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).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
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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.
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For info about Biozantium by Paeon Laboratories: http://www.biozantium.com |
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 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).
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).
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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-
FU with YB1 and their effects on tumour growth
respectively.
![]() |
Graph copied from Yu et al., 2012 which demonstrates
remarkable tumour growth inhibition as a combination therapy
with 5FU
|
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 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.
Hey Patrick, great article. Looking forward to the next instalment.
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