Recombinant attenuated Salmonella Typhimurium as vaccine vectors to treat cancers

Current cancer treatments including radiotherapy and chemotherapy have many shortcomings, such as high toxicity, low tissue permeability, low specificity, and weak target sites. Many years ago, people have begun to use bacteria for cancer prevention and treatment research. For example, Wiliam B. Coley found that if cancer patients contract bacterial diseases after surgery, the cure rate can be increased; current treatments for bladder cancer often use injections after surgery. A vaccine BCG to prevent tuberculosis to increase the cure rate. Although the mechanism by which bacteria increase the cure rate is not very clear, the above examples show that bacteria can be used to treat cancer and have many advantages.

A variety of bacteria can be used to treat cancer, for example, Bifidobacterium, Escherichiacoli, Listeria, Clostridium, Proteus, etc. The most studied bacteria used to treat cancer is Salmonella enterericaserovar Typhimurium, which is a facultative anaerobic bacteria, and its genome sequence has been Announced, and genetic manipulation is convenient. Other advantages as a cancer treatment include

1) Salmonella typhimurium itself can not only be used to treat cancer and improve the cure rate. Because Salmonella typhimurium is a complete metabolizing organism, it can continuously synthesize anti-cancer proteins in its body, so it can be used as a carrier to deliver some anti-cancer drugs, cells Factors, or small RNAs to interfere with the growth of cancer cells;

2) After oral, intravenous injection or direct injection into the cancer site, it can proliferate at the cancer site and cause systemic infection at the cancer site. Low-dose bacterial injection can achieve effective treatment effects. This advantage overcomes the disadvantage that the drug concentration gradually decreases at the cancer site;

3) Salmonella can accumulate to a high concentration at the cancer site and compete with cancer cells for nutrition, while the concentration in normal cells is very low, which is nearly 1000 times different. This advantage can prevent bacteria from causing systemic infections throughout the body and leading to sepsis In addition, Salmonella can infect most cancers, such as lung cancer, breast cancer, melanoma cancer, colon cancer and other tumor-like cancers;

4) The bacterium is a natural cytotoxicity inducer. Typhoid bacteria itself can produce toxic factors, causing cytotoxicity, attracting immune cells to the surrounding cancer cells. In addition, Salmonella infection can induce the expression of Cx43 protein, form functional Gap-junctions between cancer cells, and promote the delivery of cancer cell antigen peptides. Dendritic cells can eventually capture CD8 T-cells to eliminate cancer cells;

5) The most important point is that after taking antibiotics, Salmonella can be easily eliminated from the body. If Salmonella expresses certain fluorescent proteins, then tumor changes can be detected in vitro.

The genetically modified Salmonella VNP20009 contains two knockout mutations purI and msbB. After purI is knocked out, Salmonella loses the synthesis of adenine, and there is a large amount of adenine in tumor cells, which is conducive to the proliferation of bacteria in tumor cells; msbB mutation changes the structure of lipid A and weakens lipid A The stimulation of endogenous immune response reduces the production of tumor necrosis factor (TNF-α), so it is safe and will not cause severe bacterial infections such as sepsis.

In mouse experiments, the genetically modified bacteria showed good bacterial specificity, that is, it can proliferate in large numbers in cancer cells, but rarely in normal cells, and the ability of bacterial colonization in the two kinds of cells can be 1000 times different; It also showed a good anti-cancer effect, and the tumor became smaller.

However, in human phase I clinical trials, the expected effect was not achieved. In 28 cancer patients, although all patients were tolerant to VNP20009, VNP20009 could only be found in 3 patients’ cancer sites, one of which had tumors. Disappeared (VNP20009 injection 3 months detection), a cancer patient's tumor became larger. There are two reasons for the poor results of clinical trials. One is that VNP20009 is too weak to adapt to the human tumor environment; the other is that the mutant strain is more sensitive to the human immune system and cannot survive long enough in the human body. Time to stimulate the immune response against tumor cells and produce enough stimulating factors.

How to transform Salmonella typhimurium is the key to applying this bacterium to treat cancer. The first thing to consider is safety. Any drug aimed at humans must be safety first. Therefore, the modification of this trait must be considered first. The mutations currently used in research include mutations related to amino acid synthesis including aroA, aroD, mutations related to global regulation, phoP/phoQ, and purine synthesis related purI. All these mutations cause the toxicity of Salmonella to weaken and affect the initial infection. And colonization. The well-known geneticist and member of the American Academy of Sciences Roy Curtiss III developed a technology called delayed attenuation system applied to Salmonella vaccine to deliver pathogenic antigens to prevent and treat infectious diseases.

