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.
BackgroundIt 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.MethodsWe 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.ResultsWe 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.ConclusionsSingle-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.Electronic supplementary materialThe online version of this article (10.1186/s40425-017-0293-7) contains supplementary material, which is available to authorized users.
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.
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 β-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.
Introduction: Autologous anti-CD19 CAR T cells have shown promising clinical efficacy in B cell malignancies, with T cell expansion and blood levels for IL-15, IL-10 and Granzyme B as correlates of objective response and toxicity (Kochenderfer et al. J Clin Oncol 2016; 34:LBA3010). It is unclear, however, which key immune programs in CAR T cells impact their in vivo expansion and clinical outcome. We evaluated in detail the functionality of anti-CD19 CAR T cells by using single-cell proteomics analysis (Lu et al. PNAS 2015;113:607-615). We explored how the polyfunctionality of pre-infusion CAR T cell products, post-stimulation with the CD19 antigen in vitro, associated with CAR T cell expansion in vivo and objective response. Methods: Product T cells were separated into CD4+ or CD8+ T cell subsets using microbeads. CD4+ or CD8+ fractions were then co-cultured with CD19-K562 targets or NGFR-K562 control cells, at a 1:2 ratio for 20 hrs. Single cells were then analyzed using a 32-plex panel of secreted cytokines, chemokines, and cytotoxic molecules. Specifically, T cells were loaded onto a single-cell barcode chip capable of assaying 32 secreted proteins/cell. The polyfunctional profile and strength (pSI) of each sample was determined (Ma et al. Cancer Discov 2013;3:418-429) and analyzed relative to in vivo expansion of the CAR T cells and patient response to the CAR T cell therapy. CAR T cell expansion in blood was measured by quantitative PCR. Results: Single-cell pSI of patient CAR T cells showed a statistically significant association (p = 0.011) with objective response (complete or partial response) to the therapy. While product pSI showed variability across patients, the median pSI was 2+ times higher for responders versus non-responders. The polyfunctional profiles for both CD4+ and CD8+ cells were dominated by effector molecules, stimulatory cytokines and chemokines. Polyfunctional CD4+ and CD8+ subsets with IFN-γ, IL-8 and/or MIP-1α correlated best with patient outcome, with CD8+ T cells showing co-expression of Granzyme B, and CD4+ T cells also comprising IL-17A+IL8+ and IL5+IL8+ subsets. While CAR expansion in vivo also correlated with objective response (p = 0.032), the association between product pSI and CAR cell expansion in vivo did not reach statistical significance (p = 0.079), suggesting that they bring independent contributions to predicting objective response. In support of that, a composite index integrating pSI and CAR T cell expansion in vivo associated best with clinical response (p = 0.005). Conclusion: Polyfunctionality of CAR T cells, in conjunction with their expansion in vivo, correlates with clinical outcome in an anti-CD19 CAR T cell clinical trial. Single-cell multiplexed proteomics measurements may provide powerful insight into the clinical performance of CAR T cell products. [J.R. and P.P. contributed equally to this study.] Citation Format: John Rossi, Patrick Paczkowski, Yueh-wei Shen, Kevin Morse, Brianna Flynn, Alaina Kaiser, Colin Ng, Kyle Gallatin, Tom Cain, Rong Fan, Sean Mackay, James Heath, Steven A. Rosenberg, James N. Kochenderfer, Jing Zhou, Adrian Bot. Polyfunctional anti-CD19 CAR T cells determined by single-cell multiplex proteomics associated with clinical activity in patients with advanced non-Hodgkin’s lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2990. doi:10.1158/1538-7445.AM2017-2990
34Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple 35 therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier 36 function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are 37 associated with a number of skin diseases. Here, we present the design of an engineered commensal 38 organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin 39 diseases. The development of novel bacterial strains whose growth can be controlled without the use of 40 antibiotics, or genetic elements conferring antibiotic resistance, enables modulation of therapeutic 41 exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that 42 requires exogenously supplied D-alanine. The S. epidermidis strain, NRRL B-4268 65 66 Staphylococcus epidermidis, recently dubbed as the "microbial guardian of skin health" (Stacy and 67 Belkaid 2019), is a strong candidate for use as a live biotherapeutic product (LBP) for skin conditions. S. 68 epidermidis is a Gram-positive bacterium that is ubiquitous in the human skin and mucosal flora. As one 69 of the earliest colonizers of the skin after birth, S. epidermidis plays an important role in cutaneous 70 immunity and maintaining microbial community homeostasis (Naik, Bouladoux et al. 2015, Linehan, 71 Harrison et al. 2018). In the clinical setting, S. epidermidis has demonstrated activity as a potential 72 therapeutic (Iwase, Uehara et al. 2010, Nodake, Matsumoto et al. 2015, Nakatsuji, Chen et al. 2018). In 73 Japan, a double-blind, randomized clinical trial, topical application of autologous S. epidermidis in 74 healthy volunteers increased lipid content of the skin, suppressed water evaporation and improved skin 75 moisture retention while showing no signs of erythema (Nodake, Matsumoto et al. 2015). Others have 76 shown that S. epidermidis is capable of producing antimicrobial peptides (AMPs) that selectively target 77
