinical trials using bNAb monotherapies with VRC01, 3BNC117, or 10-1074 led to viral rebound upon antiretroviral therapy (ART) interruption in people with chronic HIV infection, but an antibody composed of multiple envelope-specific scFvs showed improved protection in a simian/human immunodeficiency virus macaque model ( 20 – 24 ). We recently reported that a hexavalent fusion protein consisting of an scFv-derived heavy chain–only domain, m36.4, which targets the highly conserved CD4-induced (CD4i) gp120 co-receptor binding site, and mD1.22, an engineered mutant of the D1 extracellular domain of CD4, mediates potent and broad in vitro and in vivo suppression of HIV infection in a humanized mouse model of HIV infection, where human HIV-infected and CAR-T cells are directly injected into the spleens of animals ( 25 ). In addition, it has been demonstrated that further combining the mD1.22-m36.4 hexavalent fusion protein with T20, which is a gp41-derived C-peptide fusion inhibitor similar to the C46 peptide, enhances its inhibitory effect against HIV-1 in vitro ( 26 ).

The application of immunotherapeutic strategies to treat HIV infection has been limited by factors unique to HIV infection including the high mutation rate of reverse transcriptase, which enables the rapid emergence of immune escape variants mutated in HIV envelope–specific epitopes ( 9 ) and recurrence of viremia ( 10 ). First-generation anti-HIV CAR approaches used the extracellular region of the CD4 receptor as the targeting domain coupled with the CD3ζ T cell signaling domain to kill productively HIV-infected cells. However, it was revealed later that CD4-based CARs render the gene-modified T cells susceptible to HIV infection ( 11 , 12 ). To overcome this limitation, several strategies to improve HIV-specific CAR-T cells were tested, including design of bispecific CAR-T cells ( 11 ), or CAR-T cells expressing a CD4ζ CAR in combination with either a gp41-derived fusion inhibitor ( 12 ) or CCR5 ablation ( 13 ). Moreover, anti-HIV CARs have been reengineered with 4-1BB or CD28 costimulatory signaling motifs to improve their in vivo persistence ( 14 ) and potency when combined with soluble broadly neutralizing antibodies (bNAbs) that recognize nonredundant gp120/gp41 epitopes ( 11 , 13 , 15 , 16 ).

Adoptive immunotherapy using chimeric antigen receptor–modified T cells (CAR-T) has shown extraordinary success for the treatment of refractory B cell malignancies that express CD19, CD20, or CD22 antigens ( 1 – 3 ). In contrast, past attempts using first-generation HIV-specific CAR-T cells for the treatment of HIV/AIDS were unsuccessful in humans despite demonstration of long-term persistence of gene-modified T cells in HIV-positive patients ( 4 – 7 ). HIV infection of susceptible cells such as CD4 + T cells or macrophages is initiated by binding of the gp120 HIV envelope (Env) glycoprotein to the CD4 receptor, which triggers conformational changes in Env that enable it to bind to CCR5 or CXCR4 proteins expressed on the surface of susceptible cells, penetrate the cell, and activate the HIV replication program ( 8 ).

RESULTS

Design and functional characterization of multispecific anti-HIV CAR-T cells We generated HIV-1–based LVs encoding mono-, bi-, and trispecific anti-HIV CARs as shown in Fig. 1A. These CARs target three putative sites on the Env trimer, which include the gp120 CD4-binding site (mD1.22), gp120 co-receptor–binding site (m36.4), and gp41 near the membrane-proximal external region (MPER) (C46 peptide). The structures of the resulting expressed anti-HIV CARs are depicted in Fig. 1B. Monospecific CAR LV constructs were generated by fusing the mD1.22 (designated “1” for mD1.22) or m36.4 (designated “3” for m36.4) domain in-frame to a single-molecule CAR architecture previously described by our group that consists of a CD8 EC followed by the CD8 TM domain, the 4-1BB costimulatory domain to support CAR-T persistence, and a CD3ζ T cell signaling chain (34). For simplicity, the single-molecule CAR architecture was designated monoCAR (M) followed by the binders used to generate the CAR construct. For instance, monoCARs containing the mD1.22 or m36.4 domain were designated M1 or M3, respectively. Conventional bispecific CARs were also generated in a monoCAR format by linking both mD1.22 and m36.4 domains together (M13) using a 