Christopher W. Peterson

Despite significant medical progress, HIV-1 remains a critical global health crisis. While combination antiretroviral therapy (cART) effectively suppresses viremia, it is not curative due to the persistence of latent viral reservoirs. Autologous hematopoietic stem and progenitor cell (HSPC) gene therapy offers a promising avenue toward sustained, drug-free remission. In this study, we investigated the feasibility and safety of a multifunctional “defend and attack” HSPC gene therapy strategy in the pigtail macaque (Macaca nemestrina) model. The species-optimized lentiviral vector, PM-MNDU-anti-HIV-1-huEGFRt, carries four therapeutic payloads: a pigtail macaque-specific CCR5 shRNA (PM-CCR5sh1005), a CD4-based chimeric antigen receptor (D1D2CAR), a C46 fusion inhibitor peptide, and a truncated human epidermal growth factor receptor (huEGFRt) serving as a cell-surface tracking marker and anti-EGFR antibody Cetuximab (CTX)-mediated negative selection marker. Following busulfan conditioning, autologous transplantation of vector-modified HSPCs in two pigtail macaques resulted in safe hematopoietic recovery. Both animals achieved sustained, multilineage gene marking in peripheral blood and lymphoid tissues without adverse effects. In one animal, upon Simian–Human Immunodeficiency Virus (SHIV) challenge, vector-modified cells underwent robust, antigen-driven multilineage expansion across both lymphoid and myeloid compartments, which contracted during cART-induced viral suppression and rapidly rebounded following treatment interruption. Administration of CTX demonstrated partial, transient reduction in huEGFRt⁺ cells, accompanied with a reciprocal increase in plasma viral load. These findings support the feasibility of evaluating a multi-pronged anti-HIV HSPC gene therapy strategy with a negative selection marker in a non-human primate (NHP) model.

Chimeric antigen receptor T (CAR T)-cell therapy has demonstrated curative potential in B-cell malignancies; yet, translating this success to chronic infections such as HIV remains a major challenge. In people living with HIV who are receiving suppressive antiretroviral therapy (ART), low-antigen levels limit CAR T-cell expansion and persistence. We previously reported data from a pilot study suggesting that HIV-targeted CD4CAR T cells could overcome this barrier through exogenous antigen supplementation, leading to robust in vivo expansion. Here we sought to comprehensively confirm and expand on those findings. We tested a broad array of strategies to enhance CD4CAR T-cell efficacy, including CRISPR-Cas9-mediated gene editing of immune checkpoint and HIV-associated genes, single and pooled competitive infusions of engineered CAR T cells, distinct CAR constructs incorporating either CD28 or 4-1BB costimulatory domains, and exogenous antigen boosting. We also developed highly sensitive droplet digital polymerase chain reaction assays to quantify CAR T-cell frequency and to corroborate the flow cytometry–based quantification of CD4CAR T-cell expansion. We evaluated these new approaches across multiple nonhuman primate (NHP) models of HIV, including both simian immunodeficiency virus– and simian-human immunodeficiency virus–infected, ART-suppressed NHPs. Although CD4CAR T-cell products exhibited antigen-specific proliferation and cytotoxicity ex vivo, they failed to expand, persist, or control viremia in vivo. We were also unable to confirm the previously observed CD4CAR T-cell expansions from our earlier studies, which have been retracted. Together, these data highlight the need for alternative strategies to potentiate anti-HIV CD4CAR T cells in the immunocompetent setting.

Despite the success of antiretroviral therapy in controlling HIV replication, latent viral reservoirs persist, presenting a major barrier to a cure. Current treatment approaches that aim to reactivate latent virus and eliminate infected cells, termed “shock and kill,” hold promise but have yet to demonstrate meaningful reservoir reduction in vivo. In this study, we explored combining ciapavir, a Smac mimetic latency-reversing agent, with adeno-associated virus–delivered (AAV-delivered) eCD4-Ig to treat antiretroviral therapy–suppressed, SHIV-infected rhesus macaques. We could demonstrate that a Smac mimetic can induce modest reactivation of the latent SHIV reservoir, as evidenced by transient increases in plasma viremia. However, while AAV-expressed eCD4-Ig conferred partial protection against intrarectal SHIV challenge in uninfected animals, neither eCD4-Ig nor ciapavir reduced the viral reservoir in SHIV-infected rhesus macaques, as determined by total SHIV DNA and a 5-target intact provirus detection assay. Animals treated with the combination showed no significant differences in viral rebound kinetics post–analytical treatment interruption compared with controls. Additionally, repeated ciapavir dosing resulted in adverse effects in some animals, suggesting potential toxicity with repeat administration. These findings highlight the challenges in reducing viral reservoirs using this shock-and-kill approach, particularly in SHIV-infected models, and suggest that further optimization of both latency-reversing agent and immune-mediated clearance strategies is required.

Hematopoietic stem cell (HSC) gene therapies provide lifelong benefit in numerous hematological diseases and disorders, but safety and toxicity remain a critical barrier for routine application. In the setting of immunodeficiency syndromes and infectious diseases such as HIV infection, conditioning regimens may exacerbate immune dysfunction, blunting, or impairing overall efficacy. Here, we conduct a head-to-head comparison of 2 novel antibody-drug conjugates (ADCs) with a pyrrolobenzodiazepine payload for autologous transplant in rhesus macaques. These ADCs target either CD117 or CD45 and are benchmarked against the clinical standard busulfan. We quantified the extent of myeloablation and immunosuppression, time to hematopoietic recovery, long-term engraftment of CCR5 CRISPR–edited autologous HSCs, and resistance to infection when challenged with increasing concentrations of an HIV-like virus. Both ADCs enabled engraftment of CRISPR-edited HSCs, although with lower levels of long-term editing compared with busulfan. We observed myeloablation with similar times to hematopoietic recovery and preserved lymphocyte counts with all 3 conditioning regimens, but neither ADC conditioning nor busulfan enabled sufficient CCR5 editing for viral immunity. Although these results only apply to the specific ADC-conditioning protocols tested here, they are a step toward developing targeted strategies to engraft cells with therapeutic edits and highlight the need for further refinement of antibody-based selection.

Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of B-cell malignancies; however, >60% of patients relapse within 1 year, often due to insufficient CAR-T persistence. Although mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted, to our knowledge, the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. After ex vivo characterization, we administered pooled autologous CAR-T arrays to 3 NHPs and tracked CAR abundance longitudinally using a novel digital droplet polymerase chain reaction assay. Ex vivo, CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo, MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral CAR-T cells and significantly outperforming canonical CD28- and 4-1BB–based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study end point, unlike other CARs, which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T activity.

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized treatment for B-cell malignancies, yet over 60% of patients relapse within one year, often due to insufficient CAR-T persistence. While mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. Following ex vivo characterization, we administered pooled autologous CAR-T arrays to three NHPs and tracked CAR abundance longitudinally using a novel digital droplet PCR assay. Ex vivo , CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo , MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study endpoint, unlike other CARs which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T cell activity. Key points: We developed the first pooled CAR-T screening platform in an immunocompetent nonhuman primate model to directly compare CAR designs. We identified MyD88-CD40 costimulatory domain as vastly superior to conventional domains in proliferation, trafficking and persistence.

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4 ⁺ T cells. We engineered an anti–PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239–infected rhesus macaques (RMs). Adoptive transfer of anti–PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti–PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1 ⁺ memory T cells in blood and tissues, including lymph node CD4 ⁺ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8 ⁺ memory T cells. These data indicate anti–PD-1 CAR T cells depleted PD-1 ⁺ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.