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44 Aoife M. Brennan et al.
3.3 Auxotrophies as a mechanism to control growth
Among the most common approaches to LBP biocontainment is engineering nutritional
auxotrophy [61–63,86], which typically entails the deletion of an essential chromosomal
gene in the chassis organism to create dependence on an exogenous supply of an essential
metabolite [81]. This strategy is not without risks, as auxotrophic microbes may still be
able to survive in the environment if the essential metabolite is provided by other organisms. Additionally, an engineered organism may regain the essential gene through a horizontal gene transfer event, although this event is likely rare [81].
An advanced synthetic auxotrophy system was recently described in a “recoded”
strain of E. coli [87]. Initially, all-natural UAG stop codons were swapped genome-wide
to UAA. The strain also expressed a heterologous tRNA and aminoacyl-tRNA synthetase pair that incorporated a novel synthetic amino acid into UAG codons strategically
placed within several essential genes. The synthetic amino acid, unlikely to be found
in the environment, was supplied exogenously; thus, the strain could not exhibit
long-term survival in the absence of nutritional intervention [87–89].
Similarly, researchers have worked toward technology for constructing auxotrophs
dependent on synthetic nucleic acids [81]. In one instance, a thymine biosynthesisdeficient strain of E. coli was subject to adaptive evolution in the presence of chlorouracil;
following the evolution, the strain could substitute chlorouracil in place of thymine in its
DNA [90]. Another approach demonstrated that modified E. coli could incorporate polymers of sugar-molecule backbones in its genetic material, termed “xeno-nucleic acids,”
that may eventually be able to functionally replace DNA or RNA [91]. Synthetic nucleic
acid auxotrophies are attractive not only because they can control bacterial growth, but
also because they are impervious to concerns of horizontal transfer to foreign microbes, as
synthetic nucleic acids would be unable to be transcribed or replicated following
acquisition [81].
Both the synthetic amino acid and synthetic DNA strategies generate strict auxotro-
phies from which strains are unlikely to escape; however, the extensive genome editing
required to the chassis currently limits applicability in LBPs beyond E. coli [81].
3.4 Kill switches as bioconta inment mechanisms
Another major strategy for the biocontainment of engineered organisms is the “kill
switch.” These mechanisms use logic-based genetic circuits programmed to sense exogenous inputs to determine whether the requirements for cell proliferation continue to be
met. In certain examples, an exogenous supply of signaling molecules is supplied to cells
to maintain the induction of genes essential for survival [81,92]. Once the signaling molecules are removed, the essential genes are no longer able to be transcribed, leading to cell
death. In other embodiments, bacterial QS systems may be reprogrammed to support
survival. In one reported example, E. coli was engineered such that the expression of
an essential gene was dependent on the presence of a QS autoinducer at concentrations
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only achievable at high cell density [93]. Cells that escaped their environment were
unable to proliferate due to low levels of autoinducer in the nonpermissive condition.
Other kill switch systems rely on layered gene circuits that maintain the transcriptional
repression of a toxic gene product [94]. In such an example, cells require the constant
presence of a regulatory element’s inducer to maintain the repression of the toxic output;
the absence of an inducer leads to the production of the toxic product and subsequent cell
death [81,94–96] (Fig. 3).
Despite the potential utility, sophisticated engineering strategies for LBP biocontainment have not been deployed in therapeutic applications to date because they generally
have escape rates higher than the 10
8
frequency recommended by the National Institutes of Health [59,60,81]. For this reason, simple nutritional auxotrophy has remained
the biocontainment strategy of choice for clinical-stage LBPs [59]. However, synthetic
biologists have conceived of several approaches to address the high escape rate of circuitbased kill switches. In one instance, researchers refined the specificity of the CRISPRCas9 system to monitor and prevent the occurrence of point mutations, which may be
applied to maintain the sequence fidelity of biocontainment modules [97]. Other
approaches look to reduce mutagenic potential in the host organism to prevent the
45Engineered microbes for cancer immunotherapy
Fig. 3 Synthetic biology components relevant to engineering microbes for cancer immunotherapy.
