Tumour-targeting bacteria engineered to fight cancer

Abstract

Recent advances in targeted therapy and immunotherapy have once again raised the hope that a cure might be within reach for many cancer types. Yet, most late-stage cancers are either insensitive to the therapies to begin with or develop resistance later. Therapy with live tumour-targeting bacteria provides a unique option to meet these challenges. Compared with most other therapeutics, the effectiveness of tumour-targeting bacteria is not directly affected by the 'genetic makeup' of a tumour. Bacteria initiate their direct antitumour effects from deep within the tumour, followed by innate and adaptive antitumour immune responses. As microscopic 'robotic factories', bacterial vectors can be reprogrammed following simple genetic rules or sophisticated synthetic bioengineering principles to produce and deliver anticancer agents on the basis of clinical needs. Therapeutic approaches using live tumour-targeting bacteria can either be applied as a monotherapy or complement other anticancer therapies to achieve better clinical outcomes. In this Review, we summarize the potential benefits and challenges of this approach. We discuss how live bacteria selectively induce tumour regression and provide examples to illustrate different ways to engineer bacteria for improved safety and efficacy. Finally, we share our experience and insights on oncology clinical trials with tumour-targeting bacteria, including a discussion of the regulatory issues.

Fig. 1.. Mechanisms of tumor destruction by live tumor-targeting bacteria.

Different bacterial species employ both shared and unique intrinsic mechanisms to destroy cancer. a. Salmonella. (1) Uncontrolled bacterial multiplication can lead to bursting of the invaded tumor cells. Intracellular bacteria may also kill tumor cells by inducing apoptosis or autophagy^-. (2) Macrophages and dendritic cells in Salmonella-colonized tumors secrete IL-1β responsible for the antitumor activity. The elevated IL-1β secretion requires both LPS-induced TLR4 signaling and inflammasome activation in macrophage following phagocytosis of Salmonella-damaged tumor cells. (3) LPS elicits TNFα expression through CD14, TLR4 and MyD88^,, (4) leading to disruption of the tumor vasculature. (5) Flagellin induces an NK cell-mediated antitumor response dependent on perforin, as well as (6) release of IFNγ, a critical cytokine for both innate and adaptive immunity, from NK cells through a TLR-independent pathway involving IL-18 and Myd88. (7) Flagellin also enhances a TLR5 and CD8⁺ T cell-dependent antitumor response in a peptide vaccine-based immunotherapeutic setting, and (8) decreases frequency of CD4⁺CD25⁺ regulatory T cells (Treg). (9) In addition, flagellin can directly suppress tumor cell proliferation through TLR5 signaling. (10) Salmonella induces upregulation of connexin 43 (Cx43)^,, , leading to gap junction formation between tumor cells and dendritic cells (DC), which promotes transfer and cross-presentation of processed tumor antigenic peptides. (11) Upregulation of Cx43 in tumor cells also reduces expression of the immunosuppressive indoleamine 2,3-dioxygenase (IDO). (12) Both tumor antigen cross-presentation by DC and decreased IDO further activate CD8⁺ T cells. b. Listeria. (13) Listeria can directly kill tumor cells through the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)-mediated production of reactive oxygen species (ROS) and intracellular calcium mobilization. (14) The immunogenic tumor cell death caused by high levels of ROS activates CD8⁺ T cells responsible for eliminating both primary tumors and metastases^,. (15) Listeria infects the immunosuppressive MDSC and alters a subpopulation of these cells into an immune-stimulating phenotype characterized by elevated production of IL-12, which is correlated with improved CD8⁺ T cell and NK cell responses. (16) Listeria vaccine strains also inhibits MDSC and Treg^,. c. Clostridia. (17) Direct tumor destruction is caused by a variety of exotoxins secreted by the colonizing clostridia, some of which (e.g. phospholipases, haemolysins, lipases) can damage membrane structures while others are internalized to interfere with critical cellular functions^-. (18) Similar to infection by other bacterial species, the clostridial infection results in an initial accumulation of granulocytes and macrophages at the infection site ^,. This first line of defense prevents the colonizing bacteria from invading into surrounding normal tissues as well as sufficiently perfused and oxygenized tumor regions^,. (19) The cellular response results in elevated cytokines and chemokines that orchestrate a concerted immune response^,. Clostridia can also trigger the release of TRAIL from neutrophils, killing cancer cells through activation of apoptosis. (20) At later time points, adaptive immune cells including CD8⁺ T lymphocytes are recruited to fight cancer.

