SBIR-STTR Award

Despite the demonstrated benefit of CD19 targeted CAR-T immunotherapeutics, two challenges remain to bringing the therapeutics to market. The first key challenge is to reproducibly manufacture the therapy so that the cells' cytokine mediated function acts in a predictably consistent fashion post manufacturing. The second key challenge is to manage the cell's adverse effect (immuno-toxicity), namely cytokine release syndrome (CRS) of IL-6 and other inflammatory cytokines, while also ensuring efficacy against the CD19 target. A more effective pre-infusion quality control test to ensure consistent and safe functioning would be not only address these two critical challenges, but help pharmaceutical firms satisfy FDA concerns in both of these areas. Such a test would have deep impact on ensuring the pharmas help these therapies to market, and in making sure that patients in need with incurable B-cell malignancies have access to these revolutionary therapies. A more reliable in vitro CAR-T functional test would allow physicians to remove or modify the inconsistent or unsafe cell therapies prior to injection, significantly reducing risk to the patient, and improving odds of therapeutic success. However, the current leading pre-infusion tests do not address the most important clinical requirements. The first requirement of such a test is to evaluate the CAR functional cell cytokine activation in the single-cell poly-functional cell subsets, a correlate of quality immune response, and also monitor for adverse functional reactions amongst these cell subsets. That requires up to 18 non-overlapping cytokines per cell. A second requirement is to measure the cells' true secretions in an “ex vivo” manner, rather than fixing the cells and manipulating their true function. IsoPlexis' microchip technology meets these two needs for the first time. It measures the range of efficacy polyfunctional cytokine markers (anti-tumor, stimulatory, chemotactic), while measuring the CRS related inflammatory and also regulatory cytokines from those subsets (up to 42 cytokines per cell). It does so in an ex vivo platform that not only measures true cell secretion to CD19 target, but can also interact with and respond to the target cell directly on device. IsoPlexis plans to use its core technology and learning to create an in vitro assessment test to measure for consistent range of function for in CAR-T cell therapies and to monitor for markers of safety and efficacy. We propose to do the following specific aims: (1) Develop a panel and reproducible assay for measurement of CD19+ CAR cells upon interaction with CD19 (immobilized) to ensure consistent function to CAR target. (2) Implement the assay above with transfected CAR-T donor samples, and demonstrate a clinically useful workflow. With the Phase I award support, we expect in the future to use it as a companion diagnostics tool to monitor the response of patients and ensure proper management of these personalized living drugs in every patient, justifying the broad impact of the proposed microdevice in immune-oncology.

Public Health Relevance Statement:
Despite the demonstrated benefit of emerging cancer immunotherapeutics, their adverse effect, e.g., immunotoxicity, is a major concern and could be potentially life threatening. We propose to further develop and validate our single cell cytokine mapping device and use it as a working platform to quality check the T cells immune effector function/immunotoxity during CAR-T treatment along with our pharmaceutical and academic partners. In the future, we expect to use our device as a companion diagnostics tool to monitor the response of patients and ensure proper management of these personalized living drugs in every patient, which has a broad impact in immuno oncology.

Project Terms:
Address; Adoptive Transfer; Adverse effects; Antibodies; Antigens; Area; Autoimmune Process; Award; B lymphoid malignancy; B-Cell Lymphomas; base; Binding; Biological Assay; CD19 Antigens; CD19 gene; cell fixing; Cell physiology; Cell secretion; Cell Therapy; Cells; Clinical; clinically relevant; companion diagnostics; cytokine; Cytokine Activation; Data; Development; Devices; Ensure; Flow Cytometry; Future; Gold; Immune; Immune response; Immunotherapeutic agent; immunotoxicity; improved; In Vitro; Inflammatory; Infusion procedures; Injection of therapeutic agent; instrument; Interleukin-6; Learning; Life; Malignant Neoplasms; Maps; Marketing; Measurement; Measures; Mediating; meetings; member; microchip; Modification; Monitor; Nature; oncology; patient stratification; Patients; personalized management; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physicians; Printing; programs; Proteins; Protocols documentation; Quality Control; Reaction; Reporting; Reproducibility; response; Risk; Safety; Sampling; Slide; Small Business Innovation Research Grant; success; Surface Antigens; Syndrome; T cell therapy; T-Lymphocyte; Technology; Testing; Therapeutic; Time; tool; Toxic effect; treatment response; tumor; Work

