Thursday, 27 March 2014

Immunotherapy - Dr Jekyll and Mr Hyde: a strange case of tumor regression and awful Salmonella

Chemotherapy vs Immunotherapy

Before I start, I thought it might be useful to remind you of this sobering statistic; World-wide 16 people die every minute, every day from cancer (that means 5 people in North America and Europe die …every minute of every hour of every day from cancer).

clinical trials - alternative cancer treatments - cancer immunotherapy - salmonella cancer vaccine
    Injection of attenuated Salmonella strain into cancer patient and the effect on 
    tumor growth (based on animal studies)

The lucky ones that survive often do so at a cost; hence the saying, "If the cancer doesn't kill you, the treatment might". The problem with conventional therapy is the “one size fits all” approach, horrendous side effects, and resistance. As such, it has prompted the need for more personalised and / or alternative cancer therapies in patients with advanced solid tumours. Unfortunately, the list of promising alternatives that potentially constitute a cure is remarkably short. However, recent scientific advances in developing attenuated (genetically modified) Salmonella strains have allowed for the creation of bacterial strains that possibly constitute such a promising alternative strategy.

The role of bacteria in tumour regression and curing cancer was recognised more than a hundred years ago. The German physicians W. Busch and F. Fehleisen separately observed that cancers regressed following accidental erysipelas (Streptococcus pyogenes) infections that occurred whilst patients were hospitalised. It wasn’t until the American physician Dr William Coley noticed that cancer could be cured following an infection with erysipelas, that it became a form of therapy. He developed a safe vaccine, composed of two killed bacterial species, S. pyogenes and Serratia marcescens which stimulates the immune system and induces a fever without incurring the risk of an actual infection. Complete and prolonged regression of advanced malignancy (long term durable remission) was documented in many cases that were treated with Coley’s Toxin (please see the extensive blog post on Coley’s Toxin if you require further information on this immunotherapy). The success of Coley's toxins provided the foundation for current research in this field. Unfortunately, many recently developed immunotherapies (including biologics such as antibodies) are unable to match the well documented cure rate of Coley’s Toxin.

Salmonella as a Cancer Fighter

This table was copied from a publication by Sznol and colleagues 
in 2000.

The success of innovative cancer therapies depends in part on their ability to selectively target cancer cells for destruction while limiting toxicity to normal tissues. Since Coley's remarkable achievements, an array of natural and genetically modified bacterial species have been investigated for their potentially tumoricidal properties. Live, genetically modified non-pathogenic Salmonella strains have been created that are capable of multiplying selectively in tumours which in turn inhibits tumour growth while not harming normal cells. In light of their selectivity for tumour tissues, these Salmonella sub species also serve as ideal vectors for transporting therapeutic proteins into cancer cells.
As a consequence of this new development, Salmonella may well be bouncing back from a long time bad reputation. Thus, even though they have generally been feared and despised by humanity, that may be about to change as a result of their cancer fighting cousins.
For info about Biozantium by Paeon Laboratories:

How does Salmonella immunotherapy work?

In line with Coley’s concept of using bacteria in cancer therapies, other bacterial species have over time been evaluated. From these studies it is clear that attenuated Salmonella Typhimurium is one of the more promising candidates. The S. Typhimurium strain and its derivatives have been used for their natural ability to colonise and destroy tumours while some scientists have utilised them as vectors to transport cytotoxic agents.

The attenuated Salmonella Typhimurium strain with which one of the earlier clinical trials was performed is called VNP20009. Initially, researchers demonstrated that this particular genetically modified form of Salmonella had a significant effect on tumour growth in mice. However, VNP20009 used in the treatment of cancer patients (during a phase I clinical trial) lacked the efficacy that was found in mice studies. Nevertheless, this investigation provided necessary evidence that Salmonella is safe for use in cancer patients in a clinical setting.
In order to understand subsequent details about why and how these attenuated Salmonella strains are capable of homing in on tumours and cause subsequent destruction, it is perhaps worthwhile to briefly review some of the basics concepts in terms of life cycle and the genetics that govern these bacteria.

