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Brain microglia (MG) may serve as a human immunodeficiency virus 1 (HIV) reservoir and ignite rebound viremia following cessation of antiretroviral therapy (ART), but they have yet to be proven to harbor replication-competent HIV. Here, we isolated brain myeloid cells (BrMCs) from nonhuman primates and rapid autopsy of people with HIV (PWH) on ART and sought evidence of persistent viral infection. BrMCs predominantly displayed microglial markers, in which up to 99.9% of the BrMCs were TMEM119+ MG. Total and integrated SIV or HIV DNA was detectable in the MG, with low levels of cell-associated viral RNA. Provirus in MG was highly sensitive to epigenetic inhibition. Outgrowth virus from parietal cortex MG in an individual with HIV productively infected both MG and PBMCs. This inducible, replication-competent virus and virus from basal ganglia proviral DNA were closely related but highly divergent from variants in peripheral compartments. Phenotyping studies characterized brain-derived virus as macrophage tropic based on the ability of the virus to infect cells expressing low levels of CD4. The lack of genetic diversity in virus from the brain suggests that this macrophage-tropic lineage quickly colonized brain regions. These data demonstrate that MG harbor replication-competent HIV and serve as a persistent reservoir in the brain.

To better understand the molecular mechanism that drives neuroinflammation, we analyzed the protein profiles of 27 brains from HIV with HIV (PWH) on antiretroviral therapy (ART), including various stages of HIV-associated neurocognitive disorders (HAND), and compared them to 9 HAND-negative controls. We found that most of the proteins that were increased—about 66.7%—were involved in immune response pathways. Of these, 23.3% were specifically related to type I interferon (IFN-I) signaling, which remains active in the brain through both HIV-related and unrelated mechanisms. Using single-cell RNA sequencing (scRNA-seq) on brain tissues collected during rapid autopsies from participants in the Last Gift cohort, we found that IFN-I signaling was especially strong in astrocytes, microglia (MG), and endothelial cells. In a mini-brain organoid model of acute HIV infection, IFN-I signaling was also highly active in astrocytes but less so in MG. Interestingly, IFN-I activation can happen without HIV being present—expression of human endogenous retrovirus-W1 (HERV-W1) Env can directly trigger this response in astrocytes, and it continues in glial cells even with effective ART. Together, our findings point to persistent IFN-I activation in glial and endothelial cells in the brain, which may contribute to neuroinflammation and cognitive disorders in PWH on ART.

The COVID-19 pandemic continues around the world, where the United States is among the worst in terms of both morbidity and fatality of the viral infection. We aim to investigate the plausible link of tissue SARS-CoV-2 viral entry gene expression, such as TMPRSS2 and ACE2, with infection and death by gender during the COVID-19 pandemic in the United States. We find a significantly higher incidence of COVID-19 death in men than in women, even though SARS-CoV-2 infection in women is higher than in men. We discover that the expression of TMPRSS2 and ACE2 in intestine, but not in lung, tends to be positively associated with the incidence of SARS-CoV-2 infection in men. In contrast, the high incidence of death in men is negatively correlated with TMPRSS2/ACE2 expression in intestine. Strikingly, the correlation of TMPRSS2/ACE2 expression with SARS-CoV-2 infection and death is the opposite in females, compared with that in males. Interestingly, male hormone signaling seems to be involved in mortality, as the low expression of testosterone receptor AR in the prostate contributes to death in men according to age. These observations point to a plausible contribution of male hormone metabolism in the regulation of TMPRSS2/ACE2 signaling to fatality by SARS-CoV-2 infection in men.

