Research progress of attenuated Salmonella as an anti-tumor and tumor gene therapy vector

With the development and improvement of molecular biology and genetic engineering technology, gene therapy has gradually developed into a new approach to tumor treatment and has become a research hotspot in the medical field. In view of the fact that one of the main problems of tumor gene therapy is the development of suitable vectors, and most of the viral vectors used in the past have restricted the development of gene therapy due to issues such as targeting, safety and feasibility. Certain bacteria that are low or non-toxic and have targeted infection characteristics are becoming more and more interesting as carriers. To this end, a systematic review of the research progress of attenuated Salmonella as an anti-tumor and tumor gene therapy vector, which is one of the current research hotspots, is reviewed.

1. Anti-tumor research of Salmonella

Salmonella is a type of intracellular invasive gram-negative facultative anaerobic bacteria that can survive in both aerobic and hypoxic environments. It is also a natural immune adjuvant, which can induce the body to produce some inflammatory factors to enhance The body's innate protective immune response. Relevant studies have shown that Salmonella can preferentially and selectively aggregate in tumor tissues of different sizes to form colonies. This is determined by the microenvironment of the tumor itself, but the mechanism of this aggregation has not been fully elucidated. It is currently believed that it may be caused by The pathophysiological characteristics of tumors that are different from normal tissues and the characteristics of Salmonella itself are the result of the combined effect. Tumor tissues are highly metabolized and rich in nutrients. Tumor cells grow rapidly but are relatively inadequate in blood supply. It is easy to form hypoxic areas and tumor necrosis areas. This provides an environment for a large number of hypoxic-promoting Salmonella to accumulate and multiply.

In the 1990s, it was discovered that highly invasive auxotrophic Salmonella strains screened by tumor cells had direct anti-tumor activity, and it was believed that this anti-tumor effect was due to the release of lipopolysaccharide (LPS) that stimulated the immune system. In 1983, Takayama et al. injected Salmonella typhimurium G30/C21 into the transplanted tumor of inbred guinea pigs, which had a certain inhibitory effect on it, but it could not be completely cured. In 1997, Pawelek et al. studied the targeting of Salmonella to multiple tumors from remote inoculation sites, selective replication within tumors, tumor arrest, and the ability to express effective genes. Although the mechanism by which Salmonella itself inhibits tumor growth is not clear, it is generally believed that it stimulates the body's immune response with bacterial metabolites, secretes a variety of cytotoxic effectors and proteolytic enzymes to kill tumor cells; Salmonella competes with tumor cells for nutrition and Growth factors, thereby inhibiting the growth of tumors, and inducing changes in tumor cytokines to coagulate tumor blood vessels and promote tumor encapsulation. In addition, Pawelek et al. also proved that the expression of Salmonella pathogenicity islands 2 (Salmonella pathogenicity islands 2, SPI2) is effective for Salmonella Anti-tumor effect is required.

2. Anti-tumor research of attenuated Salmonella

Early studies have shown that not only Salmonella itself has a direct oncolytic effect on tumors, but also that attenuated strains have better targeted colonization than wild strains. Therefore, using various genetic engineering techniques to randomly insert a transposon or delete one or more pathogenic genes into the Salmonella chromosome genome can reduce the virulence and obtain an attenuated strain, which can reduce the pathogenicity to the host while still retaining it. Its high immunogenicity provides a safety guarantee for clinical applications. It is possible to use attenuated Salmonella to selectively replicate and proliferate in tumor tissues to inhibit the growth of a variety of tumor cells and prolong the survival time of animals.

