Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor

<div><p>HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent <i>in vitro</i> at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy.</p></div

CD4 CARs respond to Env⁺ cells and not MHC class II⁺ cells.

<p><b>(A)</b> Primary human CD8 T cells were activated with either left NTD or transduced with the indicated CD4 CARs. Two weeks post activation, the CD8 T cells were co-cultured for 6 hours at a 1:1 ratio with unmodified K562 cells, K562 cells expressing high levels of HLA-DR, or K562 cells expressing HIV-1 YU2 GP160. Intracellular IFNγ and MIP-1β expression is shown on the left, and intracellular IL-2 expression and CD107a surface mobilization is shown on the right. <b>(B)</b> A co-culture assay was designed to demonstrate that CD4 CAR⁺ CD8 T cells do not kill MHC class II-expressing target cells. Briefly, NTD or CD4 28z CAR transduced CD8 T cells from <b>(A)</b> were co-cultured with K562 cells expressing both HLA-A2 and GFP as well as K562 expressing both HLA-DR*0401 and mCherry at a 1:1:1 ratio. Flow cytometry measuring GFP and mCherry expression was performed immediately after mixing (0 hr) and after 3 days of co-culture (72 hr). <b>C)</b> Summary data for a single experiment performed in triplicate, measuring the ratio of HLA-A2/GFP-expressing cells to HLA-DR*0401/mCherry-expressing cells after 24, 48, and 72 hours of culture. Error bars indicate SEM. Data is representative of three independent experiments.</p

CD4-based CARs control HIV more effectively than broadly neutralizing antibody-based CARs.

<p><b>(A)</b> Specific lysis of Cr⁵¹ labeled K562 target cells expressing HIV-1 YU2 GP160. Significance was detected using a 1-way ANOVA test on the 30:1 E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). Data plotted shows the average of three independent experiments. Error bars indicate SEM (n = 3). <b>(B)</b> Gag staining on day 6 of co-culture for CD8 negative T cells and <b>(C)</b> the CD8 positive T cells. The data from the best (PGT128) and one of the worst (PG9) scFv-based CARs are compared to the CD4 CAR here, but the complete construct comparison is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s006" target="_blank">S6 Fig</a>. (<b>D)</b> Summary data for a single experiment performed in triplicate, gating on the CD8 negative cells. Error bars indicate SEM. Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). Data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s017" target="_blank">S17 Fig</a> shows each of the 3 independent experiments.</p

CAR T cells containing 4-1BB outperform CAR T cells containing CD28 in a humanized mouse HIV-treatment model.

<p>The experimental timeline and detailed description is provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s008" target="_blank">S8 Fig</a>. CD4 T cell counts per μl of blood are shown (<b>A</b>) 3 days post ART initiation, prior to CD8 T cell injection (<b>B</b>) 18 days post ART removal and (<b>C</b>) at the endpoint termination bleeds (21 or 24 days post ART removal). For logistical reasons the mice had to be terminated in two groups, with BBz mice terminated 21 days post ART removal and the NTD and 28z terminated 24 days post ART removal. CD8 T cell counts are shown (<b>D</b>) 10 days post ART removal and CD8 T cell injection (<b>E</b>) 18 days post ART removal and (<b>F</b>) at the endpoint termination bleeds (21 or 24 days post ART removal). HIV RNA copies per μl plasma were determined by qPCR and normalized to CD4 T cell counts (<b>G</b>) 10 days post ART removal (<b>H</b>) 18 days post ART removal and (<b>I</b>) the endpoint bleed. The non-normalized viral loads are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s012" target="_blank">S12 Fig</a>. Mann Whitney Test was used to determine statistical significance (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001).</p

Re-directed T cells expressing a CD4 CAR are 100-fold more potent than re-directed T cells specific for B57-KF11.

<p><b>(A)</b> Gag staining on day 6 of co-culture for CD8 negative T cells. <b>(B)</b> Summary data for a single experiment performed in triplicate, gating on the CD8 negative T cells. Error bars indicate SEM. Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). This data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s016" target="_blank">S16 Fig</a> shows each of the 3 independent experiments.</p

CCR5-edited CD4+ T cells augment HIV-specific immunity to enable post-rebound control of HIV replication.