Adeno-associated virus (AAV)-mediated genetic targeting of microglia remains a challenge. Overcoming this hurdle is essential for gene editing in the central nervous system (CNS). Here, we characterized the minimal/native promoter of the HEXB gene, which is known to be specifically and stably expressed in the microglia during homeostatic and pathological conditions. Dual reporter and serial deletion assays identified the critical role of the natural 5’ untranslated region (-97 bp related to the first ATG) in driving transcriptional activity of the mouse Hexb gene. The native promoter region of mouse, human and monkey HEXB are located at -135, -134 and -170 bp to the first ATG, respectively. These promoters were highly active and specific in microglia with strong cross-species transcriptional activities, but did not exhibit activity in primary astrocytes. In addition, we identified a 135 bp promoter of CD68 gene that was highly active in microglia but not in astrocytes. Considering that HEXB is specifically expressed in microglia, these data suggest that the newly characterized microglia-specific HEXB minimal/native promoter can be an ideal candidate for microglia-targeting AAV gene therapy in the CNS.

BackgroundExpansion and persistence of CAR-T products has historically correlated with patient outcomes. CAR-T expansion and pharmacokinetics (PK) are typically monitored by flow cytometry or by transgene qPCR, using detection reagents that are bespoke to individual products, limiting universal application across products or platforms. In monitoring allogeneic CAR-T products, these methods also usually lack capabilities for distinguishing multi-donor origin. Here, we present qualification and clinical application of a quantitative and universal solution (AlloCell) for monitoring of ADI-001, a first-in-class CD20-targeted allogeneic γδ1 CAR T cell therapy.1 MethodsAlloCell was analytically validated for 2-genome-based (1 donor) and 3-genome-based (2 donors) analyses. To additionally qualify AlloCell for ADI-001 monitoring, genomic DNA (gDNA) mixtures were prepared using 1) gDNA from peripheral blood of two healthy individuals, or 2) gDNA from 2 FFPE blocks (to establish feasibility for application in solid tissues). The mixtures mimicked cell product levels of 3%, 1%, 0.3%, 0.1%, and 0.03%.Applying this method, clinical blood samples from the Phase 1 evaluation of ADI-001 (NCT04735471)2 were processed and gDNA was extracted for analysis. gDNA samples were analyzed using the AlloCell assay workflow. Briefly, quality control (QC) was performed using Nanodrop and Qubit. Next, gDNA was used in NGS library preparation and sequenced using NextSeq technology. Sequencing data were analyzed using the AlloCell bioinformatics pipeline.ResultsThe levels of cell product measured for qualification closely matched the expected levels of donor-derived gDNA in cell mixtures, with a high degree of linearity (n=3, R2=1), and was reproduced in FFPE samples (n=3, R2=0.996). We observed high precision across triplicates, with CVs ranging from 0.31% for 3% mixture, to 5.3% for the 0.03% mixture, or for FFPE samples, from 0.93% for 1% mixture to 23.6% for the at the lowest bound mixture of 0.03%.Data from ADI-001 patient samples demonstrated AlloCell’s ability to track cell product kinetics in a clinical setting and determined dose-dependent exposure for ADI-001 across four dose levels evaluated. Finally, in one patient receiving two separate and sequential doses of ADI-001, each manufactured from distinct donors, AlloCell successfully distinguished independent expansions of the first and second donor-sourced cell products.ConclusionsAlloCell was validated as a precise and sensitive analytical platform that can quantitatively monitor PK of allogeneic cell therapies. Application of AlloCell to monitor clinical exposure of ADI-001 successfully demonstrated dose-dependent expansion and persistence across multiple dose levels and resolved donor origin for distinct ADI-001 expansions associated with a multiple dosing regimen.Trial RegistrationNCT04735471: A Safety and Efficacy Study of ADI-001, an Anti-CD20 Allogeneic Gamma Delta CAR-T, in Subjects With B Cell Malignancies (GLEAN-1)ReferencesNishimoto KP, Barca T, Azameera A, et al. Allogeneic CD20-targeted γδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models. Clin Transl Immunology. 2022;11(2):e1373. Published 2022 Feb 2. doi:10.1002/cti2.1373Neelapu SS, Stevens DA, Hamadani M, et al. A Phase I Study of ADI-001: Anti-CD20 CAR-Engineered Allogeneic Gamma Delta 1 T cells in Adults with B-cell Malignancies. Blood 2022;140(Supplement 1):4617–1619. https://doi.org/10.1182/blood-2022–157400Ethics ApprovalEthics approvals have been obtained from each clinical site enrolling patients on NCT04735471, A Safety and Efficacy Study of ADI-001, an Anti-CD20 Allogeneic Gamma Delta CAR-T, in Subjects With B Cell Malignancies (GLEAN-1): Stanford University Medical Center, Stanford, CA; University of Miami-Sylvester Comprehensive Cancer Center, Miami, FL; Northside Hospital Blood and Marrow Transplant Group of Georgia, Atlanta, GA; The State University of Iowa, Iowa City, IA; Norton Cancer Institute, Louisville, KY; Baylor Scott & White Research Institute, Dallas, TX; MD Anderson Cancer Center, Houston, TX; Medical College of Wisconsin, Milwaukee, WIConsentInformed consent was obtained from all patients prior to enrollment, treatment and sample analyses.

