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34 Aoife M. Brennan et al.
bacterial signatures [2], and bacteria have been incorporated into the treatment of
established tumors [3].
1.1 History of microbes as tumor immunotherapy
The earliest reports of bacterial infection resulting in tumor shrinkage date back to
ancient Egypt [4], but the physician-scientist most associated with this field is William
Coley. In 1891, Coley began injecting Streptococcus pyogenes intratumorally, and he later
developed and refined his approach by heat-killing a mixture of gram-negative and grampositive organisms to create “Coley’s toxin” [5]. In the past 100 years, our understanding
of the role of the immune system in response to cancer has advanced tremendously, and
more recently, effective therapies, including immune checkpoint inhibitors, have been
developed based on this understanding [6]. Despite this progress, only a subset of tumor
types demonstrates responsiveness, and the search for treatments capable of triggering an
innate response in immunologically “cold” tumors continues.
Several groups have explored attenuated pathogenic bacteria as a treatment for various
cancers. The most advanced program is for Bacille Calmette-Guerin (BCG), an attenu-
ated strain of Mycobacterium bovis that is widely used for the adjuvant treatment of
nonmuscle-invasive bladder cancer. Multiple clinical trials have demonstrated that
BCG reduces the risk of recurrence with a 2-year disease-free survival of 57% compared
with 45% for chemotherapy [3,7]. The initial clinical trial reporting success using BCG in
the treatment of bladder cancer was published in 1976 [8]; however, the precise mechanism of this beneficial therapeutic effect is still not completely understood. Based on
preclinical data, BCG appears to induce an adaptive, tumor-specific immune response
and requires a competent immune system [3]. The live organism enters macrophages
and bladder cancer cells and stimulates cytokine production (tumor necrosis factor-alpha
[TNF-α], interferon-gamma [IFN-γ], and interleukins [IL] 12 and 18) and tumor antigen
presentation. This initial inflammatory response leads to natural killer (NK) cell and cluster of differentiation (CD)8 and CD4 T-cell activation, tumor cytotoxicity response, and
tumor cell lysis [9]. Local adverse events from treatment are common but manageable,
while systemic infection with BCG, described in 1%–5% of patients across studies, is
more troublesome [10]. A significant challenge for BCG as a treatment for bladder cancer
is the ability to maintain a consistent supply due to a long and complicated manufacturing
process, resulting in frequent shortages that interrupt treatment courses [11]. The lesson
for the next generation of bacterial therapeutics is to consider the manufacturability of the
chassis organism before advancing programs to the clinic.
1.2 Mechanistic underpinnings
Several bacterial species are intrinsically capable of colonizing tumors selectively and
localizing primarily to the hypoxic tumor core [12–17]. While bacteria in circulation
and normal healthy tissue are typically cleared by the immune system within hours, the
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tumor microenvironment (TME) provides a unique immunosuppressive, hypoxic, and
nutrient-rich environment for bacteria to proliferate and avoid immune clearance
[13,17]. This selective colonization is an attractive feature for the development of local
tumor-targeting therapeutic approaches; however, bacteria still engage the immune system within the tumor, specifically through activation of innate immunity. Cells of the
innate immune system (macrophages, dendritic cells, neutrophils) express a large variety
of pattern recognition receptors, which allow these cells to recognize microbial pathogens through their evolutionarily conserved structures, so-called pathogen-associated
molecular patterns (PAMPs) [18] (Table 1). Activation of these receptors by bacterial
ligands triggers phagocytosis and inflammatory response, which leads to immunomodulation of the TME through the production of proinflammatory cytokines and
chemokines, interferons, and pyroptosis [19,20]. This response prevents bacterial dissemination into viable tumor cell areas [21] and may lead to tumor regression by subsequent
activation of antitumor adaptive immune responses.