Low et al. used attenuated Salmonella through ear vein injection to treat adenomas and achieved good results, but systemic administration of attenuated Salmonella is prone to septic shock. VNP20009 is an attenuated Salmonella strain with in-depth clinical research, and its clinical phase I experiments have confirmed that it has high tumor targeting and safety. Zheng et al. injected the attenuated Salmonella strain VNP20009 into a variety of tumor-bearing mice (mice, human melanoma, human colon cancer, lung cancer, breast cancer, pulmonary sarcoma, etc.), and found that the bacteria clustered at the tumor site was 200 to other normal sites. 1,000 times, and the growth of tumor tissue was significantly delayed. Using a variety of animal models, it has been observed that the bacterium is mainly distributed in the liver, spleen and lymph nodes of the animal. After 30 days, only the liver can be detected, but after 40 days it cannot be detected. The green fluorescent protein and deaminase expressed by this strain are also only expressed in tumor cells, but not in normal tissues. At the same time, it was also found that only live bacteria can inhibit the growth of tumors, while bacteria killed by acetone did not produce this effect. This shows that the reason why tumor necrosis is only caused by lipopolysaccharide is insufficient. And Low et al. also proved that the anti-tumor effect of these attenuated Salmonella may not depend on TNF-α.

Tumor tissue hypoxia often leads to failure of radiotherapy and chemotherapy. Therefore, combining attenuated Salmonella with radiotherapy and chemotherapy can compensate for the defects of radiotherapy and chemotherapy, reduce tumor microvessel density and vascular endothelial growth factor (VEGF) levels, and promote the complete elimination of tumor cells. Most chemotherapeutic drugs cannot penetrate deep tumor tissues and are often ineffective against resting cells. Salmonella can overcome these obstacles. These characteristics of Salmonella may make Salmonella a powerful tool for human anti-tumor, and it has certain advantages in anti-tumor gene therapy. Pawelek et al. used lipid-mutated Salmonella combined with chemotherapy to treat malignant melanoma in mice and found that the combined application of the two can significantly inhibit the growth of tumors in mice and prolong the survival time of mice. Platt et al. conducted animal experiments on VNP20009 and X-ray alone or in combination, and the results proved that the combined treatment can produce a synergistic effect. And attenuated Salmonella combined immunotherapy can overcome the obstacle of tumor evading the body's immune system. Therefore, attenuated Salmonella combined with conventional treatment is a development model of tumor gene therapy.

3. Research progress of attenuated Salmonella as a tumor gene therapy vector

3.1 Attenuated Salmonella

Attenuated Salmonella has many characteristics as an ideal gene delivery vector for anti-tumor. Attenuated Salmonella mediates the reaction with the host through the type III secretion system, can deliver effector genes and express a variety of therapeutic proteins on the cell surface or in the cytoplasm. Induce the body to produce corresponding specific humoral immunity, cellular immunity and local mucosal immune response to resist the invasion of pathogens carrying corresponding foreign proteins. The attenuated Salmonella carrying foreign genes disintegrates and die after entering macrophages and dendritic cells, releasing multiple copies of foreign genes. Exogenous gene expression induces the body's MHC-I molecule-mediated antigen-specific CD4+ and CD8+ T cell tumor protective immune response, CTL and antigen-presenting dendritic cells are activated, and their respective activation markers CD2, CD25, CD28 , CD48 and CD80 have a decisive increase.

Because attenuated Salmonella can not only grow under aerobic and anaerobic conditions, but also can selectively accumulate in tumor tissues, the hypoxic necrosis area in the tumor can make these bacteria easy to multiply and spread in tumor tissues; at the same time, it is sensitive to antibiotics. , It can be eliminated by antibiotics if necessary, and does not produce tolerance; coupled with good invasiveness and specificity, it can be displaced from the remote inoculation site to and accumulate at the tumor site, replicating the level in the tumor tissue It is 1 000~10 000 times higher than normal tissue; and the cost is low, it can be administered by oral route, has little side effect, is suitable for long-term treatment and can transmit and express multiple effect genes, and combined with other methods to exert the greatest anti-tumor effect, long-term maintenance Until tumor regression and many other features as an ideal gene delivery vector for anti-tumor, these features provide the possibility to use Salmonella as a targeted vector for tumor gene therapy. Kochi et al. pointed out that attenuated Salmonella may become a new type of anti-tumor agent and a carrier for targeted tumor therapy.