FT596 is an induced pluripotent stem-cell (iPSC)-derived chimeric antigen receptor (CAR) natural killer (NK) cell therapy with three antitumour modalities: a CD19 CAR; a high-affinity, non-cleavable CD16 Fc receptor; and interleukin-15-interleukin-15 receptor fusion. In this study, we aimed to determine the recommended phase 2 dose (RP2D) and evaluate the safety and tolerability of FT596 as monotherapy and in combination with rituximab. We also aimed to evaluate the antitumour activity and characterise the pharmacokinetics of FT596 as monotherapy and in combination with rituximab. In this phase 1, first-in-human trial, we evaluated FT596 in patients with relapsed or refractory B-cell lymphoma at nine sites in the USA. Patients who had received at least one previous systemic therapy and had no curative treatment options were eligible for inclusion. FT596 was administered after conditioning chemotherapy without rituximab (regimen A) or combined with rituximab (regimen B). The study consisted of a dose-escalation phase using a 3 + 3 design, with dose escalation commencing at 3 × 107 viable cells as a single dose on day 1 and done independently for individual regimens. A treatment cycle consisted of conditioning chemotherapy with cyclophosphamide (500 mg/m2) and fludarabine (30 mg/m2) intravenously on days –5 to –3, followed by FT596 administered at various doses and schedules, without (regimen A) or with (regimen B) a single dose of rituximab (375 mg/m2) intravenously on day –4. Supportive care was determined by the treating investigator. Patients were observed for dose-limiting adverse events for 28 days. Patients who tolerated therapy and derived clinical benefit could receive subsequent cycles of study treatment, with modification of conditioning chemotherapy dose if clinically indicated. The dose-expansion phase evaluated additional patients at selected doses and dosing schedules that had been found to be tolerable. The primary endpoints of the study were the incidence and nature of dose-limiting toxicities within each dose-escalation cohort to determine the maximum tolerated dose or maximum assessed dose to establish the RP2D and the incidence, nature, and severity of adverse events, with severity determined according to National Cancer Institute Common Toxicity Criteria and Adverse Events version 5·0. The trial was registered with ClinicalTrials.gov, NCT04245722. Between March 19, 2020, and Jan 12, 2023, 86 patients with B-cell lymphoma received FT596 on regimen A (n=18) or regimen B (n=68). 22 (26%) of 86 patients were female and 72 (84%) of 86 patients were White. Patients had received a median of four previous lines of therapy (range 1–11) and 33 (38%) of 86 patients had received previous CAR T-cell therapy. The maximum tolerated dose was not reached. Cytokine release syndrome was reported in one (6%) of 18 patients (maximum grade 1) on regimen A and nine (13%) of 68 patients on regimen B (six with maximum grade 1 and three with grade 2). Neurotoxicity was not observed. FT596 was well tolerated as monotherapy or with rituximab and induced deep and durable responses in patients with indolent and aggressive lymphomas and the RP2D was preliminarily identified to be 1·8 × 109 cells for three doses per cycle. This study supports that cell therapy using iPSC-derived, gene-modified NK cells is a potent platform for cancer treatment and suggests that such a platform might address limitations of currently available immune cell therapies, including manufacturing time, heterogeneity, access, and cost. Fate Therapeutics.