46 Aoife M. Brennan et al.
selection of escapees. Modification of endogenous DNA repair, mutagenesis systems, and
DNA polymerases, as well as deletion of insertion-sequence elements, are all promising
strategies to reduce the escape rate in E. coli by orders of magnitude [81,94,98]. However,
similar to the issues raised with synthetic auxotrophy, the extensive genome editing
required for a low-escape kill switch system currently limits applicability in chassis other
than E. coli, underscoring the need for the development of genetic tools in other potential
LBP chassis.
4. Application of synthetic biology to engineer microbial
therapeutics for cancer
When William Coley began his work on immunotherapy in cancer in the 1800s,
he was fascinated with the observation that patients who recovered from cancer coincident with bacterial infection were able to return to a normal life, unmaimed. This is
in contrast to the other available treatments of the day such as surgery and radiotherapy.
These deep and durable remissions continue to be the goal of immunotherapy today and,
while available to a proportion of patients with certain tumor types, remain difficult to
predict. Among the current goals of the field are predicting which patients will respond
positively, broadening the potential benefit to other tumor types that have not
demonstrated responsiveness (i.e., turning “cold” tumors “hot”), and elucidating the
other immune checkpoints that tumors employ to evade the immune response. A key
challenge for those working in the field of bacterial therapeutics is how to incorporate
bacteria to address these issues.
The ideal bacterial approach would be a bacterium engineered to function at the site
of action but that spares invasion of healthy tissue and does not cause intolerable systemic
cytokine reactions. At the site of the tumor, the bacteria would address the specific
immune-evading mechanisms of that particular tumor, triggering a tumor-specific
T-cell response that could maintain immune surveillance for other tumor sites and
micro-metastasis. While multiple attempts have been made using unmodified bacteria,
synthetic biology allows us to now apply design principles to engineer effector functions
into bacteria based on mechanistic understanding of the human immune response, engineer safety features to limit toxicity, and address manufacturing and delivery challenges.
4.1 Engineering effector functions
Understanding the specific mechanisms of tumor immune evasion, as well as the mechanisms linking bacterial infection to tumor regressions, may allow a rational approach to
developing an engineered microbial therapeutic. This approach may begin with a pathogenic chassis or with an organism with low pathogenicity into which effector functions
are engineered. The bacterial PAMPs such as lipopolysaccharides, the bacterial
double-stranded DNA, and any bacterial toxins that are part of the chassis organism may
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be part of the antitumor drug upon which additional effectors are built [63].
4.2 Enzymatic and metabolic conversion
Several classes of effectors have been explored and, in certain cases, tested clinically (see
Table 3). One such category that leverages the ability of bacteria to localize to the TME
and continue to be metabolically active is based on enzymatic conversion. For example,
engineering bacteria that convert a chemotherapeutic prodrug into an active chemotherapeutic at the site of the tumor would avoid off-target toxicity. Consistent with this
approach, multiple bacterial strains have been engineered to express cytosine deaminase,
which converts the prodrug 5-fluorocytosine to the potent metabolite fluorouracil, a
common chemotherapeutic agent administered to patients with cancer [103,104]. This
approach may also be extended to the conversion of naturally occurring immunosuppressive metabolites within the TME. Kynurenine is a metabolite that has been mechanistically linked to tumor immune suppression [105]. Interfering with the generation of
kynurenine through inhibition of indoleamine 2,3-dioxygenase (IDO) was considered
an attractive target for drug development; however, this approach proved unsuccessful
in the clinic, likely due to redundancy in IDO enzymes, where inhibition of one or
two IDOs was likely insufficient to provide a clinical benefit [106]. Engineering bacteria
to consume kynurenine within the TME may be able to overcome this clinical limitation,
since this mechanism removes the offending metabolite directly [107].
Considerations for the enzymatic/metabolic conversion approach include evaluating
the potency required by bacteria in vivo, transport of the target metabolite into the
engineered bacteria, the requirement for enzyme cofactors, energy requirements and
redox balancing of the proposed engineered pathway, and the toxicity potential of the
product metabolites to both the host and the bacteria itself. While a number of these
design considerations may present challenges, potential synthetic biology tools and solutions also exist, e.g., expressing an enzyme on the cell surface if intracellular transport of
the target metabolite is limiting [108]. As further work elucidates the presence and
function of metabolites in the TME, engineered microbial therapeutics serves as a tool
to validate their mechanistic role and potential translation to human therapeutics.