While BCG is well established, several other attempts have been made to use path-
ogenic bacteria in cancer therapeutics, including bacteria from genera Clostridia, Listeria,
and Salmonella. These bacterial species employ both immunomodulation and unique
mechanisms of pathogenicity to destroy cancer cells and induce tumor regression; however, the use of pathogenic bacteria therapeutically requires engineered attenuation to
prevent severe toxicities and side effects in the host (Table 2). Strict anaerobes such as
Clostridia were selected based on a preference for the oxygen-deficient, necrotic core
of solid tumors that limits replication in healthy, vascularized tissue. In clinical trials,
an attenuated form of Clostridium novyi in which the alpha-toxin had been deleted,
referred to as C. novyi-NT, demonstrated shrinkage in the injected lesions [46], but local
reactions in surrounding tissue were problematic. Approaches with other organisms have
yielded similar results, with the challenge of achieving an appropriate benefit-risk balance
through destruction of the injected tumor and/or stimulating a tumor immune response
without causing infection or intolerable side effects. In the case of Salmonella, using attenuation or early molecular biology techniques to limit pathogenicity resulted in difficulty
in retaining the immune-stimulating properties of the strain. Further, a clinical trial of
S. typhimurium with several genetic modifications failed to demonstrate clinical activity
in a small study in patients with melanoma [47].
Recent advances in our ability to engineer bacteria with specific safety and efficacy
features using synthetic biology-based techniques, advances in our understanding of the
immune system, and an appreciation that multiple modes of action may be required to
treat most tumors have resulted in a resurgence of interest in engineered microbes in cancer immunotherapy. This work holds the potential to bring the most ancient cancer
immunotherapeutic into the modern age.
35Engineered microbes for cancer immunotherapy
36 Aoife M. Brennan et al.
Table 1 Bacterial ligands and the innate immune receptors they activate.
Bacterial ligands Receptors Cellular location
Toll-like Receptor family
Triacetylated lipopeptides TLR1 Cell surface
Peptidoglycans,
TLR2 Cell surface
Lipoproteins/lipopeptides
Lipopolysaccharides TLR4 Cell surface
Flagellin TLR5 Cell surface
Lipoteichoic acid TLR6 Cell surface
Unmethylated CpG DNA TLR9 Endosomal
NOD-like Receptor family
Peptidoglycans NOD1 Intracellular (cytoplasm, plasma, and
endosomal membrane associated)
T3SS proteins NLRC4/NAIP2,
NLRP12
Intracellular (cytoplasm, plasma, and
endosomal membrane associated)
Flagellin NLRC4/NAIP4 Intracellular (cytoplasm, plasma, and
endosomal membrane associated)
Lipopolysaccharides NLRP3 Intracellular (cytoplasm, plasma, and
endosomal membrane associated)
Listeriolysin O NLRP3 Intracellular (cytoplasm, plasma, and
endosomal membrane associated)
Hemolysins NLRP3 Intracellular (cytoplasm, plasma, and
endosomal membrane associated)
Aerolysin or other
pore-forming toxins
NLRP3 Intracellular (cytoplasm, plasma, and
endosomal membrane associated)
Anthrax lethal toxin NLRP1 Intracellular (cytoplasm, plasma, and
endosomal membrane associated)
RIG-I like Receptor family
Bacterial RNA/DNA RIG-I Intracellular, cytoplasm
Bacterial RNA/DNA MDA5 Intracellular, cytoplasm
Bacterial RNA/DNA LGP2 Intracellular, cytoplasm
AIM2- like Receptor family
DNA AIM2 Cytoplasm
Bacterial DNA IFI16 Nucleus
STImulator of INterferon Genes (STING) pathway
Bacterial DNA cGAS/STING Cytoplasm
Bacterial CDNs
STING Cytoplasm
(cyclic dinucleotides)
Glucans C-type lectins Cell surface
Sources: Ahn J, Barber GN. STING signaling and host defense against microbial infection. Exp Mol Med 2019;51:1–10;
Brubaker SW, Bonham KS, Zanoni I, Kagan JC. Innate immune pattern recognition: a cell biological perspective. Ann
Rev Immunol 2015;33:257–90; Ferrand J, Ferrero RL. Recognition of extracellular bacteria by NLRs and its role in the
development of adaptive immunity. Front Immunol 2013;4:344; Imani Fooladi AA, Mousavi SF, Seghatoleslami S, et al.
Toll-like receptors: role of inflammation and commensal bacteria. Inflamm Allergy Drug Targets 2011;10(3):198–207;
Mnich ME, van Dalen R, van Sorge NM. C-type lectin receptors in host defense against bacterial pathogens. Front Cell
Infect Microbiol 2020;10:309.
Table 2 Pathogenic bacteria used in cancer treatment.
