CAR-T Cell Products Validation
- 1. CAR-T Cell Products Validation In Vitro and In Vivo Assay
- 2. CAR-T Technology as a new therapy is still have many uncertainties. The therapeutic mechanism of CAR-T technology is not be fully elucidated. Personalized therapy. Patient individual variation. No universal standard as reference. Immune cell therapy as a new therapeutic method has no standard evaluation protocol from similar products as reference. Although no standard protocol. CAR-T cell products validation assays both in vitro and vivo are still essential for car t cell therapy development 01 02 03
- 3. • Most therapies to date have been experimental and performed under academic standards • Quality control testing uses mainly biological assays CAR-T cell Products SafetyPotency Purity Identity
- 4. Sterility & Safety Test Using preclinical experiments and in-process or final product testing to ensure the removal of reagents that were used in the manufacturing process, and the final product is free from contaminating microorganisms. Sterility: Ensure that raw materials are sterile, free of endotoxin, bacteria, fungi and mycoplasma etc. Replication competent lentivirus (RCL): p24 ELISA; psi-gag PCR; tet PCR etc. Cytokine Release Syndrome (CRS): Multi-cytokine ELISA kits. On-target, Off-tumor toxicity: Cell content analysis; histological analysis, animal model; Neurological toxicity: unknown mechanism, no specific assay method.
- 5. Purity: To ensure the removal of any extraneous matter. Product related impurities: cell marker antibody, FACS etc. • Define intended target cell population • Evaluate subtypes of cell populations • Remove/deplete irrelevant contaminating cell types Process related impurities: multiple detection method • Residual ancillary materials • Typically removed by washing multiple times Dynamics of cell populations may change during cell expansion, but final cell product should be well defined
- 6. Potency: to examine whether the therapeutic capability of the cell product will be as it was intended. Measures multiple product Critical Quality Attribute (CQAs): • Transduction efficiency: flow cytometry, PCR • CAR expression level: flow cytometry • Cytokine production: ELISA, ELISPOT • T cell proliferation & expansion: MTT, MTS, XTT assay, animal model • Target tumor cell killing: T cell killing assay, flow cytometric assays • Potential to persist/engraft post infusion this may not be confirmed until later phase 2 or phase 3 clinical trials.
- 7. Identity: To establish and certify the product characteristics • Detection of specific CAR sequences: PCR, FACS • Cell type distribution: additional cell surface markers, ELISA, FACS • CAR transduction efficiency: mRNA by qPCR
- 8. Laboratory techniques in development to increase manufacturing efficiency Artificial APCs
- 9. CAR Cell In Vitro Assay Service CAR Expression Test Cytokine Release Test Cytokine Release Test Viability and Bio-distribution Analysis In Vitro Cytotoxicity Test CAR-T Preclinical In Vivo Assay Service Construction of Xenograft Animal Model Efficacy Test of CAR-T In vivo Toxicity Evaluation of CAR-T
- 10. Address: 45-1 Ramsey Road, Shirley, NY 11967, USA Tel: 1-631-871-5806 Fax: 1-631-614-7828 Email: info@creative-biolabs.com Web: www.creative-biolabs.com/car-t/
Editor's Notes
- #3: CAR-T cells are typically assessed for their therapeutic potential first in vitro, then in mouse models, and finally in Phase I clinical trials, with financial and time commitments rising exponentially at each transition. As such, there is a need for in vitro assays that can reliably identify promising CARs at early stages of the bench-to-bedside pipeline. In vitro quantifications of cytokine production, T-cell proliferation, and target-cell lysis are the standard assays by which CARs are evaluated for basic function. However, these assays often fail to predict relative in vivo performance when comparing multiple CARs that demonstrate basic in vitro function
- #9: Artificial aAPCs have been developed from K562 cells, a chronic myelogenous leukemia cell line that does not express the major histocompatibility complex or T-cell-related costimulatory ligands.53–55 These cells have been transduced with lentiviral vectors, resulting in the specific expression of stimulatory and costimulatory molecules for the activation and expansion of different subsets of T cells. In addition to expressing CD32 or CD64, the high-affinity Fc receptor that can bind anti-CD3 and anti-CD28 mAbs, K562 cells can be modified to express other molecules on their surface, such as 4-1BB or a wide variety of other costimulatory receptors. These aAPCs have have been shown to result in increased activation and expansion of T cells compared with the magnetic bead–based aAPC.55 K562 cells may also be engineered to express cytokines and have a history in clinical trials as tumor antigen vaccines.56,57 Therefore, K562 cells may be an ideal cell scaffold on which the desired major histocompatibility molecules and costimulatory ligands can be expressed for the use of T-cell activation and expansion.