Fig. 1.. Mechanisms of tumor destruction by live tumor-targeting bacteria.

Different bacterial species employ both shared and unique intrinsic mechanisms to destroy cancer. a. Salmonella. (1) Uncontrolled bacterial multiplication can lead to bursting of the invaded tumor cells. Intracellular bacteria may also kill tumor cells by inducing apoptosis or autophagy^-. (2) Macrophages and dendritic cells in Salmonella-colonized tumors secrete IL-1β responsible for the antitumor activity. The elevated IL-1β secretion requires both LPS-induced TLR4 signaling and inflammasome activation in macrophage following phagocytosis of Salmonella-damaged tumor cells. (3) LPS elicits TNFα expression through CD14, TLR4 and MyD88^,, (4) leading to disruption of the tumor vasculature. (5) Flagellin induces an NK cell-mediated antitumor response dependent on perforin, as well as (6) release of IFNγ, a critical cytokine for both innate and adaptive immunity, from NK cells through a TLR-independent pathway involving IL-18 and Myd88. (7) Flagellin also enhances a TLR5 and CD8⁺ T cell-dependent antitumor response in a peptide vaccine-based immunotherapeutic setting, and (8) decreases frequency of CD4⁺CD25⁺ regulatory T cells (Treg). (9) In addition, flagellin can directly suppress tumor cell proliferation through TLR5 signaling. (10) Salmonella induces upregulation of connexin 43 (Cx43)^,, , leading to gap junction formation between tumor cells and dendritic cells (DC), which promotes transfer and cross-presentation of processed tumor antigenic peptides. (11) Upregulation of Cx43 in tumor cells also reduces expression of the immunosuppressive indoleamine 2,3-dioxygenase (IDO). (12) Both tumor antigen cross-presentation by DC and decreased IDO further activate CD8⁺ T cells. b. Listeria. (13) Listeria can directly kill tumor cells through the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)-mediated production of reactive oxygen species (ROS) and intracellular calcium mobilization. (14) The immunogenic tumor cell death caused by high levels of ROS activates CD8⁺ T cells responsible for eliminating both primary tumors and metastases^,. (15) Listeria infects the immunosuppressive MDSC and alters a subpopulation of these cells into an immune-stimulating phenotype characterized by elevated production of IL-12, which is correlated with improved CD8⁺ T cell and NK cell responses. (16) Listeria vaccine strains also inhibits MDSC and Treg^,. c. Clostridia. (17) Direct tumor destruction is caused by a variety of exotoxins secreted by the colonizing clostridia, some of which (e.g. phospholipases, haemolysins, lipases) can damage membrane structures while others are internalized to interfere with critical cellular functions^-. (18) Similar to infection by other bacterial species, the clostridial infection results in an initial accumulation of granulocytes and macrophages at the infection site ^,. This first line of defense prevents the colonizing bacteria from invading into surrounding normal tissues as well as sufficiently perfused and oxygenized tumor regions^,. (19) The cellular response results in elevated cytokines and chemokines that orchestrate a concerted immune response^,. Clostridia can also trigger the release of TRAIL from neutrophils, killing cancer cells through activation of apoptosis. (20) At later time points, adaptive immune cells including CD8⁺ T lymphocytes are recruited to fight cancer.

Fig. 1.. Mechanisms of tumor destruction by live tumor-targeting bacteria.

