Cell Therapy
Biocytogen offers advanced platforms to support the development of diverse cell therapies, including CAR-T, CAR-Macrophages, CAR-NK, TILs, and TCR-T, providing both in vitro services and in vivo models for comprehensive research.
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- Introduction
- Preclinical Capabilities for Cell Therapy Evaluation
- Examples of Cell Therapy Preclinical Research
Publication
- January 15, 2021Science Advances
- May 20, 2019Nature Communications
Introduction
Cell therapy involves transferring live cells into a patient to treat or cure diseases, using either the patient’s own cells (autologous) or donor cells (allogeneic). Immune cell therapies, especially CAR-T therapy, have revolutionized cancer treatment, achieving remarkable success in hematological malignancies. CAR-T cells are engineered T-cells modified to express chimeric antigen receptors that recognize and attack tumor antigens.
Biocytogen offers advanced platforms to support the development of diverse cell therapies, including CAR-T, CAR-Macrophages, CAR-NK, TILs, and TCR-T, providing both in vitro services and in vivo models for comprehensive research. We have developed a series of immunodeficient mice and established CDX models along with immune reconstitution models to facilitate the evaluation of cell therapies.
Preclinical Capabilities for Cell Therapy Evaluation
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Examples of Cell Therapy Preclinical Research
A Raji lymphoma mouse model was established using B-NDG mice and the efficacy of anti-human CD47 antibodies were verified. B-luc-GFP Raji cells (5×105) were injected via tail vein of B-NDG mice. In vivo imaging systems (IVIS) was used to observe tumor growth. When the fluorescence intensity of the tumor reaches about 1×106 p/sec, the animals were divided into one control group and three treatment group (n=6). (A) Fluorescence intensity curve of tumor cells; (B) Body weight. The results showed that all three anti-human CD47 antibodies could significantly inhibit tumor growth. B-NDG mice is a powerful model for efficacy verification of anti-human CD47 antibodies. Values are expressed as mean ± SEM.
Severe xenogeneic graft versus host disease (xeno-GVHD) would shorten treatment window and hamper efficacy evaluation of transplanted cell therapy. β2 microglobulin (B2M) is a small protein as a component of MHC class I molecules, which are present on all nucleated cells, and that are involved in self-recognition and in defense against micro-organisms. So knockout MHC class I β2-microglobulin (β2m) is an effective way to decrease GVHD.
(A) B2M is involved in the composition of MHCI molecules; (B-C) B-NDG B2m KO plus mice (blue line) showed decreased GVHD scores and prolonged survival compared to WT B-NDG mice(orange line) after implanted PBMCs; (D-E) CAR-T in vivo efficacy study in B-NDG B2m KO plus mice, tumor volume(D) and body weight(E).
- CD19-CAR T cells or untransfected T cells were co-cultured with target cells (Daudi, Raji, K562) at different ratios of 5:1, 10:1, and 20:1 for 24 hours. Tumor cytotoxicity was evaluated with Fixable Viability Dye by flow cytometry.
- The results showed that compared to untransfected T cells, CD19-CAR T cells exhibited significant cytotoxicity against Daudi and Raji cells, which expressed CD19. However, CD19-CAR T did not show specific cytotoxicity towards K562 cells, which was CD19 negative.
- CD19-CAR T labeled with CFSE and were co-cultured with target cells (Raji, K562) at different ratios of 5:1, 10:1, and 20:1 for 72 hours. CD19-CAR T proliferation was evaluated by flow cytometry.
- The results showed that CD19-CAR T cells exhibited significant proliferation when co-culture with Raji cells, which expressed CD19, compared with K562 cells, which was CD19 negative.
- CD19-CAR T were co-cultured with target cells (Raji, K562) at different ratios of 5:1, 10:1, and 20:1 for 24 hours. The expression of CD25 and CD69 of CD19-CAR T was evaluated by flow cytometry.
- The results showed that CD19-CAR T cells exhibited significantly increased expression of CD25 and CD69 when co-culture with Raji cells, which expressed CD19, compared with K562 cells, which was CD19 negative.
- CD19-CAR T were co-cultured with target cells (Raji, K562) at different ratios of 2:1, 5:1, and 10:1 for 24 hours. After co-incubation，supernatant was collected and detected IFN-γ and IL-2 by Bio-Lite Luciferase Assay System.
- The results showed that only the CD19-CAR T + Raji group exhibited a dramatic increase in secretion of IFN-γ and IL-2, compared with CD19-CAR + K562 group or untransfected groups.
- DNA was extracted from CD19-CAR T or untransfected T cells, collected on day 10 and day 13 respectively. Each well of the standard and sample plates contained two replicate wells. A standard curve concentration of 105 copies/µL, 104 copies/µL, 103 copies/µL, 102 copies/µL and 10 copy/µL was plotted.
The y-axis represents the Ct value, the x-axis represents the logarithm (base 10) of the concentration of standard copies. The slope of the standard curve represents the efficiency of real-time PCR amplification. When the amplification efficiency is 100%, the slope is -3.32.
- Amplification efficiency E=10−1/slope－1 ，The amplification efficiency for this experiment was 85%, we are trying more specific primers for further experiments.
