For short-term contact links, such as collected tissues or cells, intermediates, finished product return, etc., the researcher should conduct safety assessment or compatibility research on the container.
The secondary packaging container or material used for transportation should also be verified, such as research and verification on its shading, sealing and resistance to mechanical pressure.

5. Pharmacology and Toxicology Research

(1) General considerations for non-clinical evaluation
1. Evaluation content
Because the material composition and mechanism of cell therapy products are different from those of small molecule drugs, macromolecular biological drugs and medical devices, traditional and standard preclinical evaluation methods may not be suitable for cell therapy products. The core of the non-clinical evaluation content of cell therapy products is the cell type and clinical use. It is also closely related to the cell source, type, production process, modification, and non-cellular ingredients in the prescription; the choice of evaluation content is also related to the research and development plan and the corresponding clinical The test plan is related; due to the wide variety of cell therapy products, there are huge differences and uncertainties in the treatment principles, biological behaviors in the body, and the pros and cons of clinical applications. The evaluation requirements for each product should follow the principle of "specific analysis", and Carry out necessary non-clinical trials in the subsequent clinical development process. At the same time, the non-clinical safety evaluation guidelines for biotechnology drugs (ICHS6) can provide a reference for the preclinical evaluation of cell therapy products.
When cell therapy products fall into the following situations, more preclinical studies are recommended:
Stem cells and pluripotent cells, including induced pluripotent stem cells (iPS);
Genetically modified cells, pay attention to the functional realization and safety risks of the introduced genes;
Products with complex production process and complex prescription composition;
Cell products for long-term, systemic medication or cells that remain in the human body for a long time.
It is used in the treatment of non-fatal diseases that currently have treatment means.
2. Test substance requirements
Non-clinical evaluation trials should try to use cell products intended for clinical use; the production process and quality of test substances used for key trials supporting registration applications should be consistent with the products intended for clinical trials (if inconsistency should be explained , And evaluate its impact on predicting human response).
If animal-derived analogs can be used for non-clinical evaluation due to the selection of relevant animals, animal-derived analogs should be as similar as possible to the production process and quality standards of cell therapy products, and necessary comparisons should be made to confirm alternatives For the quality attributes of, consider the following comparison parameters:
Procedure for organization or sample acquisition
Cell identification, isolation, expansion and in vitro culture procedures
Cell growth kinetic parameters (such as cell doubling time, cell growth curve, cell proliferation peak time)
Phenotypic and functional characteristics (such as secretion of growth factors and cytokines, cell population-specific phenotype/genotype markers)
Final product formula / cell scaffold planting method (if any)
Storage conditions and cell viability of the final product.
The parameters of the above-mentioned similar cell therapy products and human-derived cell therapy products should be as close as possible to maximize the accuracy of data obtained from animal experiments. When the non-clinical safety data of animal-derived analogs are extrapolated to the initial safe dose of clinical trials, the data needs to be fully and scientifically considered. Any similarities and differences between pre-clinical samples and clinical samples should be explained during the new drug application.
3. Animal species selection
For non-clinical evaluation, suitable species of animals should be selected for testing. The selected animals should have a biological response similar to the expected human response for cell therapy products. The following aspects should be considered in the selection of animal models:
Ÿ Physiology and anatomy are comparable to humans, the anatomical part of the injection/transplantation site;
Ÿ Tolerance/susceptibility to infection and replication of viral or microbial vectors used in gene therapy;
Ÿ Immune tolerance to human cell products or cell products carrying human transgenes;
Ÿ The feasibility of the clinical drug delivery system/process, the feasibility of transporting a specific dose of cells to the therapeutic target;
Ÿ The availability of immunodeficiency animals can realize long-term safety assessment of products.
If there is a lack of relevant animals, in vitro experiments, homologous animal models, gene knockout and transgenic models, humanized animals, etc. can be used to conduct experiments to study the pharmacological and toxicological properties of one or more aspects of cell therapy products. In addition, animal models of diseases can be considered for pharmacological and toxicological evaluation. Due to the characteristics of cell therapy products (such as prolonged product effect duration, persistence in the body, complex mechanism of action between cell products and the disease environment, and invasive route of administration), disease animal models may be more suitable for evaluating the activity of these products And security.
4. Method of administration (route)
In non-clinical evaluation, the administration method of cell therapy products should be able to simulate the clinical administration method to the greatest extent. If the clinical administration method cannot be simulated in animal experiments, the alternative administration method/method should be clarified in preclinical research, and its scientificity and rationality should be clarified.
When a special drug delivery device (device) is used for drug delivery, the drug delivery device system used in non-clinical trials must be consistent with the clinic, otherwise the reasons for the actual drug delivery device system used should be explained. The applicant should provide sufficient data to judge the safety of the drug delivery device system. The evaluation of the drug delivery device system can be evaluated separately or together with the cell therapy product.
5. Test substance analysis
Taking into account the complexity of cell characteristics analysis methods, on the basis of the quality inspection report provided by the production unit, at least the cell count and the determination of the proportion of living cells should be carried out, consistent with the requirements of pharmaceutical research. Other cell attributes and preparation quality analysis shall be based on specific conditions. The researcher decides.