47Engineered microbes for cancer immunotherapy
4.3 Cytotoxic agents
Another category of LBP effector function is the production of cytotoxic agents. Shiga
toxin (Stx2) has been engineered into Salmonella strains under the control of a
pH-sensitive promoter and was shown to induce tumor necrosis in preclinical models
[109]. Bacterial strains have also been engineered to express pore-forming bacterial
toxins, S. aureus α-hemolysin or E.coli cytolysin A, under the control of tumor-specific
promoters to provide localized toxin expression [110–112]. Similarly, expression of the
Table 3 Summary of select clinical studies using bacteria.
Strain
Chassis
organism
Indication
evaluated
Route of
administration Clinical data available Reference
VNP20009 Salmonella
typhimurium
C. novyi-NT Clostridium
novyi
CRS 207 Listeria
monocytogenes
ADXS-HPV Listeria
monocytogenes
APS001F Bifidobacterium
longum
bacTRL-IL-12Bifidobacterium
longum
SYNB1891 E. coli Nissle Solid tumors IT
VXM01 Salmonella
typhimurium
SalpIL2 Salmonella
χ4550
Metastatic
melanoma
Solid tumors IT • Tumor destruction in the injected lesion
Pancreatic IV
HPV-associated
cancer
Solid tumors IV
Solid tumors IV
Glioblastoma Oral
Liver metastases
of solid
tumors
IV • Tumor colonization in some cases
IV
Oral
[47]
• No clinical response
[46]
• Sepsis and infection in surrounding tissue
at higher doses
• Did not prolong survival compared to
chemotherapy in a phase 2b study
• Well tolerated
[99]
[100]
[101]
• Overall survival improved over historical
benchmarks
• Phase 1/2 clinical trial ongoing https://clinicaltrials.
gov/ct2/show/
NCT01562626
• Phase 1/2 clinical trial ongoing https://www.
clinicaltrials.gov/
ct2/show/
NCT04025307
• Phase 1/2 clinical trial ongoing https://clinicaltrials.
gov/ct2/show/
NCT01562626
• Three of nine patients had objective
responses when oral vaccine used in
combination with a checkpoint inhibitor
[102]
• Phase 1 completed http://www.
clinicaltrials.gov/
ct2/show/
NCT01099631
major virulence factor from Listeria monocytogenes, Listeriolysin O (LLO), a pore-forming
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hemolysin responsible for lysis of the phagolysosome vacuole, demonstrated a reduction
in tumor mass in mice [113]. This toxin may also be fused with known tumor antigens to
more effectively trigger the antitumor effect [114,115].
4.4 Microbial antitumor vaccines
Vaccination is an alternative antitumoral strategy that employs live-attenuated pathogenic
bacteria or genetically engineered nonpathogenic bacteria to codeliver an antigen in the
presence of bacterial ligands that serve as innate immune adjuvants [116]. Listeria mono-
cytogenes is a preferred vector for such therapy due to the selective infection of antigenpresenting cells and its ability to enter the cytosol of the infected cell, which allows efficient
delivery of tumor-associated antigens to both the major histocompatibility complex
(MHC) class I and II antigen presentation pathways that drive antitumor CD8 T-cell activation. Fusion of natural tumor antigens such as PSA, Mage-b, HER-2/neu, tyrosinase,
and HPV-16 E7 to LLO toxin significantly increases the antigenicity of these tumorassociated proteins. A number of Listeria-based antitumor vaccines have been evaluated
based on this principle [43,117]. Attenuated Salmonella has also been investigated in the
delivery of tumor-associated antigens to the immune system. Since it has evolved to interact with the host through the gastrointestinal tract, Salmonella was used as an oral delivery
agent for tumor antigens encoded in cDNA plasmids. These Salmonella-based vaccines,
targeting tumor antigens such as alpha-fetoprotein [118], gp 100 [119], or CEA [120],
induced antitumor immunity and significantly reduced tumor growth.
