Objective: To investigate the anti-tumor efficacy of chimeric antigen receptor-nature killing cells (CAR-NK cells) against prostate stem cell antigen (PSCA) via DAP12 co-stimulation signal. Methods: CAR-NK cells were generated via lentiviral transfection and the expression of CAR in NK cells was evaluated using flow cytometry. The anti-tumor activity of CAR-NK cells was further assessed in both cellular and animal models. Results: The prostate cancer cell DU145 expressed PSCA with a positive rate of about 98.50%. Flow cytometry revealed that (64.07 ± 3.01)% of CAR-NK cells were positive. Furthermore, the killing rate of CAR-NK cells was approximately 1.5 times higher than that of control NK cells. ELISA tests showed that CAR-NK cells released significantly higher levels of TNF-α, IFN-γ, CD107α, Granzyme B, and Perforin-1, when compared to control NK cells in the process of killing DU145 cells. Additionally, animal experiments show that CAR-NK cells had a better inhibiting effect in the growth of DU145 tumor cells in mice compared with control NK cells as significant difference was found between the two groups (P<0.000 1). Conclusion: The findings suggested that PSCA-targeting CAR-NK cells with DAP12 had a greater anti-prostate tumor effect, providing a potential cellular therapeutic option for the treatment of prostate cancer.
Objective: To construct T cells encoding chimeric antigen receptor (CAR) targeting glypican-3 (GPC3) and chimeric antigen costimulatory receptor (CCR) targeting epithelial cell adhesion molecule (EPCAM), and analyze their activities in vitro. Methods: Gene fragments encoding EPCAM-CCR and GPC3-CAR was cloned into lentivirus vector plasmid. The recombinant CCR+CAR pCDH vector was identified by enzyme digestion, PCR and sequencing. T lymphocytes were isolated, activated and expanded. A stable combinatorial CAR modified cell line was generated using the lentivirus. Western blot, RT-PCR and flow cytometry (FCM) were used to verify the expression of CCR+CAR in CCR+CAR T cells. The secretion of cytokines IL-2, IL-4 and INF-γ was detected by ELISA. Results: The CCR+CAR pCDH lentiviral recombinant plasmid was successfully constructed. Human T lymphocytes were successfully isolated, activated and expanded. Human T lymphocytes were successfully infected with CCR+CAR pCDH lentivirus. RT-PCR and Western blot showed that the target protein was successfully expressed, and FCM analysis showed that the expression rate of CCR+CAR on CCR+CAR T cells was about 42%. ELISA was used to detect CCR+CAR co-culture with HepG2 and Lu-7 expressing GPC3 and EPCAM, the secretion levels of IL-2, INF-γ and IL-4 were higher, and the co-culture of human normal liver cells L-02 did not find effective activation of CCR+CAR T cells. Conclusions: The combined CCR+CAR T cells were successfully obtained, and the immune effect of CAR T combined with the two antigen recognition signals was attempted in hepatocellular carcinoma cells. The result laid a foundation for the immune effects in the subsequent in vitro experiments, and provided a potentially novel approach to augment the off-target effect of CAR-T immunotherapy for solid tumors.
Objective: To isolate a fully human anti-PD-L1 antibody from a naïve phage-display human Fab library and increase its affinity through in vitro antibody affinity maturation, and then modify the antibody with disulfide bond stability to obtain a humanized anti-PD-L1 disulfide-stabilized diabody with high affinity and stability. Methods: First, the PD-L1 recombinant protein was used as the antigen in the naïve phage Fab antibody library to screen Fab antibodies. Then the hot spots in the variable region gene of Fab antibody with good binding ability against PD-L1 were analyzed, and the phage antibody mutation library was constructed by randomly mutating 7 hot spots in the light chain and heavy chain CDR3 region, from which antibodies with improved affinity were screened. Finally, two disulfide bonds were introduced in the antibody backbone region to construct disulfide-stabilized anti-PD-L1 ds-Diabody, which was expressed in GS115. Results: Six specific anti-PD-L1 phage Fab antibodies were screened and obtained, the CDR3 region hot spot of one of the antibodies with better binding ability was randomly mutated to successfully construct a phage antibody mutant library with a library capacity of 1.14×108 CFU/mL, and phage antibody mutant strains with increased affinity of about 6 times were screened. The disulfide bond was introduced to the backbone region of the antibody, and the ds-Diabody with stable disulfide bond was successfully constructed and expressed. Conclusion: The ds-Diabody has higher binding affinity and better stability than Fab antibody to PD-L1, which provides a strong basis for drug development, tumor treatment and other research on PD-1/PD-L1 pathway.
