On August 31, 2017, the US FDA approved the first gene therapy method for CAR-T cell therapy. The drug targets the antigen CD19, which is an antigen protein expressed on the surface of a variety of blood tumor cells, including B-cell lymphoma and leukemia cells, and is used for the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia under 25 years of age patient.
CAR-T therapy uses the patient’s own immune cells to eliminate cancer cells. Strictly speaking, this is a cell therapy, not a drug. As a representative of cell therapy, chimeric antigen receptor T cell immunotherapy can isolate T cells from patients and introduce chimeras targeting tumor antigens. Antigen receptor (CAR). These receptors can guide T cells to attack cancer cells, thereby killing the latter.
CAR-T cell therapy products are completely different from other non-in vivo expansion drugs. This difference is not only reflected in the aspects of individualization, small yield and limited batches, but also large differences in starting materials, immature preparation processes, and biological efficacy. And the complexity of safety evaluation and other issues.
On April 16, 2020, Dr. Wei Yi, the senior director of Medicilon’s Pharmacology Department, met the call and answered in detail the four major stages of CAR-T cell therapy non-clinical research: in vitro pharmacodynamic research, in vivo pharmacodynamic research, Various issues in pharmacokinetic research and safety research. Medicilon’s pre-clinical research service has established a research operation process and quality system that is in line with international standards. It has international AAALAC certification and China NMPA certification. The GLP laboratory also meets the US FDA, Australian TGA and other international standards. At the same time, Medicilon has grasped the new trends of immunotherapy and targeted tumor therapy, and continuously explored and improved the preclinical research platform for emerging tumor treatment.
A CAR-T in vitro pharmacodynamic study
- What in vitro tests can detect the effectiveness of CAR-T? Incubate CAR-T cells and tumor target cells in vitro to detect tumor killing rate or proliferation inhibition rate;
The expression level of IFN-γ;
Changes in the phenotype of CAR-T cells related to killing, etc.
- How to judge whether the selected in vitro efficacy test method is effective?
In the early methodological verification, it is necessary to analyze the specificity of the method to prove that its biological efficacy has a certain dose-effect relationship with CAR-T cells, and consider using in vivo methods to verify the effectiveness of in vitro methods.
Two CAR-T in vivo pharmacodynamics study
- What are the sources of test substances for non-clinical studies in vivo? Animal source, Proof-of-Concept;
Blood donated by healthy volunteers, products for clinical use;
The patient’s blood is generally unnecessary to use.
- What are the commonly used non-clinical research animal models for CAR-T drugs? Homologous mouse model
With a complete immune system, mouse-derived CAR-T cells can target and bind to tumor-associated antigens (TAA) in vivo. Limited to CAR-T products made from mouse-derived cells.
Transgenic mouse model
Rats with normal immune system express human TAA, and the research object needs to be mouse-derived cells.
Transplanted tumor mouse model
Transplanting human tumors into immunodeficient mice can be used to study the effects of human CAR-T cells on human tumors.
Humanized mice with immune system reconstruction
It is prepared by transplanting human immune cells into immunodeficient mice.
*At present, transplanted tumor mouse models are commonly used to study the inhibitory effect of CAR-T cells on tumors.
- What are the commonly used detection methods and evaluation indicators for in vivo testing?
Detection method Evaluation index
Bioluminescence imaging technology BLI fluorescence intensity indicates tumor load
Flow cytometry The number of tumor cells in the body
Immunological methods ELISA, MSD, etc. Changes in serum cytokines related to tumors
Routine pharmacology, pathology, tumor-related parameters
- What problems should be paid attention to in the design of in vivo drug efficacy testing? Contain different dose levels to reflect the dose-effect relationship;
Need to set up a T cell control group (untransfected T cells);
It is recommended to use CAR-T cells produced from different donors to evaluate the stability of the drug effect;
Add or set satellite group.
Three CAR-T pharmacokinetic studies
- The importance of CAR-T therapy PK research
CAR-T cell products have strong targeted cell killing activity, and can cause serious off-target side effects when combined with non-tumor cells in the human body.
The in vivo efficacy of CAR-T cells depends on the effective proliferation of cells and the formation of immune memory.
- How to choose an animal model for pharmacokinetic research?
At present, the most commonly used pharmacokinetic research model for CAR-T cells is the transplanted tumor model.
- What are the detection technologies for CAR-T pharmacy?
Quantitative PCR technology, etc.
Four CAR-T non-clinical safety studies
- The safety risks of CAR-T cell products mainly include: Cytokine Release Syndrome (CRS);
Decrease of B cells;
Targeting and off-target (on-target/off-tumor);
The tumorigenicity/tumorigenicity of CAR-T cells.
- Does the non-clinical safety study of CAR-T cells need to be conducted under GLP conditions?
The non-clinical safety evaluation research of CAR-T cell therapy products should also comply with the “Non-clinical Drug Research Quality Management Practice” (GLP).
Medicilon has obtained the AAALAC certification and the National Medical Products Administration (NMPA) GLP certificate. The GLP laboratory also meets the GLP standard of the US FDA, and the research data is recognized by the Australian Drug Administration. At the same time, we have established a research operation process and quality system that is in line with international standards, and meets domestic and foreign research and development standards and regulatory requirements.
- How to prepare the test substance in the non-clinical safety study of CAR-T cells?
In view of the particularity of the preparation process of CAR-T products, the entrusting party shall provide a complete quality analysis data report of the test substance, and shall provide coverage of all dose concentrations and simulation of all transportation processes, handling operations, and acceptance of the animal pre-dose process. Research data on stability of test substance.
After the test substance arrives at the research institution, if it needs to undergo processing operations such as resuscitation and resuspension before administration, the research institution must at least check the cell shape, total number of living cells, cell viability, color, and other foreign sources except cells. Observe or detect sexual foreign bodies.
If the test substance can be directly administered without treatment after arriving at the research institution (within the time allowed by stability), the above-mentioned testing can be carried out by the client.
- How to use existing human test data?
If the existing human body data after scientific evaluation can indicate the effectiveness and safety of cell therapy products, and can ensure the safety of clinical subjects, then non-clinical research can be based on the principle of specific species and specific analysis to avoid unnecessary Animal testing.
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