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Cancer is the leading cause of death in domestic canines, and current standard anti-cancer therapies are both costly and hindered by the complexity of tumor biology, furthermore, it also may have unwanted side effects. Bacterial cancer therapy is some concept more than 100 years. With the development of modern biotechnology, biological products often represent the cutting-edge of biomedical research and, in time, may offer the most effective and economy means to treat a variety of cancer types that presently have no other treatments available. Microbial metabolism and sensitivity can lead to cancer site-specific treatment, which are highly focused on the tumor and safe to other tissues. Activation of tumor-specific immunity is another important mechanism of such therapies. Salmonella Typhimurium have been evaluated as one of the most promising cancer therapeutics
After over ten years of small animals study in the University of Hong Kong, such as types of mice and cancer models, BACMUNO Biotechnology Limited have devised an innovative experimental method that involves the genetic modification of the Salmonella bacteria strain into an “obligate” anaerobe, termed Salmonella typhimurium vaccine YB1 strain. This enables the modified bacteria YB1 to target the hypoxic regions of solid malignant tumors. During this process, the immune system will also be activated, which, in conjunction with bacteria can both repress the growth of the primary tumor and cancer metastasis.
YB1 is live attenuated bacteria as biological drugs, including those manufactured by biotechnology, tend to be heat sensitive and susceptible to microbial contamination. Biological drugs include a wide range of products such as vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins. In contrast to most drugs that are chemically synthesized and their structure is known, most biologics are complex mixtures that are not easily identified or characterized. Therefore, it is necessary to use biological medicinal principles from initial manufacturing to be evaluating effect steps, which is also in contrast to most conventional drugs.
1.Introduction of YB1 program
Detailed introduction
Infections with some Salmonella strains such as VNP20009 have long been known in some cases to cause partial or complete regression of malignant tumors by targeting the hypoxic region in a tumor. Although treated with VNP20009 can achieve tumor/normal tissue ratios of 1,000:1, the precise number in tumor is 109 CFU/g verse the normal tissue number is 106 CFU/g [1]. The problem is 106 CFU/g of live Salmonella bacteria may cause damages in its function, and lead to systemic toxicity. This remains a major obstacle to the successful use of these bacteria as therapeutic agents for cancer.
The contribution of YB1 is to bypass the problem of attenuation.
Facultative anaerobes, such as Salmonella, have considerable advantages as agents for bacterial therapy due to their ease of culturing and manipulation as well as their capacity for targeting and killing tumors. However, their ability to invade normal aerobic tissues makes the consequences of infection potentially more serious, leading investigators to make efforts to attenuate the bacteria.
The YB1 was used recombineering technology to convert a Salmonella typhimurium strain into an “obligate” anaerobe without otherwise interfering with the function of the bacterium. This avoids the problem of infection in aerobic normal tissues, as the modified strain will lyse under these conditions. When in the hypoxic regions of a tumor, the bacterium can thrive and act as the wild type form as it is not compromised by an attenuation process（2）
2. Biological drug YB1
Detailed introduction
The microenvironment of solid tumor is unique and abnormal. The basic structure of tumor microenvironment can be roughly divided to 3 layers. Around the blood vessels, a center layer is well-oxygenated and with nutrient supplies which allow cancer cells to accumulate around tumor abnormal vessels. Outside is hypoxia region containing hypoxic cancer cells. Much further is tumor necrosis region, which has no living cells (Fig.1). Therefore, it is possible to distinguish a solid tumor from surrounding normal tissues. The major differences were the characteristic of cancer cells and the formation of tumor vasculature system [3], which results in hypoxia and necrosis region [4].
Fig. 1. Microenvironment of solid tumor (Adapted from online materials www.mc.pref.osaka.jp/omc2/eng/biochemistry.html). Due to inefficient supplement of blood vessel network, there will be hypoxia and necrosis region in solid tumor. Inside the hypoxia region, the nutrition and oxygen level are low.
The oxygen level varies across different environments. In the air, it is about 21% (150 mm Hg) and in mammalian tissues and organs, it ranges between 2%-9% (15-70 mm Hg) [5]. In hypoxic regions of most tumors, the oxygen level is approximately less than 0.7% (5 mm Hg) [6]; in some strict hypoxia is lower than 0.02% [5]. Therefore, hypoxia is the perfect target for cancer targeting therapy.
3 Design of Salmonella YB1 strain
3.1 Hypoxia region- the hallmark of tumor Detailed introduction
Fig. 1.
The basic concept of YB1 simple. For the hypoxia hallmark of tumor is obvious, if we can somehow engineer facultative anaerobe Salmonella only survival under oxygen level below 0.5%, the tumor targeting purpose will be achieve, and the damage to the normal tissues could be avoided (Fig. 2).
Fig. 2.
3 Design of Salmonella YB1 strain
3.2 The concept of YB1 Detailed introduction
SL7207 is an attenuated strain of wild type Salmonella typhimurium with the deletion in aroA gene, resulting in bacterial auxotrophy for compounds: p-aminobenzoic acid and 2,3-dihydroxybenzoate [7]. SL7207 attenuated in the mammalian host and applied in tumor targeting researches by several groups, although it can affect the health of immune-compromised mice [8-11] .