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Bacteria Mechanism of action Engineered attenuation
37Engineered microbes for cancer immunotherapy
Salmonella An intracellular gram-negative pathogen,
Salmonella kills invaded tumor cells
through lysis by uncontrolled
proliferation, induction of apoptosis, or
autophagy [14,22–24]. Bacterial
PAMPs induce proinflammatory
response in macrophages and dendritic
cells. Following TLR activation, cells
produce IL-1b and TNFα and induce
antigen cross-presentation of tumor
antigenic peptides [25], leading to
tumor disruption and stimulation of
tumor-specific CD8 + T-cell responses
[26–28]. Additionally, flagellin activate
NK cells to induce perforin and release
IFNγ and IL-18 [29,30].
Clostridia A gram-positive endospore-forming
bacteria that secretes a number of
exotoxins in colonized tumors leading
to damage of tumor cell membranes,
interference with critical cellular
functions, and subsequent tumor cell
lysis [33–35]. Inflammatory responses
induced by bacterial PAMPs lead to
upregulation of cytoki nes and
chemokines in the tumor environment
and recruitment of CD8 T cells [36].
Listeria A gram-positive intracell ular pathogen
that infects professional antigenpresenting cells such as monocytes/
macrophages and dendritic cells. In the
tumor, Listeria resides in MDSCs where
it is somewhat protected from immune
clearance [38,39]. However, infection
with Listeria alters the immunological
state of subpopulation MDSCs, leading
to production of IL-12, which may
improve CD8 T-cell and NK cell
responses [38]. Once in the tumor,
Listeria directly kills tumor cells
through NADPH-activated
production of ROS and apoptosis
[40,41], which also adds to the
activation of antitu mor CD8 + T-cell
responses.
Deletion of the msbB gene from Salmonella
resulted in modification of lipid A, a
critical component of LPS, and
minimized TNFα-mediated septic
shock [31]. Salmonella was also made
defective in the synthesis of signaling
molecule ppGpp (thus named
ΔppGpp) required for induction of
numerous virulence genes, which
impacted the bacterial ability to enter
and replicate in the tumor cells [32].
Salmonella A1-R is engineered to be
auxotrophic for leucine and arginine,
which are likely enriched in the tumor
but not in normal tissues [16].
Clostridium novyi (C. novyi)was
transformed to a significantly safer
strain (C. novyi-NT) by deleting the
gene for a lethal exotoxin [37].
Deletion of master virulence regulator
gene prfAin Listeria [42] made bacteria
unable to escape into a cytosol of
infected cells. To compensate for this
inability and to preserve delivery of
tumor antigens to cytosol for crosspresentation, the prfA
-deficient strains
were engineered to express low levels
of prfA and truncated Listeriolysin
O that may be fused with the antigens
of choice for enhanced
immunogenicity [43]. Attenuation of
Listeria is also achieved by deleting
genes actA and inlB, responsible for
bacterial dissemination [44] or by
inactivation of the dal and dat genes
required for the synthesis of bacterial
cell wall [45].
Abbreviations: APCs, antigen-presenting cells; CD, cluster of differentiation; IFN, interferon; IL, interleukin; LPS, lipopolysaccharides; MDSC, myeloid-derived suppressor cell; NADPH, reduced nicotinamide adenine dinucleotide; NK, natural killer; PAMP, pathogen-associated molecular pattern; ROS, reactive oxygen species; TLR, toll-like receptor; TNF,
tumor necrosis factor.
38 Aoife M. Brennan et al.
2. Application of synthetic biology to therapeutics
2.1 History of synthetic biology
Synthetic biology is a multidisciplinary area of research that seeks to control cellular
behavior through the design, construction, and incorporation of logic-based genetic elements. The past few decades have witnessed the emergence and rapid expansion of
molecular and computational biology toolkits that have enabled the precise programming
of cell systems to perform prescribed functions [48]. This dramatic growth has enabled
synthetic biology to be applied to therapeutics, where it has the potential to be transformative. The focus of this section will be on microbial cell systems; the application of
synthetic biology to mammalian cells has been described elsewhere [49,50].
