Non-clinical Research for Cellular Immunotherapies

Medicilon has built a one-stop research platform for the preclinical R&D of cellular immunotherapies, covering a variety of immunotherapy methods including CAR-T, TCR-T and CAR-NK. Using a wealth of animal models and a variety of advanced analysis techniques, comprehensively considering the characteristics of different research projects, Medicilon has completed numerous pre-clinical projects for clients worldwide.

• Cellular immunotherapy has developed by leaps and bounds in recent years, providing a cure for many difficult-to-treat cancers.  With the continuous discovery of tumor targets and mechanisms, the track is still full of vitality and hope bringing new breakthroughs in tumor treatment and human welfare.
  Medicilon has built a one-stop research platform for the preclinical R&D of cellular immunotherapies, covering a variety of immunotherapy methods including CAR-T, TCR-T and CAR-NK.  Using a wealth of animal models and a variety of advanced analysis techniques, comprehensively considering the characteristics of different research projects, Medicilon has completed numerous pre-clinical projects for clients worldwide.

Introduction to the Test Product

• Category：
  CAR-T CellTCR-T CellCAR-NK CellDNT CellTIL Cell
• The complexity of Immune Cells:
  Proliferation in vivoIndividualization and heterogeneityLimited batches and productionComplicated preparation processRaw materials with huge variationsComplex biological efficacy and safety evaluation

Pharmacology and Pharmacodynamics of Cell & Gene Therapy Drugs

• Safety Pharmacology
  Potential undesired effects of the study drug on physiological function at doses within or above the therapeutic rangeGenerally include effects on the central nervous system, cardiovascular system, and respiratory systemResearch on other organ systems may need to be supplemented depending on product characteristics
• In vitro Pharmacodynamic Studies
  Efficacy testing of cell therapy (such as CAR-T):Tumor killing rate or proliferation inhibition rateIFN-γ expression levelPhenotype changes of CAR-T cells
  Preparation and Evaluation of CAR-T
  
  Plasmid Vector Construction
  The PD-1 shRNA was integrated into the CAR plasmid, and then transduced into T cells by a lentiviral vector to obtain CAR-T cells with PD-1 silencing function.  The results showed that effective silencing of PD-1 significantly suppressed the immunosuppressive effect of the tumor microenvironment and prolonged the activation time of CAR-T cells, resulting in a longer tumor killing effect.  PD-1-silenced CAR-T cells significantly prolong the survival of mice with subcutaneous prostate and leukemia xenografts.  This demonstrated that PD-1 silencing technology is a suitable solution to promote the therapeutic effect of CAR-T cells on subcutaneous prostate and leukemia xenografts[1].  The plasmid sequencing work in this experiment was completed by Medicilon.
  
  CAR-T Cell Killing Assay
  
  CAR-T cell killing experiments show that CAR-T cell-dependent killing is increased compared with Mock-T cells
• In vivo Pharmacodynamic Studies
  Cell Therapy Test Substances
  Can be prepared from blood donated by healthy volunteersSome proof-of-concept studies could be done with alternative products of animal originThe similarities and differences between non-clinical test substances and clinical samples should be explained in the new drug application
  Gene Therapy Test Substance
  Consider factors such as production process, key quality characteristics (such as titer), preparations for clinical useIf there is species specificity, the activity of the test substance in non-clinical research should be evaluatedIf the vector uses an expression tag, the impact of the tag on the supportability of non-clinical trials should be analyzed
  Detection Methods and Evaluation Indicators
  Bioluminescent Imaging (BLI)Flow Cytometry: Detecting the number of tumor cells in animalsFlow Cytometry, ELISA, MSD: Changes in tumor-related cytokinesRelated Parameters: Tumor volume, tumor weight, tumor cell colonization site in animals and median survival period of animals
  Model Resources：
  Homologous Mouse ModelTransgenic Mouse ModelTransplanted Tumor Mouse Model

                  

  

  The picture shows the pharmacodynamic study of dual-target CAR-T cells in K 562 subcutaneously transplanted tumor mouse model
  Humanized Mouse Model of Immune System Reconstruction
  
  
  △The picture shows the pharmacodynamic study of the mouse pharmacodynamic model of hPBMC immune system reconstruction induced by Raji-luc fluorescein-labeled lymphoma cells
  Bispecific CAR-T Efficacy Research: CD19/BCMA
  
  CAR-T cell killing experiments show that CAR-T cell-dependent killing is increased compared with Mock-T cells

Pharmacokinetic Study of Cell & Gene Therapy Drugs

• Points to Consider for Pharmacokinetic Study:
  The parmacokinetc research studies sample in vivo behaviors, such as  distribution, proliferation and survival time. We also track transgenic product expressed and secreted outside the immune cells, if any, to provide its local and/or systemic exposure characteristics.
  Exposure: Gene therapy products should be analyzed and evaluated according to the specific characteristics of the product considering the actual exposure in non-clinical researchBiodistribution: Biodistribution of gene therapy products refers to the distribution, persistence and clearance of gene therapy products in target and non-target tissues in vivoShedding: Shedding assays should include testing for infectivity of excreted components
• Pharmacokinetic (Biodistribution) Detection Technology：
  Imaging TechnologyFlow CytometryImmunohistochemical TechniqueQuantitative PCR TechnologyddPCRELISPot
  Detection of Distribution of Lung Cell Therapy
  
  The detection results obtained by qPCR and flow cytometry are consistent
  

Nonclinical Safety Evaluation of Cell & Gene Therapy Drugs

• In toxicology research, a comprehensive safety analysis and evaluation of gene therapy products should be conducted, and the safety of the expression products of introduced genes should also be evaluated if necessary.  Gene therapy products should be effectively introduced/exposed in relevant animal species.
  The non-clinical safety risks of cell therapy (such as CAR-T cells) mainly include:
  Cytokine release syndrome (CRS)Graft-versus-host disease (GVHD)Reversible neurotoxicityReduction of B cellsOn-target/off-tumorTumorigenicity/tumorigenicity
  Model Resources:
  Homologous Mouse ModelTransgenic Mouse ModelTransplanted Tumor Mouse ModelNon-Tumor-Bearing Immunodeficiency MiceHIS Mouse ModelVarious methods of administration such as intravenous injection, intratumoral injection, and joint cavity injection are available.
  Evaluation Content：
  Single Dose Toxicity TestRepeated Dose Toxicity TestImmunogenicity / ImmunotoxicityIn vitro Soft Agar Clone Growth TestIn vivo Tumorigenicity / TumorigenicityFormulation SafetyOther: Express Product Safety
  Reference：

  [1] Jing-E Zhou, et al. ShRNA-mediated silencing of PD-1 augments the efficacy of chimeric antigen receptor T cells on subcutaneous prostate and leukemia xenograft. Biomed Pharmacother. 2021 May;137:111339. doi: 10.1016/j.biopha.2021.111339.