3xG 4 S motif. Fig. 1 Illustration of the multispecific anti-HIV CAR architecture. (A) Gene structure of multispecific anti-HIV CARs encoded by LVs. The term monospecific, bispecific, or trispecific refers to the total number of binding specificities contained in the CAR molecule(s) within a single construct. For simplicity, each anti-HIV binder is assigned a number. For instance, mD1.22 = 1, m36.4 = 3, and C46 peptide = 4. The term monoCAR (M; one-molecule CAR architecture) or duoCAR (D; two-molecule CAR architecture) refers to the number of CAR molecules encoded by an LV and expressed in a single cell. For instance, a monoCAR containing the mD1.22 domain is referred to as M1, or a duoCAR containing the mD1.22 and m36.4 domains is called D13. An illustration of the Env gp120/gp41 trimer and putative target sites for each anti-HIV binder is shown on the right. (B) Cartoon illustration of each anti-HIV CAR used in the study. Monospecific CARs contain either mD1.22 (1) or m36.4 (3) fused to a CD8 ectodomain (EC), CD8 transmembrane domain (TM), 4-1BB costimulatory domain, and CD3ζ T cell signaling chain to form M1 or M3, respectively. The conventional bispecific CAR contains mD1.22 fused to m36.4 via a 3xG 4 S motif to form a one-molecule CAR construct (M13). The bispecific duoCAR contains the mD1.22-CAR and m36.4-CAR coexpressed in T cells using a two-molecule architecture (D13). In another iteration, the D134Δ construct contains the bispecific CAR (M13) in addition to the C46 peptide that is anchored to the T cell membrane but does not contain a CD3ζ T cell signaling chain (4Δ). Last, the trispecific duoCARs contain three specificities across two CAR molecules within a single cell. The first CAR contains the C46 peptide (4) fused to the N terminus of the mD1.22 (1) domain via a 3xG 4 S or 5xG 4 S linker (designated “S” for short or “L” for long) and a second CAR containing the m36.4 domain (3) to form D413S or D413L, respectively. CAR-T cell therapies currently in the clinic for cancer treatment use a conventional monoCAR design whereby a single molecule with one or more binding domains targets exposed epitopes on antigens that are located on the surface of target cells [reviewed in (35)]. However, the gp120-binding site for certain highly conserved neutralizing antibodies may not be exposed before the conformational change that is induced by the interaction between CD4 and gp120. To facilitate sequential targeting, first, the mD1.22 domain to its CD4-binding site and, subsequently, the m36.4 domain to its conformationally exposed binding site (28, 36, 37), we also designed LVs encoding a unique two-molecule CAR architecture designated duoCAR (D). The duoCAR can be engineered with two or more Env specificities via a bicistronic P2A comprising LV to allow for simultaneous expression of both CARs in a single T cell. We therefore constructed an LV-encoding bispecific duoCAR that contained the mD1.22-CAR and the m36.4-CAR (D13). To further potentiate multitargeting and protection from HIV infection, we engineered the anti-HIV CARs with a third anti-HIV domain based on the FDA-approved fusion inhibitor enfuvirtide or T20. As previously reported, T20 and similar gp41-derived C peptides (e.g., C46) block HIV infection by binding to the gp41 six-helix bundle locking the HIV-1 envelope glycoprotein into a fusion-incompetent state (38, 39). For trispecific CARs, we considered that because the C46 peptide targets gp41 and mD1.22 targets gp120, it would be more logical to fuse the C46 peptide to the N terminus of the mD1.22 domain using different linker lengths. As previously reported by Liu et al., bispecific anti-HIV CAR function is influenced by the spatial separation between two antigen-binding domains (11). Therefore, we tested a short (“S” for 3xG 4 S linker) and long (“L” for 5xG 4 S linker) flexible linker between the C46 peptide (designated as “4” for C46) and mD1.22 domain (1) in trispecific duoCARs to generate D413S and D413L, respectively. Last, to determine the optimal location of the C46 peptide, we constructed an additional trispecific construct containing a bispecific CAR and a membrane-anchored C46 peptide (D134Δ) fused together in a different orientation and without the CD3ζ T cell signaling motif on the C46 peptide. Therefore, the membrane-anchored C46 peptide should lack effector function.