The origins of synthetic biology originated with the landmark studies of the lac
operon conducted by Jacob and Monod in 1961 [51,52]. These pioneering researchers
described the ability of Escherichia coli to regulate the expression of lactose-metabolizing
genes in response to the presence of galactosides in culture media. This natural process,
which Jacob and Monod termed “induction,” laid the foundational groundwork for
future studies designed to elucidate the ability of bacteria to alter their pattern of gene
expression in response to environmental and chemical signal sensing. In the following
decades, this field progressed with the advent of molecular cloning, polymerase chain
reaction, and automated DNA sequencing [48]. These advances allowed researchers
to rationally engineer DNA into patterns not normally found in nature, allowing, for
the first time, human control over bacterial gene expression, which afforded researchers
a simplified method for elucidating the phenotypic and biochemical effects of genes,
enzymes, and pathways. In commercial biotechnology applications, this control of gene
expression allowed for standardized production of recombinant proteins and other molecules of interest.
Around the turn of the century, a formal discipline began to emerge from these basic
research efforts that sought to fully harness the power of rational design and construction of biological systems. Engineers, physicists, biochemists, and computer scientists
joined forces with molecular biologists to understand how to manipulate and tune biological parts, how to perform and improve genetic engineering using standardized and
modular components, and how to accurately model transcription and translation of
DNA sequences in silico [48,53,54]. The focus of the field has been on the implementation of “genetic circuits,” which comprise DNAs that encode biological parts
designed to perform logic functions akin to a computer program executing an algorithm
[55–57]. In simple terms, a genetic circuit receives and converts an input signal into a
quantifiable output. Through the implementation and layering of such controllable
logic elements, genetic circuits may be utilized to perform an array of useful biological
functions [58].
2.2 Sense and respond
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Genetic circuits are characterized by two main features: the input, which senses and integrates an external signal, and the output, which encodes the circuit’s mechanistic activity
or “effector function.” The latter feature will be described in detail in a later section of
this chapter. The input, or “switch,” is typically controlled by an inducer-promoter pair.
Jacob and Monod described a naturally occurring genetic switch when they demonstrated that E. coli detected the presence of lactose and its analogs in culture media
and produced enzymes responsible for lactose metabolism in response [52]. The lac
operon consists of three genes controlled by an upstream promoter element. Promoters
are responsible for the recruitment of RNA polymerase, which subsequently directs
downstream gene transcription. The lac promoter also contains regulatory DNA motifs,
called operators, that direct the binding of the LacI repressor. When bound to its operators, LacI blocks gene expression through occlusion of RNA polymerase binding.
When present in culture media, lactose or its analogs bind to LacI, preventing operator
binding and allowing access to RNA polymerase with consequent gene expression
[51,52]. This simple mechanism paved the way for the more intricate synthetic biological
systems that were later developed. Most of the control of bacterial gene expression is at
the level of transcription, and for this reason, genetic switches remain at the core of the
synthetic biologist’s toolbox [59,60].
Bacterial promoters display species-specificity. While historical studies of gene regu-
lation have been performed in workhorse organisms including E. coli, biological parts from
this organism are unlikely to function in distant relatives, such as Bacteroides or Clostridium.
Synthetic biology tools must therefore be tailored to the microbe of interest. Advances in
next-generation sequencing have been integral in helping scientists predict regulatory
sequence elements across the genomes ofmany previously unstudied bacterial species, with
efforts focused on the construction of modular libraries of inducer-promoter pairs spanning a comprehensive range of regulation and expression strength [56,60].
The switch composition of a genetic circuit is largely dependent on the level of effector activity required and the environment in which the circuit is expected to function.
For industrial applications, strong promoters are controlled by small-molecule inducers,
such as isopropyl β-D-1-thiogalactopyranoside (IPTG) or anhydrotetracycline (aTc), are
typically used, as they are well characterized and known to provide superior control of
expression. However, for therapeutic purposes, where engineered live biotherapeutic
products (LBPs) may be expected to function in unique microenvironments, IPTG
and aTc may not be the most practical options. For this reason, synthetic biologists have
characterized so-called “in vivo” switches that are capable of sensing and responding to
signals encountered within the human body. Living biotherapeutics may be designed
to detect changes in pH, temperature, or oxygen tension [56,58,60]. For example, promoters regulated by the fumarate and nitrate reductase regulator (FNR) of E. coli,an
oxygen-responsive transcription factor, have been investigated in clinical-stage LBPs
39Engineered microbes for cancer immunotherapy
40 Aoife M. Brennan et al.
to activate the expression of effector functions in the largely anaerobic gastrointestinal
tract [61,62] or the hypoxic core of solid tumors [63]. Disease-specific switches have also
been constructed, such as those sensing inflammation-dependent signals like reactive
oxygen species, tetrathionate [60,64], or nitrate [65].
