Lilliput

Catalog for the exhibition Lilliput held at the Seton Hall University Walsh Gallery, June 8 - July 24, 2009. Curated by Jeanne Brasile and Asha Ganpat. Includes an essay by Jeanne Brasile and Asha Ganpat. Includes color illustrations

Engineering Chimeric Antigen Receptors for Durable Control Over HIV-1 Replication

This thesis project aimed to develop chimeric antigen receptors (CARs) capable of durably suppressing the Human Immunodeficiency Virus Type 1 (HIV) replication, by building upon a previous CD4-based CAR that was employed in several clinical trials. We applied lessons learned from cancer-targeting CARs to optimize the CAR vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains, in an effort to determine which components augmented the ability of CD8 T cells to control HIV replication. CD8 T cells expressing the optimized CARs were at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR or TCR-based approaches and substantially better than broadly neutralizing antibody-based CARs. We then utilized a humanized mouse model of HIV infection to demonstrate superior control over HIV replication, better protection of CD4 T cells, and greater CAR T cell expansion with the optimized vectors compared to the original clinical trial vector. Compared to optimized CD4 CARs containing the CD28 costimulatory domain, CARs containing 4-1BB expanded better in vivo in the absence of antigen and resulted in greater control over HIV replication. We found that the CD4 CAR promoted infection of transduced CD8 T cells and employed CCR5 zinc finger nucleases (ZFNs) or a GP41-based fusion inhibitor to protect the CAR T cells. We employed ZFN-pretreated, CAR-transduced CD8 T cells in our mouse models and saw an enrichment of the disrupted alleles in HIV-infected mice compared to mock controls. In humans, a functional cure will require CAR T cells to prevent the spread of HIV following virus reactivation from the latent reservoir. We modeled this scenario in vitro using ART patient T cells and latency reversing agents (LRAs). Preliminary data suggest that CD4 CAR T cells can respond to low levels of antigen produced by resting ART patient cells in the presence of LRAs. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and provide optimism that CAR T cells could help achieve a functional cure

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

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

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

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

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