Bispecific T cell-redirecting antibodies are designed to bind to selected tumor-associated antigens and to CD3 of the T cell receptor. Linking tumor cells and T cells results in activating T cells and inducing targeted T cell-mediated killing of the recognized tumor cells. They have become one of the most promising approaches in tumor immunotherapy. Although this therapy was successfully applied in hematological malignancies therapy, no significant progress in the treatment of solid tumors have been achieved. In this article, we review the major challenges of bispecific T cell-redirecting antibodies, and novel strategies to overcome these hurdles as well as to broaden the indications for this therapy, particularly to solid cancers.
In recent years, the incidence rate of hematological malignancies has increased year by year in China. At present, chemotherapy and bone marrow transplantation are the main methods for treating hematological malignancies. Chemotherapy has significant side effects and damages the normal tissues and organs of patients, and bone marrow transplantation faces a series of problems such as typing and rejection reactions. Chimeric antigen receptor modifies T cells, namely CAR T cell immunotherapy, which has made great breakthrough and progress in the treatment of malignant tumors in recent years, especially in the clinical treatment of hematological tumors. However, CAR-T cell immunotherapy also faces the problems of a lack of specific targets, inhibition of tumor microenvironment and poor effect of solid tumor invasion. This article briefly introduces the principle, development history, current research progress and existing problems of CAR-T cells. Meanwhile, the clinical application status and prospect of CAR-T cells in hematologic malignancies are summarized and discussed in detail. Finally, we summarize and discuss the research difficulties and directions of CAR-T therapy in solid tumors, so as to provide a reference for subsequent related studies.
Traditional cancer therapies such as chemotherapy and radiotherapy have problems such as poor targeting and high toxic side effects. Tumor-targeting bacteria can specifically colonize in the solid tumor microenvironment, and can be genetically engineered to synthesize anti-cancer drugs, improve the selectivity of drugs for tumor tissues and avoid the damage to normal tissues by chemotherapeutic drugs. These advantages make tumor-targeting bacteria a research hot spot in targeted cancer therapy in recent years. Escherichia coli Nissle 1917(EcN), a probiotic bacterium that has been widely studied and used, has attracted much attention in bacterial cancer therapies. EcN is non-pathogenic and non-immunotoxic and has a highly effective tumor-targeting ability. It can be rapidly cleared in normal tissues. Here, the latest advances in engineering of EcN for tumor-targeting therapy were reviewed. The applications of EcN in adjuvant therapy were also discussed. With the advancement of genetic engineering and synthetic biology, scientists’ ability to design and synthesize bacteria is growing stronger. EcN as a programmable living drug holds the promise of becoming a powerful weapon against cancer.
Malignant tumor is considered to be one of the main causes of human death, and its morbidity and mortality are increasing by years. Tumor-targeted drugs can act precisely on tumors and have gained a lot of attention in recent years. Specific identification and extensive binding to tumor cells are key to achieving tumor-targeted therapy, and the lack of effective and specific pan-tumor targeting tools has greatly limited the further development of tumor-targeted therapy. Compared with existing tumor targeting approaches, Plasmodium recombinant protein rVAR2 can specifically bind Oncofetal chondroitin sulfate (ofCS), which is widely expressed on the surface of tumor cells, and has the advantage of pan-tumor targeting, providing a new idea for tumor targeting therapy. In this paper, we analyze and summarize the characteristics of rVAR2 and its progress in tumor-targeted therapy in recent years, and discuss its future development direction, in order to provide a reference for the development of clinical tumor-targeted therapy and diagnosis methods.