Other synthetic switches have been developed that permit self-sensing by engineered
bacteria through the repurposing of bacterial quorum sensing (QS) systems [56,60].QS
systems typically depend on the production of small molecules or peptides, termed
“autoinducers,” that accumulate in the environment. As the cell population increases,
so does the concentration of the autoinducer, which in turn modulates gene expression
when the bacterial population/autoinducer reaches a critical density (Fig. 1). In this manner, bacteria are programmed to switch on their effector function only when an effective
cell concentration has been established [60], which may be useful in LBPs in which the
effector function is toxic or results in unwanted side effects, ensuring that the engineered
activity is delivered only to disease-specific environments that support LBP growth and
division [60,66,67].
In contrast to delivering effectors only when needed, providing a continuous response
to a transient signal may prove advantageous in certain applications [60]. For this purpose,
synthetic biologists may choose to deploy a “memory device.” Among the earliest
memory devices constructed in the synthetic biology field was the toggle switch [48].
The toggle function was dependent on the stable output states achieved by the mutual
repressive ability of the phage lambda cI/Cro genetic switch. The toggle switch is adapted
to sense a particular molecule or environmental condition by layering additional switches
that control the expression of the cI and Cro transcription factors themselves [68,69].
Serine integrases have also been deployed as memory elements. These enzymes catalyze
Fig. 1 Design considerations in engineering microbes for immunotherapy.
the unidirectional inversion of DNA between two recognition sites and cause a perma-
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nent switch to the “on” state upon sensing a transient induction event, akin to the literal
“flipping” of a switch, with DNA being the element flipped [65,70]. Memory devices,
when coupled with the appropriate sensors, serve as powerful biodiagnostic tools due to
their ability to stably report a condition sensed in vivo [69].
Another method used by synthetic biologists to alter genetic circuit expression is
through manipulation of bacterial translation, which is commonly performed by modifying ribosome binding sites (RBS). The RBS is a noncoding region of mRNA that
coordinates both recruitments of the bacterial ribosome and the strength of translation
initiation. In the past, RBS optimization was largely an empirical endeavor; however,
advances in omics and in silico modeling have led to highly predictive algorithms that
allow fully automated RBS design [54,71–73].
2.3 The intersection of synthetic biology and therapeutics
Genetically engineered microbes are the source of many industrially-produced large and
small molecule therapeutic compounds, including antibiotics, enzymes, peptide hormones, and antigens. For this reason, they are often described as “living factories.”
The stage has now been set for synthetic biologists to leverage such industrial learnings
for the development of LBPs, with the end goal of using synthetic biology to program
and administer “living factories” to treat human disease [56,58,60] (Fig. 2).
41Engineered microbes for cancer immunotherapy
2.4 Chassis selection
Among the most important considerations for the development of LBPs is the choice of
chassis organism into which genetic circuits can be engineered. Unsurprisingly,
laboratory-domesticated E. coli strains such as K-12 have been used for the development
Fig. 2 Synthetic biology tools.
42 Aoife M. Brennan et al.
of many in vitro synthetic biological systems. However, commensal bacteria may represent a superior choice for in vivo applications since they are presumed to be benign and to
have evolved to appropriately interact with the host and its immune system. Additionally,
strains already in use as probiotics may earn greater levels of acceptance by the public and
the United States Food and Drug Administration (FDA) [58]. Still, the environment in
which the chassis is expected to function (e.g., skin, gastrointestinal tract, tumor, etc.), the
ease of its genetic manipulation, and its ability to be manufactured at scale are also crucial
factors that must be appropriately balanced.
E. coli Nissle 1917 is a commercially available probiotic strain that has been used for
over 100 years in its unengineered form to treat various gastrointestinal conditions [74].