At present, relevant experts have used attenuated Salmonella as a gene transfer vector in the gene therapy of solid tumors, deep tumors and metastatic tumors. In vitro experiments and animal model experiments have achieved satisfactory therapeutic effects. For example, VNP20009 is a clinical research In-depth attenuated Salmonella strains have been confirmed in clinical phase I experiments to have high tumor targeting and safety. And VNP20009 can inhibit the growth of a variety of mouse and human transplanted tumors, including mice, human melanoma, human colon cancer, lung cancer and breast cancer. Part of the results have been initially applied in clinical practice, showing a strong prospect for development.

3.2 Advances in research on the treatment of tumors with cytokines

Cytokines are an important part of the body's anti-tumor effect mechanism. Cytokines can activate anti-tumor effects by directly killing tumor target cells. The anti-tumor research of IL-2, IL-12, IL-18, INF-γ, TNF and GM-CSF has achieved a lot of success. Saltzman and Al-Ramadi found that Salmonella typhimurium carrying IL-2 has a strong inhibitory effect on tumors in mice and dogs, and can effectively reduce the formation of lung metastases.

Low et al. constructed Salmonella typhimurium with Escherichia coli cytosine deaminase (CD) to efficiently express active CD in tumor cells and enhance the effect of inhibiting tumor growth. Studies have found that both IL-12 and GM-CSF have good anti-tumor effects, and the combination of the two can significantly enhance the anti-tumor effect. However, there are reports that GM-CSF can inhibit the secretion of IL-12. This indicates that Salmonella can not only inhibit tumor growth, but also can serve as a good expression vector for foreign genes to present drug proteins to tumors in vivo.

Attenuated Salmonella typhimurium with human LIGHT is enriched in mouse tumor tissues and expresses LIGHT, which can effectively inhibit primary breast cancer, colon cancer and metastatic lung cancer. The attenuated Salmonella typhimurium carrying FasL and IL-18 genes has also been confirmed to have significant anti-tumor effects. Yu et al. proved that oral administration of attenuated Salmonella expressing murine granule-monocyte colony stimulating factor (mGM-CSF) made Ewing's sarcoma regression in mice. This cytokine can increase the effect of cytotoxic T cells in peripheral blood. Ursshi-ma et al. found that oral attenuated Salmonella expressing CD40 ligand (CD40L) can effectively inhibit the growth of B-cell lymphoma in mice.

3.3 Research on the treatment of tumors with other exogenous effector proteins

Salmonella can effectively transmit multiple effector genes and express therapeutic proteins. Lee et al. used the attenuated Salmonella strain carrying the TSP-1 gene to treat melanoma and lung metastasis, and found that the bacteria that accumulate in the tumor site is 1000 to 10,000 times that of the liver and spleen, and the expression level of TSP-1 in the tumor site is also liver and lung metastasis. 1800 times the spleen. This indicates that recombinant Salmonella can significantly inhibit tumor growth, prolong the survival time of mice and reduce the density of microvessels in tumors. Feltis et al. found that cyclooxygenase-2 inhibitors and oral attenuated Salmonella vaccines have a synergistic effect in resisting liver metastasis of colon cancer. The VNP20009 recombinant bacteria containing CEA can increase tumor-specific localization, laying a foundation for improving the immunogenicity of cancer vaccines expressing Salmonella tumors.

Jiang Zhongming and others constructed a recombinant attenuated Salmonella carrying TIP30 and human INF-γ genes, which has a significant therapeutic effect on adenoid cystic carcinoma. Attenuated Salmonella carrying siRNA-Stat3 plasmid has a significant inhibitory effect on the growth of orthotopic liver transplantation tumors in mice. After immunizing mice with recombinant attenuated Salmonella human papillomavirus HPV6b, the vaginal mucosa can secrete specific anti-human papillomavirus HPV6bSigA. In addition, the recombinant attenuated Salmonella expressing VEGFR2 can induce a specific immune response of VEGFR2, can kill tumor vascular endothelial cells, can treat mouse gliomas, and can inhibit the growth of subcutaneous tumors in mice.