Multispecific anti-HIV duoCARs are efficiently expressed on the surface of primary human T cells Previously, we have shown that HIV-1–based LVs safely and efficiently deliver an HIV-specific genetic payload to primary human T cells for the treatment of HIV-1–infected patients (5, 40–43). To test whether anti-HIV CARs could be efficiently delivered and expressed on the surface of primary T cells by LVs, we transduced HIV-naïve CD4+ and CD8+ T cells with the anti-HIV CAR LVs and evaluated the cells for CAR expression by flow cytometry detecting either the mD1.22 domain (Fig. 2A) or the C46 peptide (Fig. 2B). A figure exemplifying the gating strategy is provided in fig. S1. For monospecific and bispecific CAR-T cells, expression was determined via the increase in mD1.22 expression on the surface of transduced CD4+ and CD8+ T cells as compared with untransduced (UTD) T cells. The CD4 antibody used for flow cytometry experiments recognizes the D1 domain of the native CD4 receptor and the mD1.22 domain (modified D1 domain) of the mD1.22-CAR. UTD T cells were used to set the gates for CAR positivity, and a shift in CD4 (mD1.22) mean fluorescence intensity on transduced CD4+ or CD8+ T cells was interpreted as the percentage of CAR+ CD4+ and percentage of CAR+ CD8+ T cells, respectively. To detect trispecific CARs (D134Δ, D413S, and D413L), we used the gp41-derived C46 peptide engineered into these constructs as a unique molecular tag recognized by the 2F5 antibody. As shown in Fig. 2B, trispecific CARs were detected by 2F5 on the surface of CD4+ and CD8+ T cells across multiple donors. LV-modified primary CD4+ and CD8+ T cells expressed anti-HIV CARs with transduction rates up to 60% across different donors after a single round of transduction (Fig. 2, C and D). Fig. 2 LV-encoded multispecific anti-HIV CARs expressed on the surface of CD4+ and CD8+ T cells capture HIV Env. (A) Representative detection of anti-HIV CARs via the mD1.22 domain on the surface of activated primary T cells using anti-CD4 flow cytometry after transduction and expansion (n = 7 donors). VioBlue-conjugated CD4 antibody recognizes the D1 domain of the native CD4 receptor expressed by CD4+ T cells and the mD1.22-CAR, which contains a modified D1 domain. UTD T cells are used as a biological control to set gates. The x axis shows CD4+ T cells (Q4) and mD1.22 expression on CD4+ T cells in the quadrant labeled CAR+ CD4+. The y axis shows CD8+ T cells (Q1). mD1.22 expression on CD8+ T cells is shown in the quadrant labeled CAR+ CD8+. FITC, fluorescein isothiocyanate. (B) Representative detection of trispecific CARs via the C46 peptide using 2F5 Mab directed against the gp41 MPER region (n = 5 donors). Graphical representation of % CAR-modified T cells from multiple donors using either (C) anti-CD4 (n = 7 donors) or (D) anti-C46 (2F5 Mab) flow cytometry (n = 5 donors). The error bars represent mean ± SD of independent donors. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Bonferroni posttest analysis (***P < 0.0001 and ****P < 0.00001). IgG, immunoglobulin G; PE, phycoerythrin; FACS, fluorescence-activated cell sorting. (E) Measurement of soluble HIV envelope (Env) capture by all T cells. Representative histogram is shown (n = 2 donors). Specific binding of anti-HIV CARs to soluble, His-tagged gp140 (clade B) relative to the endogenous CD4 receptor expressed on CD4+ T cells in the total T cell population. UTD T cell control and CD19-CAR-T cells serve as a control for specificity. (F) Measurement of soluble HIV envelope capture by CD8+ T cells. Representative histogram is shown (n = 2 donors). The ability of anti-HIV CARs to bind to Env was demonstrated by their capacity to capture soluble gp140 [His-tagged soluble version of gp120/gp41 ecto derived from a clade B isolate (28)], indicating that the targeting domains were active on the surface of modified T cells and not altered by the binder architecture (Fig. 2E). The m36.4-CAR (M3) was detectable on the surface of genetically modified T cells but bound more gp140 when expressed on CD4+ T cells than on CD8+ T cells. The capture of gp140 by the m36.4-CAR on CD8+ T cells was increased by the presence of the mD1.22-containing CARs (Fig. 2F). These data indicate that the ability of the m36.4-CAR to bind to the HIV envelope is enhanced in the presence of the CD4 receptor as well as the CD4-like mD1.22 domain and supports a sequential targeting mechanism as previously described for the soluble m36 domain (37).