e15010 Background: Circulating biomarkers have the potential to detect cancer in its earliest stages and monitor patients in remission. The integration of proteogenomics in circulating biomarkers may transform the molecular diagnostics of cancer and accelerate basic and clinical oncology research. Proteomics bridges the gaps of functional information lost due to post-transcriptional and post-translational modifications in a genomic approach. A recent study showed that adding just 8 protein biomarkers to a panel of circulating DNA biomarkers increased the diagnostic accuracy up to 98% sensitivity and 99% specificity. However, the proteogenomic approach normally requires the use of multiple different assay technologies and laboratory workflows, including mass spectrometry. Methods: MosaicNeedles are densely integrated nanoneedle sensors fabricated on a planar substrate that integrates proteogenomic analysis in one platform. 94,000 sensors with more than 2 billion total nanoneedles can be integrated on to a standard SBS plate, which can be configured into 96, 384 or 1536 well format. Each sensor contains an array of nanoneedles, dedicated to detecting one analyte of interest. All nanoneedles comprising the same sensor are functionalized with the same capture probes. The capture probe can be either an antibody for protein detection or an oligonucleotide with a specific target sequence to a DNA fragment, mRNA, or miRNA of interest. Results: At low analyte concentration, the binding of proteins to the nanoneedles follows a Poisson distribution. Therefore, statistically, no more than one molecule is bound per nanoneedle. A further addition of aptamers or antibodies will form a sandwich complex with the target analyte. Since each of the nanoneedles has an intrinsic optical resonance spectrum and will red-shift as the sandwich complex forms on its surface, the number of analytes can be quantitated by simply counting the number of nanoneedles that display a color change. At high analyte concentration, each nanoneedle has more than one analyte, so the number of analytes can be calculated by averaging the spectrum shifts of all nanoneedles. This combined single molecule counting (digital) and spectrum shift (analog) analysis allows the platform to detect both high abundance and low abundance protein analytes in one reaction. A 10,000-plex study can be achieved with a total of 2.5 billion nanoneedles on a 50mm by 50mm consumable. In this consumable, a 2,000-plex proteome and 8,000 cell-free DNA fragments can be detected. Conclusions: A full proteogenomic quantification can be performed on the NanoMosaic platform in one reaction with high sensitivity and large dynamic range. It simplifies the workflow and allows users to integrate proteomic and genomic information to discover new circulating biomarkers.
Proteomics have the potential to transform biomarker discovery, diagnostics, and personalized medicine as it provides functional and actionable information linked to disease phenotypes. However, mass spectrometry (MS) – based proteomics has not been possible at large scale due to limited dynamic range, complex workflow and high experimental cost per sample. Antibody-based methods, although widely used in targeted proteomics for their better sensitivity, have been limited to the profiling of a few hundred selected proteins, partly due to the limited availability of antibody pairs. NanoMosaic has demonstrated antibody-aptamer sandwich assays on the MosaicNeedle࣪ platform. This expands the content size from a few hundred to a few thousand, achievable in one reaction with high sensitivity and specificity and low cross-reactivity. MosaicNeedles࣪ are densely integrated nanoneedle sensors fabricated on a planar substrate. 94,000 sensors with more than 2 billion total nanoneedles can be integrated on to a standard SBS plate, which can also be grouped into 96, 384 or 1536 formats. Each sensor contains an array of nanoneedles, dedicated to detecting one analyte of interest. All nanoneedles comprising the same sensor are functionalized with the same capture antibodies or aptamers. At low analyte concentration, the binding of proteins to the nanoneedles follows a Poisson distribution. Therefore, statistically, no more than one molecule is bound per nanoneedle. A further addition of aptamers or antibodies will form a sandwich complex with the target analyte. Since each of the nanoneedles has an intrinsic optical resonance spectrum and will red-shift as the sandwich complex forms on its surface, the number of analytes can be quantitated by simply counting the number of nanoneedles that display a color change. At high analyte concentration, each nanoneedle has more than one analyte, so the number of analytes can be calculated by averaging the spectrum shifts of all nanoneedles. This combined single molecule counting (digital) and spectrum shift (analog) analysis allows the platform to detect both high abundance and low abundance protein analytes in one reaction. We have demonstrated that the aptamer-antibody assay on the MosaicNeedle࣪ platform achieves better sensitivity than the well-developed antibody-pair assay. It also achieves larger dynamic range and alleviates the antibody cross-reactivity by refining aptamers during the SELEX process. The scalability in manufacturing nanoneedles combined with the low sample requirement and no fluorescent labeling all help to drive down the cost-per-protein to enable large-scale, unbiased proteomics. We envision researchers will use MosaicNeedle࣪ platform for both the upstream target discovery phase and then transfer seamlessly to the downstream validation phase by building customized and targeted panels for proteins of high interest that emerge from the discovery phase. Citation Format: Qimin Quan, Alaina Kaiser, Joshua Ritchey, Joe Wilkinson, John Geanacopoulos, John Boyce. MosaicNeedles: A tool for large-scale proteomics combining antibodies and aptamers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3919.
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