4. Outlook

Although attenuated Salmonella has the advantage of a good gene delivery vector, it has achieved good results in animal experiments and clinical phase I and II experiments, but tumor gene therapy is still in the early stage of development, and many results observed in animal experiments are still It cannot be fully realized on the human body, and there are still many difficulties in clinical application. For example, the most feasible way of tumor gene therapy is systemic administration. Salmonella is a pathogenic bacteria. The pathogenic effect of its endotoxin should not be ignored, and there is a risk of bacteremia caused by proliferation in the blood; Salmonella belongs to facultative anorexia The specificity of aerobic bacteria for targeted colonization of hypoxic tissues needs to be further improved. In addition, how to efficiently transfer Salmonella vectors into human tumor tissues and enter quiescent tumor cells, how to effectively reduce the human body’s immune function to recombinant vectors, and how to effectively combine gene therapy with other treatment methods are all tumor genes. Difficulties in treatment. However, in fact, these key issues are the hotspots of current tumor gene therapy research, and new developments continue to appear. Future research should focus on improving the expression efficiency of therapeutic genes through molecular biology technology and genetic engineering technology, and screening out more targeted and safer attenuated Salmonella strains. Tumor gene therapy has broad development prospects in modern clinical medicine, and it will certainly play an important role in tumor treatment.

At present, the world's first oncolytic bacterial carrier product is a synthetic biology modified Salmonella YB1 invented by the Hong Kong Medicine Oncolytic Biopharmaceutical R&D team. YB1 can identify tumor areas according to the concentration of oxygen and achieve targeted colonization of tumor areas. Compared with oncolytic viruses, oncolytic bacteria can survive and proliferate without relying on host cells. Our self-developed bacterial vector YB1 relies on the tumor’s hypoxic microenvironment and can also invade host cells in the hypoxic microenvironment. Including but not limited to tumor cells. Therefore, while ensuring the targeting of YB1, we also increase the adaptability of YB1 in tumors.

In addition, due to the large genome load, oncolytic bacteria can also carry large amounts of drugs. Taking YB1 as an example, this innovative carrier product can synthesize drugs without relying on host cells. Therefore, YB1 can directly synthesize antibodies, protein drugs, mRNA vaccines, etc., and can achieve a large area of the drug to cover the entire tumor area. In fact, oncolytic bacteria and oncolytic viruses belong to the category of oncolytic vectors, so they are also a large class of new immunotherapies. Loss of control in the body is difficult to stop or effectively control the situation, and if the oncolytic bacteria YB1 is out of control in the body, you can simply use sensitive antibiotics to directly stop the study, and as a bacterium, there is no risk of integrating the human genome.

At present, the company has deployed multiple product pipelines for the application of oncolytic bacteria YB1. In the field of YB1 oncolytic bacteria cancer immunotherapy, the company has 7 specific pipelines of drugs under research. YB1 carries protein drugs, antibodies, and mRNA vaccines. And oncolytic viruses, etc., can be applied to the clinical treatment of a variety of solid tumors. At present, the clinical trial of pet primary cancer treatment has been completed, and the effect is very significant, and it has entered the application stage of its human clinical trial. In addition, YB1 can be used in the field of tumor treatment as well as various thrombosis treatments. We have deployed three pipelines of products under development for the application of YB1 in the field of antithrombotic therapy. They are the first-generation targeted thrombosis product YB1-rt-PA and YB1 with recombinant defibrase.

As the most cutting-edge research results in the field of emerging oncolytic vector technology, the successful application of YB1 in the field of tumor therapy is expected to provide new solutions for cancer treatment. We also hope that through professional scientific research and the development of innovative drug product pipelines, we will help realize humanity A century-old vision of conquering cancer.