Different bacterial species employ both shared and unique intrinsic mechanisms to destroy cancer. a. Salmonella. (1) Uncontrolled bacterial multiplication can lead to bursting of the invaded tumor cells. Intracellular bacteria may also kill tumor cells by inducing apoptosis or autophagy^-. (2) Macrophages and dendritic cells in Salmonella-colonized tumors secrete IL-1β responsible for the antitumor activity. The elevated IL-1β secretion requires both LPS-induced TLR4 signaling and inflammasome activation in macrophage following phagocytosis of Salmonella-damaged tumor cells. (3) LPS elicits TNFα expression through CD14, TLR4 and MyD88^,, (4) leading to disruption of the tumor vasculature. (5) Flagellin induces an NK cell-mediated antitumor response dependent on perforin, as well as (6) release of IFNγ, a critical cytokine for both innate and adaptive immunity, from NK cells through a TLR-independent pathway involving IL-18 and Myd88. (7) Flagellin also enhances a TLR5 and CD8⁺ T cell-dependent antitumor response in a peptide vaccine-based immunotherapeutic setting, and (8) decreases frequency of CD4⁺CD25⁺ regulatory T cells (Treg). (9) In addition, flagellin can directly suppress tumor cell proliferation through TLR5 signaling. (10) Salmonella induces upregulation of connexin 43 (Cx43)^,, , leading to gap junction formation between tumor cells and dendritic cells (DC), which promotes transfer and cross-presentation of processed tumor antigenic peptides. (11) Upregulation of Cx43 in tumor cells also reduces expression of the immunosuppressive indoleamine 2,3-dioxygenase (IDO). (12) Both tumor antigen cross-presentation by DC and decreased IDO further activate CD8⁺ T cells. b. Listeria. (13) Listeria can directly kill tumor cells through the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)-mediated production of reactive oxygen species (ROS) and intracellular calcium mobilization. (14) The immunogenic tumor cell death caused by high levels of ROS activates CD8⁺ T cells responsible for eliminating both primary tumors and metastases^,. (15) Listeria infects the immunosuppressive MDSC and alters a subpopulation of these cells into an immune-stimulating phenotype characterized by elevated production of IL-12, which is correlated with improved CD8⁺ T cell and NK cell responses. (16) Listeria vaccine strains also inhibits MDSC and Treg^,. c. Clostridia. (17) Direct tumor destruction is caused by a variety of exotoxins secreted by the colonizing clostridia, some of which (e.g. phospholipases, haemolysins, lipases) can damage membrane structures while others are internalized to interfere with critical cellular functions^-. (18) Similar to infection by other bacterial species, the clostridial infection results in an initial accumulation of granulocytes and macrophages at the infection site ^,. This first line of defense prevents the colonizing bacteria from invading into surrounding normal tissues as well as sufficiently perfused and oxygenized tumor regions^,. (19) The cellular response results in elevated cytokines and chemokines that orchestrate a concerted immune response^,. Clostridia can also trigger the release of TRAIL from neutrophils, killing cancer cells through activation of apoptosis. (20) At later time points, adaptive immune cells including CD8⁺ T lymphocytes are recruited to fight cancer.

Fig. 2.. Inducible promoters used for targeted colonization and payload expression.

a. Various inducible promoters can be used for either tumor-selective expression or temporally or spatially controlled expression. (1) A Salmonella strain was engineered such that an essential gene was placed under the control of a hypoxia-inducible promoter, while expression of an inhibitory antisense RNA for this gene was activated by an oxygen-inducible promoter to minimize basal level expression in oxygenated normal tissues. This strain showed a robust tumor colonization and greatly enhanced clearance from normal tissues, thus resulting in a substantially improved safety profile compared to the parental strain. Hypoxia-inducible promoters have also been used to direct the expression of effector genes such as those encoding cytotoxic proteins, which requires tighter control for safety reasons. (2) Promoter elements responsive to low pH were among the ones identified to be active in tumor microenvironment in studies using “promoter traps” (see below). (3) A genetic circuit that can be triggered by glucose gradients often present in solid tumors has also been used to engineer bacteria, enabling them to express antitumor proteins in metabolically more active tumor regions. (4) Exogenously applied transcriptional inducers such as L-arabinose, acetyl salicylic acid and doxycycline can tightly regulate the relevant inducible promoters introduced into bacteria^,,,-, providing a means to control the expression of effector genes in a temporal fashion. (5) Ionic radiation at as low as 2 Gy has also been shown to activate the recA promoter on a plasmid transfected into Clostridium^-, raising the possibility to regulate effector gene expression with focused radiation treatment at clinically relevant doses (2 Gy is similar to a typical fractionated dose used in radiation therapy in an adjuvant setting for solid tumors). b. “Promoter traps” have been employed to identify promoter elements active in the tumor microenvironment^,,. “Promoter trap” libraries can be constructed by transforming bacteria with either (6) plasmids containing random genomic DNA fragments cloned upstream of a promoterless reporter gene, or (7) transposons containing a promoterless reporter gene which integrate randomly into the bacterial genome. These “promoter trap” libraries can be either (8) injected into experimental tumors or (9) co-cultured with tumor cells. Bacteria are then recovered and analyzed for reporter activities. Clones with high reporter activities are likely to contain promoter elements active in the tumor microenvironment.

