Abstract B074: A recall antigen-based potency assay for immunomodulatory biologics that could discriminate responders from nonresponders
| Autor: | Pirouz Daftarian, Marc C. Delcommenne, Eden Kleiman, Wushouer Ouerkaxi |
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| Rok vydání: | 2019 |
| Předmět: | |
| Zdroj: | Cancer Immunology Research. 7:B074-B074 |
| ISSN: | 2326-6074 2326-6066 |
| Popis: | The surge of therapeutic regimens involving immune checkpoint antagonists and co-stimulatory pathways (ACP) antagonists used as single agents or in combination necessitates better functional screening assays. In vitro functional screening assays need to be non-invasive and high throughput. The recall antigen assay is arguably the one that, in an antigen specific controlled system, directly assesses the activity of potential immune checkpoint inhibitors (ICI) candidates, in a relatively physiologically relevant manner. The recall antigen assay has been often used in the past to prove the potency of ICI or ACP as indicated in publications and submissions to regulatory bodies. We have conducted a thorough optimization combined with modifications to the recall antigen assay to enable the need for a more effective and efficient assay.Our recall antigen assay has been optimized across multiple parameters including donor selection based on expression of co-inhibitory/co-stimulatory molecules. This facilitates the selection of the right donor for the right candidate. We have optimized both stimulation and readout components to enhance signal to noise ratio. For example, we reduced the duration of in vitro antigen specific CD8+ T-cell clonotype expansion from 2 weeks to 5 to 7 days. To maximize the direct readout, we have used an MHC Tetramer guided analysis, using a cocktail of MHC tetramers made with stimulating peptides. To further empower the tetramer assay with functionality, we also have co-stained tetramer positive cells with activation markers. Finally, we verified the assay using various activity-confirmed ICIs and controls. The assay uses characterized PBMCs stimulated with a pool of peptides with positive and negative treatments (e.g., PD-1 blockades and IgG4) and “test samples,” which are the candidates to be tested. Depending on the donor’s recall responses, a typical assay results in two to 6-fold increase of the antigen specific MHC tetramer positive population. Furthermore, after developing this recall antigen assay, we validated it by performing the assay in different laboratories, using different reagents lots, different instrumentations, and different operators. Recall antigen assay data indicated that not all donors respond to approved immune checkpoint blockades. Healthy donors stimulated with anti CD3 or cytokine cocktails varied showed a wide a wide variability of immune checkpoint molecules (ICM) cell surface levels. Further studies using characterized PBMCs in our recall antigen potency assay revealed that the expression of immune checkpoint molecules may contribute in unresponsiveness to a given ICI. In conclusion, we have developed an improved in vitro recall antigen potency assay for immunomodulatory biologics functional screening including ICIs and APCs. Furthermore, we observed that in our studies this potency assay divides hosts into responders and nonresponders. Interestingly, we also showed significant variation of persons’ PBMC responses to ICIs, which may be related to their inherent expression levels of immune checkpoint molecules. We intend to confirm this finding with further studies using well-annotated clinical samples. Citation Format: Marc Delcommenne, Eden Kleiman, Wushouer Ouerkaxi, Pirouz Daftarian. A recall antigen-based potency assay for immunomodulatory biologics that could discriminate responders from nonresponders [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B074. |
| Databáze: | OpenAIRE |
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