Full-process Service Drives Cell & Gene Therapy in Drug Development

 

• March 18, 2023

Table of Contents

1. Introduction to Cell & Gene Therapy (CGT)
2. CGT's Global Market Size
3. CAR-T Therapy
4. PD-1 silencing enhances anti-tumor efficacy in CAR-T cells
5. Summary and Outlook
6. Medicilon Gene Therapy Product R&D Service Platform

Cell & Gene Therapy (CGT) has developed quickly in recent years, providing the possibility of curing many difficult-to-treat malignant tumors. The global CGT industry is heating up rapidly due to technology, capital, and policies. Many CGT drug research and development have entered the clinical stage, providing new treatment concepts and ideas for refractory diseases.

Medicilon has established a complete cell & gene therapy drug research and development platform, which can provide one-stop services such as pharmacology, biodistribution, and safety evaluation research for cell & gene therapy products. Medicilon has completed many preclinical research projects for gene and cellular immunotherapy programs for clients using animal models and various advanced analytical techniques by considering the characteristics of different R&D projects.

Learn more about “Medicilon’s Cellular Immunotherapies platform”

Introduction to Cell & Gene Therapy (CGT)

What is Cell & Gene Therapy (CGT) ?

Cell therapy refers to using bioengineering methods to obtain cells with specific functions, and through in-vitro expansion, unique culture, etc., to make these cells have the parts of enhancing immunity, killing pathogens and tumor cells, and achieving the purpose of treating a particular disease.

Gene therapy can be divided into in-vitro (ex-vivo) therapy and in-vivo therapy. The research and development pipelines for in-vivo and in-vitro gene therapy are divided equally, and viruses are still the most commonly used gene carriers. The in-vivo or in-vitro method is determined by many factors, such as the location of the disease, the difficulty of obtaining cells in the body, etc. Generally speaking, there is no significant difference between in-vivo and in-vitro methods.

Whether it is in-vivo or in-vitro therapy, the vast majority of R&D pipelines use viral vectors. The most widely used platforms are adeno-associated viruses, lentivirus, and adenovirus. Among them, adeno-associated virus (AAV) is a non-enveloped virus with a size of about 26nm, containing only a single-stranded linear DNA gene and a protein capsid. AAV is widely used in gene therapy due to its low toxicity, high transduction rate, controllable cost, and low risk of insertion.

In-vivo and ex-vivo gene therapy[1]

The types of CGT drugs generally include gene therapy vectors carrying specific genes (such as virus AAV); gene-modified cell therapy products (such as CAR-T, CAR-NK); oncolytic virus products with specific functions; mRNA products; Oligonucleotide products, etc.

CGT's Global Market Size

According to the analysis of Coherent Market Insights, the global gene cell therapy market will grow from US$6,020.0 Million in 2017 to US$35.4 Billion in 2026, with a compound annual growth rate of 21.9%.

From the Coherent Market Insights official website

FDA-approved CAR-T therapy

CAR-T Therapy

Chimeric Antigen Receptor T (CAR-T) cells are T cells engineered to express a chimeric antigen receptor (CAR) on the cell surface to target tumor-associated antigens (TAAs), which in turn activate CAR-T cells to kill tumor cells. The CAR structure has evolved from the first-generation CAR (only CD3ζ signaling domain) to the second-generation CAR (CD3ζ+41BB or CD28 signaling domain) and the third-generation CAR (CD3ζ+41bb and CD28 signaling domain).

CAR-T therapy has shown promising clinical results in hematological malignancies such as B-cell non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, and Hodgkin’s lymphoma. Still, it has limited efficacy in solid tumors. This may be attributed to immunosuppressive mechanisms in the solid tumor microenvironment; immunosuppressive cells and molecules allow tumor cells to evade immune recognition during therapy. The effect of immune escape makes it difficult for CAR-T cells to penetrate tumor tissue and kill tumor cells.

Inhibition of the PD-1/PD-L1 axis plays a crucial role in the immune evasion of tumor cells. PD-1, a member of the CD28 family, is expressed in various immune cells, particularly CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, natural killer cells, and dendritic cells. PD-1 has two binding ligands, PD-L1 (B7-H1 or CD274) and PDL2 (B7-DC or CD273).

PD-L1 is expressed in various tumor cells, such as human lung cancer, ovarian cancer, colon cancer, kidney cancer, and melanoma, as well as in T cells, B cells, macrophages, dendritic cells, mesenchymal stem cells surface.

Compared with PD-L1, the expression of PD-L2 is limited, mainly expressed on the surface of activated macrophages, dendritic cells, and a small number of tumor cells. PD-1/PD-L1 binding can inhibit T cell activation and cytokine production, resulting in immunosuppression. PD-1/PD-L1 binding can also inhibit the activity of PI3K molecules and block CD28 signaling, thereby reducing the proliferation ability, tumor-killing ability, and cytokine release of T cells.