Another approach to immunization is targeting tumor stroma and vasculature
required for tumor growth, where different stromal components may be targeted. For
example, an orally administered live Salmonella vaccine strain, VXM01, carrying an
expression plasmid encoding vascular endothelial growth factor receptor-2
(VEGFR2), an antigen expressed on proliferating endothelial cells within tumor tissue,
elicited VEGFR2-specific polyfunctional mixed CD8+ and CD4 + effector T-cell
responses, leading to effective protection against tumor challenges [121] and demonstrating promising potential in clinical studies with pancreatic tumors and glioblastoma
[102,122].
Two key challenges to this strategy are the selection of tumor antigens and the potency
generated by pathogenic or attenuated pathogenic bacterial chassis adjuvants, which represent an even greater concern when applied to cancer patients who are largely immunocompromised. The solution may be a more efficiently designed adjuvant combination
together with advanced tumor antigen selection or prediction [123] to achieve satisfactory
potency as well as minimizing the potential for cross-reactivity with healthy tissue.
49Engineered microbes for cancer immunotherapy
50 Aoife M. Brennan et al.
4.5 Cytokine delivery
Attenuated pathogenic bacteria and probiotic strains require additional engineered
immune effectors to drive notable antitumor activity. Thus, mammalian cytokines
may be expressed by the bacteria and delivered locally to tumor tissue upon colonization.
Promising preclinical data have been generated with a number of cytokines engineered
into Salmonella [124,125]. Attenuated S. typhimurium with plasmid encoding hIL-2 significantly decreased hepatic metastases in a murine MC-38 tumor model in comparison
to the attenuated strain alone. While no toxicities were observed, the strain still failed to
eliminate tumors [126]. Depletion of NK and CD8 T-cells significantly inhibited the
antitumor effects of this strain [127]. Another attenuated S. typhimurium (aroA
strain was engineered with plasmids containing mouse IL-4 or IL-18. Compared to the
control strain, the cytokine-encoding plasmid strains resulted in significantly increased
survival time and increased levels of IFNγ in sera of animals with B16-F1A melanoma.
However, these strains only delayed tumor growth and did not lead to tumor elimination
[124]. Another study with attenuated S. typhimurium engineered to synthesize IL-18 also
inhibited the growth of primary subcutaneous tumors (CT26, D2F2) as well as pulmonary metastases. This activity was associated with increased intratumoral production of
inflammatory cytokines, increased accumulation of T-lymphocytes and NK cells in
tumors, and a massive infiltration of granulocytes [128]. A lipid A-negative strain of
S. typhimurium was engineered with a plasmid encoding human LIGHT, a TNF-family
cytokine serving as a growth factor for dendritic cells and a stimulator of chemokines and
immune cell migration. Unlike control bacteria, attenuated S. typhimurium expressing
LIGHT inhibited the growth of primary tumors, as well as the dissemination of pulmonary metastases, in various carcinoma mouse tumor models. This antitumor activity was
achieved without significant toxicity and was associated with infiltration of inflammatory
cells [129]. Other examples of engineered S. typhimurium include production of IL-12,
granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand, IFN-γ and
CCL21 [130–133]. In all cases, the use of engineered bacteria resulted in significant inhibition of tumor growth and prolonged mouse survival [134,135].
A challenge in engineering bacteria to express bioactive mammalian cytokines is the
need for post-translational modifications. Many cytokines function as a dimer or trimer,
and multimerization may be challenging to achieve through bacterial protein translocation machinery. Fusion proteins may be considered with linkers designed between each
monomer subunit to facilitate the correct folding and multimerization during bacterial
production. If intramolecular or intermolecular disulfide bonds exist, a more oxidative
environment may be required during cytokine folding, such as that found within the
periplasmic region of gram-negative bacteria. Additional chassis modifications, such as
balancing the expression of disulfide bond-facilitating proteins and removal of proteases,
may provide a means for enhancing the bioactivity of effector proteins [136].
mutant)
Protein secretion is less problematic in gram-positive bacteria, which only contain a
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single lipid bilayer for payloads to traverse [137]. For gram-negative bacteria, secretion is
achieved by either co-opting an existing dedicated dual membrane-spanning secretion
system or guiding the protein to the periplasmic region using sec secretion signal peptides
and modifying the outer membrane to generate leakiness [138,139]. A combination of
secretion signal peptide optimization and other chassis modifications may be necessary to
optimize secretion for each unique effector [140].
