This slides briefly introduced why car t cell validation assay is essential for car t therapy development and which assays should be done to validate the safety, identity, purity and potency of car t cell products.
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
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02
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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
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
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
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.