Search

Enzyme-linked immunosorbent assays (ELISAs) have traditionally been used to detect alloantibodies in patient plasma samples post hematopoietic cell transplantation (HCT); however, protein microarrays have the potential to be multiplexed, more sensitive, and higher throughput than ELISAs. Here, we describe the development of a novel and sensitive microarray method for detection of allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome, called HY antigens. Six microarray surfaces were tested for their ability to bind recombinant protein and peptide HY antigens. Significant allogeneic immune responses were determined in male patients with female donors by considering normal male donor responses as baseline. HY microarray results were also compared with our previous ELISA results. Our overall goal was to maximize antibody detection for both recombinant protein and peptide epitopes. For detection of HY antigens, the Epoxy (Schott) protein microarray surface was both most sensitive and reliable and has become the standard surface in our microarray platform. [ABSTRACT FROM AUTHOR]

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery. • Single assay to identify and characterize all major human immune cell lineages • Readily available and extensively validated antibody panel • Additional (>10) targets can be added to meet specific hypotheses • Allows identification of disease-associated immune signatures and biomarkers Hartmann et al. provide an experimental framework to identify and characterize all major human immune cell lineages in a single assay using mass cytometry (CyTOF). This validated and readily available workflow ensures comprehensive immunophenotypic analysis, improves data comparability, and allows identification of disease-associated immune signatures and biomarkers for human immunotherapy. [ABSTRACT FROM AUTHOR]

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches. [Display omitted] • Tumors and CAR T cell effectors can be simultaneously profiled using cell-free DNA • Alterations in multiple classes of genes influence outcomes after CAR19 therapy • Tumor genotype and phenotype influence CAR19 T cell expansion, and vice versa • A multivariable model incorporating tumor and effector features predicts outcomes Sworder et al. develop and apply a tool to simultaneously profile tumor and effector-mediated determinants of resistance to anti-CD19 CAR T cells using cell-free DNA. The authors profile two independent cohorts of patients with relapsed/refractory large B cell lymphoma and identify genomic alterations, molecular thresholds, and microenvironmental changes associated with resistance. [ABSTRACT FROM AUTHOR]

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1–6 × 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors. [Display omitted] • GPC2-CARs with standard 41BB designs require >3 × 104 molecules for full activation • Metastatic neuroblastomas express ∼5 × 103 GPC2 molecules/cell • CAR antigen density threshold is highly tunable using modular engineering and c-Jun overexpression • Tuned GPC2 CAR T cells mediate potent neuroblastoma regression without toxicity Heitzeneder et al. develop potent chimeric antigen receptor (CAR) T cells targeting glypican-2 (GPC2), an oncofetal antigen expressed in neuroblastoma. Accurate quantification of antigen density on clinical samples demonstrated that potency of traditionally designed GPC2-CARs was limited due to inadequate antigen sensitivity. Iterative engineering of CARs overcomes this limit, achieving profound anti-tumor efficacy without compromising safety. [ABSTRACT FROM AUTHOR]