BackgroundWe conducted a phase I clinical trial that infused CCR5 gene-edited CD4+ T cells to determine how these T cells can better enable HIV cure strategies.MethodsThe aim of trial was to develop RNA-based approaches to deliver zinc finger nuclease (ZFN), evaluate the effect of CCR5 gene-edited CD4+ T cells on the HIV-specific T cell response, test the ability of infused CCR5 gene-edited T cells to delay viral rebound during analytical treatment interruption, and determine whether individuals heterozygous for CCR5 Δ32 preferentially benefit. We enrolled 14 individuals living with HIV whose viral load was well controlled by antiretroviral therapy (ART). We measured the time to viral rebound after ART withdrawal, the persistence of CCR5-edited CD4+ T cells, and whether infusion of 10 billion CCR5-edited CD4+ T cells augmented the HIV-specific immune response.ResultsInfusion of the CD4+ T cells was well tolerated, with no serious adverse events. We observed a modest delay in the time to viral rebound relative to historical controls; however, 3 of the 14 individuals, 2 of whom were heterozygous for CCR5 Δ32, showed post-viral rebound control of viremia, before ultimately losing control of viral replication. Interestingly, only these individuals had substantial restoration of HIV-specific CD8+ T cell responses. We observed immune escape for 1 of these reinvigorated responses at viral recrudescence, illustrating a direct link between viral control and enhanced CD8+ T cell responses.ConclusionThese findings demonstrate how CCR5 gene-edited CD4+ T cell infusion could aid HIV cure strategies by augmenting preexisting HIV-specific immune responses.REGISTRATIONClinicalTrials.gov NCT02388594.FundingNIH funding (R01AI104400, UM1AI126620, U19AI149680, T32AI007632) was provided by the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute on Drug Abuse (NIDA), the National Institute of Mental Health (NIMH), and the National Institute of Neurological Disorders and Stroke (NINDS). Sangamo Therapeutics also provided funding for these studies

EF1α promoter and CD8α transmembrane domains improve CAR expression and control over HIV.

<p><b>(A)</b> Schematic of the constructs compared in this figure. (<b>B)</b> CD4 CAR expression 8 days after activation. Median fluorescence intensity (MFI) is indicated on each graph. (<b>C</b>) Intracellular Gag staining on day 7 of co-culture, for CD8 negative T cells and <b>(D)</b> for CD8 positive T cells. <b>(E)</b> Summary data for a single experiment, performed in triplicate, gating on the CD8 negative cells. Error bars indicate SEM. Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). This data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s015" target="_blank">S15 Fig</a> shows each of the 3 independent experiments. (<b>F)</b> The levels of intracellular Gag in CD8 negative T cells over the time course of an experiment. Each graph represents a different E:T ratio. Error bars indicate SEM (n = 3).</p

Lentiviral backbone augments CAR expression and control over HIV replication.

<p><b>(A-D)</b> Primary human CD8 T cells were activated with αCD3/αCD28 coated beads and were either left <b>(A)</b> nontransduced (NTD), <b>(B)</b> transduced with the original MMLV-based CD4 CAR, or <b>(C)</b> transduced with the same CAR placed in a HIV-based lentiviral vector, both driven by the PGK promoter. After eight days T cells were stained for CD4 and CD8 by flow cytometry. Median fluorescence intensity (MFI) is indicated on each graph. (<b>D)</b> Overlying histograms of the data shown in <b>(A-C). (E)</b> Eight days post activation, qPCR was performed and the number of integrated vector copies per cell was calculated. (<b>F</b>) Schematic of experimental design to study the control over HIV replication by T cells expressing HIV-specific CARs. Briefly, following activation with αCD3/αCD28 coated beads, CD4 T cells were infected with HIV Bal, and 24 hours later the indicated CD8 T cells were mixed at the indicated effector to target (E:T) ratios. After 7 days of co-culture, the expression of surface CD4, CD8, and intracellular Gag was measured by flow cytometry. <b>(G)</b> Intracellular Gag staining on CD8 negative cells, and <b>(H)</b> Intracellular Gag staining on CD8 positive cells. <b>(I)</b> Summary data for a single experiment, performed in triplicate, gating on the CD8 negative cells. Error bars indicate standard error of the mean (SEM). Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). This data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s014" target="_blank">S14 Fig</a> shows each of the 3 independent experiments. (<b>J)</b> Measurement of levels of intracellular Gag in CD8 negative T cells over the time course of an experiment. Each graph represents a different E:T ratio. Error bars indicate SEM (n = 3).</p

Optimized CAR T cells control HIV-1 replication better and expand to greater levels <i>in vivo</i> than first generation CAR T cells.

<p>Cohorts of NSG mice were infused with 8 million human CD4 T cells and 2 million human CD8 T cells (50% CAR transduction efficiency). CD8 T cells were either left NTD, transduced with optimized CD4 CARs containing either 4-1BB or CD28 intracellular costimulatory domains, or the clinical trial, MMLV-based CAR, denoted in as NTD, BBz, 28z, and MMLV-CD4z, respectively. Three weeks post injection, engraftment was measured to determine <b>(A)</b> baseline peripheral CD4 T cell counts and <b>(C)</b> baseline CAR⁺ CD8 T cell counts. Two days later mice were infected with HIV-1 Bal via tail vein injection. 22 days post infection, <b>(B)</b> endpoint peripheral CD4 T cell counts and <b>(D)</b> CAR⁺ CD8 T cell counts were obtained. <b>(E)</b> Seven and <b>(F)</b> eighteen days post infection mice were bled and HIV RNA copies per μl plasma were determined by qPCR and normalized to CD4 T cell counts. The non-normalized viral loads are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s008" target="_blank">S8 Fig</a>. Mann Whitney Test was used to determine statistical significance (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001).</p