(2) Pharmacodynamic research
Pharmacodynamic research should adopt reliable methods to verify the basic treatment principles of cell therapy products, study the homing ability and immune regulation function of cell therapy products, observe the expression of specific cytokines and/or specific genes, and determine biological effect markers. The design of the experiment should consider factors such as the mechanism of action of the cell therapy product, the length of the disease cycle, and the method of administration, as well as the characteristics and survival time of the cell therapy product.
A variety of animal models should be used to complete the pharmacodynamic evaluation of cell therapy products, and the selected animal models should be close to experimental animals in the pathophysiological process of human diseases. When animal models cannot fully reflect the pathophysiological process of human diseases, alternative models and in vitro experiments should be considered; if alternative models cannot be found, the preparation of similar cell therapy products from animal sources should be considered for pharmacodynamic evaluation. When the clinical need to adopt a special method of administration can not be achieved in the small animal experiment, and when the clinically similar dosage is used, small animals cannot be administered, it is recommended to use large animals.

(3) Pharmacokinetic research
Pharmacokinetic studies should be able to clarify the in vivo processes of cells and the accompanying biological behaviors. Traditional pharmacokinetic research methods may not be suitable for the research of cell therapy products. Appropriate animal models should be selected according to the types and characteristics of cells. Generally consider that there are half males and half males, such as choosing single-sex animals, the reasons need to be explained; appropriate biological analysis methods should be established according to the research purpose and the clinical value of the test indicators and necessary verifications; human-derived cells entering the animal body may be affected due to their immunogenicity Cell assays should be considered when establishing detection methods and analyzing results. The content of pharmacokinetic research includes but is not limited to the following:
1. Biodistribution
Distribution refers to the initial location of cells after they are given to the body, including the presence in blood and various tissues; the initial distribution is greatly affected by blood flow, and is also related to the specificity of cells; biodistribution studies must consider the representativeness of sampling and Consistency, select at least three time points in the experiment cycle for research. One or more cell tracking methods should be used, and the scientific rationality of the methods should be explained. The available technical methods are imaging technology, PCR technology, immunohistochemistry, etc.
2. Cell migration, colonization (homing), differentiation
After the initial distribution, cells will migrate, colonize in certain tissues or further differentiate into functional cells to exert their therapeutic effects; the distribution and persistence of cell therapy products are the most important factors affecting the effectiveness and safety of cell therapy products Factors should be observed dynamically, if necessary, until the disappearance or loss of function of these cells.
3. The biological behavior of cells in the body
Metabolism research also pays attention to the proliferation of cells in the body, the secretion of biomolecules, and the interaction with host tissues. The interactions also include the media (excipient components) of cell therapy products and the surrounding tissue reactions caused by secreted biologically active molecules.
4. Special considerations for genetically modified cells
For genetically modified cells, in order to prove the therapeutic principles of genetic modification, it is necessary to conduct research on the existence, expression, and biological effects of the expressed product of the target gene.