The main principle of this system is to replace the promoters of key pathogenic genes with adjustable promoters. Under in vitro induced conditions, the pathogenic factors can be expressed and can infect the host like wild bacteria. However, once it reaches the body, it will cause Disease factors are no longer expressed, causing weakening of toxicity.

This system can also be applied to the construction of anti-cancer Salmonella. In addition, it is necessary to consider how Salmonella can accumulate into tumor cells. Two issues need to be considered. One is that the active swimming of Salmonella is controlled by the flagella of Salmonella, but the expression of flagella in the host is weak or even not expressed. In order to overcome this shortcoming, The method of genetic modification can be used to promote flagella to increase swimming; in addition, the tropism of Salmonella can express some receptors on the surface of Salmonella. For example, TAR receptor can sense aspartic acid produced by tumor cells; TRG receptor senses The ribose produced by tumor cells accelerates the accumulation of tumor cells. Also consider how to make Salmonella express foreign proteins or transcribe small RNAs.

Generally, the expression of foreign proteins requires the use of plasmids as vectors. At present, plasmids containing drug resistance genes are the most widely used. However, resistance plasmids cannot be used in clinics, so auxotrophic plasmids can be considered. If the expressed protein is toxic to the bacteria itself, you can consider using the delayed expression system developed by Curtiss or some promoters induced in vivo to overcome this shortcoming.

In addition, the endotoxin of Salmonella must be considered. Endotoxin can cause sepsis and inflammatory complications, which can be life-threatening in severe cases. MsbB mutation reduces the structure of endotoxin by one chain and reduces the inflammatory response induced by it. However, human clinical trials have shown that bacteria containing MsbB cannot colonize cancer cells well, nor can they inhibit the proliferation of cancer cells. It may also be associated with msbB mutations. Salmonella can not induce the production of strong tumor necrosis factor (TNF-α).

How to ensure the safety of Salmonella and induce inflammation well, it is necessary to systematically study which endotoxin structure is more suitable for the transformation of anti-cancer Salmonella.

In short, although Salmonella typhimurium has been used as a carrier to deliver foreign antigens to prevent and treat diseases for many years, there is still no effective vaccine for clinical application. Salmonella typhimurium has been studied for many years as a method of cancer treatment, but clinical trials have not achieved the expected results.

Although there are many difficulties, the application of Salmonella typhimurium for cancer treatment has many advantages, and it is still worthy of in-depth systematic research. Salmonella Typhimurium can cause systemic infection in mice, but it can only infect humans locally, not systemically. In future studies, genetic modification of SalmonellaTyphiorParatyphi may be considered, which may be more suitable for the treatment of human cancer.

At the same time, the global oncology drug market has achieved sustained and rapid scale growth, attracting more companies in the biotechnology field to enter the market, hoping to occupy a favorable position in the future hunting of the huge market. Hong Kong Medicine Oncolytic Biopharmaceutical Co., Ltd., which successfully developed the world’s first oncolytic bacterial carrier product YB1, has gained widespread attention in the industry market by virtue of its breakthrough progress in the research of innovative cancer immunotherapy-oncolytic bacterial therapy .

The core technology product of Hong Kong Medicines oncolytic oncolytic bacteria YB1 is a genetically programmed strain of Salmonella typhimurium. It can be used as a carrier to efficiently present antibodies, mRNA, protein drugs, etc., accurately target the hypoxic area inside the tumor, and A large number of replication and amplification within the tumor can greatly increase the concentration of YB1 vector at the target site of solid tumors, and release a variety of therapeutic "warhead" drugs, which can inhibit tumor growth and cause tumor dissolution, and at the same time, it can eliminate tumor metastasis. Clinical application potential. To

YB1 uses the world's first tumor hypoxia specific targeting technology to achieve tumor targeting, and has obtained the only patent for oxygen-regulated oncolytic bacteria in the world. YB1 technology also has strong compatibility, and is compatible with chemotherapeutic drugs, immune checkpoint antibodies, CAR-T and other cell technologies. The company currently has 7 innovative product pipelines that combine oncolytic bacteria with other drugs. In addition to its application in the field of tumor treatment, YB1 technology can also be applied to the treatment of various thrombotic diseases. At present, the company has deployed three thrombolysis-related product pipelines.