Multispecific duoCAR-T cells exert strong and specific cytotoxic effects against HIV Env+ targets The major goal of our study was to identify the most favorable CAR construct and architecture having the most potent and broad anti-HIV effects. To screen for functional anti-HIV CARs, we generated a surrogate HIV-1–infected cell line from human embryonic kidney (HEK) 293T cells, designated 293T-Env-Luc (Env+), which constitutively and highly expresses Env glycoprotein derived from an HIV-1 clade B isolate on its surface, as detected by the 2G12, b12, and 2F5 antibodies using flow cytometry (Fig. 3A). Using this cell line, we screened more than 40 different anti-HIV CAR iterations, which enabled us to quickly rule out nonfunctional and suboptimal CARs to select the most potent CARs to evaluate within this study and further interrogate in later described HIV-1 challenge assays. Envelope negative HEK 293T-Luc or Raji-Luc cells (Env−) were used as negative controls to test the specificity of CAR-T cells, which, after transduction, contain a population of CAR-modified and unmodified T cells. We subsequently cultured the total CAR-T cell population with Env+ or Env− target cells at varying effector-to-target (E:T) ratios. Twenty-four hours later, cocultures were lysed, and reduction of luciferase activity in the presence of CARs was quantified to determine CAR-mediated cytolysis. As a control, we also constructed a CD4-CAR and compared its function to the mD1.22-CAR. As shown in Fig. 3B, the mD1.22-CAR was as active as the conventional CD4-CAR and demonstrated no off-target killing of Env− targets (Fig. 3C). The specificity of CAR-mediated killing against Env+ targets was shown using UTD T cells or T cells modified to express an mCherry reporter, for which no cytotoxicity was observed. Comparative analysis revealed that among monospecific CARs, the mD1.22-CAR (M1) outperformed the m36.4-CAR (M3) (Fig. 3D). The conventional bispecific CAR (M13) was more potent than monospecific CARs (M1 or M3). The bispecific duoCAR (D13) was notably more potent than effectors expressing a conventional single (M1; P < 0.0001) or bispecific single-molecule CAR (M13; P < 0.01; Fig. 3D) despite lower transduction efficiency for this CAR among different donors. As shown in Fig. 3E, these CARs exhibited no off-target killing of targets that lacked HIV Env expression, even in the presence of major histocompatibility complex (MHC) class II molecules, which could interact with the mD1.22 domain, a modified D1 domain derived from the CD4 receptor (Fig. 3F). Last, as shown in Fig. 3G, culturing anti-HIV CAR-T cells with Env+ target cells (E:T ratio 10:1) triggered interferon-γ (IFN-γ) secretion, whereas no cytokine production was detected from UTD T cells or T cells modified to express the mCherry reporter. Minimal cytokine production was induced in the presence of Env− targets. Fig. 3 Cytotoxicity of anti-HIV CAR-T cells against HIV-1 Env-expressing target cells. (A) BnAb detection of the HIV-1 Env glycoprotein on the surface of cell lines used for cytotoxicity assays. (B) Cytotoxicity of conventional CD4-CAR versus mD1.22-CAR-T cells against Env+ target cells (representative figure, n = 2 donors) and (C) Env− target cells (293T-Luc). The mCherry reporter–modified donor T cells serve as a negative control. (D) Cytotoxicity of monospecific and bispecific anti-HIV CAR-T cells against Env+ and Env− target cells, (E) 293T-Luc, and (F) Raji-Luc. (G) IFN-γ secretion from monospecific and bispecific anti-HIV CAR-T cells upon challenge with Env+ target cells. (H) Cytotoxicity of multispecific anti-HIV CAR-T cells against Env+ and Env− target cells, (I) 293T-Luc, and (J) Raji-Luc. (K) IFN-γ secretion from multispecific anti-HIV CAR-T cells in the presence of Env+ target cells. n = 3 donors for monospecific versus bispecific CARs; n = 2 donors for bispecific versus trispecific CARs. The error bars represent ±SEM. Statistical analyses were performed by two-way ANOVA followed by Bonferroni posttest analysis performed for all comparisons (***P < 0.0001, **P < 0.001, and *P < 0.01; n.s., not significant). Next, we screened trispecific CARs for their ability to specifically kill HIV Env+ targets. As shown in Fig. 3H, the trispecific duoCAR vector, D413S, was more cytotoxic on target cells than trispecific CAR vectors, D134Δ and D413L, and bispecific CAR vectors, M13 and D13. As expected, bispecific and trispecific CARs maintained their specificity with no off-target killing observed on 293T-Luc and Raji-Luc Env− targets, respectively (Fig. 3, I and J). Trispecific anti-HIV duoCAR-T cells also secreted IFN-γ when challenged with Env+ cells as compared with Env− targets (Fig. 3K). Together, multispecific CAR-T cells exert potent and specific cytotoxic effects against HIV Env-expressing cells.