As a natural human isolate, this gram-negative strain is an attractive platform for the further development of engineered LBPs, including clinical-stage programs in metabolic
disease [59,61,62] and oncology [63]. Salmonella typhimurium is a second gram-negative
organism that has made an attractive chassis for cancer therapy, although, unlike the commensal E. coli Nissle, Salmonella strains must be genetically attenuated for safety before
in vivo administration [56]. Facultative anaerobes such as E. coli and Salmonella represent
attractive engineering platforms due not only to the repertoire of tools available for their
genetic manipulation but also to their metabolic adaptability, displaying robust growth
during aerobic high cell density manufacturing as well as within the hypoxic TMEs
[56]. Bacteroides are a gram-negative genus comprising the most abundant colonizers
in the human gut, and strains such as B. thetaiotaomicron and B. fragilis have recently
emerged as a viable LBP chassis following the introduction of tools allowing their genetic
manipulation [60,75]. However, the obligate anaerobic nature of these organisms may
impose challenges in large-scale manufacturing campaigns.
Gram-positive bacteria have also been used for LBP development, though the suite of
synthetic biology tools available for their genetic manipulation is generally less advanced
than that for E. coli. In particular, the Lactobacillus and Lactococcus genera have attracted
significant attention as gut-based engineered LBPs owing to their intrinsic probiotic
properties [59,60]. Additionally, the structure of the gram-positive cell envelope, comprising only a single lipid bilayer, is advantageous for the engineered secretion of effector
proteins, and several L. lactis-based LBPs have been advanced to the clinical stage for this
function [59]. Bifidobacterium longum is a probiotic strain derived from the gut but repurposed as a chassis for tumoral-acting LBPs, with one LBP reaching the clinical stage
for the conversion of 5-fluorocytosine into 5-fluorouracil, an established chemotherapeutic agent [76–78]. Nonprobiotic gram-positive strains have also been used in LBP
development. C novyi-NT, an obligate anaerobe known to be effective in eradicating
treatment-resistant tumors in preclinical models, has spawned an active area of research
for synthetic biologists looking to enhance its therapeutic performance via the development of genetic engineering technology [56,79]. Listeria monocytogenes, a gram-positive
strain known mostly for its ability to cause foodborne illness, have been recognized for
their potential use in cancer immunotherapy [56,59]. This organism has been primarily
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investigated in vaccine applications, and several engineered LBP vaccines have been
advanced to clinical trials [59]. Additionally, the intracellular lifestyle of Listeria indicates
its potential utility as a gene delivery vector [80]. Listeria is genetically tractable and, as a
facultative anaerobe, intrinsically more manufacturable than obligate anaerobic grampositive, making it a more appealing chassis for LBP development.
3. Synthetic biology techniques to engineer safety and control
growth
3.1 Biocontainment of genetically modified live biotherapeutic
products
Concerns have been raised by both scientists and the public over unintended environmental impacts following the potential escape of genetically modified organisms [81]. Additionally, if engineered LBPs are to be used for human administration, safeguards must
be put in place to control and prevent unwanted cell growth in vivo [61]. For this reason,
the adoption of biocontainment measures is important when developing engineered LBPs,
with efforts in place to prevent the spread of not only the engineered organism but also its
genetic material [59]. For this purpose, synthetic biologists have implemented several
innovative biocontainment strategies that may be incorporated into LBP design.
43Engineered microbes for cancer immunotherapy
3.2 Genetic stability
Plasmids are excellent tools for quickly manipulating and interrogating genes of interest;
however, plasmid DNA is not particularly stable and will typically be lost in the absence
of antibiotic selection. The FDA also discourages the use of antibiotic markers in LBPs
[59,82], further discouraging the use of plasmids to carry engineered genetic circuits.
Synthetic biologists have developed techniques to overcome these limitations. For example, an essential gene may be deleted from the chromosome of the chassis organism and
cloned into a plasmid for complementation instead of antibiotic selection [83,84]. Toxin-
antitoxin systems have also been deployed, where a toxin is encoded on a plasmid with
the cognate antitoxin encoded on the chassis chromosome [85]. In the event of an unintentional plasmid transfer, the encoded toxin would kill the new host and prevent the
further spread of engineered DNA.
Plasmids do suffer a key liability: they are prone to unequal segregation during rounds
of cell division [60]. For this reason, if possible, avoiding plasmid use altogether may be
the ideal choice for LBP engineering and may be achieved through the integration of
engineered circuits directly into the chassis chromosome [59,61]. Though this approach
may be considered optimal, the genetic tools required for chromosomal integration may
not be available for all potential chassis organisms.
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