3.3 Stepwise of YB1 construction / 3.3.2 Hypoxia induce promoter PpepT Detailed introduction
3 Design of Salmonella YB1 strain
3.3 Stepwise of YB1 construction / 3.3.1 SL7207 - the parental Salmonella strain of YB1 Detailed introduction
In Salmonella, the switch between aerobic and anaerobic growth is regulated by fumarate and nitrate reduction gene (fnr). When oxygen is absent, the monomer FNR forms iron-sulphide clusters and generate homodimers which specifically bind promoters to start transcription [12]. When oxygen is present, the iron-sulphide clusters will be oxidized, and the dimmers will be disassembled. Thus, the transcription is terminated. Promoter containing FNR binding sites are activated under hypoxia[13].
Promoter PpepT was chosen to be the hypoxia induce promoter of YB1. It is the promoter region of gene pepT in Salmonella, which has double promoter sequences. The upstream promoter P2 is a constitutive promoter and downstream promoter P1 is FNR regulated promoter (Fig. 3A). Promoter PansB is an artificial promoter modified from ansB gene of E.coli, it was changed one cyclic AMP receptor protein binding sites into FNR binding sites to generate two FNR binding sites [14] (Fig. 3B). The PansB was applied as a experimental control in YB1 development.
Fig.3 Genetic structure of hypoxia induced promoters. A, PpepT from SL7207; B, Artifical PansB from E.coli.
Fig. 3.
3.3 Stepwise of YB1 construction / 3.3.3 Essential gene selection- asd
To achieve engineered Salmonella death under certain condition, the important part is to find a way to kill it. Essential genes are those genes of an organism that are thought to be critical for its survival. However, being essential is highly dependent on the circumstances in which an organism lives [15]. The perfect candidate for tumor targeting should meet the following conditions: (a) Efficient to induce lysis when lack of its expression; (b) Cannot obtain supplementary ingredients in mammalian tissues; (c) Supplied with certain ingredients could easily be cultured in medium.
The asd mutation meets all above requirements. Asd gene encodes aspartic semialdehyde dehydrogenase. Asd mutations have an obligate requirement for diaminopimelic acid (DAP) and will undergo lysis in environments deprived of DAP [16]. DAP is a characteristic of certain cell walls of Salmonella. DAP is often found in the peptide linkages of NAM-NAG chains that make up the cell wall of gram-negative bacteria, and not present in mammalian systems (Fig. 4). When provided or synthesized by asd gene, they exhibit normal growth. When in deficiency, they still grow but with the inability to make new cell wall proteoglycan (Fig. 4), resulting bacteria quick lysis.
Fig. 4. The chemical structure of pepitdoglycan in cell wall of Salmonella
Fig. 4.
In the resulting YB1 strain, the FNR related anaerobic capable promoter PpepT controls asd transcription while an aerobic promoter. To prevent possible leakage from hypoxia induced promoter, an antisense promoter of sodA gene, PsodA, which is negatively regulated by FNR, was added to the end of asd gene [17]. PsodA, facilitates transcription of antisense asd that blocks any leakage of asd expression under aerobic conditions (Fig.5).
Fig. 5. YB1 design. Construction of the hypoxia conditioned asd gene expression cassette. The sense promoter PpepT contains the constitutive promoter P2 and the FNR regulated promoter P1 and PsodA is the antisense promoter.
Fig. 5.
3.3 Stepwise of YB1 construction / 3.3.4 Genetic circuit regulation of YB1
Alternate asd gene expression cassettes were constructed as control strains. The PW construct uses the sense PpepT promoter to control asd gene. In the EW construct a sense FNR regulated promoter PansB and antisense promoter PSodA were used (Fig. 6). Furthermore, YB-asd was generated with the deletion of asd gene from SL7207.
Fig. 6 Construction of alternate asd gene expression cassettes
3.3 Stepwise of YB1 construction / 3.3.5 Genetic circuit regulation of YB1 variants (PW and EW
Fig. 6.
In the YB1 mechanism of action, asd is under the control of a hypoxia-conditioned promoter. In normal tissues or aerobic conditions, PpepT is inactive due to high oxygen level, asd is not expressed, any leak expression will be neutralized by antisense PsodA promoter. Diaminopimelic acid (DAP) is not synthesized and the bacterium will lyse during growth unless DAP is supplied by the environment (Fig. 7A). In tumor or anaerobic condition, asd will be expressed, and bacteria will survive (Fig. 7B). Thus, Salmonella YB1 is converted from a facultative to an ‘‘obligate’’ anaerobe, rendering it safe in normal tissues.
Fig. 7 Diagram of YB1 action mechanism.
Fig. 7.
3 Design of Salmonella YB1 strain
3.4 Mechanism of Action of YB1
YB1 and other derivative strains were tested under high and low oxygen levels. Of the engineered strains in the absence of DAP, only YB1 showed the combination of growth under anaerobic culture conditions (oxygen levels below 0.5%) and repression in the aerobic environment (oxygen level higher than 0.8%). SL7207 and PW showed growth in all conditions. YB-asd and EW showed growth only with the addition of DAP. Serial reductions in the oxygen level and bacterial concentration were used to establish the range of conditions under which YB1 and the other strains could survive in the presence or absence of DAP (Fig. 8).[18]
Fig. 8.
Fig. 8 Test of YB1 and other strains under serial reductions of oxygen level conditions. Different mutant strains at serial dilutions under decreasing oxygen levels were cultured for24 hours and bacterial growth was observed. Columns:1. SL7207; 2. YB-asd;3. YB1;4. PW;5. EW.
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