Anti-HIV duoCAR molecules induce potent CD107a expression and T H 1 cytokine production from primary human T cells We next compared the relative potency of anti-HIV conventional and duoCAR-T cells by measuring expression of CD107a, a functional marker of T cell degranulation, and intracellular T helper type 1 (T H 1) cytokine production. To better assess the differential effects of the conventional CAR versus duoCAR architecture, we generated a more physiologically relevant target cell population by infecting human PBMCs from a healthy donor with an HIV-1 replication-competent IMC expressing the C.Du422.1 Env glycoprotein and Renilla luciferase reporter (25). Seven days after infection, infected PBMCs were cocultured with M1-, M13-, or D13-engineered donor-matched CAR-T cells, followed by flow cytometry analysis of the above-mentioned T cell activation markers in CAR+ CD8+ T cells. We selected these three constructs, because they differ from each other by the number of binders (M1: mD1.22 versus M13: mD1.22-m36.4) or by the number of CAR polypeptide molecules expressed within the CAR-T cell (M13: bispecific monoCAR versus D13: bispecific duoCAR). Given the similar magnitude of cell surface expression (Fig. 4A) and binding of these three constructs to Env, we could effectively differentiate the effect of binder and CAR architecture on T cell activation. As shown in Fig. 4B, we observed that the bispecific duoCAR (D13) strongly induced T cell activation, leading to high CD107a expression and a two- to threefold increase in IFN-γ and interleukin-2 (IL-2) production as compared with either single-molecule mono- or bispecific CAR-T cells (M1 or M13, respectively). M13 was inferior to M1 for CD107a expression, IL-2, and IFN-γ production (Fig. 4B), although the binding of M13 and M1 CAR to soluble HIV gp140 protein was very similar. This shows that placing the mD1.22- and m36.4-binding domains on a single CAR resulted in diminished CD107a and T H 1 cytokine expression and, therefore, some loss of functionality. In contrast, although the D13 CAR showed similar binding to both M1 and M13 CARs, the expression of CD107a, IFN-γ, and IL-2 production was increased in T cells expressing the duoCAR. Although these data are observational, the results suggest that the differential expression of CD107a, IFN-γ, and IL-2 is not due to differences in Env binding on infected cells but may be due to their ability to transmit two signals independently via simultaneous or sequential gp120 engagement. Given the high amount of cytokine production, we tested whether the duoCAR architecture induced any potential deleterious tonic signaling or exhaustion effects that could potentially affect the ability of these cells to persist long term in the body. Typically, antigen-independent CAR-T signaling (tonic signaling) is characterized by the presence of multiple cell surface expressed co-inhibitory receptors (e.g., PD-1, Lag-3, and Tim-3) (44). We determined that duoCAR-T cells displayed no appreciable tonic signaling in the absence of antigen demonstrated by the lack of multiple coexpressed exhaustion markers on these cells (Fig. 4C). These data show that the duoCAR architecture does not impair T cell functionality and is similar to conventional CAR-T cells. Together, these data indicate that the two-molecule architecture of anti-HIV duoCAR-T cells affords superior effector functionality over similar single-molecule CAR-T cells. Fig. 4 Anti-HIV duoCAR strongly induces T cell activation without impairing functionality. (A) Expression profile of the anti-HIV CARs used to determine CD107a and cytokine production upon challenge with HIV-infected donor-matched PBMCs. (B) CAR-T cell activation profile 6 hours after challenge with HIV-infected PBMCs. The graph shows the percentage of UTD T cell control or CAR-T cells producing the indicated T cell activation marker (CD107a) or intracellular cytokine (IL-2, IFN-γ, or double-positive for IL-2 and IFN-γ) in the presence of donor-matched PBMCs infected with Du422.1-IMC-LucR virus. The graph shows pooled data from combined triplicate wells of a single donor. (C) Exhaustion marker profile of anti-HIV CAR-T cells in the absence of antigen stimulation. The coexpression of Lag-3, PD-1, and Tim-3 on the surface of anti-HIV CAR-T cells in the absence of antigen stimulation was quantified by flow cytometry and analyzed in SPICE 6 software. The pie slices show the proportion of cells expressing 0, 1, 2, or 3 inhibitory receptors. The arc above the pie slice indicates the inhibitory receptor(s) expressed by effector T cells. Representative data are shown (n = 2 donors).

