Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL.

After treatment with chimeric antigen receptor (CAR) T cells, interleukin-15 (IL-15) elevation and CAR T-cell expansion are associated with non-Hodgkin lymphoma (NHL) outcomes. However, the association of preinfusion CAR product T-cell functionality with clinical outcomes has not been reported. A single-cell analysis of the preinfusion CD19 CAR product from patients with NHL demonstrated that CAR products contain polyfunctional T-cell subsets capable of deploying multiple immune programs represented by cytokines and chemokines, including interferon-γ, IL-17A, IL-8, and macrophage inflammatory protein 1α. A prespecified T-cell polyfunctionality strength index (PSI) applied to preinfusion CAR product was significantly associated with clinical response, and PSI combined with CAR T-cell expansion or pretreatment serum IL-15 levels conferred additional significance. Within the total product cell population, associations with clinical outcomes were greater with polyfunctional CD4+ T cells compared with CD8+ cells. Grade ≥3 cytokine release syndrome was associated with polyfunctional T cells, and both grade ≥3 neurologic toxicity and antitumor efficacy were associated with polyfunctional IL-17A-producing T cells. The findings in this exploratory study show that a preinfusion CAR product T-cell subset with a definable polyfunctional profile has a major association with clinical outcomes of CAR T-cell therapy. This trial was registered at www.clinicaltrials.gov as #NCT00924326.

Controlling the Growth of the Skin Commensal Staphylococcus epidermidis Using d-Alanine Auxotrophy.

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d-alanine. The S. epidermidis NRRL B-4268 Δalr1 Δalr2 Δdat strain (SEΔΔΔ) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SEΔΔΔ colonized and increased expression of human ß-defensin 2 in cultured human skin models in vitro. SEΔΔΔ showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SEΔΔΔ as a live biotherapeutic strain whose growth can be controlled by d-alanine.IMPORTANCE The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d-alanine. The basis of this strain will support future clinical studies of this strain in humans.

CD8+ T-cell mediated anti-malaria protection induced by malaria vaccines; assessment of hepatic CD8+ T cells by SCBC assay.

Malaria is a severe infectious disease with relatively high mortality, thus having been a scourge of humanity. There are a few candidate malaria vaccines that have shown a protective efficacy in humans against malaria. One of the candidate human malaria vaccines, which is based on human malaria sporozoites and called PfSPZ Vaccine, has been shown to protect a significant proportion of vaccine recipients from getting malaria. PfSPZ Vaccine elicits a potent response of hepatic CD8+ T cells that are specific for malaria antigens in non-human primates. To further characterize hepatic CD8+ T cells induced by the sporozoite-based malaria vaccine in a mouse model, we have used a cutting-edge Single-cell Barcode (SCBC) assay, a recently emerged approach/method for investigating the nature of T-cells responses during infection or cancer. Using the SCBC technology, we have identified a population of hepatic CD8+ T cells that are polyfunctional at a single cell level only in a group of vaccinated mice upon malaria challenge. The cytokines/chemokines secreted by these polyfunctional CD8+ T-cell subsets include MIP-1α, RANTES, IFN-γ, and/or IL-17A, which have shown to be associated with protective T-cell responses against certain pathogens. Therefore, a successful induction of such polyfunctional hepatic CD8+ T cells may be a key to the development of effective human malaria vaccine. In addition, the SCBC technology could provide a new level of diagnostic that will allow for a more accurate determination of vaccine efficacy.

Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response.

BACKGROUND: It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies. METHODS: We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods. RESULTS: We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors. CONCLUSIONS: Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.