Salmonella belongs to the Enterobactericae family, which is a group of Gram-negative pathogenic, facultative intracellular anaerobic bacteria.
In humans, the Salmonella enterica, subspecies enterica serovars Typhimurium and Typhi (species that are normally encountered in the environment) are the causative agents of gastroenteritis and typhoid fever, respectively.

Depending on the serotype of Salmonella strain and several host organism factors, the infecting bacteria may colonise solely the intestinal epithelium which would lead to gastroenteritis or it may spread beyond the gut (to the liver and spleen predominantly, causing typhoid fever). 
Salmonella Typhimurium infection in humans is usually restricted to the digestive tract, with the exception of infants, the elderly or immunocompromised individuals (such as transplant patients or HIV infected individuals) in whom it can spread.
Salmonella Typhi on the other hand causes typhoid fever in humans while it is not pathogenic to animals. Interestingly, serotypes that lack host specificity, such as Salmonella Typhimurium, are more frequently associated with disease in young rather than in adult animals. This would suggest that the Typhimurium strain of bacteria has been unable to adapt to the mature immune system.

To understand the genetic engineering that underpins the attenuated variants of this bacteria, it is important to know some of the genes, their functions, and how they are regulated which I will briefly describe in the section below. 

Approximately 90% of the genes found in the Salmonella Typhi strain are identical to the Salmonella Typhimurium serovar (the two strains that have served as the starting point for therapeutic attenuated strains). However, of the 4000 genes that these bacteria share some 200 genes in the Typhi strain are non-functional or have been inactivated, while most of these genes are fully functioning in the Typhimurium strain (genes that are similar and that are found in another species are called homologs).

Many of these aforementioned non-functional homologs are genes that govern virulence factors (which are genes that make a micro-organism (e.g.bacteria) more pathogenic). Most of these virulence factors are contained in clusters on the Salmonella genome. These clusters are called Salmonella Pathogenicity Islands (SPI). The Salmonella Typhimurium and Typhi strain genome share 11 SPIs. However they each also express virulence genes from specific SPIs unique to each strain., SPI14 in the case of the Typhimurium strain and SPI7, SPI15, SPI17, and SPI18 in the case of the Typhi strain.

Some of these virulence genes that are expressed by Salmonella govern its ability to multiply inside a range of cells such as epithelial cells (skin cells), macrophages, dendritic cells, and neutrophils (i.e. cells of the immune system). In order for Salmonella to get inside these host cells and survive a relatively hostile environment, it utilises two “Type III Secretion Systems” (otherwise known as T3SS). These T3SS consist of a number of proteins and form a structure on the outside of the bacterial wall that can be thought of as a needle. The SPIs that contain the genes (genes are essentially the “blueprint”) for the building blocks of this T3SS structure are SPI1 and SPI2. The genes contained on these SPIs include InvG, InvJ, PrgH, PrgI, PrgK, SpaO, SipB, SipC and SipD.

Invasion and intracellular survival is regulated by a number of different systems, including PhoQ/PhoP, and OmpR-EnvZ, while maturation of the Salmonella Containing Vacuole (SCV) is regulated by genes found on the SPI2 (to avoid phagosome-lysosomefusion and degradation). 

These regulatory events (T3SS1 and T3SS2 modulation) happen within a timeframe of hours (0-4 hours) upon entering the cell (see figure 1 near the top in this blog post).

Why are there different cancer fighting Salmonella strains?

As mentioned above, attenuation of virulence factors is a key step in the process of creating Salmonella strains that can be utilised in a clinical setting.  Approximately fifty Salmonella genes have been identified that can be modified and allow for the creation of viable attenuated strains with altered virulence and metabolic functions.
There are several techniques to generate strains with altered genetics. This includes the most basic technique of passaging Salmonella through selective media (after which you screen for surviving “mutants”), and more modern techniques like site-directed mutagenesis (precise and targeted molecular laboratory methods).