Anti-HIV duoCAR-T cells eliminate PBMCs infected with broad Env-IMC-LucR viruses We evaluated the anti-HIV activity of duoCAR-T cells against several different HIV-1 strains to assess their ability to broadly eliminate productive HIV-infected cells. To do so, we adapted a previously described neutralization assay using replication-competent IMCs expressing different Envs that also encode a luciferase reporter (hereafter referred to as Env-IMC-LucR). The Env-IMC-LucR viruses allow for a highly quantitative readout of HIV-1 infection in primary target cells (45, 46), and when cocultured with CAR-T cell effectors, the luciferase activity can be used to monitor the inhibitory activity of different CAR constructs. We cultured anti-HIV CAR-T cells in the presence of donor-matched HIV-infected PBMCs at an E:T ratio of 1:1 for 7 days followed by quantification of luciferase activity as a measurement of HIV infection and assessment of CAR-mediated elimination of HIV-infected target cells (Fig. 5A). These CAR-T effectors were generated from different donors enriched for both CD4+ and CD8+ T cells, with the composition being slightly more CD4+ T cells (fig. S2). We hypothesized that multispecific anti-HIV CAR-modified CD4+ T cells in combination with CD8+ T cells would eliminate HIV-infected cells and protect the CAR-T cells from HIV infection. Given that the mD1.22-CAR exhibited similar cytotoxic effects to the CD4-CAR in our cytotoxic T lymphocyte (CTL) assays, we used the mD1.22-CAR (M1) as a control for our in vitro HIV challenge assay to further interrogate our multitargeting duoCAR approach. As shown in fig. S3, anti-HIV CAR-T cells potently eliminated donor PBMC targets infected with an IMC expressing Env from clade B viruses NL4-3 (X4-tropic), BaL (R5-tropic), or SF162 (R5-tropic). Overall, the mD1.22-CAR (M1) was less potent than a conventional bispecific CAR (M13). To account for donor variability, we compared the CAR constructs within each donor group. The bispecific duoCAR (D13) was consistently more potent than conventional monoCARs (M1 or M13) despite their valency (P < 0.05, pairwise Student’s t test). Given that the potency of the mD1.22-CAR increased upon the addition of the m36.4 domain as demonstrated by the M13 or D13 vectors indicates that both domains are active and capable of triggering CAR-mediated cytolysis of HIV-infected PBMCs. Conversely, the trispecific (D413S and D413L) and bispecific duoCAR-T cells (D13) exhibited similar potencies. Only modest improvements were seen for a trispecific CAR (D134Δ) construct that is composed of the membrane-anchored C46 peptide and the bispecific M13 CAR. Fig. 5 Broad and potent in vitro killing of HIV-infected PBMCs by multispecific duoCAR-T cells. (A) Schematic of the in vitro HIV-LucR PBMC killing assay. IMCs of HIV-1 encoding a heterologous HIV-1 Env glycoprotein and a Renilla luciferase cassette (generically referred to as Env-IMC-LucR or HIV-LucR) were used to infect donor-matched PBMCs followed by coculture with UTD T cell control or CAR-T cells for 7 days. After 7 days, the cocultures were lysed, and luciferase activity was assessed to quantify HIV-1 infection. (B) In vitro HIV challenge of anti-HIV CAR-T cells with PBMCs infected with Du422.1-IMC-LucR virus (VRC01/3BNC117-resistant Env, clade C). T, targets (Du422.1-IMC-LucR–infected PBMCs). Data shown are ±SD of triplicate wells for four different donors. Statistical analysis was performed using a pairwise Student’s t test for each donor. Significance is considered P < 0.05. (C) In vitro HIV challenge of anti-HIV CAR-T cells with PBMCs infected with Env-IMC-LucR viruses expressing Env found worldwide. The figure shows averaged log HIV-1 inhibition relative to UTD T cells (n = at least 3 donors tested in triplicate; n = 2 donors tested in triplicate for AE.CNE55). Log inhibition of HIV-1 infection was calculated by the following formula using background-corrected relative light units (RLUs) obtained for each sample: log inhibition = log 10 (RLU CAR /RLU UTD ) and then averaged across donors. Statistical analysis was performed by pairwise comparison of CAR constructs across multiple donors via a fixed-effects ANOVA model of background-corrected log inhibition of HIV-1 infection adjusting for donor and treatment. To account for multiple comparisons, we chose a more conservative P value of ≤0.01 for statistical significance. To further interrogate multispecific CAR constructs, we tested their anti-HIV effect against three IMCs expressing Env from HIV-1 clade C viruses (Du422.1, Du172.17, and Cap45) that are resistant or partially resistant to two bNAbs that target the CD4-binding site, VRC01 and 3BNC117, a potential limitation for mD1.22-based CARs. Among the CARs tested, the IMC expressing the VRC01/3BNC117-resistant Du422.1 Env discriminated