Single-chain fragment variable (scFv) is a small molecule recombinant antibody consisting of a variable heavy chain (VH) and a variable light chain (VL) linked together by a flexible peptide junction. The mRNA of single chain antibodies is isolated mainly from hybridomas and reverse transcribed into cDNA as a template for scFv gene amplification, resulting in a gene library containing a large number of different VH and VL fragments of scFv. Screening and identification of the affinity and specificity of the scFv is accomplished using different display techniques, and the resulting scFv can be used to successfully express its proteins through various expression systems. Despite its small molecular weight, scFv contains the antigen-binding domain of intact antibodies and has high specificity and affinity for antigens, as well as low immunogenicity, and also has a strong ability to penetrate and spread through tumor tissue. Therefore, scFv has become a hot research topic in the development of oncology therapeutic approaches. This review details the methods and problems in the preparation of scFv and focuses on the research progress of scFv in tumor diagnosis and treatment, with a view to providing a theoretical basis for the preparation of scFv and its application in diagnosis and treatment of tumors.
Oncolytic viruses, capable of infecting and destroying malignant tissues, are becoming new biological therapies for treating cancers. Their ability in selectively replicating inside cancer cells and inducing lysis of certain cancer cells leads to the releasing of tumor-specific antigens for anti-cancer immune responses, acting as in situ cancer vaccines. The insights into the mechanisms of virus replication, entry, induction and inhibition of immune response have promoted the technology development of virus utilization in the treatment of certain human diseases. In the past decade, the progress of clinical trials in the field of oncolytic viruses has confirmed the therapeutic benefits in cancer therpy. Using oncolytic virus as a carrier of foreign genes to treat specific types of cancer is a feasible method, which could become new growth avenue of the cancer immunotherapy market. This article provides a comprehensive and multidimensional analysis of the niche areas and the dynamic changes from several aspects such as the oncolytic virus immunotherapy market, key technology progress, major enterprise competition pattern and product R&D progress, combined with a forecast of the prospects for the development of oncolytic viruses as a cancer therapy, for the purpose of providing reference in research and development direction selection and regional industrial decision-making for related companies.
Gene therapy provides a new strategy for the treatment of major refractory diseases with a promising market outlook. By of the end of 2022, a total of 45 gene therapy drugs around the world were approved to be listed, and more than 3 000 drugs were in clinical trials. There are also 4 types of domestic gene therapy products available in China. Currently, the top three leading enterprises in the world account for more than 50% of the market share. As there is an increasing number of patients with rare diseases and tumors in China year by year, the domestic gene therapy market continues to grow. Driven by industry development policies, a number of enterprises such as Yao Ming Ju Nuo, Legend Biology, and Keji Biology have emerged and achieved certain progress. With the gradual improvement of policies, the deepening of scientific research, and the lowering of treatment costs, gene therapy is expected to become a conventional and safe treatment.
Messenger RNA (mRNA) is a kind of nucleic acid sequence which can express any protein of interest after its translation and modification in cells. Because of its inherent feature of protein of interest production, mRNA has potential to be used as drugs to treat various diseases, including viral infections, tumors, diseases caused by deficiency or abnormality of a certain protein in vivo, as well as genetic diseases by encoding Cas9 protein which is involved in gene editing. With the great success of mRNA vaccines, mRNA drugs have drawn increasing attention for their application potential. In addition, due to their advantages such as rapid research and development cycle, the ease and rapid large-scale production at low cost, and elimination of insertional mutagenesis and integration risk for the host, mRNA drugs have become the third generation of drugs after small molecule and antibody drugs. In this review, the structural properties and the delivery system of mRNA are introduced, the clinical progress of mRNA therapeutics and domestic mRNA vaccine development are summarized, and the remaining problems to be solved in the market application including research, manufacture and logistics of mRNA therapeutics are discussed, hoping to provide a reference for mRNA drug discovery and manufacturing.
Human immune-related synthetic biology has always been one of the hot spots in the international biomedical field, which has shown great application potential in such fields as immunotherapy for critical diseases and preventive medicine. Moreover, human immune-related synthetic biology and biological safety will increasingly become an important health research topic for science and technology to benefit human welfare. This cutting-edge research field will also affect the overall national security and the future fate of mankind. By focusing on the analysis of possible biosafety risk factors in the field of synthetic biology related to human immunity, the research was conducted from five aspects: the impact of microorganisms on immune function, immunosuppression, immune overreaction, autoimmune response and human genomic immunity, and the biosafety issues related to synthetic biology were proposed and the corresponding strategies were proposed as safeguard measures for the scientific and technological innovation and development of biosafety and synthetic biotechnology in the field of human health.