Adoptive transfer of autologous T cells engineered to express chimeric antigen receptors (CARs) has emerged as a promising immunotherapy for patients with hematologic malignancies, such as CD19 CARs in leukemias and lymphomas. However, challenges remain in terms of manufacturing consistency and the functional profile of the CAR-T cell product, since infused cells, as “living drugs”, can be activated and release a variety of cytokines upon specific antigen recognition. These secreted cytokines may result in not only therapeutic efficacy, but also life-threatening immunotoxicity, such as cytokine release syndrome and neurologic toxicity. It is essential for cancer patients to have a full spectrum potency and toxicity profiling of CAR-T cell products before infusion. We have developed a single-cell barcode chip (SCBC) technology, which enables (a) assaying 30+ secreted proteins per individual live cell, (b) a broad range of immune cell functions covering efficacy and safety, (c) is designed to fit various types of immune cells, (d) is the first quantitative metric of these proteins per cell, and (e) requires samples sizes totaling only 5000 cells, all critical leaps over flow cytometry based platforms. With the collaboration of two leading CAR-T pharmaceutical companies, IsoPlexis’ single-cell deep profiling has revealed, for the first time, a pre-infusion correlate to post-infusion patient response. The SCBC analysis of 20 patients with lymphoma demonstrates a significant correlation of the polyfunctional strength of the CAR-T cells with objective response (complete or partial) to the CAR-T cell therapy (p = 0.012), indicating a powerful metric of quality assessment, where the other gold standard technologies did not detect any significant correlations. Based on the clinical data and the automation progress in our SBIR Phase I grant, we are proposing the following specific aims in this two-year Phase II application: AIM 1: Develop automated “flow cell” consumable compartment, which captures 32-plex single-cell cytokine response, to prepare for full automation required by large CAR-T trial (months 1-10); AIM 2: Produce a fully automated cartridge analysis and workflow system, to allow 10 samples assayed in parallel with minimal user interaction, allowing easy introduction into clinical core labs (months 1-15); AIM 3: Produce a comprehensive informatics suite, and fully test our automated system and informatics in two 30 patient cohort trials, providing robust and predictive biomarkers for pre-infusion CAR-T quality. Establish MSKCC and UCLA beta sites for post-phase II transition (months 7-24). With this Phase II submission, we will deliver the first effective CAR-T pre-infusion quality check assay to predict objective response in patients, in an easy-to-use, automated system that can be used throughout all cellular immunotherapy trials.

Public Health Relevance Statement:
Adoptive transfer of autologous T cells engineered to express chimeric antigen receptors (CARs) has emerged as a promising immunotherapy for patients with hematologic malignancies, such as CD19 CARs in leukemias and lymphomas. However, challenges remain in terms of manufacturing consistency and the functional profile of the CAR-T cell product, as cytokines secreted by these cells, post-infusion, may result in not only therapeutic efficacy, but also life-threatening immunotoxicity. We have developed a single-cell, highly- multiplexed, barcoding device that measures all safety and efficacy functional cytokines secreted from single live cells, to meet the need of cancer patients to have a full spectrum potency and toxicity profiling of CAR-T cell products before infusion.

Project Terms:
Address; Adoptive Transfer; Antibodies; Antigens; Area; Autologous; Automation; base; Biological Assay; Businesses; Cancer Patient; CD19 Antigens; CD19 gene; Cell physiology; Cell Therapy; Cells; cellular engineering; chimeric antigen receptor; chromatin immunoprecipitation; Clinical; Clinical Data; Clinical Trials; cohort; Collaborations; cytokine; Cytometry; Data; design; Devices; Ensure; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Goals; Gold; Grant; Hematologic Neoplasms; Heterogeneity; HIV; Image; Immune; Immunosuppressive Agents; Immunotherapeutic agent; Immunotherapy; immunotoxicity; improved; Individual; Informatics; Infusion procedures; Legal patent; leukemia/lymphoma; Life; Lymphoma; Measures; member; Memorial Sloan-Kettering Cancer Center; Neurologic; Pancreas; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phenotype; phenotypic biomarker; Population; predictive marker; Proteins; Proteomics; Protocols documentation; Receptor Cell; response; Safety; Sample Size; Sampling; Site; Slide; Small Business Innovation Research Grant; Solid Neoplasm; Staging; Surface; Syndrome; System; T-Lymphocyte; Technology; Testing; Time; Toxic effect; Treatment Efficacy; tumor