the relative anti-HIV activity of duoCAR-T cells from other CAR constructs across four different donors (Fig. 5B). Further CAR-T titration studies revealed that even at lower E:T ratios, multispecific duoCAR-T cells sustained their ability to eliminate HIV-infected target cells better than monoCAR-T cells (fig. S4). These data effectively demonstrated the synergistic effect of the mD1.22 and m36.4 domains within the bispecific monoCAR and duoCAR-T cells. In the presence of PBMCs infected with an IMC expressing Env from other VRC01-resistant HIV strains, anti-HIV duoCAR-T cells nearly completely eliminated HIV-infected donor PBMCs as measured by virus-encoded reporter gene expression relative to other CARs (fig. S5), indicating that duoCAR-T cells, irrespective of their valency, are the most potent anti-HIV CAR architecture. Last, we investigated the ability of anti-HIV CAR-T cells to eliminate PBMCs infected with different IMCs expressing Env from diverse HIV-1 clades representing the genetic diversity of the HIV Env found in different regions of the world. We selected a global Env representative for each clade tested based on the phylogenic characterization of its envelope and neutralization profile as previously described (47). As shown in figs. S6 and S7, we demonstrate that anti-HIV duoCAR-T cells potently eliminated HIV-infected PBMCs infected with an IMC expressing Env from genetically distinct clades. The results from these studies are summarized as averaged log HIV-1 inhibition relative to UTD T cells from multiple donors in Fig. 5C. To determine statistical significance across multiple PBMC donors, we evaluated CAR constructs via ANOVA and chose a stricter P value cutoff of ≤0.01 to account for multiple pairwise comparisons. Overall, we found that D13 duoCAR-T cells were more potent than monoCAR-T cells for most Env-IMC-LucR viruses tested at the more stringent P value cutoff and consistent with the Student’s t test analysis for individual donors. Notably, the bispecific duoCAR-T cells (D13) were as potent as the trispecific duoCAR-T cells (D413L). However, the capacity of bispecific duoCAR-T cells to eliminate >99% of HIV infection in PBMC cocultures makes it difficult to discern whether the potency or breadth of CAR-T cells may be further increased by trispecific duoCAR-T cells. Figure S8 shows the percentage of HIV-1 inhibition via CAR-mediated elimination of infected cells by conventional monoCAR-T cells versus duoCAR-T cells. Anti-HIV duoCAR-T cells comprising two functional CAR molecules, each with its own CD3ζ T cell signaling domain, were more effective at eliminating HIV-1 infection originating from PBMCs infected with an IMC expressing Env from distinct clades than CAR-T cells expressing a single CAR molecule, where the second polypeptide chain did not contain an intracellular CD3ζ T cell signaling domain (D413S or D413L versus D134Δ; Fig. 5C and fig. S8). This further supports the added impact of the two-molecule architecture to provide improved effector functionality over conventional CAR designs and the notion that both major gp120 targeting domains are active. To better distinguish between suppression of infection and elimination of cells already infected with HIV by the anti-HIV CAR-T cells, we infected phytohemagglutinin-activated PBMCs with an Env-IMC-LucR virus encoding the env gene from a clade C strain, Du422.1, which was potently inhibited by CAR-T cells in our standard in vitro HIV challenge assay shown in Fig. 5A. After 4 days of culture to enable initiation and spread of infection, we added the HIV integrase inhibitor Raltegravir to the test cell cultures to prevent further spread of the infection followed by the addition of autologous UTD T cells or a select group of CAR-T cells (M1, M13, and D13). Three days later, we quantified the LucR activity in the cocultures treated with and without Raltegravir and in the absence or presence of CAR-T cells. We postulated that if CAR-T cells were eliminating infected cells, then we would observe a further reduction of LucR activity in cell cultures containing infected cells treated with Raltegravir plus CAR-T cell effectors as compared to the cell cultures containing infected cells treated with Raltegravir alone (no added effectors) or with Raltegravir plus UTD T cells. In contrast to the added UTD T cell control, the addition of D13 CAR-T cells to HIV-infected PBMCs also treated with Raltegravir resulted in marked further reduction in LucR activity in contrast to single-domain monoCAR-T cells (M1) and slightly better than bispecific monoCAR-T cells (M13), demonstrating its capacity to eliminate HIV-infected cells (fig. S9). Together, we show that multispecific anti-HIV duoCAR-T cells not only reduce HIV infection but also can additionally target and eliminate HIV-infected cells.