(4) Safety evaluation
1. GLP compliance requirements
The safety evaluation of cell therapy products should comply with the requirements of the "Quality Management Practice for Non-clinical Drugs" (GLP). It is recommended to follow the GLP specifications to the greatest extent. However, due to the obvious technical complexity in the safety evaluation of cell therapy products, for some indicators to be tested under non-GLP conditions, the reliability and reliability of the test results should be explained and evaluated. The impact of integrity, and then to evaluate its impact on the overall safety evaluation of cell therapy products.
2. Safety pharmacology test
The active substances secreted by cells in the body may affect the functions of the central nervous system, cardiovascular system, and respiratory system; the cells themselves are distributed or colonized in vital organs, and the prescription ingredients of cell products may also affect organ functions. Therefore, for cell therapy Products should be considered for safety pharmacological testing. If the toxicity test finds potential risks, relevant safety pharmacological tests should be supplemented to confirm its unexpected pharmacological effects.
3. Single dose toxicity test
Cell therapy products intended for use in humans require a single dose toxicity test. Through this test, the dose-response relationship between the dose and systemic and/or local toxicity can be obtained, which is helpful to understand the target organ of toxicity, and provides an important reference for the dose design of repeated administration toxicity test. The observation time of a single dose should be longer than that of a conventional single dose toxicity test, because the cells survive for a long time in the body and their biological effects will last longer.
4. Repeated administration toxicity test
The trial design should include the basic elements of conventional toxicology research trials, combined with the special design of cell therapy, in order to obtain as much safety information as possible.
Selection of animal species: Generally, the principle of specific analysis of specific issues should be followed. For products that may not survive in animals due to immunogenicity, immunodeficient mice or rats can be selected for testing. If there is enough information to support that cell products will not cause cells to survive in animals due to immunogenicity, then suitable non-immune-deficient animals can be selected for testing. If cell therapy products involve stents or drug delivery devices, and they cannot be administered to rodents due to their small size, large animals can be considered for testing. Under normal circumstances, male and female animals should be used for testing. If there are special indications, single-sex animals can be selected according to the actual situation. If there are no related animals, non-related animal tests are also valuable for evaluating the production process, the safety of the full prescription, and non-target effects.
Dosage group design: refer to the guidelines for repeated administration toxicity studies, but at least include the clinical dose or effective dose, and the group higher than this dose, combined with the prescription composition and production process, set up an appropriate control group.
Observation indicators: In addition to the conventional observation indicators, combined with product characteristics, select appropriate observation indicators, such as: selective tissues, such as administration site and possible proliferative lesions, to determine whether there are human cells; evaluate species-specific primary Tumor (rodent or human). Special indicators of cell therapy should be observed, such as the presence of input cells, secretion of biologically active molecules, immune response, interaction with host tissues, etc.; if combined with other tests, corresponding indicators should be set.
5. Immunogenicity and immunotoxicity test
Immunogenicity is affected by a variety of factors, including homologous or non-homologous treatments, administration site, cell maturation status, administration times, immune diseases, and immune system aging, etc., which need to be combined with product characteristics and clinical applications Design the immunogenicity evaluation. In the immunogenicity evaluation, the difference between animal models and clinical patients should be fully considered, and it is necessary to select animal species that are close to the human body's response to the test substance for immunogenicity evaluation. Since human-derived cell therapy products are foreign objects for laboratory animals, they are very easy to produce immunogenic reactions in animals, although such immunogenic reactions do not predict that similar immunogenic reactions will also occur in humans. , But it can indicate the existence of such risks. In addition, in addition to the immunogenicity caused by the cells in cell therapy products, attention should also be paid to the substances used or produced in the preparation process of cell therapy products such as induced pluripotent stem cells, such as culture media and forced expression gene products. Possible immunogenic reaction. Therefore, it is necessary to evaluate the new components of cell therapy products separately.
In addition to inducing the body to produce an immunogenic response, cell therapy products can also produce immunotoxicity by acting on or regulating the immune system. For cell therapy products that mainly act on or modulate the immune system to exert pharmacological effects, the immunotoxicity induced by cell therapy should be particularly worthy of attention. Taking tumor immunotherapy as an example, the immunotoxicity risks induced by cell therapy products mainly include three types: targeted toxicity of non-tumor tissues, off-target toxicity of tumor tissues, and cytokine release syndrome. Since animals in different disease states may have different responses to cell therapy products, normal animals and disease model animals can be selected for immunotoxicity evaluation.
6. Tumorogenicity test
The tumorigenicity risk of cell therapy products depends on the differentiation status of different cells in the product, the cell culture method used in the production process causes changes in growth kinetics, transgene expression of genetically modified cells (such as multiple growth factors), and induction or enhancement of the host The possibility of tumor formation in the body and the target patient population need to be comprehensively considered based on the above characteristics.
The tumorigenicity test must use the clinically intended product for the test. Due to immune rejection, human-derived cell therapy products are generally recommended to use immunosuppressed rodents for tumorigenicity tests.
7. Reproductive toxicity test
The evaluation of reproductive and developmental toxicity of cell therapy products mainly depends on the characteristics of the product, clinical indications and the population to be used clinically, and should be analyzed according to specific conditions.
8. Genotoxicity test
For human-derived cell therapy products, unless the product has a direct interaction with DNA or other chromosomal materials, no genotoxicity test is required.
9. Special safety test
Local tolerance, tissue compatibility and tolerance to secreted substances should be evaluated.
10. Other toxicity tests
For cell therapy products that use genetic modification, attention should be paid to replication-competent virus production (Replication-competent Viruses, RCV) and insertion mutations, especially the activation of oncogenes. The risk of RCV formation can be assessed during the production quality assessment stage, and the risk of insertion mutations should be evaluated.

Six, clinical research

When the cell preparation enters the clinical trial stage, it should follow the requirements of the "Quality Management Practice (GCP) for Drug Clinical Trials". In principle, the research content of clinical trials should include clinical safety evaluation, pharmacokinetics research, pharmacodynamics research, dose exploration research and confirmatory clinical trials