The ability to engineer a combination of effectors with different antitumor mechanisms into a single bacterium is an advantage for engineered microbes as we now understand that tumors use multiple mechanisms to evade the immune response. Multiple
genes may be engineered simultaneously and switched on at different times, with some
focusing on initial stimulation to turn a cold tumor hot and others ensuring a sustained
antitumoral effect to best balance immediate and long-term efficacy. Future engineering
approaches will likely focus on engineering circuits, timing, and “dose” based on an
understanding of the mechanism of tumor immune evasion, or even direct feedback from
factors within the TME itself. An engineered microbe that specifically responds to realtime factors within the TME to produce the correct effector at the correct time and the
correct dose will be the ultimate embodiment of synthetic biology in cancer therapeutics.
The studies with engineered Salmonella described herein reflect relatively simple engineering of the bacteria by introducing plasmids with constitutive promoters to produce
desired effectors in the tumor. Recently, the development of synthetic biology and multiple bacterial engineering tools have allowed for the development of bacterial therapeutics that sense and respond to their environment.
One of the strategies to control the timing and duration of the effector secretion is to
build gene oscillator circuits using quorum sensing, where the accumulation of
autoinducer triggers synchronous lysis of engineered bacterial population in the colonized tumor. This approach allows local release of oncolytic factors as well as immunomodulatory proteins. Nonpathogenic E. coli was engineered to lyse and release a
nanobody recognizing CD47 into the TME to block the anti-phagocytic CD47 signal
commonly overexpressed in several human cancer types [141]. Another example with a
similar lysis circuit built in E. coli Nissle is a controlled release of nanobodies targeting
programmed cell death-ligand 1 and cytotoxic T lymphocyte-associated protein-4 to
reverse T-cell exhaustion as well as the release of a cytokine, GM-CSF [142]. In both
cases, treatment with engineered bacteria led to robust tumor regression and systemic
antitumor immunity and memory.
51Engineered microbes for cancer immunotherapy
4.6 Innate immune effectors
While the bacterial chassis itself exhibits immunomodulatory properties in the tumor by
engaging innate immune signaling through bacterial PAMPs, such as lipopolysaccharides
52 Aoife M. Brennan et al.
through TLR4 or bacterial DNA through TLR9 receptor, the addition of the specific
innate immune effectors significantly increases the antitumor activity of a strain. One
example is the use of nonvirulent tumor-targeting bacteria releasing multiple TLR
ligands. An attenuated S. typhimurium strain was engineered to secrete Vibrio vulnificus
flagellin B (FlaB), a TLR5 ligand, under the inducible
L-arabinose promoter to restrict
flagellin expression to tumor tissue after bacterial colonization. Engineered FlaBsecreting bacteria effectively suppressed tumor growth and metastasis in mouse models
and prolonged survival through activation of innate immune receptors TLR5 (flagellin)
and TLR4 (lipopolysaccharides), which induced the infiltration and activation of
immune cells such as monocytes/macrophages and neutrophils [143].
A second example is the use of E.coli Nissle engineered to produce STImulator of
INterferon Genes (STING) agonist c-di-AMP (SYNB1891) through heterologous
expression of L. monocytogenes dacA (encoding c-di-AMP-synthase) under the control
of a hypoxia-inducible promoter. Intracellular STING activation by this strain resulted
in the production of type I interferons from macrophages and dendritic cells and drove
cytotoxic T-cell activation. SYNB1891 was able to drive robust tumor eradication and
induce long-term immunological memory in mice [63]. SYNB1891 is currently undergoing evaluation in phase 1 clinical trial.