Protection of multispecific anti-HIV duoCAR-T cells from HIV-1 infection Next, we sought to understand whether the genetically modified CAR-T cells, which are CD4-enriched, were susceptible to HIV-1 infection. In contrast to reports that use flow cytometry to detect intracellular p24 as a measure of HIV-1 infection within CAR-T cells (11, 12), the intracellular Renilla luciferase that is produced upon infection of T cells by Env-IMC-LucR viruses is an extremely sensitive indicator of HIV infection upon the addition of its substrate to lysed cocultures. In addition, the Renilla luciferase reporter has a relatively short half-life in cells (~3 hours) and allows for quantification of HIV infection in CAR-T cells before cell killing occurs. Therefore, we directly challenged anti-HIV CAR-T cells with several Env-IMC-LucR viruses expressing Env from different HIV-1 clades to assess protection of multispecific CAR-T cells from HIV infection. After 7 days of infection, we quantified luciferase activity in the effector cells. Direct challenge of anti-HIV CAR effectors (both CD4+ and CD8+ T cells) revealed that in some donors, the mD1.22-CAR (M1) failed to fully suppress viral infection (Fig. 6). This loss of suppression was most pronounced in certain donors infected with Env-IMC-LucR viruses expressing BaL, NL4-3, SF162, Cap45, C.Du172.17, C.Du422.1, and AE.CNE55 HIV-1 Env glycoproteins. In contrast, HIV infection was not detected in all anti-HIV CAR-T cells that additionally contained m36.4 and/or the C46 peptide. This protective effect was essentially architecture independent (M13 versus D13) because the additional presence of the m36.4 domain was sufficient for CAR-T protection from infection in both constructs. This result proves that the m36.4 domain is active in the bispecific CAR and the duoCAR as demonstrated by its consistent ability to protect mD1.22-CAR-T cells from HIV-1 infection. Furthermore, we observed no immediate additional benefit with the addition of the C46 peptide, at least for the period of this short-term assay. These data support other reports showing that CAR-T cells engineered with the CD4 receptor and domains such as the mD1.22 domain are susceptible to HIV infection (11, 12), and our findings demonstrate that the addition of the m36.4 domain appears to fully protect the mD1.22-CAR-T cells from infection by several IMCs containing Env found in different regions of the world. Fig. 6 Multispecific anti-HIV CAR-T cells are protected from HIV-1 infection. CD4-enriched anti-HIV CAR-T cells were directly challenged with Env-IMC-LucR viruses encoding env from strains (A) BaL (clade B, R5-tropic), (B) NL4-3 (clade B, X4-tropic), (C) SF162 (clade B, R5-tropic), (D) CAP45 (clade C, partially resistant to VRC01), (E) C.Du172.17 (clade C, resistant to VRC01), or (F) C.Du422.1 (clade C, resistant to VRC01/3BNC117). Anti-HIV CARs were also challenged with Env-IMC-LucR viruses expressing Env from representative HIV-1 clade AC (G) AC.246-F3, clade AE (H) AE.CNE8, a second clade AE (I) AE.CNE55, clade BC (J) BC.CH119.10 (partially resistant to VRC01), and clade G (K) GX1632_S2_B10. Donor-matched HIV-negative PBMCs (HIV− PBMC) and HIV-infected PBMCs (HIV+ PBMC) serve as negative and positive controls for the assay, respectively. The error bars shown are ±SD of three independent donors tested in triplicate. Statistical analysis was performed by pairwise comparison of CAR constructs across multiple donors via a fixed-effects ANOVA model of log-transformed RLU values adjusting for donor and treatment. Significance is considered P ≤ 0.01.