List of attenuated Salmonella strains used in cancer therapies, including info on the genes that 
have been modified.
Targeting genes that regulate virulence is the most straightforward choice of modification. This involves inactivation of genes that encode for proteins which facilitate interaction with the host organism (human) or modification of factors (e.g. transcription factors) that regulate the expression of those genes. Examples of such genes includes phoP, phoQ (these regulate the expression of many genes that confer resistance to antimicrobial peptides), or the htrF gene which facilitates survival under conditions of stress.

Also, genes that encode for proteins involved in the metabolic pathway can be inactivated which subsequently results in strains that are dependent on external sources for compounds or nutrients (auxotrophic strains). An example of such a strain is the aro mutant or the pur mutant. 

Please consult the table next to this section for a brief summary of other mutations found in attenuated Salmonella strains that may be used as a cancer therapy.

What scientific and clinical evidence exists for the efficacy of Salmonella therapy?

In this section I will highlight some interesting examples of research that has been conducted with various attenuated strains of Salmonella including some background on how scientists created them.

The first example relates to the auxotrophic strain A1-R (see table with list of strains above). A1-R was developed and created by Zhao and colleagues in Robert Hoffmann’s research group by modifying the parental strain (ATCC 14028). This particular strain is a Green Fluorescent Protein (GFP) expressing bacteria, a feature they used to their advantage during the tumour targeting optimisation process in mice. They essentially injected the intermediate strain (called A1) into H-29 human colon adenocarcinoma bearing mice and subsequently isolated GFP expressing bacteria from these tumours. In vitro experiments demonstrated that this newly isolated strain (called A1-R) had an increased affinity for these cancer cells (which was approximately six times stronger than the original strain).

This A1-R strain was subsequently tested in a range of human tumours that had been grafted onto nude mice. Types of cancers that were investigated include tumours of the breast, prostate, pancreas, and lung. These studies all resulted in significant tumour growth inhibition and in some cases even resulted in complete eradication of the cancer. For detailed scientific reports on these studies please consult the folowing publications: Kimura et al., 2010, Zhang et al., 2012, Hayashi et al., 2009, Hayashi et al., 2009.
Interestingly Hayashi and colleagues utilised the A1-R strain in a metastatic cancer of the pancreas (tested in mice bearing metastasised human pancreatic tumours). In five of six mice this metastasised cancer in the lymphnodes was eradicated within 7-21 days following intravenous injection of A1-R, while mice in the control group (the ones that did not get an injection with A1-R) suffered from increased levels of metastases.

Given that attenuated Salmonella strains have a natural preference for colonising tumours rather than normal host tissues (we are talking about orders of magnitude difference here), and that without any further modifications they inhibit tumour growth, some researchers have used such strains to exert tumour directed cytotoxic effects by inducing the immune system in order to mount an immune response against the tumour.

Also, as mentioned before, less than optimal tumour targeting, and level of toxicity associated with current standard cancer therapies makes Salmonella based therapies a serious alternative. What's more, solid tumours harbour hypoxic regions that are often resistant to many forms of therapy (including radiation and other treatments). As such a recently developed facultative anaerobic strain by Yu and colleagues at the Huang lab may well offer a potential solution to this major issue in cancer therapy. In their relatively recently published work they show that  in breast tumour bearing mice, their YB1 strain targeted the tumour and inhibited its growth considerably. Importantly, this particular strain was rapidly eliminated from normal tissues and blood (3 days post infection Salmonella was barely detectable in the liver). This would suggest that YB1 is a safe bacterial vector for anti-tumour therapies without the trade-off of reduced tumour fitness (which has often been the case with other attenuated Salmonella strains (compared to the parental strain)).

These researchers used a recombineering approach to create a Salmonella strain that is not viable in normal tissues by placing an essential gene, asd, under the control of a hypoxia-induced promoter (the FNR binding site containing pepT promoter (PpepT) was used to drive expression of asd, while an antisense promoter of the sodA gene, which is negatively regulated by FNR, was added to the PpepT-asd construct to make the strain YB1 (the sodA gene promoter was added to the construct to prevent leakage from the pepT promoter)). The asd gene encodes an enzyme that is essential for the synthesis of DAP (a fundamental component of the bacterial cell wall). As a result this Salmonella species only expresses asd in hypoxic conditions and as such it grows well under hypoxia, but will lyse under conditions experienced in normal tissue.