4.7 Engineering safety and tumor targeting
Tumors contain resident bacteria that likely invade the circulation from mucosal surfaces,
circulate in low numbers, and identify a niche within the tumor [2]. What these bacteria do
within the tumor and whether they impact outcomes and response to treatment is
unknown, but they do offer hints that may be helpful to understand the safety and delivery
challenges of engineered microbes. The approaches that may be considered when using
engineered bacteria for cancer immunotherapy are broadly classified into three main
groups:
1. Local. These approaches are designed to deliver an effector that works predominantly within the tumor. This approach is never absolute, as even radiotherapy has
been described to result in responses in distant tumors, i.e., abscopal effect. An example of a locally acting program is the C. novyi-NT approach (see Table 1). The strain is
designed to colonize the necrotic core of tumors and induce lysis of tumor cells,
thereby causing a local immune response. A phase 1 clinical trial has evaluated the
safety of this approach in patients with solid tumors following a single intratumoral
injection. In approximately half of the patients who received an injection, colonization of the injected tumor and tumor lysis occurred, and in 21% of patients, shrinkage
of the injected tumor by > 10% was demonstrated. A delivery challenge with this
approach is that not all solid tumors have an anaerobic core, therefore replication
and colonization may be inconsistent [99]. In addition, since Clostridia are spore
formers, the risk of seeding poorly oxygenated but noncancerous areas may necessi-
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tate long-term antibiotic treatment following dosing.
2. Systemic. These approaches are based primarily on triggering a systemic immune
response, i.e., vaccination, in which the bacteria are not required to be near the tumor
site itself. In this setting, bacteria are delivered as an adjuvant to a separate antigen,
engineered to express a tumor-related antigen or rely on the presence of sufficient
tumor antigen present in vivo. A good example of this approach is VXM01 (see
Table 3), which is an attenuated strain of Salmonella containing a plasmid expressing
VEGFR2. VEGFR2 is overexpressed in several tumor types and plays an important
role in angiogenesis. VXM01 is taken orally, where the plasmid is delivered to lymphoid tissue in the gastrointestinal tract and results in the development of a
VEGFR2-specific T-cell response [144]. This approach targets angiogenesis in tumors
and thereby an antitumor response. In a clinical trial in patients with advanced glioblastoma, VXM01 in combination with avelumab resulted in partial responses in three of
nine patients [102].
3. Local-systemic. These approaches are designed based on the delivery of bacteria to
the site of the tumor to illicit a local immune response and rely on subsequent systemic
T-cell responses to control noninjected lesions and prevent a long-term recurrence.
Delivery of the microbe to the tumor itself may result in superior priming of the antitumor response and induce stronger antitumor activity. Two examples of this approach
are bacTRL-IL-12 and SYNB1891. bacTRL-IL-12 is based on modified
Bifidobacterium longum encoding IL-12. Following intravenous administration, the bacteria are designed to colonize tumors, deliver the IL-12 transgene to mammalian cells
within the tumor, increase local expression of IL-12, and drive a local response
followed by a systemic antitumor immune response. In contrast, SYNB1891 is an
engineered strain based on E. coli Nissle that was designed to express a STING agonist.
This LBP is delivered by intratumoral injection, where the bacteria are hypothesized to
live in the TME, stimulate a local immune response, and, following engulfment of the
strain by antigen-presenting cells, generate a tumor-specific T-cell response [63].
The most direct route to delivering bacteria to the TME is by intratumoral (IT) injection.
The advantage to this approach is that high numbers of bacteria are delivered consistently,
resulting in less dependence on growth within the tumor, thereby enabling auxotrophs
and other safety features. Disadvantages of this route include its limitation to tumors that
are accessible via IT injection and its reliance on a robust immune response to address
noninjected lesions, realizing that most tumors affect multiple sites. However, advances
in the procedure of injecting tumors using interventional radiology approaches have
increased the number of tumors that are accessible for IT injection [145]. The advantage
of an intravenous route of administration is that bacteria are delivered to, and able to colonize multiple tumor sites. The disadvantage is that a number of the nonpathogenic chassis organisms used for engineered microbial approaches are serum sensitive and may be
53Engineered microbes for cancer immunotherapy
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