Fig. 2.. Inducible promoters used for targeted colonization and payload expression.

a. Various inducible promoters can be used for either tumor-selective expression or temporally or spatially controlled expression. (1) A Salmonella strain was engineered such that an essential gene was placed under the control of a hypoxia-inducible promoter, while expression of an inhibitory antisense RNA for this gene was activated by an oxygen-inducible promoter to minimize basal level expression in oxygenated normal tissues. This strain showed a robust tumor colonization and greatly enhanced clearance from normal tissues, thus resulting in a substantially improved safety profile compared to the parental strain. Hypoxia-inducible promoters have also been used to direct the expression of effector genes such as those encoding cytotoxic proteins, which requires tighter control for safety reasons. (2) Promoter elements responsive to low pH were among the ones identified to be active in tumor microenvironment in studies using “promoter traps” (see below). (3) A genetic circuit that can be triggered by glucose gradients often present in solid tumors has also been used to engineer bacteria, enabling them to express antitumor proteins in metabolically more active tumor regions. (4) Exogenously applied transcriptional inducers such as L-arabinose, acetyl salicylic acid and doxycycline can tightly regulate the relevant inducible promoters introduced into bacteria^,,,-, providing a means to control the expression of effector genes in a temporal fashion. (5) Ionic radiation at as low as 2 Gy has also been shown to activate the recA promoter on a plasmid transfected into Clostridium^-, raising the possibility to regulate effector gene expression with focused radiation treatment at clinically relevant doses (2 Gy is similar to a typical fractionated dose used in radiation therapy in an adjuvant setting for solid tumors). b. “Promoter traps” have been employed to identify promoter elements active in the tumor microenvironment^,,. “Promoter trap” libraries can be constructed by transforming bacteria with either (6) plasmids containing random genomic DNA fragments cloned upstream of a promoterless reporter gene, or (7) transposons containing a promoterless reporter gene which integrate randomly into the bacterial genome. These “promoter trap” libraries can be either (8) injected into experimental tumors or (9) co-cultured with tumor cells. Bacteria are then recovered and analyzed for reporter activities. Clones with high reporter activities are likely to contain promoter elements active in the tumor microenvironment.

Fig. 3.. Gene circuit for a transcriptional program regulating bacterial activities at the population level.

Illustrated is an example of a sophisticated gene circuit for a transcriptional program enabling synchronized population control and therapeutic payload release in repeated cycles. (1) The AHL-bound transcription factor LuxR interacts with and activates promoter PluxI that drives the expression of (from top to bottom) the AHL synthase LuxI to establish a positive feedback loop, the therapeutic payload, and the bacteriophage φX174 protein E to lyse the bacteria. CDS, coding sequence. (2) The AHL signaling molecules diffuse freely across the cell membranes, enabling synchronization of neighboring bacterial cells in the population for a concerted action. At low densities of the bacterial population, AHL molecules diffuse predominately out of bacterial cells, leaving the gene circuit inactive. Increased population density allows AHL molecules inside the majority of the bacterial cells to accumulate and reach a threshold concentration required to activate the gene circuit. (3) Synchronized activation of the transcriptional program leads to simultaneous lysis of bacterial cells in the population by protein E as well as a burst of therapeutic payload release. (4) The small number of bacteria surviving the lysis repopulate and kick off another cycle of lysis and payload release.