At Creative Biolabs, our scientists will professionally perform multiple evaluations on the post-transfection CAR-T cells to verify the solid success of CAR-T construction and superior function. With advanced technologies and experienced researchers, scientists from Creative Biolabs can design and operate a full range of validation assay services according to your T cells engineered objective. In detail, the following assays are adopted for this purpose:
In order to obtain high-quality CAR-T cells prior to scientific studies, preclinical researches, and clinical trials, transgene expression is evaluated for gene transfection efficiency and the reactivity of engineered lymphocytes to antigen. Ideal gene transfer means that exogenous DNA can be incorporated into the host genome and stable long-term expression. To ensure the best outcome, the expression level of CAR gene should be tested at different stages during the whole process of CAR-T cell therapy, including post-transfection with CAR gene, cell preparation, and clinical treatment period. Generally, the methods include western blot (WB), enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), immune-histochemical (IHC), etc.
To verify the integrity of immune functions of the CAR-T cells, the level of cytokines (mainly IFN-ɣ, IL-12, and GM-CSF) released by modified T cells is one of the indicators reflecting the functional activity of T cells in vivo. And the widely used methods include enzyme-linked immuno spot assay (ELISpot), intracellular cytokine staining assay (ICS), and flow cytometry.
Since the CAR-T cell therapy may cause some acute on-target/on-tumor toxicity (cytokine release syndrome, tumor lysis syndrome) or on-target/off-tumor toxicity, and in some cases, CAR-T cells undergo antigen-independent proliferation that could increase cell-mediated toxicity and raise the concern of immortalization of infused CAR-T cells, evaluation of the cytotoxicity of the CAR-T cells and the possible adverse effects is the most important validation step for the safety consideration.
Our scientists have developed the second and the third generation CAR products, which can enhance the persistence of CAR-T cells or -NK cells proliferation. Meanwhile, this activation and proliferation process can also be evaluated via our different proliferation assay approaches such as MTT, MTS, Cell Counting Kit-8 (CCK-8), 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in combination with flow cytometric analysis, [3H] thymidine incorporation and other techniques. At the same time, cytokines concentration (e.g. IL-2) assay is also available for determining T cell proliferation.
Since a major obstacle associated with current CAR-T cell therapy is that the limited replicative lifespan of CAR-T cells prohibits the in vivo expansion and long-term persistence of these cells, potentially hindering the long-term therapeutic efficacy of CAR-T cells, verification of the normal ability of CAR-T cells to proliferate in cultural and physiological conditions manifests as the first critical evaluation parameter. As a frontier biotech service provider, Creative Biolabs now provides preclinical services for CAR-T cell therapy. All tests are conducted by experienced technicians on the most appropriate models with the most advanced techniques.
Preclinical in vivo therapeutic efficacy test of CAR-T cells in animal models provides valuable information for their later clinical application. With years of preclinical in vivo research and project management experience, Creative Biolabs is able to provide therapeutic efficacy test service for a proof-of-concept or therapeutic efficacy validation, by utilizing appropriate animal models, designing comprehensive research practices and handling high-quality in vivo studies.
In addition, the customized tests are also welcome. Meanwhile, we also provide versatile testing assay kits as the following:
Copyright © 2007 - 2018 Creative Biolabs. All Rights Reserved