As bacteria are expected to induce a host immune response, the scientists observed that neutrophils were found in the YB1 infected tumours. This would suggest YB1 may enhance tumour killing by strongly attracting neutrophils to the tumour.
However, it is important to note that YB1 did not completely inhibit breast tumour growth.

Nevertheless, in subsequent experiments they compared the widely used anti-cancer drug 5-
Graph copied from Yu et al., 2012 which demonstrates 
remarkable tumour growth inhibition as a combination therapy 
with 5FU
FU with YB1 and their effects on tumour growth respectively.
YB1 retarded tumour growth with an effectiveness greater than that of the drug 5-FU alone. When the investigators combined YB1 and 5-FU treatment they showed a synergistic effect where tumour growth was almost brought to a complete standstill (see graph in Figure 1 at the top of this article)

In summary, YB1-like bacteria could have the advantages of an obligate anaerobic bacterium (in tumour targeting) while maintaining the chemotaxic properties and ability to target metastasis.

As such the recombineered ‘‘obligate’’ anaerobe, YB1, represents a new direction in producing bacterial therapeutic agents for cancer.

Where can you get access to Salmonella immunotherapy for cancer?

Unfortunately, you can only get access to this therapy via clinical trials. This type of therapy is not yet approved.
Currently the following clinical trials are still recruiting patients for their studies:

There are currently no ongoing studies in the USA (see the clinical trial data base for the USA)
In the EU (Europe) one clinical trial is currently being conducted:

A pilot phase II study of a combined immunotherapy protocol based on oral vaccination and direct intratumoral injection of Salmonella Typhi Ty21a Vivotif in metastatic cutaneous melanoma patients. The medical condition of interest is Metastatic stage III and IV M1a melanoma. This study is managed by the Istituto Europeo di Oncologia in Milan, Italy (see the clinical trial data base for the EU).

Please keep in mind that there are several physicians / oncologists that want to conduct clinical trials with this type of therapy. So please check the databases regularly for updates. If you are a medical doctor and would like to conduct a clinical trial investigating attenuated Salmonella, please get in touch.

Also, if you are a physician or sponsor and are conducting a clinical trial in which Salmonella is used to treat cancer, then please contact me if you would like to have your trial listed on this page.

New developments in Salmonella based Immunotherapies?

Given the intracellular lifestyle and immunomodulatory properties associated with attenuated Salmonella strains, they are also well suited to deliver therapeutic molecules right into the cancer cells.
There are a range of cargo molecules, but all are based on inserting genetic material which codes for proteins such as tumour antigens (e.g. HSPPC-96), cytokines (e.g. IL-18), apoptosis-inducing factors (e.g. TRAIL), prodrug-converting enzymes, or genetic material that encodes for short hairpin RNAs (shRNAs) which are able to silence expression of a gene of choice (e.g. a gene such as ERBB2 or EGFR that is normally upregulated in cancer).

In addition, recent reports indicate that Salmonella Drug Conjugates (SDCs) may soon become reality and potentially common place in the treatment of diseases such as cancer. Park and colleagues, from the Chonnam National University in South Korea, developed this SDC they dubbed “bacteriobot” which in simple terms is an attenuated salmonella strain covalently linked to a robotic device, 3 micrometers in size, that automatically sprays anticancer drugs when it reaches a cancer cell. The creation has already been patented in dozens of countries, including the United States, Japan and all members of the European Union.

In summary, bacterial cancer therapy based on various Salmonella strains is supported by solid preclinical and early phase clinical data. 

Please note: Any medical or scientific information published on this website is not intended as a substitute for informed medical advice from a physician and you should not take any action before consulting with a health care professional. For more information, please read my terms & conditions.

1 comment:

  1. Hey Patrick, great article. Looking forward to the next instalment.