PD-1 silencing enhances anti-tumor efficacy in CAR-T cells

In this study, shRNA (short-hairpin RNA)-mediated gene silencing technology was used to block the influence of PD-L1/PD-1 immunosuppression axis on the proliferation and anti-tumor effect of CAR-T cells, thereby enhancing its therapeutic effect on subcutaneous prostate and leukemia xenograft.

The PD-1 shRNA was integrated into the third-generation of CAR plasmid, which was then transduced into T cells by lentivirus to obtain CAR-T cells with PD-1 silencing function. The results showed that the efficient silencing of PD-1 significantly inhibited the immunosuppressive effect of the tumor microenvironment, and prolonged the activation duration of CAR-T cells, resulting in a long tumor-killing effect. The PD-1 silenced CAR-T cells significantly prolonged the survival period of subcutaneous prostate and leukemia xenograft bearing mice. This study proved that PD-1 silencing technology is a suitable solution for promoting the therapeutic effect of CAR-T cells on subcutaneous prostate and leukemia xenograft.

The plasmids sequenced were fully identified by Medicilon.

Lentivirus vector plasmid construction[2]

The anti-tumor effect of CD19/△PD-1 CAR-T cells was evaluated in vivo. The results showed that CD19/△PD-1 CAR-T cells effectively and continuously inhibited tumor growth as marked by a decrease in tumor volume immediately after administration.

The tumor volume in CD19/△PD-1 CAR-T group was significantly smaller than those in CD19 CAR-T, NC-T, and saline groups, demonstrating that PD-1 silencing is a critical factor for the anti-tumor effect. The median survival times of mice treated with saline, NC-T, CD19 CAR-T, and CD19/△PD-1 CAR-T cells were 21, 23, 37, and 50 days, respectively. The mice treated with CD19/ΔPD-1 CAR-T cells survived for significantly more extended periods than CD19 CAR-T and NC T cells, demonstrating that blocking the PD-1/PD-L1 pathway reduces immunosuppression in vivo and increases the anti-tumor effect of CAR-T cells.

CD19/△PD-1 CAR-T cells inhibited the growth of K562-CD19 subcutaneous tumors in vivo[2]

Summary and Outlook

From traditional small molecules, protein drugs to nucleic acid drugs to cell & gene therapy and other new types of treatment have become the focus of the biomedical industry. The innovation of pharmaceutical companies is constantly upgrading, and Medicilon is also making continuous efforts to lay out the field of cell & gene therapy CDMO. It has established a complete cell & gene therapy drug research and development platform, which can provide cell & gene therapy products, a one-stop service for pharmacological efficacy, biodistribution, and safety evaluation research.

Medicilon Gene Therapy Product R&D Service Platform

Medicilon’s preclinical research services cover pharmacodynamics research, drug safety evaluation, pharmacokinetics research, biological analysis, etc. Medicilon has established a complete gene therapy product research and development platform, which can provide cell and gene therapy products with a one-stop shop pharmacological efficacy, distribution and safety evaluation research.

Service Qualification

Medicilon’s animal facilities have been certified by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) and the NMPA GLP certificate and successfully passed the GLP audit of the US FDA.

Service Module

• Pharmacological effects
• Biodistribution
• Immunogenicity
• Safety evaluation

Type of Drugs

• Cell therapy products
• Oncolytic virus products
• mRNA products
• Oligonucleotide products
• Gene editing products

Model

• Orthotopic tumor model
• Xenograft model
• Syngeneic model
• Humanized tumor model
• Transgenic model
• PDX model
• Other disease models

Analysis Platform

• Nucleic acid level analysis platform
• qPCR, RT-qPCR, dPCR
• Cell-level analysis platform
• Cell counter, FACS, ELISPOT, TCID50
• Protein level analysis platform
• ELISA, FIA, CLIA, ECL, HTRF, RIA, Gyrolab, Bio-plex

Advanced Instruments and Equipment

• CytoFLEX flow cytometer
• QIAcuity one digital PCR instrument
• Luminex, MSD Electrochemical luminescence analyzer
• MD SpectraM4/M5, ABI7500 qPCR
• Covaris E220, Kingfisher

Reference:
[1]. Kerstin B Kaufmann, et al. Gene therapy on the move. EMBO Mol Med. 2013 Nov;5(11):1642-61.
[2]. 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.
[3]. Y Agata, et al. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol, 8 (1996) 765-772.
[4]. D.M. Pardoll. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 12 (2012) 252-264.
[5]. Janis M Taube, et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med, 4 (2012) 127ra37.

FCM-Data-Analysis(BD-FACSCelesta)

BD-FACSCelesta-Flow Cytometry-12 channels