Oral mezigdomide is a cereblon-modifying agent that results in degradation of transcription factors essential for myeloma cell survival. Given with dexamethasone, it induced responses in 40% of heavily pretreated patients.

Induced pluripotent stem cell (iPSC)–derived natural killer (NK) cells offer an opportunity for a standardized, off‐the‐shelf treatment with the potential to treat a wider population of acute myeloid leukaemia (AML) patients than the current standard of care. FT538 iPSC‐NKs express a high‐affinity, noncleavable CD16 to maximize antibody dependent cellular cytotoxicity, a CD38 knockout to improve metabolic fitness, and an IL‐15/IL‐15 receptor fusion preventing the need for cytokine administration, the main source of adverse effects in NK cell–based therapies. Here, we sought to evaluate the potential of FT538 iPSC‐NKs as a therapy for AML through their effect on AML cell lines and primary AML cells. We observed that FT538 iPSC‐NKs induce effector‐to‐target cell ratio dependent apoptosis in cell lines and primary AML cells, including cells from high‐risk patients. Flow cytometric analysis revealed that FT538 iPSC‐NKs induce AML cell death when combined with the AML therapies: cytarabine, venetoclax and gilteritinib. Moreover, cytarabine did not affect FT538 iPSC‐NK viability, suggesting that iPSC‐derived NK therapies and chemotherapy may be a promising treatment combination. This study provides the basis for further study of iPSC‐derived NK cell therapies as a treatment option for high‐risk AML patients, particularly those with disease resistant to standard therapies.

Cluster of differentiation 47 (CD47) is a transmembrane protein ubiquitously expressed on human cells but overexpressed on many different tumor cells. The interaction of CD47 with signal-regulatory protein alpha (SIRPα) triggers a “don’t eat me” signal to the macrophage, inhibiting phagocytosis. Thus, overexpression of CD47 enables tumor cells to escape from immune surveillance via the blockade of phagocytic mechanisms. We report here the development and characterization of CC-90002, a humanized anti-CD47 antibody. CC-90002 is unique among previously reported anti-CD47 bivalent antibodies that it does not promote hemagglutination while maintaining high-affinity binding to CD47 and inhibition of the CD47–SIRPα interaction. Studies in a panel of hematological cancer cell lines showed concentration-dependent CC-90002-mediated phagocytosis in acute lymphoblastic leukemia, acute myeloid leukemia (AML), lenalidomide-resistant multiple myeloma (MM) cell lines and AML cells from patients. In vivo studies with MM cell line-derived xenograft models established in immunodeficient mice demonstrated significant dose-dependent antitumor activity of CC-90002. Treatment with CC-90002 significantly prolonged survival in an HL-60-disseminated AML model. Mechanistic studies confirmed the binding of CC-90002 to tumor cells and concomitant recruitment of F4-80 positive macrophages into the tumor and an increase in expression of select chemokines and cytokines of murine origin. Furthermore, the role of macrophages in the CC-90002-mediated antitumor activity was demonstrated by transient depletion of macrophages with liposome-clodronate treatment. In non-human primates, CC-90002 displayed acceptable pharmacokinetic properties and a favorable toxicity profile. These data demonstrate the potential activity of CC-90002 across hematological malignancies and provided basis for clinical studies CC-90002-ST-001 (NCT02367196) and CC-90002-AML-001 (NCT02641002).